"j^'jir"ti«v,3'% Hsitt Ofallege of Agriculture At ({(acneU Iniuetaitg Dtltata. ^. ^. Htbrarg Cornell University Library QH 231.L5 The microtomist's vade-mecum; a handbook 3 1924 003 021 494 Cornell University Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003021494 THE MICROTOMIST'S VADE-MECUM A HANDBOOK OF THE METHODS OF MICROSCOPIC ANATOMY BY ARTHUR BOLLES LEE, Hon. F.R.M.S. EIGHTH EDITION EDITED By J. BRONTE GATENBY, B.A,, B.Sc, D.PhU. (Oxon.), D.Sc. (Lond.), F.R.M.S., SOMETIME LEOTUREIl IN HISTOLOGY, OXITOKD ; LECTUEBR IN OTTOIOGY AND SENIOR ASSISTANT IS ZOOIOGT, UNIVERSITY COLLEGE, LONDON, AND SENIOK DEMY, MAGDALEN COLLEGE, OXFORD. • With the collaboration of W. M. PAYLISS, M.A., D.Sc. (Oxon.), F.R.S., F.R.M.S., PROFESSOR OF GENERAL PHYSIOLOGY IN UNIVERSITY COLLEGE, LONDON: C. DA FANO, M.D., F.R.M.S., L.D. ON MORBID ANATOMY, UNIVERSITY OF PAVIA (HALT), LECTURER IN HISTOLOGY, KING'S COLLEGE, LONDON ; A. DREW, D.Sc. (Lond.\ F.R.M.S., IMPERIAL CANCER RESEARCH FUND, LONDON ; W. CRAMER, Ph.D., D.Sc., M.R.C.S., L.R.C.P., F.R.M.S., IMPERIAL CANCER RESEARCH FUND, LONDON; AND J. THORNTON GARTER, F.R.M.S., F.Z.S., HON. RESEARCH ASSISTANT, UNIVERSITY COLLEGE, LONDON. ..It PHILADELPHIA BLAKISTON'S SON & CO. 1012 WALNUT STREET 1921 Printed in Great Britain PEEFACE TO EIGHTH EDITION. In the preparation of this new edition of Dr. Belles Lee's well- known book I have received the assistance of Professor W. M. Bayliss, Dr. C. Da Fano, Dr. A. Drew, Dr. W. Cramer, and Mr. J. Thornton Carter. It has been almost entirely due to these workers that my plans for the new edition have been able to be carried out in the way I wished. Thanks to them, this book may be considered a most complete and stimulating book of reference for the research worker. Professor W. M. Bayliss, to whom I am especially grateful, has rewritten Chapter XI. on " Staining." Dr. C. Da Fano has revised, and in some cases almost completely rewritten, the difificult chapters on " Neurological Techniques," which constitute indeed a special branch of microtomy. Dr. A. Drew has completely rewritten the valuable section on " Protozoa " ; while from Dr. W. Cramer's pen has come most of the article on " Fatty Substances," which will bring to the notice of embryologists and histologists the newest advances in the micro-chemistry of this subject. Mr. J. Thornton Carter has revised the section on "Teeth and Bone." I have personally been responsible for the rest of the book, besides having written special chapters or sections on " Chromatin, Chromosomes, Nucleoli, Glycogen, Iron, Yolk, Fat, Mitochondria, Golgi Apparatus and Benzidine Dyes " ; I have completely rewritten the section on " Mammalian Embryological Methods." In the sections dealing with the cytoplasmic inclusions, most of the various structures, known as "attraction sphere rodlets," "idiozome," " Golgi-Kopsch apparat," " nebenkern batonettes," etc., are grouped under the term " Golgi apparatus," as now seems justifiable in view of the results of modern researches on the subject. A small, yet very important, addition to the present volume has been the inclusion of two new methods for staining bacteria in tissue (§§ 689, 761). The histologist and cytologist are often puzzled to know whether certain enigmatic bodies they find are or are not bacterial in nature. vi PREFACE. An addition which is likely to' be welcomed, and to make the book more useful, is a special chapter for students (Chapter XXXVII.). I had especially desired to have a chapter dealing with " Tissue Culture," and had asked my friend Mr. H. M. Carleton, of Oxford, to undertake it ; but as illness prevented his carrying out the work, I was myself obliged at the eleventh hour to write the article. Dr. A. Drew was most helpful here, and Dr. Strangeways, of Cambridge, lent me some literature on the subject. For advice and encouragement in this arduous task I have to thank my many friends. Dr. BoUes Lee sent me his blessing and three hundred references ; Professor J. P. Hill placed his note- books and experience at my disposal; Dr. J. A. Murray, of the Cancer Research Fund, proved a perfect mine of suggestions, and I dug" freely at this source. I am very grateful to both Dr. Murray and his amiable staff, of whom Dr. Drew and Dr. Cramer have helped me most. Professor Starling, of University College, Pro- fessor B. S. Goodrich, of Oxford, Professor Sherrington, of Oxford, Professor Boycott, of University College Medical School, Professor E. B. Wilson, of Columbia University, and Professor D. M. S. Watson, of University College, London, helped me in one way or another. Professor W. M. Bayliss and Dr. Oscar Brady gave me valuable information on certain chemical aspects. My friends at the Rothamsted Station, especially Mr. Ward Cutler and Dr. Imms, were very helpful. Dr. Allen, of Plymouth, and Dr. Orton kindly answered inquiries. Great care has been taken in the preparation of the Index, and I have to thank my sister for much assistance. In writing a book of this kind, it is difficult at times to ascertain exactly who did originate some one method, and if we have occa- sionally overlooked the original source we will be glad to have such lapsus calami rectified in some future edition. Finally, it must be remarked that the arrangement and method of exposition of the new material closely follows that of Dr. BoUes Lee, while the bulk of the book is still largely due to him. J. BRONTE GATENBY. University College, London. CONTENTS PAET I, PAGE CHAPTER I. Introductory ......... i CHAPTER II. Killing . . . . . . . . . .11 CHAPTER III. Fixing and Hardening . . . . . . .18 CHAPTER IV. Fixing and Hardening Agents — ^Mineral Acids and their Salts .......... 29 CHAPTER V. Fixing and Hajidening Agents — Chlorides, Organic Acids, AND Others ......... 44 CHAPTER VI. De-alcoholisation and Clearing Agents .... 65 CHAPTER VII. Imbedding Methods — Introduction 71 CHAPTER VIII. Imbedding Methods : Paraffin and other Fusion Masses . 76 Paraffin, 76 ; Gelatin, 92. CHAPTER IX. Collodion (Celloidin) and other Imbedding Methods . 95 Collodion or Celloidin, 95 ; other Cold Masses, 106 ; Grind- ing Masses, 108 ; Freezing, 109. viii CONTENTS. PAGE CHAPTER X. Serial Section Mounting . . . . . . .111 Methods for Paraffin Sections, 111 ; Methods for Watery- Sections, 116 ; Methods for Celloidin Sections, 116. CHAPTER XI. Staining . . . ... 120 CHAPTER XII. Carmine and Cochineal Stains . . . . . .135 Theory of Carmine Staining, 135 ; Aqueous Carmines, Acid, 136 ; Neutral and Alkaline, 140 ; Alcoholic Carmines and Cochineals, 141. CHAPTER XIII. HjEMATEIN (HiEMATOXTLIN) StAINS ..... 145 Theory of Staining with Hsematoxylin, 145 ; Iron-hsematein Lakes, 147 ; Aluminium-hsematein Lakes, 151 ; other Hsema- tein Compounds, 155. CHAPTER XIV. Nuclear Stains with Coal-tar Dyes ... . 159 Progressive Stains, 159 ; Regressive Stains, 162. CHAPTER XV. Plasma Stains with Coal-tar Dyes . . . .171 CHAPTER XVI. Methtlen Blue . . . . . . . . .186 CHAPTER XVII. Metallic Stains (Impregnation Methods) .... 197 Silver, 198 ; Gold, 202 ; other Metallic Stains, 208. CHAPTER XVIII. Other Stains and Combinations . . . . . .211 Carmine Combinations, 212 ; Hsematein Combinations, 213. CHAPTER XIX. Examination and Preservation Media . . . .216 Aqueous Liquids, 217 ; Mercurial Liquids, 220 ; other Fluids, 220 ; Glycerin Media, 222 ; Glycerin Jellies, 223 ; High Refractive Liquids, 224 ; Resinous Media, 225. CONTENTS. ix CHAPTER XX. Cements and Varnishes PAGE 229 PAET II. SPECIAL METHODS AND EXAMPLES. CHAPTER XXI. Injection — Gelatin Masses (Waem) 232 Carmine, 234 ; Blue, 236 ; other Colours, 237. CHAPTER XXII. Injections — other Masses (Cold) 238 CHAPTER XXIII. Maceration, Digestion, and Corrosion .... 243 Maceration, 243 ; Digestion, 248 ; Corrosion, 249. CHAPTER XXIV. Decalcification, Desilicification, and Bleaching . . 251 Decalcification, 251 ; Desilicification, 255 ; BleacMng, 255. CHAPTER XXV. Embrtolosical Methods ....... 258 Mammalia, 263 ; Aves, 271 ; Reptilia, 274 ; Amphibia, 276 ; Pisces, 279 ; Tunicata, 281 ; Bryozoa, 282 ; Mollusca, 282 ; Arthropoda, 284 ; Vermes, 288. CHAPTER XXVI. Cttological Methods ........ 292 Glycogen, 294 ; Iron, 297 ; Chromosomes, 303 ; Cytoplasmic Inclusions, 316. CHAPTER XXVII. • Tegumentary Organs ........ 339 CHAPTER XXVIII. Muscle and Tendon (Nerye-endings) ..... 344 Striated Muscle, 344 ; Electric Organs, 345 ; Tendon, 347 ; Smooth Muscle, 348. M. b X CONTENTS. PAGE CHAPTEE XXIX. Connective Tissues ........ 350 Connective Tissue, 350 ; Elastic, 352 ; Plasma Cells 354 ; JPatty Substances, 356 ; Bone and Cartilage, 369 ; Skele- tons of Embryos, 377. CHAPTER XXX. Blood and Glands ........ 379 Blood, 379 ; Benzidine Dyes, 388 ; Glands, 391. CHAPTEE XXXI. Nervous System — General Methods ..... 397 CHAPTEE XXXII. Nervous System — Special Methods, chiefly Cytological . 410 Cells, 410 ; Cells and Fibres, 416 ; Golgi's Internal Apparatus, 435 ; Medullary Sheath, 439. CHAPTEE XXXIII. Myelin Stains (Weigert and others) . . . . 442 CHAPTEE XXXIV. Axis-Cylinder and Dendrite Stains (Golgi and others) . 454 CHAPTEE XXXV. Neuroglia and Sense Organs ...... 479 Neuroglia, 479 ; Eetina, 493 ; Inner Ear, 496. CHAPTEE XXXVI. Methods for Invertebrates ...... 499 Tunica ta, 499 ; Molluscoida, 500 ; MoUusca, 500 ; Arthropoda, 504 ; Vermes, 509 ; Echinodermata, 518 ; Coelenterata, 521 ; Porifera, 525 ; Protozoa, 526. CHAPTEE XXXVII. Cultivation of Tissue " in vitro " and its Technique . . 560 CHAPTEE XXXVIII. A Guide foe Students of Microtomy . . . 556 APPENDIX 563 INDEX ... 564 THE MICROTOMIST'S VADE-MECUM. PART I. CHAPTER I. INTRODUCTOEY. 1. The General Method. — The methods of modern microscopic anatomy may be roughly classed as General and Special. There is a General or Normal method which consists in carefuUy fixing the structures to be examined, staining them with a nuclear stain, dehydrating with alcohol, and mounting series of sections of the structures in balsam. It is by this method that the work is blocked out and very often finished. Special points are then studied, if necessary, by Special Methods, such as examination of the living tissue elements, in situ or in " indifferent " media ; fixation with special fixing agents ; staining with special stains ; dissociation by teasing or maceration ; injection ; impregnation ; and the like. There is a further distinction which may be made, and which may help to simplify matters. The processes of the preparation of tissues may be divided into two stages, Preliminary Preparation and Ulterior Preparation. Now the processes of preliminary preparation are essentially identical in all the methods, essential divergences being only found in the details of ulterior preparation. By prelimi- nary preparation is meant that group of processes whose object it is to get the tissues into a fit state for passing unharmed through all the ulterior processes to which it may be desired to submit them. It comprehends the operations of (1) kilUng ; (2) fixing ; (3) the washing and other manipulations necessary for removing thejfixing agent from the tissues, and substituting for it the preservative liquid or other reagents which it is desired to employ. Ulterior preparation comprehends the processes sketched out in §§ 3 et seq. 2. Preliminary Preparation. — The first thing to be done with any structure is to fix its histological elements. (This statement applies equally to all classes of objects, whether it be desired to cut them into sections or to treat them in any other special way.) Two 2 INTRODUCTORY. tilings are implied by the word " fixing " : first, the rapid killing of the element, so that it may not have time to change the form it had during Ufe, but is fixed in death in the attitude it normally had during life ; and second, the hardening of it to such a degree as may enable it to resist without further change of form the action of the reagents with which it may subsequently be treated. Without good fixation it is impossible to get good stains or good sections, or prepara- tions good in any way. The structure, having been duly fixed by one of the processes described in the chapter on Fixing Agents, is, except in special cases, washed in order to remove from the tissues as far as possible all traces of the fixing reagent. The kind of liquid with which washing out is done is not a matter of indiflerence. If corrosive subKmate (for instance), or osmic acid, or a solution into which chromic acid or a ohromate enters, have been used for fixing, the washing may be done with water. But if certain other agents, such as picric acid, have been used, the washing should be done with alcohol. The reason for this difference is that the first-named reagents (and, indeed, all the compounds of the heavy metals used for fixing) cause certain of the elements of the tissues to become less reactive, and partly or wholly insoluble in water. The combinations of picric acid and the elements of the tissues, on the other hand, are generally much more soluble in watery fluids. These operations having been duly performed, two roads become open; The object may be further prepared by what may be termed the wet method, in which all subsequent operations are performed by means of aqueous media. Or it may be further prepared by the dehydration method, which consists in treatment with successive alcohols of gradually increasing strength, final dehydration with absolute alcohol, imbibition with an essential oil or other so-called clearing agent which serves to remove the alcohol, and lastly either mounting at once in balsam or other resinous medium or imbedding in paraffin for the purpose of making sections. The dehydration method is the course which is generally preferred, chiefly because of its great superiority as regards the preservation of tissues. For the presence of water is the most important factor in the conditions that bring about the decomposition of organic matter, and its complete removal is the chief condition of permanent preservation. 3. Dehydration. — The further course of preparation by' the dehydration method is as follows :— At the same time that the superfluous fixing agent is being removed from the tissues, or as Boon as that is done, the water of the tissues must be removed. This is CHAPTER I. 3 necessary for two reasons : first, in the interest of preservation, as above explained ; and secondly, because all water must be removed in order to allow the tissues to be impregnated with the imbedding material necessary for section-cutting, or with the balsam with which they are to be finally preserved. This dehyd/ralion is per- formed as follows ; — The objects are brought into weak alcohol, and are then passed through successive alcohols of gradually increased strength, remaining in each the time necessary for complete satura- tion, and the last bath consisting of absolute or at least very strong alcohol. In dealing with, delicate objects, it may be necessary to take special precautions in order to avoid injury to them through the violent difEusion-curreiits that are set up in the passage from water to alcohol, or from one bath of alcohol to another of considerably different density. Some kinds of diffusion-apparatus may conveniently be used in these oases. The objects may be placed with some of their liquid in a tube corked at one end and closed at the.other by a diaphragm of muslin or chamois skin or other suitable membrane, the tube being then immersed in a vessel containing the grade of alcohol that it is desired to add to the liquid in the tube, and the whole allowed to remain until by di:Susion through the diaphragm the two liquids have become of equal density. Or, Cobb's differentiator (Proc. Liim. Soc, N.S.W., v, 1890, p. 157 ; Journ. Roy. Mic. Soc, 1890, p. 821) may be employed. Or, the apparatus of Haswell (Proc. Linn. Soc, N.S.W., vi, 1891, p. 433 ; Journ. Boy. Mic. Soc, 1892, p. 696). Or that of Cheatle, described in Journ. Pathol, and Bacteriol., i, 1892, p. 263, or Journ. Boy. Mic Soc, 1892, p. 892. Seealso ScHULTZE (Zeit ws«. Jlf*fc.,ii, 1885, p. 637) ; and SusuKi, ibid., 1909, p. 211 ; Kolstee (ibid., xvii, 1900, p. 294). The " Siebdosen," or sieve-dishes of Steinach, Zimmbkmann, and SucHANNEK (vide Zeit. wiss. Mih., iv, 1887, p. 433, and vii, 1890, p. 158), are useful for many purposes. See also Tisohatkin, ibid., xxiii, p. 46. PAXBCHrLD's perforated porcelain cylinders for washing {ibid., xii, 1896, p. 301) seem to be a very neat idea. See also the similar device of ScHAFPEB {ibid., xvi, 1900, p. 422 ; Journ. Boy. Mic. Soc, 1900, p. 394). For Ewalk'S' section -washing apparatus, see Zeit. Biol., xxxiv, 1897, p. 264. That of SoHOEBEL {ibid., xx, 1903, p. 168) is simple and efficient ; as also that of Keeigbaum {ibid., xxvii, 1910, p. 604). A capiUary siphon for the aspiration of liquids in the fixing, staining, and washing of suspended blood-corpuscles, sperm-cells, protozoa, and the Uke, is described by Ewaid, ibid., p. 263. It is sometimes stated that it is necessary that the last alcohol- bath should consist of absolute alcJohol. This, however, is incorrect, a strength of 95 per cent, being sufficient in most cases. For the small amount of water that remains in the tissues after. treatment with these grades of alcohol is efficiently removed in the bath of 1— z 4 INTRODUCTORY. clearing agent if a good clearing agent be employed. Oil of cedar will remove the remaining water from tissues saturated "with 95 per cent, alcohol ; oil of bergamot wiU " clear " from 90 per cent, alcohol, and anilin oil will clear from 70 per cent, alcohol. I am not aware of any substance that can entirely take the place of alcohol for dehydration and preservation. Acetone and methylal have been substituted for alcohol in the dehydration of methylen- blue preparations (Parker, Zool. Anz., 403, 1892, p. 376), and aniUn oil can be made to dehydrate watery sections if they be first mopped up with blotting-paper ; but a really efficient substitute for alcohol in general work remains yet to be discovered. 4. Preservation. — Considered as a mere dehy5.rating agent, alcohol fulfils its functions fairly well. But considered as a histo- logical preservative agent, it is far less satisfactory. If tissues be left in alcohol for only a few days before further preparation, injurious effects wiU perhaps not be very disagreeably evident. But it is otherwise if they are put away in it for many weeks or months before the final preparation is carried out. The dehydrating action of the alcohol being continuously prolonged, the minute structure of tissues is sometimes considerably altered by it ; they become over- hard and shrink, and become brittle, and their capacity for taking stains well becomes seriously diminished. Kultschitzky (Zeit. iviss. Mik., iv, 1887, p. 349) has proposed to remedy this by putting up objects after fixation and washing out with alcohol in ether, xylol, or toluol. Flemming (Arch. mik. Anat., xxxvii, 1891, p. 685) advises putting up objects after fixation in a mixture of alcohol, glycerin, and water, in about equal parts, pointing out that objects thus preserved may be at any moment either prepared for sectioning by treatment with pure alcohol or softened for dissection or teasing by a Uttle soaking in water, and that they do not become so hard and brittle as alcohol specimens, and retain their staining power much better. After extensive experience of this plan I can recom- mend it, and would only further suggest that the action of the liquid seems to me to be in many cases much improved by addition of a little acetic acid (say 0-5 to 0-75 per cent.). For material that is intended only for section-cutting, I find that by far the best plan is to clear (next §) and imbed at once in paraffin. This affords, as far as I can see, an absolutely perfect preservation. Cedar-wood oil is, I find, nearly, if not quite, as good as paraffin, so far as the preservation of the tissues is concerned, but of coxirse it is not so handy for storage. CHAPTER I. 5 5. Removal of Alcohol ; Clearing.— The water having been suflaciently removed, as described in § 3, the alcohol is in its turn removed from the tissues, and its place taken by some anhydrous substance, generally an essential oil, which is miscible with the material used for imbedding or mounting. This operation is generally known as Clearing. It is very important that the passage from the last alcohol to the clearing agent be a gradual one. This is effected by placing the clearing medium under the alcohol. A sufficient quantity of alcohol is placed in a tube (a watch-glass will do, but tubes are generally better), and then with a pipette a suffi- cient quantity of clearing medium is introduced at the bottom of the almhol. Or you may first put the clearing medium into the tube, and then carefully pour the alcohol on to the top of it. The two fluids mingle but slowly. The objects to be cleared, being now quietly put into the supernatant alcohol, float at the surface of separation of the two fluids, the exchange of fluids takes place gradually, and the objects slowly sink down into the lower layer. When they have sunk to the bottom, the alcohol may be drawn off with a pipette, and after some further lapse of time the objects will be found to be completely penetrated by the clearing medium. This method of making the passage from one fluid to another applies to all cases in which objects have to be transferred from a lighter to a denser fluid — for instance, from alcohol, or from water, to glycerin. This is a convenient stage for carrying out minute dissections^ if any such have to be done, a drop of clearing agent being a most helpful medium for carrying out such dissections (see § 9). At this point the course of treatment follows one of two different roads, according as the object is to be mounted direct in balsam (§ 8), or is first to be sectioned (§ 6). 6. Imbedding, and Treatment of Sections. — The objects are now imbedded. They are removed from the clearing medium, and soaked until thoroughly saturated in the imbedding medium. This is, for small objects, generally paraffin, liquefied by heat, and for large objects either paraffin or a solution of collodion or " celloidin " (in this last case the clearing may be omitted and the tissues be imbedded direct from the alcohol). The imbedding medium con- taining the object is then made to solidify, and sections are made with a microtome through the imbedding mass and the included objects. The sections are then mounted on a slide by one of the 6 INTRODUCTORY. methods described in the chapter on Serial Section Methods, the imbedding material is removed from them (in the case of parafl&n), they are stained in situ on the slide, dehydrated with alcohol, cleared, and mounted in balsam or damar. Or they may be stained, washed, dehydrated, and cleared in watch-glasses, and afterwards mounted as desired— the imbedding medium being first removed if desirable. Or, the material may be stained in bulk, before cutting the sections. In this case the object, after having been fixed and washed out, is taken from the water, or while still on its way through the lower alcohols (it should not be allowed to proceed to the higher grades of alcohol before staining, if that can be avoided), and passed through a bath of stain, then dehydrated with successive alcohols, passed through a clearing medium into paraffin, cut, and treated as above described, the sections in this case being mounted direct from the chloroform, xylol, or other solvent with which the parafiin is removed. If aqueous staining media be applied (and this is sometimes desir- able), the structures should either be stained in toto immediately after fixing and washing out, or sections may be stained on the slide, the objects, if delicate, being passed through successive baths of alcohol of gradually decreasing strength before being put into the aqueous stain. In my opinion it is generally advisable not to stain in bulk material that is intended to be sectioned ; by staining it as sections the staining can be much better controlled, and many excellent stains can in this way be employed that are not available for staining in bulk ; and of course sections can be stained much more rapidly than material in bulk. Balsam mounts of which the stain has faded, or which it may be desired to submit to some other staining process, or mount in some other medium, may often with great advantage be re-stained and re-mounted. All that is necessary is to put the slide into a tube of xylol or benzol till the cover falls off (about two days), wash well for some hours in clean xylol, and pass through alcohol into the new stain. Since this was pointed out to me by Dr. Henneguy I have unmounted and re-stained a large number of old preparations, and have succeeded in every case with series of sections mounted on Mayer's albumen, or by the water method. For shellac-mounted series, see E. Meyer, Biol. Centralb., x, 1890, p. 509, or last edition. The most convenient vessels, I find, in whicli to perform the various operations of staining, difEerentiating, dehydrating, clearing, etc., on the slide, are flat-bottomed corked glass tubes. I have mine made 10 CHAPTER I. 7 centimetres high and 27 millimetres internal diameter. Each of these wiQ then take two slides, Enghsh size, placed back to back. 7. R6sum6 of the General Method,— To sum up, you may either fix, wash out, stain, wash, dehydrate, clear, imbed, cut sections, clear and mount them in balsam ; or fix, wash, dehydrate, clear, imbed, cut, stain, wash, dehydrate, clear, and moimt — according to choice. 8. Preparation of Entire Objects, or of Material that is not to be sectioned. — The treatment of objects which can be studied without being cut into sections is identical with that above described, with the omission of those passages that relate to imbedding processes. Its normal course may be described as fixation, washing out, staining, treatment with successive alcohols of gradually increasing strength, final dehydration with absolute alcohol, clearing, and mounting in balsam. In the preparation of entire objects or structures that are intact and covered by an integument not easily permeable by Hquids, special care must be taken to avoid swelling from endosmosis on the passage of the objects from any of the liquids employed to a liquid of less density, or shrinkage from exosmosis on the passage to a liquid of greater density. This applies most specially to the passage from the last alcohol into the clearing medium. A slit should be made in the integument, if possible, so that the two fluids may mingle without hindrance.* And in all cases the passage is made gradual by placing the clearing medium under the alcohol, as described (§ 5). Fluids of high difEusibility should be employed as far as possible in all the processes. Fixing agents of great penetrating power (such as picric acid or alcoholic sublimate solution) should be employed where the objects present a not easily permeable integument. Washing out is done with successive alcohols, water being used only in the case of fixation by osmic acid, or the chromic mixtures or other fixing solutions that render washing by water imperative. Staining is done by preference with alcoholic staining media. The stains most to be recommended are Grenacher's borax-carmine, or one of Mayer's alcohoUc carminic acid or hsematein stains. Aqueous stains are more rarely indicated, though there are many cases in which they are admissible, and some in which they are preferable. 9. Minute Dissections.— These are best done, if necessary, in a drop of clearing agent. I recommend cedar-wood oil for this purpose as it gives to the tissues a consistency very favourable for dissection, whilst its viscosity serves to lend support to deUcate structures. Clove oil has a tendency to make tissues that have lain in it for r.ome 8 INTRODUCTORY. time very brittle. Tte brittleness is, however, sometimes very helpful in minute dissections. Another property of clove oil is that it does not easily spread itself over the surface of a slide, but has a tendency to form very convex drops, and this also makes it frequently a very convenient medium for making minute dissections in. If it be desired to dissect in a watery fluid, such as glycerin, it may be well to prepare the slide by spreading on it a thin layer of Mayer's albumen, and on this place a small drop of glycerin, or other dissect- ing medium. As soon as the dissection has been accomphshed, a cover may be let fall, horizontally, on to the preparation to keep the parts in place, and a weight placed on it. Then the moimt may be filled up with glycerin, or other mounting medium, run in under the cover, and closed, if desired, or instead of the albumen a solution of gelatin may be taken, and hardened in formol with the objects on it. For a balsam mount, after clove or cedar oil, Schallibaum's collodion may be taken, and the organs fixed in situ on this by adding xylol. 10. Instruments. — For all that concerns the mechanism and manipulation of the Microscope, see vol. i of Carpenter's The Microscope, eighth edition, 1891 ; paying particular attention to all that is said concerning the English and the Continental Models, pp. 254 to 261, the Substage, pp. 184 to 189, Condensers, pp. 289 to 316, and Tube Length, pp. 158 to 159. For information concerning the principles of construction and manipulation of the Microtome, see also Carpenter's The Micro- scope. Microtomes are instruments for the accurate production of thin slices of tissues. They are used both for cutting tissues that have acquired a certain favourable consistency through having been imbedded in paraffin, and also for cutting tissues that have been imbedded in softer masses, such as collodion, and tissues that have not been imbedded at aU. Not all microtomes are equally well adapted for all these three classes of work. The microtome of the zoologist should at all events be one that is well adapted for cutting imbedded material. Now there are two methods of imbedding in general use — the paraffin method and the celloidin method. In. the paraffin method the object is cut dry, frequently with the knife set square to the hne of section. In the celloidin method, as in the cutting of unimbedded tissues, it is generally cut wet, and'always with the knife set slanting. Some microtomes that are well adapted for the paraffin method are ill adapted for the celloidin method or the cutting of unimbedded CHAPTER I. 9 material, and vice versa. It may be well to possess the two sorts of instrument ; but if only one can be afforded it should be such as will give good work in either way. Microtomes fall further into two classes according as the knife and the surface of section of the object are (a) in a horizontal plane, or (b) in a vertical plane. The former offer greater facility for the orientation of the plane of section, which is an important point for the zoologist and embryologist. Amongst these may be mentioned (a) The " Sliding " Microtomes, in which the knife is carried on a sledge and moved against the object (those of Thoma, Schanze, EEiCHEE"'r, and others). The Thoma, of medium size, as made by R. Jung, Hebelstrasse, Heidelberg (No. 56 of his catalogue for 1911, which may be obtained from Mr. C. Baker, 244, High Holborn, London), is very suitable for the zoologist. It works equally well with either paraffin or celloidin, and can be adapted as a freezing microtome. But this (as is the case with the others above mentioned) will not always furnish work of the highest accuracy ; for the knife being only clamped at one end is liable to spring, and to give sections of unequal thickness. This defect is remedied in (6), a type of sliding microtomes in which the knife is clamped at both ends and is a fixture, the object being carried on a sledge and moved against it (Cambridge Scientific Instrument Company's large microtome, the Minot precision microtome, Leitz's, db G-root's, Jitng's " Tetrander." This last seems to be near perfection ; see the description by Mayer in Zeit. wiss. Mik., xxvii, 1910, p. 52 ; but is more cumbrous than is desirable for ordinary work. Class a also includes some instruments in which the knife is carried on a horizontal arm and swung against the object by a rotary movement (Jung, Roy, Fromme, Reichbrt, Thate, and others). I know nothing of these personally, but doubt their constant accuracy. Class B contains some very fine instruments, admirably adapted for the production of continuous ribbons of sections by the paraffin method, but not so well adapted for celloidin or other work in the wet way, or for soft objects. Amongst these are the New and Old Rocking Microtome, made by The Scientific Instrument Co., Cambridge, or by Swift & Son, or by Jung, or by van der Stad, Amsterdam ; the Minot, made by Bausch & Lome and the Spencer Model, or by Becker (Gottingen), or by Zimmermann (21, EmiUen- strasse, Leipzig) ; the Reinhold-Giltay, made by J. W. Giltay, Delft. For descriptions of the multitudinous models on the market see 10 INTRODUCTORY. the reports in the Zeit. wiss. Mik. and Journ. Roy. Micr. Soc, and the price lists of the instrument makers. 11. Staining Reagents and Chemicals. — For some years it has been difficult to obtain good dyes, but recently several new firms have succeeded in manufacturing materials suitable for the most critical work. The pre-war continental dyes were generally impure products, and since the new dyes being made are mostly purer, it is often necessary to make some slight alteration in the quantity of dye added to a staining mixture before the optimum staining 6ondition is reached. Details of such alteration necessary for British dyes should be sent to the Journal of the Royal Microscopical Society. I advise the reader to get his reagents and dyes from some well-known British firm which has specialised in the matter. Great Britain. — ^Flatters and Garnett, Ltd., 309, Oxford Road, Manchester (all sorts of chemicals, dyes and apparatus ; makers of Gilson's " Euparal ") ; the British Drug Houses, Ltd., Graham Street, City Road, London, N. 1 (makers of many sorts of dyes) ; The British Dyestuffs Corporation, Ltd., 70, Spring Gardens, Manchester (important makers of dyes ; London agents are, Baird and Tatlock, 14, Cross Street, Hatton Garden, B.C. 1) ; Hawksley & Sons, 83, Wigmore Street, Cavendish Square, W. 1 ; and Charles Baker, 244, High Holborn, W.C. (both firms are agents for Griibler, and for most microscope and accessory apparatus makers). For incubators, etc., for embedding and tissue-culture work, Charles Hearson & Co., Ltd., 235, Regent Street, W. 1. United States of America. — Eimer and Amend, 205 — 211, Third Avenue, New York ; Palo Company, 90, Maiden Lane, New York ; Edward Pennock, 3609, Woodland Avenue, Philadelphia, Pa. ; General Biological Supply House, 5508, Kimback Avenue, Chicago, 111. ; The Will Corporation, Rochester, N.Y. ; Paul Weiss, 1620, Arapahoe Street, Denver, Colorado. Germany. — G. Griibler and HoUborn, Chemiker, Leipzig, Germany. CHAPTER II. KILLING. 12. In the majority of cases, tlie first step in the preparation of an organ or organism consists in exposing it as rapidly and as com- pletely as possible to the action of one of the Fixing Agents that are discussed in the next chapter. The organ or organism is thus taken in the normal living state ; the fixing agent serves to bring about at the same time, and with sufficient rapidity, both the death of the organism and that of its histological elements. It should be noted that narcotisation generally implies some change in the cells, and most narcotics have to be applied for a long time. Such treatment is absolutely barred in material destined for careful cytological study. This applies especially to ether and chloroform, which are extremely injurious to cells : in the case of larger mammals like the cat and dog a preliminary treatment in ether or chloroform may be necessary, but directly after ansesthesia the animals' throats should be cut or they should be killed by a blow, if possible. Coal gas chambers are good for killing all mammals, and I do not believe carbon monoxide is hurtful to cells. Amphi- bians killed by chloroform are often completely spoilt for cytological purposes ; if the brain is not wanted, pith the animal. For birds the time-honoured custom of wringing their necks is recommended. In the case of small lizards, newts and such live stock it is a gdfcd plan to cut off their heads quickly with strong scissors. If the material is wanted for chromosome or mitochondria work look up these sections for special directions. But these methods are by no means applicable to all cases. There are many animals, especially such as are of a soft consistence, and deprived of any rigid skeleton, but possessing a considerable faculty of contractility, which if thus treated contract violently, and die in a state of contraction that renders them unfit for study. In these cases special methods of killing must be resorted to. Speaking generally, there are two ways of dealing with these difficult cases. You may kill the animal so suddenly that it has not time to con- tract : or you may paralyse it by narcotics before killing it. See also under " Chromosomes," § 652, and " Mitochondria," § 673. 12 KILLING. Sudden Killing. 13. Heat.— Tie application of Heat afEords a means of killing suddenly. By it the tissues are more or less fixed at the same time that somatic death is brought about. The difficulty consists in hitting off the right temperature, which is of course different for different objects. I think that 80° to 90° C. will generally be amply sufficient, and that very frequently it will not be necessary to go beyond 60° C. An exposure to heat for a few seconds will generally suffice. Small objects (Protozoa, Hydroids, Bryozoa) may be brought into a drop of water in a watch-glass or on a slide, and heated over the flame of a spirit-lamp. For large objects, the water or other liquid employed as the vehicle of the heat may be heated beforehand and the animals thrown into it. As soon as it is supposed that the protoplasm of the tissues is coagu- lated throughout, the animals should be brought into alcohol (30 to 70 per cent, alcohol) (if water be employed as the heating agent). An excellent plan for preparing many marine animals is to kill them in hot fresh water. Some of the larger Nemertians are better preserved by this method than by any other with which I am acquainted. 14. Slowly Contracting Animals. — ^Animals that contract but slowly, such as Alcyonium and Yeretillum, and some Tunicates, such as Pyrosoma, are very well killed by throwing them into some very quickly acting fixing liquid, used either hot or cold. Glacial or very strong acetic add (van Beneden's method) is an excellent reagent for this purpose ; it may be used, for example, with some Medusae. After an immersion of a few seconds or a few minutes, according to, the size of the animals, they should be brought into alcohol of at least 50 per cent, strength. Lemon juice employed in this way has gii^n me very good results with small Annelids and Hirudinea. Corrosive sublimate is another excellent reagent for this purpose. Narcotisation. 15. Narcotisation is performed by adding some anaesthetic sub- stance very gradually, in very small doses, to the water containing the animals, and waiting patiently for it to take effect slowly. Menthol. — Now used with great success for ansesthetising large marine animals. Place latter in clean vessel, and sprinkle over surface of water, menthol crystals. As the latter dissolve the animals expand. In from twelve to twenty-four hours they may be transferred to a fixer. Very good for Anemones, Holothuria, Asoidia and many MoUusca. (Personal communication from Dr. E. J. Allen, Plymouth.) 16. Nicotin in solution (Andres, Atti R. Accad. dei Lined, v, 1880, p. 9). Andres employs a solution of 1 gramme of nicotin in a litre CHAPTER II. 13 of sea water. The animal is placed in a jar containing half a litre of sea water, and the solution of nicotin is gradually conducted into it by means of a thread, acting as a syphon, of such a thickness as to be capable of carrying over the whole of the solution of nicotin in twenty- four hours. See also Mitth. Zool. Stat. Neapel, Bd. ii, 1880, p. 123. 17. Chloroform may be employed either in the liquid state or in the state of vapour. The animals being extended, a watch-glass containing chloroform may be floated on the surface of the water in which they are contained, and the whole covered with a bell-glass. As soon as they have become insensible they are killed by means of hot subUmate or chromic acid solution plentifully poured on to them. (KoROTNEFF, Mitth. Zool. Stat. Neapel, v, 1884, p. 233.) Liquid chloroform is employed by squirting it in small quantities on to the surface of the water containing the animals. A syringe or pipette having a very small, orifice, so as thoroughly to pulverise the chloroform, should be employed. Small quantities only should be projected at a time, and the dose should be repeated every five minutes until the animals are anaesthetised. I have seen large Medusae very completely anaesthetised in ex- tension in an hour or two by this method. Andres finds that it does not succeed with Actiniae, as with them maceration of the tissues supervenes before anaesthesia is estabhshed. Preyee (Mitth. Zool. Stat. Neapel, Bd. vii, 1886, p. 27) recommends chloroform water for star -fishes. Waddington employs a mixture of equal parts of 1 per cent. sol. of cocaine (or eucain) and saturated sol. of chloroform in water (sea or fresh), according to the habitat. 18. Ether and Alcohol may be administered in the same way. Andres has obtained good results with Actiniae by the use of a mixture (invented by Salvatore lo Bianco) containing 20 parts of glycerine, 40 parts of 70 per cent, alcohol, and 40 parts of sea water. This mixture should be carefully poured on to the surface of the water containing the animals, and allowed to diffuse quietly throiigh it. Several hours are sometimes necessary for this. EisiG (Fauna u. Flora Golf. Neapel, xvi, 1887, p. 239) benumbs CapiteUidae by putting them into a mixture of 1 part of 70 per cent, alcohol with 9 parts 6f sea water. .; Oestbrgren (Zeit. wiss. Mik, xix, 1903, p. 300) makes a saturated (7 to 8 per cent.) solution of ether in sea or soft water, and uses it either concentrated or diluted to about 1 per cent., and finds it to succeed with all classes of aquatic animals. 14 KILLING. CoEi (Zeit. wiss. Mik., vi, 1890, p. 438) recommends a mixture composed of 10 c.c. methyl-alcohol (of 96 per cent, strength), 90 c.c. water (fresh or sea water), and 0-6 grm. of sodium chloride (to be added only when fresh water is taken, the addition of the salt having for its object to prevent maceration). It may be weU to add to this mixture a very few drops of chloroform (for Cristatella ; Zeit. wiss. Zool, Iv, 1893, p. 626). 19. Chloreton (Aceton Chloroform) is reconamended for inverte- brates and larvae of Rana by Eaistdolph {Zool. Anz., xxui, 1900, p. 436). Kbecker {Zeit. wiss. Zool., xcv, 1910, p. 383) takes solutions of ^ to 1 per cent. for.Ohgochseta. Sulima {Zeit. Biol. Techn., Strasburg, i, 1909, p. 379) takes a mixture of 99 parts of sea water and 1 of 10 per cent. sol. of chloreton in absolute alcohol, for Scyllium and Anguilla. For Bryozoa, see Bessie Green, Joum. Roy. Mic. Soc, 1914. 20. Hydrate of Chloral. — Foettinger {Arch, de Biol., vi, 1885, p. 115) operates by dropping crystals of chloral into the water containing the animals. For Ahyonella he takes 25 to 80 centi- grammes of chloral for each hundred grammes of water. It takes about three-quarters of an hour to render a colony sufficiently insensible. He has obtained satisfactory results with marine and fresh- water Bryozoa, with AnneUda, MoUusca, Nemertians, Actiniae, and with Aster acamthion. He did not succeed with Hydroids. Lo Bianco {Mitth. Zool. Stat. Neapel, Bd. ix, 1890, p. 442) employs for various marine animals freshly prepared solutions of chloral in sea water, of from •j's to ^ per cent, strength. I have never had the slightest success with Nemertians. Verworn {Zeit. wiss. Zool., xLvi, 1887, p. 99) puts Cristatella for a few minutes into 10 per cent, solution of chloral, in which the animals sooner or later become extended. KuKENTHAL {Jena Zeit. NaPurw., Bd. xx, 1887, p. 611) has obtained good results with some Annelids by means of a solution of 1 part of chloral in 1,000 parts of sea water. The chloral method gives rise to maceration with some subjects, as I can testify, and has been said to distort nuclear figures, 21. Cocaine (Richards, Zool. Anz., cxcvi, 1885, p. 332). — Richards puts a colony of Bryozoa into a watch-glass with 5 c.c. of water, and adds gradually 1 per cent, solution of hydrochlorate of cocaine in water. After five minutes the animals are somewhat numbed; half a cubic centimetre of the solution is added, and ten minutes later the animals should be found to be dead in a state of extension. This method is stated to succeed with Bryozoa, Hydra, and CHAPTER II. 15 certain worms. It is the best method for Rotifers (Rousselet). It has also been recommended for Aplysia. It has been pointed out (by Coei, in the paper quoted § 18) that, unfortunately,' when fixing agents, such as sublimate solution, are added to the animals, the cocaine is thrown down on them as a white precipitate. This precipitate, however, may be redissolved afterwards in alcohol (EisiG). Cocaine solutions cannot be depended on to keep for more than a few days. 22. Eucain. Harris (Journ. Roy. Mic. Soc, 1900, p. 404) recommends a 1 per cent, solution of eucain hydrochloride, as giving far better results, with Vorticelhdae, Rotatoria, and Vermes. Rousselet {ibid.) reports favourably as to its action on Floscularise. It is stated to be perfectly stable in aqueous media. It dissolves in sea water to about 0-5 per cent. 23. Hydroxylamin. — Hofer {Zeit. wiss. Mik., vii, 1890, p. 318). Either the sulphate or, preferably, the hydrochlorate may be used. This should be dissolved in water (spring or sea water, according to the habitat) and exactly neutralised by addition of carbonate of soda. The organisms are placed in a solution diluted to about 0-1 per cent., for thirty minutes or less (as for Infusoria), to 0-26 per cent., for from fifteen minutes to one hour (Hydra), 1 per cent., one half to two hours {Hirudo), or as much as ten to twenty hours (Helix and Anodonta). Hydroxylamin is a powerful reducing agent, and should therefore be well washed out before treating with easily reducible fixing agents. 24. Chloride or Sulphate of Magnesium. — Tullberg {Arch. Zool. Exper. et Gen., x, 1892, p. 11). For Actiniae, a 33 per cent, solution of the chloride should be very slowly added to the water containing the expanded animal, imtil the vessel contains 1 per cent, of the salt (thus for 1 litre of sea water 33 c.c. of the solution must be added). The addition must be completed within haK an hour, and thirty minutes later the animal may be fixed. For terrestrial and fresh-water Invertebrates rather stronger solutions shoiild be used. Redenbaugh {Amer. Natural., xxix, 1895, p. 399) takes the sulphate, either added in crystals to the sea water containing the animals imtil a saturated solution is obtained, or in the shape of a saturated solution into which they are thrown (Annelids). See also Mayer, Biol. Bull. Wood's Hole, xvii, 1909, p. 341 (puts direct into sol. of f strength). 25. Poisoning by small doses of some fixing agent is sometimes good. Lo Bianco MUs Asddia and Bhopalcea in an extended state (Mitth. Zool. Stat. Neapel,ix, 1890, p. 471) by pouring a little 1 per cent. 16 KILLING. chromic acid on to the surface of the water containing them, and allowing it to diffuse slowly into it. About twelve to twenty -four hours is necessary. He kills Oiona in a similar way with a mixture of 1 part of 1 per cent, chromic acid and 9 parts of 49 per cent, acetic acid. Osmic acid, or Kleinenberg's solution, is sometimes employed in the same way. I have seen Medusse kiUed in a satisfactory manner by means of crystals of corrosive sublimate added to the water containing them. Morphia, Curare, Strychnin, Prussic Acid, and other paralysing drugs, have also been employed. 26. Asphyxiation may be sometimes successfully practised. Terrestrial Gastropods may be kiUed for dissection by puttiag them into a jar quite full of water that has been deprived of its air by boiling, and hermetically closing it. After from twelve to twenty- four hours they are generally found dead and extended. The effect is obtained somewhat quicker if a little tobacco be added to the water. Good results are sometimes obtained with aquatic animals by simply leaving them to exhaust the oxygen of the water in which they are contained. I have sometimes succeeded with Holothurise and other Bchinoderms in this way. Ward (see Amer. Nat., xxv, 1891, p. 398) has succeeded with Hydroids, Actiniae, and similar forms, and Uexkull [Mitth. Zool. Stat. Neapel, xii, 1896, p. 463) with Echinids. Marine animals are sometimes successfully killed by simply putting them into spring water. Warm Water wiU sometimes serve to immobilise and even kill both marine and fresh-water organisms. Carbonic Acid Gas has been recommended (by Fol, Zool. Anz., cxxviii, 1885, p. 698). The water containing the animals should be saturated with the gas. The method is stated to succeed with most Ccelenterata and Echinodermata, but not with Molluscs or Fishes. I have had most excellent results with small Annelids and Hirudinea. It is not necessary to employ a generator for obtaining the gas. It suffices to take an ordinary " soda-water " syphon, and squirt its contents into the water containing the animals. Narcotisation is very rapidly obtained with very small animals, but much more slowly with larger ones. For instance, Stylaria proboscidea, I find, is paralysed in a few seconds ; a small Nephelis of 15 or 20 millimetres in length, will require aibout five minutes ; and a large Nephelis, of from 10 to 15 centimetres, will require as many hours. Uexkull [Mitth. Zool. Stat. Neapel, xii, 1896, p. 463) has paralysed CHAPTER II. 17 Echinids very rapidly witli carLonic acidj likewise a small Teleostean fish ; whilst Scyllium and Crustaceans were affected much more slowly, and mussels not at all. 27. Peroxide o£ Hydrogen.— Volk {Zool. Anz., xix, 1896, p. 294) kiUs Rotatoria by means of one or two drops of a 3 per cent, solution added to 1 c.c. of the water containing them. CHAPTER III. FIXING AND HARDENING. 28, The Functions of Fixing Agents. — The meaning of the term " fixing " has been explained above (§ 2). Here is an example showing the necessity of fixation. If a portion of living retina be placed in aqueous humour, serum, or other so-called " indifferent " medium, or in any of the media used for permanent preservation, it will be found that the rods and cones will not preserve the appearance they have during life for more than a very short time ; after a few minutes a series of changes begins to take place, by which the outer segments of both rods and cones become split into discs, and finally disintegrate so as to be altogether unrecognisable, even if not totally destroyed. Further, in an equally short time the nerve-fibres become varicose, and appear to be thickly studded with spindle- shaped knots ; and other post-mortem changes rapidly occur. If, however, a fresh piece of retina be treated with a strong solution of osmic acid, the whole of the rods and cones will be found perfectly preserved after twenty-four hours' time, and the nerve-fibres will be found not to be varicose. After this preliminary hardening, portions of the retina may be treated with water (which would be ruinous to the structures of a fresh retina), they may even remain in water for days without harm ; they may be stained, acidified, hardened, imbedded, cut into sections, and mounted in either aqueous or resinous media without suffering. This example shows that one of the objects aimed at in fixing is to impart to tissues the degree of hardening necessary to enable them to offer such mechanical resistance to 'post-mortem change and to the processes of after-treatment as not to suffer change of form. Another important function of fixing is to render insoluble elements of cells and tissues that would otherwise be more or less dissolved out by the liquids employed in the after-treatment. A third and highly important function of fixing agents consists in producing optical differentiation in structures. By coagulating the elements of tissues and cells, fixing agents alter their indices of refraction, raising them in varying degrees. They do not act in an equal degree on all the constituent elements of cells and tissues, but raise the index of CHAPTER in. 19 some more than that of others, thus producing optical differentiation where there was little or none before. Compare the aspect of the epithelium of the tail of a living tadpole, observed in water, with its aspect after the action of a little diluted solution of Flemming. In the living state the protoplasm of its cells has a refractive index little superior to that of water, and consequently so low an index of visibility that hardly any structure can be made out in the object. But as soon as the protoplasm has been sufficiently coagulated by the reagent the refractive indices of some of its elements will have been raised to above that of balsam, the chromatin of the nuclei will be brought out, and other structures be revealed where none was visible before. 29. The Action of Fixing Agents consists in coagulating and rendering insoluble certain of the constituents of tissues. This is effected sometimes without any chemical action being involved, as when alcohol is employed, which acts by simple withdrawal of the water of the tissues. But in the majority of cases the fixing agents enter into chemical cmnbination with certain of the elements of the tissues. The compounds thus formed are sometimes unstable and soluble, so that they are removable by washing, as is the case with several of those formed by picric acid. It is found in practice, however, that those formed by chromic acid and its salts, and the salts of the heavy metals, as mercury, iron, platinum, gold, and silver, are mostly insoluble. The insolubihty of these bodies is an advantage in that it ensures that the tissues shall not be robbed of their essential constituents, nor deprived of their desired consistency and optical differentiation, by the reagents subsequently employed. It is also sometimes an advantage in that certain of the compounds in question have the property of combining with certain colouring matters, and thus affording important stains which could not otherwise be obtained ; or in other words, of acting as mordants. But it is sometimes a disadvantage, inasmuch as these same compounds which render possible the production of some stains are hindrances to the production of others. Tissues that have been fixed with osmic or chromic acid or their salts are in general not easily to be stained with carmine or similar colouring matters, unless the metals have been previously removed by special chemical treatment ; though they may generally be stained with hssmalum, or, after sectioning, with iron hsematoxylin or tar colours. According to Fischer {Fixirung, Fmbung, und Bau des Proto- 20 FIXING AND HARDENING. plasmas, Jena, G. Fischer, 1899), the coagulation which constitutes fixation is, in the case of the liquid and semi-liquid constituents of tissues, always a phenomenon of precipitation. The more solid constituents (suck as fibrils that are visible during life, nucleoli, and the like) he admits may be acted on by fixing reagents without the formation of any visible precipitates. But all the liquid ones, in so far as they are fixed at all, are visibly precipitated in special precipi- tation forms, which vary according to the precipitant. Each fixing agent gives its own cha,ia.ctenstic fixation image, which may be more or less lifelike, but can never be absolutely so. Fischer gives copious descriptions of the precipitation forms of the chief organic compounds found in tissues, and of the precipitation powers of the chief fixing agents, which the reader will do well to study. It seems to be a consequence of Fischer's theory of fixation by precipitation that the most energetic fixing agents should always be found amongst the most energetic precipitants. But on the showing of his experiments this is not so. For instance, it is allowed on all hands that osmic acid is a most energetic fixative. But Fischer finds (op. cit., pp. 12 — 14, 27) that it is a very incomplete and weak precipitant. Or, to take a contrary instance, he finds that picric acid is an energetic precipitant of the majority of cell constituents ; but surely every cytologist must admit that it is not a highly energetic fixative ! It would seem to foUow, from these instances and from other similar ones, that Fischer's tables of precipitating power cannot be taken as a measure of the fixing power of the reagents. And further, the study of the fixation images of tissues afforded by osmic acid, formaldehyde, and other reagents, seems to show that the coagula- tion brought about by them is in part accompanied by the formation of visible precipitates, but in part not so, and that they may do their work to a larger extent than he seems to admit through a homogeneous coagulation. Fischer, studying the effects of certain fixatives on albumose, states that mixing 10 per cent. sMghtly acid deutero- albumose with Altmann's bichromate-osmic fluid causes a precipitate of granules of from 1 to 3 ju in diameter, while corrosive sublimate of 7 per cent, causes granules of 0-4 to 1 ju in size ; one might be led away, as was Fischer, to consider that Altmaim's fluid used on cells therefore causes artifacts to appear. As a matter of fact corrosive sublimate is much more dangerous than Altmann's fluid, in this respect, while Altmann's fluid merely preserves cell granules which are visible intra vitam. The ground protoplasm after corrosive is more granular and coarsely reticulate than after Altmann ; this CHAPTER III. 21 does not apply to mitochondria or Golgi elements which are often very badly preserved with corrosive. Fischer {pf. cit.) says, " Many kinds of cell contents, indeed the majority, have an alkaline reaction, and are thereby quite inacces- sible to the precipitating action of certain agents, such as osmic acid, or bichromate ; and the action of certain other fixatives, such as platinum chloride or chromic acid, is more or less hindered by the presence of free alkalies. For neither the chromic acid (of the Flemming), nor the platinum chloride (of the Hermann) would be adequate to act as acidifiers to the osmic acid of the mixtures." My own experience is directly contrary to these conclusions of Fischer : it is common knowledge among modern workers that a cell fixed in acetic acid-containing solutions has a more " raked out " appearance than when the acetic acid is omitted : this applies not only to cell granules, but to the appearance of the ground cytoplasm, nucleoli, and chromatin filaments. The statement that osmic acid must be acidified before it will fix all parts of the cell is also contrary to general experience. Formalin neutraUsed gives a gentler and more precise fixation than acid formalin. While Fischer's results may be excellent so far as concern his theoretical conclusions on the fixation of weak solutions of egg-white, etc., too much attention should not be paid to one who is not thoroughly acquainted with practical cytology and histology. But from his very suggestive observations it certainly appears that the formation of visible precipitates is a very widespread, if not universal concomitant of fixation ; and that the wider the precipitating power of a fixative {i.e. the greater the number of organic liquids that it can precipitate), the greater will be the number of artifacts to which it can give rise. It has lately been pointed out by some workers (e.g. Unna, Arch, f. Mihr. Anat., Ixxviii), that many of the fixing reagents come under the category of oxidisers ; Unna places great importance on the fact that some of the most successful fixers are oxidisers, e.g. OSO4, KjCrjOy, CrOa ; but formalin, admittedly a splendid reagent, is a reducer, while picric acid and corrosive sublimate are feeble oxidisers, and that only under special conditions. Of the ten common reagents used for fixing, only four are marked oxidisers, and Unna's generalities with reference to the significance of oxidisers may not be the correct explanation. Generalities such as made by Unna with reference to the rdle of oxidisers in fixation resemble like claims which have been made with reference to the supposed necessity for the constant use of an acid in fixatives (and preferably acetic acid which is reaUy a dangerous reagent). 22 FIXING AND HARDENING. Witt regard to the relative values of oxidisers or reducers in fixing cytoplasm or nucleus, formalin (reducer)* and OSO4 (oxidiser) are both famous cytoplasm fixers, while acetic acid (neither oxidiser nor reducer), or alcohol (reducer) and GrOg (oxidiser) are well-known nuclear fixatives. Helly's fluid, formol-bichromate or formol-Flemming are all splendid fixatives, and mixtures of both oxidisers and reducers ; it is difficult to see how Unna's theories can apply here. In the case of Flemming fluid, without acetic acid, it is certain that the fixation process in so far as it concerns the OSO4 in this mixture, is not solely an oxidising process, at least of the same nature as the fixation reaction by the chromic acid (CrOg). In a word, fixation of the cell by various kinds of chemical reagents is an extremely complicated matter concerning a large number of organic substances whose re- actions to the chemical used are probably different in most cases. 30. The Characters of the Usual Fixing Agents. — These agents are as follows : — 1. Osmiura tetroxide, 2. Formaldehyde gas, 3. Chromium trioxide, 4. Bichromate of potassium, 5. Platinum chloride, 6. Mercury bichloride, all in water. 7. Picric acid in water, 8. Alcohol, 9. Nitric acid and 10. Acetic acid in water. Chloroform and urea are also used. In the group marked A are arranged the more valuable reagents, in B the less valuable or destructive ones. Good fixatives can be made from the substances in group A without using any of the reagents in group b. The latter contain most of the reagents useful for chromosome work, the former, reagents useful for fixing the cytoplasm and " resting " nucleus. From group a have been made the following mixtures : Altmann, Ghampy, and Flemming and Hermann-without-acetic acid ; these are among the best mixtures known. Then there are formol (5 per cent, to 10 per cent.), Eegaud, Helly, formol-Miiller and formol- Flemming, which are so good for mammals. Good general micro- anatomical fixatives from both groups a and e, are Zenker, Bouin, * See, however, Blum, Enzykl. d. mikr. Tech., 1910. A. i CHAPTER III. 23 Gilson-Petrunkewitsch and corrosive acetic, but these all destroy- much of the cell-contents, and give an incorrect picture of the cell, excepting of chromatinic structures, for which they are indicated. A good fixing agent should first of all preserve all the elements it is desired to fix. But that is not enough ; it should also give good optical differentiation, and should have sufiicient power of penetra- tion to ensure that small pieces of tissue be equally fixed by it throughout. No single substance or chemical compound fulfils all that is required of a good fixing agent ; hence it is that all the best fixing agents are mixtures. Osmic acid, for instance, fulfils some of these conditions, but not all of them. It kills rapidly and preserves admirably the elements of cytoplasm, but nuclei not so well. But the optical differentiation that it gives, though sometimes good, is often very inferior. For osmic acid, by coagulating in nearly equal degrees alike the spongioplasm (the plastin reticulum) and the hyaloplasm (the enchylema) of the cell-body, and the chromatin of nuclei, raises alike the refractive indices of all of them ; so that if the fixing action have been in the least degree overdone, the cells acquire a homogeneous aspect in which the finer details are obscured by the general refractivity of the whole. If now, instead of using it pure, it be used in combination with chromic acid, a better differ- entiation is obtained ; for chromic acid, whilst enhancing, and at all events not interfering with the fijxation of chromatin, serves to facilitate penetration and to counteract the excessive action of the osmic acid on the protoplasm, so that the cells come out less homo- geneous and with more detail observable in them. Descriptive embryologists often use strange illogical mixtures containing both reducible substances and violent reducers, both fat-solvents and fat-preservers, mixed together without regard for the chemistry of fixation. It is only the logically planned fixative that is foimd generally useful, and which stands the test of time. Fixation falls under three broad headings : — 1. Micro-anatomical, in which correct preservation of cell aggre- gates, without shrinkage or expanding, is the desideratum. Such is the aim of most descriptive embryologists. 2. Cytological from the point of view of the chromosome or nucleus. 3. Cytological from the point of view of fixing the cell in a state which most resembles its condition when alive ; also so as to identify the ceU elements, especially in the cytoplasm. In most cases the results attained by workers belonging to sections 1 and 2 can truly be said to give a caricature of the cell intra vitam. I give below a general classification of fixatives, those in (a) being 24 FIXING AND HARDENING. fixatives causing the maximum disturbance and destruction in the individual cell, those in (c) the least. A great deal, however, depends on the accessibility of the cells to the fixative, and as to whether vertebrate or invertebrate material is being used. (a) Carnoy, Petrunkewitsch, alcohol, Gilson, picro-nitric, etc. Fat, mitochondria, Golgi apparatus, and often delicate yolk discs do not show after these. (Using alcohols and xylol subsequently.) (b) Bouin, Zenker, corrosive acetic, Flemming-with-acetic acid, etc. Mitochondria and Golgi apparatus rarely show after these, except possibly in mammals, where these cell inclusions are more resistant than in invertebrata. Fats show with the last-mentioned fixative. (c) Osmic acid, Flemming-without-acetic, Champy, Altmann, formalin, Mann's mercuiy-osmic liquid, Sjovall's method, etc. Preserve all formed granules (except glycogen). (Using fluids subsequently as above.) In section (c) the formol alone wiU not preserve fat ; but see Sjovall's method (§ 696). The fixatives have not been classed according to how they them- selves alone affect the contents of the cell, but according to how they preserve the cell preparatory to its treatment in the liquids necessary for embedding and sectioning. Injurious Kquids which should never be used in cytological fixation (3, vide supra) are acetic acid, chloroform and alcohol. Acetic acid is nearly the most destructive to deUcate lipins, and its use, except where chromosomes are being studied, is rarely indicated ; any worker who uses acetic acid in his fixing mixtures cannot hope to get a correct picture of any part of his cell, possibly excepting the chromo- somes (not the resting nucleus). The most valuable fixatives are osmium - tetroxide, bichromate of potassium, chromium - trioxide, and formaldehyde, possibly in the order named ; the most valuable mixtures are Miiller-formol (or Helly), Flemming-without-acetic, Altmann, and Champy ; the three latter approach as near perfection as present-day technique allows. Altmann's fluid (KaCraO, -f OSO4) I find to be a splendid mixture. In no case, except in small invertebrates, do these fixatives (excluding formol) give a true fixation of cell aggregates ; this is due to their inferior penetrating powers, and to an unevenness of penetration. Small invertebrates, both marine and fresh- water, and small pieces of tissue, are usually exquisitely preserved in chrome-osmium mixtures, but are not then generally suitable for staining and mounting whole, especially for staining in carmine mixtures. CHAPTER in. 25 For routine zoological work Bouin's picro-formalin-acetic is recommended. Grilson-Petruakewitsch is a fixative which is easy to -work and generally better than corrosive sublimate acetic. For routine vertebrate histological work Zenker and Helly's Zenker-formol are indicated. I think the beginner should avoid such things as liquid of Flemming and similar mixtures. Picric acid gives a fair though weak fixation, with very good penetration, is easy to manage, and does not make tissues brittle, whioh sublimate easily may do. Pure formol is not bad, and very easy to manage. Speaking generally, osmio acid, chromic acid, bichromates, chloride of platinum, and the majority of the compounds of the heavy metals, are hindrances to staining ; whilst heat, alcohol, trichloracetic acid, formol, corrosive subKmate, picric acid, and acetic acid, are neutral, or even favourable, in this respect. 31. The Practice of Fixation.— See that the structures are fer- fectly living at the instant of fixation, otherwise you will only fix pathological states or post-mortem states. Some observers have made special observations on the effect of delay in fixation; J. Thornton CabteS (Phil. Trans. Boy.Soc., Series B,vol. ccviii, 1917) has made some interesting experiments on the finely granular ameloblasts in the developing teeth of the pike. He noticed that the cytoplasm gave evidence of marked changes unless fixed within three minutes of " death " ; these changes were manifested by the behaviour of the cyioplasmic granules to stains ; the selectivity of the latter was progressively altered as the rapid post-mortem changes were set in action. Fixation is generally performed by immersion of the objects in the fixing liquid. In this case, everything should be done to facilitate the rafid penetration of the fiixing agent. To this end let the struc- tures be divided into the smallest portions that can conveniently be employed, and if entire organs or organisms are to be fixed whole, let openings, as large as possible, be first made in them. The penetration of reagents is greatly facihtated by heat. You may warm the reagent and put it with the object to be fixed in the paraffin stove, or you may even employ a fixing agent heated to boiling-point (as boihng sublimate solution for certain corals and Hydroids, or boiling absolute alcohol for certain Arthropods with very resistent integuments). But this should only be done as a last resource. Let the quantity of fixing agent employed be ma,ny times 26 FIXING AND HARDENING. the volume of the objects to be fixed. If this precaution be not observed the composition of the fixing liquid may be seriously altered by admixture of the liquids or of the soluble substances of the tissues thrown into it. For a weak and slowly acting fixing agent, such as picric acid, the quantity of liquid employed should be in volume about one himdred times that of the object to be fixed. Eeagents that act very energetically, such as Flemming's solution, may be employed in smaller proportions. But fixation may also be performed by injection of the fixing liquid into the objects, thus ensuring a more rapid and tborough penetration of voluminous objects. See for this practice the methods of fixation by injection of Golgi, De Quervain, Mann, and others, given under Nervous System. Beaus and Dbuenek {Jena Zeit. Naturw., Bd. xxix, 1895, p. 435) fix fishes by injection through the bulbus aortce. The vessels are first washed out with normal salt solution, and the fixing liquid is then thrown in. KoLMEE (Anat. Am., xlii, 1912, p. 47) fixes thus even large mammals (Chimpanzee, Groat). He first washes out with Ringee's solution. It is well not to leave specimens in fixing Uquids longer than is sufiicient to obtain the desired reaction. Sublimate, for instance, soon makes tissues brittle. But long immersion may be neces- sary to produce the desired optical differentiation with some reagents. Careful washing out (by which is meant the removal from the tissues of the excess of uncombined fixative) is necessary in order to get tissues to stain properly. But it is not always equally impera- tive. Alcohol and formaldehyde do not require washing out before staining ; acetic and picric acid only for some stains ; sublimate will allow of staining even if not washed out, but allows of a sharper stain if well washed out ; aU osmic, chromic, and platinic liquids require very thorough washing out. It is important to use the appropriate liquid for washing out the fixing agent after fixation. It is frequently by no means a matter of indifEerence whether water or alcohol be employed for washing out. Sometimes water will undo the whole work of fixation (as with picric acid). Sometimes alcohol causes precipitates that may ruin the preparations. Objects fixed in alcohol, formol, acetic acid, picric acid, or nitric acid require to be washed out with alcohol, or at least with some hardening liquid, whilst those that have been fixed with osmic or chromic acid, or with one of the other compounds of the heavy metals, require in general to be washed out with water. Sublimate, however, is best washed out with alcohol. CHAPTER III. 27 Use liberal quantities of liquid for washing. Change the liquid as often as it becomes turbid, if that should happen. The process of washing out is greatly facilitated by heat. Picric acid, for instance, is nearly twice as soluble in alcohol warmed to 40° C. as in alcohol at the normal temperature (Fol). 32. Fixation of Marine Animals. — The tissues of marine organisms are as a general rule more refractory to the action of reagents than those of corresponding fresh-water or terrestrial forms, and fixing solutions should in consequence be stronger (about two to three times). Marine animals ought to he. freed from the sea water adherent to their surface before treating them either with alcohol or any fixing reagent that precipitates the salts of sea water. If this be not done, the precipitated salts will form on the surfaces of the organisms a crust that prevents the penetration of reagents to the interior. Fixing solutions for marine organisms should therefore be such as serve to keep in a state of solution, and finally remove, the salts in question. If alcohol be employed, it should be acidified with hydro- chloric or some other appropriate acid. Picro-nitric acid is a fixing reagent that fulfils the conditions here spoken of. (On this subject see Mayer, in Mitth. Zool. Stat. Neapel, ii (1881), pp. 1 et seq., and Allen and Browne in " Science of the Sea," John Murray, 1912). 33. Hardening. — The process of hardening is distinguished from that of fixing as being directed to the attainment of a degree of consistency sufficient to allow of soft tissues being cut into sections without imbedding. It is an after-process, and only ranks as a special method. Methods of imbedding have now been brought to such a degree of perfection that the thorough hardening of soft tissues that was formerly necessary in order to cut thin sections from them is, in the majority of cases, no longer necessary. But there are some excep- tions. Such are, for instance, the cases in which it is desired to cut very large sections, such as sections of the entire human brain. The reagents employed for hardening are for the most part of the same nature as those employed for fixing. But it does not follow that all fixing agents can be employed for hardening. Corrosive subUmate, for instance, would be most inappropriate as a hardening agent. 84. The Practice of Hardening. — Employ in general a relatively large volume of hardening liquid, and change it very frequently. If 28 FIXING AND HARDENING. the volume of liquid be insufficient, its composition will soon become seriously altered by the diffusion into it of the soluble substances of the tissues ; and the result may be a macerating instead of a harden- ing liquid. Further, as soon as, in consequence of this diffusion, the liquid has acquired a composition similar in respect of the propor- tions of colloids and crystalloids contained in it to that of the liquids of the tissues, osmotic equiUbrium wiU become established, and diffusion will cease ; that is to say, the hardening liquid will ceaSe to penetrate. This means, of course, maceration of internal parts. On the other hand, it appears that a certain slight proportion of colloids in the hardening Hquid is favourable to the desired reaction, as it gives a better consistency to the tissues by preventing them from becoming brittle. Hence the utility of employing a certain proportion of hardening agent. Hardening had better be done in tall cylindrical vessels, the objects being suspended by a thread, or muslin bag, or otherwise, at the top of the liquid. This has the advantage of allowing diffusion to take place as freely as possible, whilst any precipitates that may form fall harmlessly to the bottom ; or, they may be laid on a layer of cotton-wool, or filter-paper, or spun glass. In general, begin hardening with a weak reagent, increasing the strength gradually, as fast as the tissues acquire a consistency that enables them to support a more energetic action of theweagent. Let the objects be removed from the hardening fluid as soon as they have acquired the desired consistency. CHAPTEE IV. FIXING AND HARDENING AGENTS — MINERAL ACIDS AND THEIR SALTS. 35. Osmic Acid. — The tetroxide of osmium (OsOj) is the substance commonly known as osmic acid, though it does not possess acid properties. It is extremely volatile, and in the form of an aqueous solution becomes partially reduced with great readiness in presence of the slightest contaminating particle of organic matter. It is generally believed that the aqueous solutions are reduced by Kght alone, but this is not the case : they may be exposed to the light with impunity if dust he absolutely denied access to them. The solution of osmic acid in chromic acid solution is not, like the solution in pure water, easily reducible, but may be kept without any special precautions. I therefore keep the bulk of my osmium in the shape of a 2 per cent, solution of osmic acid in 1 per cent, aqueous chromic acid solution. This solution serves for fixation by osmium vapours, and for making up solution of Flemming, which is the form in which osmium is most generally employed. A small quantity of osmic acid may also be made up in 1 per cent, solution in distiUed water, and kept in a drop-bottle with grooved stopper, from which quantities can be obtained when required without removing the stopper. CoEi {Zeit. wiss. Mik., vi, 1890, p. 442) finds that solutions in distilled water keep perfectly if there be added to them enough permstoganate of potassium to give a very slight rosy tint to the liquid. From time to time, as the solution becomes colourless, further small quantities of the salt should be added, so as to keep up the rosy tint. BuscH finds that the addition of sodium iodate hinders reduction {Neurol. Centralb., xvii, 1898, p. 476). ^PiNTNER finds that a slight addition of corrosive sublimate has the same effect, e.g. 10 drops of 5 per cent, solution of sublimate added to 100 c.c. of 1 per cent, solution of osmic acid. For the Kopsch, Mann-Kopsch and Sjovall methods the osmic acid solution mu^t be free from all traces of chrome and platinum salts, etc. 30 FIXING AND HARDENING AGENTS. For the so-called " regeneration " of reduced solutions, see previous editions. Osmic acid is met with in commerce in the solid form in sealed tubes. The assigned weights shoidd be checked, as they may vary greatly. ^Fixation by the Vapours. — This is indicated in most of the cases in which it is possible to expose the tissues directly to the action of the vapour. The tissues are treated as described in § 695. Very small objects, such as isolated cells, are simply placed on a slide, which is inverted over the mouth of the bottle. They remain there until they begin to turn brown (isolated cells will generally be found to be sufficiently fixed in thirty seconds : whilst in order to fix the deeper layers of relatively thick objects, such as retina, an exposure of several hours may be desirable). It is well to wash the objects with water before staining, but a very slight washing will suffice. For staining, methyl-green may be recommended for objects destined for study in an aqueous medium, and, for permanent preparations, alum-carmine, pioro-carmine, or hsematoxylin. In researches on nuclei, it may be useful to employ the vapours of a freshly prepared mixture of osmic and formic or acetic acid (Gilson, La Cellule, i, 1885, p. 96). The reasons for preferring fixation by the vapour are that osmic acid is more highly penetrating in vapour than in solution ; that the arduous washing out required by the solutions is done away with ; and that all possibility of 'deformation through osmosis is elminated. See also under " Cramer's Method," § 695. Fixation by Solutions. — Osmic acid is now very seldom used pure in the shape of solutions. When, however, it is so employed it is used in strengths varying from Jq to 2 per cent. I should say myself that, as a rule, not more than 0-1 per cent., and never more than 2 per cent., should be used. On account of its feeble penetrating power the objects to be fixed should be as small as possible. The solutions should be kept protected from bright light during the immersion of tissues. (This precaution is not necessary if Flemming's or Hermann's solution be used.) If the immersion is to be a long one the tissues must be placed with the solution in well- closed glass-stoppered vessels. The objects may be deemed to be fixed as soon as they have become brown throughout. But see " Mann-Kopsch Methods," § 693. After-Treatment.~The excess of osmic acid must be well washed out before proceeding to any further steps in preparation ; water should be used for washing. Notwithstanding the greatest care in CHAPTER 77. 31 soaking, it frequently happens that some of the acid remains in the tissues, and causes them to blacken in time, and in any case hinders staining. To obviate this blackening it has been advised to wash them out in ammonia-carmine or picro-carmine (not very effectual), or to soak them for twenty-four hours in a solution of bichromate of potash (Miiller's solution or Erlicki's will do), or in 0-5 per cent, solution of chromic acid, or in Merkel's solution. The treatment with bichromate solutions has the great advantage of highly facili- tating staining with carmine or hsematgxylin. Max Schultze recommended washing, and mounting permanently in acetate of potash ; Fol, treatment with a weak solution of carbonate of ammonia. But the best plan of all is to properly bleach the prepara- tions. See " Bleaching." This may be done by means of peroxide of hydrogen. Overton (Zeit. wiss. Mih., vii, 1890, p. 10) finds that it is completed in a few minutes in a mixture of 1 part commercial peroxide with 10 to 25 parts 70 per cent, alcohol. (The commercial peroxide, slightly acidulated with HCl, will keep well in the dark ; but the mixture with alcohol must be made fresh for use.) Accord- ing to Bristol {Amer. Natural, xxvii, 1893, p. 176) the peroxide acts best in the sun. Binet {Journ. de I'Anat. et de la Physiol., xxx, 1894, p. 449) has successfully used permanganate of potash. Mann (Methods, etc., p. 83) takes a solution of 0-25 per cent., and treats the browned tissues with 1 part of saturated solution of sulphurous acid to 9 of normal salt solution. . . . Monckeberg and Bethe (Arch. Mik. Anat., liv, 1899, p. 135) have succeeded in satisfactorily restoring the staining susceptibiUty of osmium material by means of sulphurous acid (obtained by adding hydrochloric acid to bisul- phite of sodium, 2 to 4 drops of the acid added to 10 c.c. of a 2 per cent, solution of the salt). Pol {Lehrh., p. 174) recommends a weak aqueous solution of ferri- cyanide of potassium. I find the sulphate of iron solution used in Benda's hsematoxylin stain has a marked bleaching eflect, and so also, though in a less degree, the iron alum of Heidenhain's process. Altmann (Die Elementarorganismen, pp. 33 and 35) puts sections overnight into gold chloride of 2 per cent., and reduces in formic acid in the sun, and removes the gold by iodised alcohol. But perhaps the best plan is the chlorine method of Mayer, or his magnesium peroxide, for both of which see " Bleaching." The same stains recommended for objects fixed by the vapours will be found useful here. For sections, of course, in both cases safranin and other anilin stains may be employed with advantage, ss may hsematoxylin. 32 FIXING AND HARDENING AGENTS. In general, osmic acid, especially when used in tlie form of vapour, fixes protoplasm very faitkfully, nuclei badly. It is pre-eminently a fixative of the hyaloplasm or enchylema of cells. The penetrating power of the solution is very low, so that if any but very small pieces of tissue be taken the outer layers become over-fixed before the reagent has penetrated to the deeper layers. Over-fixed cells have a certain homogeneous, glassy, or colloid look, and are unfit for study, and attention should be confined to cells four or five layers deeper down, which wijl generally be found to present the required intensity of fixation. In these the fixation is admirable, with no shrinkage and next to no swelling of anything. 36. The Osmium Tetroxide Reaction. — Mann beheved that during the osmic reaction on fatty substances the OSO4 was reduced to osmium tetra-hydroxide Os(OH)4. Other observers have assumed the reaction to be the reduction of the OsO^ to some lower oxide. The matter has recently been reviewed by Professor J. R. Partington and Mr. D. B. Huntingfoed, who find that the reduced substance is a hydrated form of OSO2, possibly OSO2, 5H2O, or OSO2, 6H2O. In all probabihty, Professor Partington informs me, the amount of water is not definite. (See also § 768 on " Fat.") 37. Osmic Mixtures. — ^Nicolas (Intern. Monatssehr., 1891, p. 3) adds J per cent, of osmic acid to nitric acid of 3 per cent. I liave employed a similar mixture and not tad good results, though I find tlie mixture keeps perfectly. BuscH (Neurol. Centralb., xvii, 1898, No. 10, p. 476 ; Zeit. wiss. Mik.,- XV, p. 373) finds that the penetration of osmic acid is enhanced by- combining it with iodate of sodium, which by hindering its too rapid decomposition in the tissues ensures a more energetic action in the deeper layers. He adds 3 per cent, of sodium iodate to a 1 per cent, solution of osmic acid. Unna (MonatsoTir. praH. Derm., xxvi, 1898, p. 602) adds 1 per cent, of alum to a 1 per cent, solution. For some mixtures of Kolossow, see 5th ed., or Zeit. iciss. Mikr., v, 1888, p. 51, and ix, 1892, p. 39. See also under " Cytology," § 677. 38. Chromic Acid.— Chromic anhydride, CrOj, is found in com- merce in the form of red crystals that dissolve readily in water, forming chromic acid, H2Cr04. These crystals are very deliquescent, and it is therefore well to keep the acid in stock in the shape of a 1 per cent, solution. Care must be taken not to allow the crystals to be contaminated by organic matter, in the presence of which the anhydride is readily reduced into sesquioxide. Chromic acid is generally employed in aqueous solution. Some CHAPTER IV. 33 observers (Klein ; Ubban Pritchard ; Perenyi) have recom- mended alcoholic solutions ; but this is evidently irrational. For in the presence of alcohol chromic acid has a great tendency to become reduced to chromous oxide or sesquioxide, neither of which appears to have any fixing power. The most useful strengths in which it is employed in aqueous solution are from 0-1 to 1-0 per cent, for a period of immersion of a few hours (structure of cells and ova). For nerve tissues weaker solutions are taken, ^ to J per cent, for a few hours. Stronger solutions, such as 5 per cent., should only be allowed to act for a few seconds. Washing out. — The general practice is to wash out very thoroughly with water (by preference running water, for many hours) before bringing into alcohol or any staining liquid. For if the objects are put direct into alcohol it is found that after a short time a fine precipitate is thrown down on the surface of the preparations, thus forming an obstacle to the further penetration of the alcohol. Previous washing by water does not prevent the formation of this precipitate, and changing the alcohol does not prevent it from forming again and again. It has, however, been found by Hans ViRCHOW {Arch. Mik. Anat., xxiv, 1885, p. 117) that it may be entirely prevented by simply keeping the preparations in the dark. The alcohol becomes yellow as usual (and should be changed as often as this takes place), but no precipitate is formed. If this precaution be taken, previous washing with water may be omitted, or at all events greatly abridged. Mayer [Grundzuge, 1st ed., p. 28) proceeds as follows : — The fixed material is merely rinsed in water and brought direct into 70 per cent, alcohol. It is washed therein, preferably in the dark, until after several changes the alcohol remains colourless. It is then either passed through higher alcohols and imbedded in paraffin, the chromous oxide (or whatever chrome compound it may be that is present in the tissues) being removed from the sections after these are made ; or this necessary removal is performed at once. If this be preferred, the material is brought into sulphuric acid diluted with twenty volumes of water, or into nitric acid diluted with ten volumes of water. After at most a few hours therein, it wiU have become of a light greyish green, and on removal of the acid may be readily stained. If it be preferred to treat the sections, it is sufiicient to put them into the usual hydrochloric acid alcohol (4 to 6 drops of HCl to 100 c.c. of 70 per cent, alcohol), in which after a short time they become almost white, and will stain excellently with any of the 34 FIXING AND HARDENING AGENTS. usual stains. So also Edinger {Zeit. wiss. Mik, i, 1884, p. 126 ; nitric acid 1 : 20 for five minutes). Unna {Arch. Mik. Anat., xxx, 1887, p. 47) holds that the chrome is present in the tissues in the form of chromic chromate, and removes it by treatment with per- oxide of hydrogen. Overton (Zeit. wiss. Mik., vii, 1890, p. 9) employs a weak solution of sulphurous acid, which converts it into a sulphate. See also the directions for bleaching osmic acid prepara- tions, § 35. Tissues that have been fixed in chromic acid may be stained in aqueous solutions, as water does not have an injurious effect on them. The best stain for chromic material that has not been treated by Mayer's special process, or by a similar one, is hsematoxylin, or, for sections, the basic tar colours. Chromic acid is not a very penetrating reagent, and for this reason, as well as for others, is now seldom used jpure ion fixing. For prolonged hardening it is generally employed in strengths of ^ to ^ per cent., the immersion lasting a few days or a few weeks, according to the size and nature of the object. Mucous membrane, for instance, will harden satisfactorily in a few days ; brain will require some six weeks. Large quantities of the solution must be taken (at least 200 grammes for a piece of tissue of 1 centimetre cube — Eanvier). In order to obtain the best results you should not employ portions of tissue of more than an inch cube. For a human spinal cord you should take 2 litres of solution, and change it for fresh after a few days. Six weeks or two months are necessary to complete the hardening. I think it is frequently useful to add a little glycerin ; there is less brittleness. The solution should be taken weak at first, and the strength increased after a time. The objects should be removed from the solution as soon as they have acquired the desired consistency, as if left too long they will become brittle. They may be preserved till wanted in alcohol (95 per cent.). It is well to wash them out in water for twenty-four or forty-eight hours before putting them into the alcohol. After a time they generally become green in the alcohol. They may be bleached if desired. Chromic acid is a most powerful and rapid hardening agent. (By it you may obtain in a few days a degree of hardening that you would hardly obtain in as many weeks with bichromate, for instance.) It has the defect of a great tendency to cause brittleness. CHAPTER IV. 35 39. Chromo-aeetic Acid (Flemming, Zellsbz., Kern. u. Zelltli., p. 382). Chromic acid . . . 0-2 to 0-25 per cent. Acetic acid . . . 0-1 per cent, in water. Flemming found this the best reagent for the study of the achromatic elements of karyokinesis. You can stain with hsematoxylin, or the basic anilin dyes. The following has been recommended for Annelids bj» Eiilebs : — To 100 c.c. of chromic acid of 0-5 to 1 per cent, add from 1 to 5 drops of glacial acetic acid. The acetic acid is said to be sufficient to counter- act any shrinkage due to the chromic acid. Fix overnight, wash out several hours in water. Similar to this is the " chromo -acetic acid, No. 1," of Lo Bianco {Mitfh. Zool. 8tat. Neapcl, ix, 1890, p. 443), viss. 1 part 50 per cent, acetic acid and 20 parts 1 per cent, chromic acid, which is found very useful for fixing marine animals. 40. Chromo-formic Acid (Rabl, Morph. Jahrb., x, 1884, pp. 215, 216). — Four or 5 drops of concentrated formic acid are added to 200 c.c. of 0-33 per cent, chromic acid solution. The mixture must be freshly prepared at the instant of using. Fix for twelve to twenty-four hours, wash out with water. Used by Rabl for the study of karyokinesis." 41. Chromo-aceto-osmic Acid (Flemming, Zellsubstanz, Kern und ZeUtheilung, 1882, p. 381). First or Weak formula : Chromic acid . . . 0-25 per cent. ] Osmic acid . . . .0-1 ,, r in water. Glacial acetic acid . .0-1 ,, ) Meves (Encycl. milcr. Techn., 1, p. 475) sometimes adds 1 per cent, of sodium chloride. FoL (Lehrb. d. vergl. mik. Anat., 1884, p. 100) recommends the following variant : 1 per cent, chromic acid . . . .25 vols. 1 per cent, osmic acid . . . . 2 „ 2 per cent, acetic acid . . . . 5 „ Water 68 „ ^that is to say, a mixture weaker in osmium than Flemming's. A mixture still weaker than this in osmium, viz. with 1 vol. osmic acid solution, instead of 2, has been recommended by CoRi {Zeii. wiss. Mik., vi, 1890, p. 441). Second or Steong formula (Zeit. wiss. Mik., \, 1884, p. 349) : 1 per cent, chromic acid . . . .15 parts. 2 per cent, osmic acid . . . . 4 ,, Glacial acetic acid .... 1 part. If this mixture be kept in stock in large quantities, it may go bad, on account of the large proportion of organic acid contained in it. 3—2 36 FIXING AND HARDENING AGENTS. I therefore recommend that the osmic and chromic acid be. kept ready mixed in the proportions given, and 5 per cent, of acetic acid added at the moment of using. Weaker Formula. — More recently, Flemming has been making up the mixture with only 2 parts of the osmic acid instead of 4, and has spoken of this modification as " weaker osmium mixture " (Meves, in Encycl. Mikr. Techn., p. 476). Meves {he. cit.) takes for delicate objects 15 parts of chromic acid of only 0-5 per cent., 2 or 4 of osmic acid of 2 per cent., and 1 of acetic acid, and thus gets less shrinkage. Under "Cytology" Sections, § 678, see Benda and Gatenby modifications. PoDwyssozKi recommends (for glands especially) the following modification : 1 per cent. CrOj dissolved in 0-5 per cent, solution of corrosive sublimate . . . . .1.5 o.c. 2 per cent, osmic acid solution .... 4 c.c. Grlacial acetic acid . . . . . 6 to 8 drops. ■ The sublimate is said to augment the penetration of tlie osmium, but is unfavourable to staining (Zieglee's Beitrdge s. path. Anat., i, 1886 ; Zeit. wiss. Mile, iii, 1886, p. 40'5). The first or weak liquid is the better for very small objects, the second or strong one for larger ones, as it has better, penetration. These liquids may be allowed to act for many hours or days, or according to some workers even weeks or months ; but this exagge- rated fixation is clearly only justifiable in very special cases, if at all. For chromosome studies some workers fix for only one hour. Others recommend cooling the Flemming on ice before using. Wash out very thoroughly in water (running, twenty-four hours), or treat as directed for chromic acid, § -38. Stain with alum hsematoxyhn if you wish to stain in toto (staining in this way with other reagents is possible, but difficult). Stain sections with safranin or other basic coal-tar colour, or with iron hsenia- toxylin. For fixing with the strong mixture you need only take a bulk of Uquid of some 4 times the volume of the objects (but with the weak mixture the proportion should be increased). Both of them are first-rate fixatives of cellular structures, both as regards their preser- vation and as regards their optical differentiation. But they must be properly used, and not appUed to objects for which they are not fitted. For instance, their poiver of penetration is extremely bad ; they will not fix properly, even in a loose-celled tissue, through more than a layer of about five cells thick. They are therefore suitable CHAPTER IV. 37 only for very small objects or for very small pieces of tissue, such as suffice for cytological or histological work. The strong liquid especially has not the character of a general reagent. As a matter of fact it was recommended by Flemming in the first instance merely for a very special purpose, the hunting for karyokinetic figures, and not for general purposes. It is still very much used, but in my opinion unadvisedly. In most cases, Bouin's picroformol will do all that it is intended to do, without its disadvantages. It may be used for prolonged hardening, e.g. of small pieces of nervous tissue, and is very good for that purpose. Fat is blackened (or browned) by it. See § 35. Chromatin is mordanted by it for basic anihn dyes, enabling them to give peculiarly sharp and powerful stains. 42. Osmic Acid and Bichromate. — Altmann {Die Elementar- organismen, Leipzig, 1890) takes for his " bioblasts " a mixture of equal parts of 5 per cent, solution of bichromate of potash and 2 per cent, solution of osmic acid. The bichromate ought not to contain any free chromic acid. Refer to § 680. Lo Bianco [Mitth. Zool. Stat. Neapel, ix, 1890, p. 443) employs for marine animals a mixture of 100 c.o. of 5 per cent, solution of bichromate and 2 c.o. of 1 -per cent, osmic acid. HoEHL (Arch. Anat. Phys., Anat. Ahih., 1896, p. 31) recommends a mixture of 80 c.c. of 3 per cent, bichromate, 20 c.c. of 1 per cent, osmic acid, and 2 c.c. of glacial acetic acid. 43. Bichromate-chromic-osmic Acid. — Champy [Arch, de Zool. Exper., 1913). — Mixture of 7 parts of 3 per cent, bichromate of potash, 7 parts of 1 per cent, chromic acid, 4 parts of 2 per cent, osmium tetroxide. This mixture keeps well. Fix for from six to twenty -four hours. Wash out in running water about the same time. You can stain in iron hsematoxylin, or less well in Altmann or Benda. See § 681 for a description of mordanting after Champy's fluid. This fluid is extremely useful, and I nearly always use it in addition to Flemming. 44. Osmic, Bichromate, and Platinic Mixture (Lindsay Johnson's Mixture). — Latest formula, 1895, communicated by Dr. Lindsay Johnson : Bichromate of potash (2-5 per cent.) . . 70 parts. Osmic acid (2 per cent.) . . . . 10 „ Platinum chloride (1 per cent.) . . . 15 „ Acetic or forinic acid . . . . 5 „ 38 FIXING AND HARDENING AGENTS. Henneguy, who has worked a great deal with this reagent, and recommends it highly, says {Lecons sur la Cellule, p. 61) that it is well only to add the acetic or formic acid just before using, as it frequently reduces the osmium and platinum very rapidly and energetically. He finds that it contracts the more spongy sorts of protoplasm less than mixture of Flemming. I think highly of it — for certain objects. Twelve hours is probably the optimum time for fixation. Wash out in water. 45. Platino-aceto-osmic Acid (Hermann's) Solution (Arch. Mik. Anat., xxxiv, 1889, p. 58). — One per cent, platinum chloride 15 parts, glacial acetic acid 1 part, and 2 per cent, osmic acid either 4 parts or only 2 parts. Hermann found that protoplasm structures are thus better preserved than with the chromic mixture. As with Flemming, the optimum time is from twelve to sixteen hours. Wash out at least three hours in running water. The after-treatment and staining should be the same as for objects treated with Flemming's solution. Eengel (Zeit. wiss. Zool., Ixiii, 1898, p. 454) washes out for half an hour to an hour with saturated aqueous sol. of picric acid, which he thinks facilitates the staining, especially of nuclei. The action of this fixative is, roughly, similar to that of Flemming's. Like Flemming's, it mordants chromatin for staining with " basic " colours, with which it affords equally fine nuclear stains. But, owing to the platinum in it, it diminishes more than Flemming's the colorability of tissues with " acid " colours, so that it is extremely difficult to obtain good plasma stains after its action. It causes a notable shrinkage in chromatin. It gives a full fixation of cyto- plasm, to which it gives a much more fine-grained aspect than liquid of Flemming does. Leaving out the acetic acid, the solution may be used for mito- chondria as in § 679. 46. Rawitz (Z&U- wiss. Milcr., xxv, 1909, p. 386) takes 4 parts of Kahlbaum's Phospho-Tungstic acid, 5 of alcohol, and 1 of acetic acid, added just before use, fixes for twenty-four hours, and washes out the sections before staining with water containing a little calcium acetate. , 47. Nitric Acid (Altmann, Arch. Armt. Phys., 1881, p. 219). — Altmann employs for fixing embryos dilute nitric acid, containing from 3 to 34 per cent, pure acid. Such a solution has a sp. gr. of about 1 -02. Stronger solutions have been used, but do not give such good final results. After extensive trial I find Altmann's solution to be a second- rate reagent, giving a weak and thin fixation. His {ibid., 1877, p. 115) recommended a 10 per cent, solution. CHAPTER IV. 39 Flemming at one time employed solutions of 40 to 50 per cent, for the ova of Invertebrates. Telltesniczkt {Arch. mik. Anat., lii, 2, 1898, p. 222) thinks that " for general oell-flxing " the proper strength is 2 to 2J per cent., as stroliger grades act too energetically on the superficial layers. Mater has had good results with 5 per cent, solution. Nitric acid has the valuable property of hardening yolk without making it brittle. Pure water should in ilo case be used for washing out ; the prepara- tions should be brought direct into alcohol. Some persons take absolute, but I should say 70 per cent, is more generally indicated. Rabl has employed a 1 or 2 per cent, solution of alum. For prolonged hardening, strengths of from 3 to 10 per cent, are sometimes employed. A strength of 12 per cent., allowed to act for two or thrse weeks, is said to afford very tough preparations of the encephalon. Benda (Verh. Anat. Ges., 1888 ; Ergeh. d. Anat, i, 1891, p. 7) fixes for twenty-four to forty-eight hours in 10 per cent, nitric acid, and then hardens in bichromate of potash. Fol's Mixture (verbally communicated to me).— Three vols, of nitric acid, with 97 vols, of 70 per cent, alcohol. 48. Chromo-nitric Acid (Pbkenti's formula, Zool. Anemg., v, 1882, p. 469) : 4 parts 10 per cent, nitric acid. 3 parts alcohol. 3 parts 0-5 per cent, chromic acid. Fix for four to five hours and pass into alcohol of 70 per cent. This mixture has been criticised (see previous editions) as irrational, the alcohol reducing the chromic acid and itself becoming etherised by the nitric acid. Some workers reject it, especially for ova, for which it is specially intended. But others speak highly of it. I myself have used it extensively for preparing objects for dissection and museum specimens, and found it admirable for these purposes-. But preparations made to test its value from a cytological point of view have given me only second-rate results. It is now little used. 49, Chromic Acid and Platinum Chloride (Merkel's Macula lutea des Menschen, Leipzig, 1870, p. 19).— Equal volumes of 1400 solution of chromic acid and 1-400 solution of platinum chloride. Objects should remain in it for several hours or even days. After washing out with alcohol of 50 to 70 per cent., objects stain excellently. If objects that have been fixed by osmic acid be put into it for some hours, blackening is said to be effectually prevented. This is an excellent hardening medium for delicate objects. Merkel allowed from three to four days for the action of the fluid for the retina ; for Annelids Eisig employs an immersion of three to five hours, and transfers to 70 per cent, alcohol ; for small leeches 40 FIXING AND HARDENING AGENTS. WWtman finds one hour sufficient, and transfers to 50 per cent, alcohol. A similar mixture, with the addition of 0-25 to 0-1 per cent, of acetic acid, is recommended by Brass for Protozoa ; and Lav- DOWSKY has used for nuclei a mixture of 10 parts of 1 per cent, chromic acid, 5 of 1 per cent, platinum chloride, and 100 of 5 per cent, acetic acid. Whitman recommends for the hardening of pelagic fish ova a stronger mixture (due, I believe, to Eisig), viz. — 0-25 per cent, solution of platinum chloride . . 1 vol. 1 per cent, solution of chromic acid . . . 1 „ The ova to remain in it one or two days (Whitman, Methods in Micro. Anat., p. 153). Salts. 50. Chromates. — The chromates are amongst the oldest and best tried of hardening agents. The bichromate of potash especially was at one time universally employed for hardening all sorts of tissues. Flemming {Arch. mik. Anat., xviii, 1880, p. 352) pointed out that though it preserves cytoplasm well it causes chromatin to swell, and therefore should not be employed /or the study of nuclei. But, duly corrected with acetic acid, it affords a correct and fine fixation of nuclei ; whilst preserving hyaloplasm, and its inclusions, secretions, etc., much better than chromic acid. For an elaborate study of the action of cliiome salts on nucleus and cytoplasm, see Burckhabdt, La Cellule, xii, 1897, p. 335. He finds that the bichromates of sodium, ammonium, magnesium, strontium, and zinc have the same destructive action on nuclei that the bichromate of potassium has ; but that the bichromates of barium, calcium, and copper have not. He concludes that acetic acid ought always to be added, not only to ensure the correct fixation of nuclei, but also to enhance penetration and the good preservation of cytoplasm. The following is recommended by him as a good combination for the fixation both of cytoplasm and nucleus : Bichromate of barium, 4 per cent, solution . . 60 vols. Bichromate of potassium, ,5 per cent, solution . . 30 ,, Glacial acetic acid . . . . . . 5 ,, (Instead of the barium you may take 4 per cent, solution of bichromate of calcium, or 6 per cent, solution of bichromate of copper.) For the demonstration of the achromatic figure of cell division he recommends — Chromic acid, 1 per cent, solution . . . .60 vols. Bichromate of potassium, 5 per cent, solution . . 30 ,, Glacial acetic acid . . . . . . . 5 ,, CHAPTER IV. 41 51. Bichromate of Potash,— Perhaps the most important of all known hardening agents, sensu stricto. It hardens slowly, much more so than chromic acid, but it gives an incomparably better consistency to the tissues. They may remain almost indefinitely exposed to its action without much hurt. The strength of the solutions employed is from 2 to 5 per cent. As with chromic acid, it is extremely important to begin with weak solutions and proceed gradually to stronger ones. About three weeks will be necessary for hardening a sheep's eye in solutions gradually raised from 2 to 4 per cent. Spinal cord requires from three to six weeks, a brain at least as many months. After hardening, the objects should be well soaked out in water before being put into alcohol, or be treated as directed for chromic acid, § 38. They had better be kept in the dark when in alcohol. See § 38. (Bohm and Oppel [Taschenbuch, 3 Auf., 1896, p. 22] fix in the dark.) If you wish to have a good stain with carmine you should not put the objects into alcohol at all, even for a second, until they have been stained. You may stain either with carmine or hsematoxylin, as well as with tar colours. Bichromate objects have an ugly yellow colour which cannot be removed by mere soaking in water. It is said that it can be removed by washing for a few minutes in a 1 per cent, solution of chloral hydrate. Prof. GiLSON writes me that alcoholic solution of sulphurous anhydride (SO.2) is very convenient for the rapid decoloration of bichromate objects. A few drops suffice. See also § 38, and " Bleaching." To facilitate staining with hematoxylin, Wolff (Zeit. wiss. Mik., xv, 3, 1899, p. 311) first stains in Boehmer's haematoxyhn for twenty-four hours, and then for a few minutes in the same hsematoxylin to which has been added 1 drop per watch -glassful of 5 per cent, solution of oxahc acid. The simple aqueous solution of bichromate is hardly to be recom- mended as a fixing agent, because not only does it not preserve nuclei properly, but also because it penetrates very slowly. The first of these defects may be overcome entirely, the second to some extent by addition of acetic acid ; whence the Uquid of Tellyes- NiczKY, next §. 52. Acetic Bichromate (TelLyesniczky, Arch. mik. Anat., Hi, 1889, p. 242) : Bichromate 3 grms. Glacial acetic acid ..... 5 c.c. Water 100 „ Smaller objects to remain in the fluid for one or two days, larger 42 FIXING AND HARDENING AGENTS. ones longer. Wash well in plenty of water, and pass through alcohols of increasing strength, beginning with 15 per cent. Mixtures of bichromate with osmic acid have been given above, §§ 42, 43 and 44. 53. MiJLLEE's Solution. — Bichromate of potash . . 2 — 2J parts.. Sulphate of soda .... 1 part. Water 100 parts. The duration of the reaction is about the same as with the simple solution of chromic salt. Eecent authors find the action of this liquid to be identical with that of plain bichromate, and doubt whether the sulphate in it has any effect whatever as regards its hardening properties. Fol says that for mammalian embryos, for which it has been recommended, it is worthless. 54. Erlicki's Solution {Warschauer med. Zeit., xxii, Nos. 15 and 18 ; Progres Medical, 1897, No. 31) : Bichromate of potash . . . 2-5 parts. Sulphate of copper .... 1-0 part. Water 100-0 parts. Here the addition of the cupric sulphate is intelligible, for this salt is itself a hardening agent of some energy. As a matter of fact, " Erlicki " hardens very much more rapidly than either simple bichromate or Miiller's solution. A spinal cord may be hardened in it in four days at the temperature of an incubator, and in ten days at the normal temperature (Fol, Lehrb. d. vergl. mik. Anat., p. 106). Human embryos of several months may be conveniently hardened in it. Nerve-centres that have been hardened in Erlicki's fluid frequently contain dark spots with irregular prolongations, simulating ganglion- cells. These are now known to consist of precipitates formed by the fluid. They may be removed by washing with hot water, or with water slightly acidified with hydrochloric acid, or by treating the specimens with 0-5 per cent, chromic acid before putting them into alcohol (TscHiSCH, Virehow's Arch., Bd. xcvii, p. 173 ; Edinger, Zeit. wiss. Mile., ii, p. 245 ; Loewenthal, Bev. mid. de la Suisse romande, 6me ann6e, i, p. 20). 55. Ktjltschitzkt's Solution (Zeit. wiss. Mik., iv, 1887, p. 348). — A saturated solution of bichromate of potash and sulphate of copper in 50 per cent, alcohol, to which is added at the instant of using a little acetic acid, 6 or 6 drops per 100 c.c. To make the solution, add the finely powdered salts to the alcohol in CHAPTER IV. 43 excess, and leave tliem together in total durlcness, for twenty-four hours. Fix for twelve to twenty-four hours in the darh. Then treat with strong alcohol for twelve to twenty-four hours. 56. Dekhutzen's Liquids {0. B. Acad. Sci., cxxxvii, 1903, pp. 415 and 445). — (a) 250 c.c. of 2-5 per cent. sol. of bichromate in sea-water, 26 CO. of 6-3 per cent, nitric acid, and 54 c.c. of 2 per cent, osmic acid. For general use with marine animals. (b) 173-1 c.c. of the bichromate sol. and 26-9 of 2 per cent. sol. of osmic acid. For objects containing calcareous elements that it is desired to preserve. These liquids are stated to be isotonic with sea-water. 57. Bichromate and Sublimate (Kultschitzkt, Arch. f. mih. Anat., xlix, 1897, p. 8). — ^Two parts bichromate, ^ part corrosive sublimate, 50 parts 2 per cent, acetic acid, and 50 parts 96 per cent, alcohol. The niixture should be filtered after twenty-four hours. Tissues of verte- brates may remain in it for four to six days. Lavdowskt {Zeit. leiss. Mil-., xvii, 1900, p. 301) takes 600 c.c. of 1 per cent, acetic acid, 20 to 26 grms. bichromate, and 5 to 10 c.c. saturated solution of sublimate in water. 58. Bichromate of Ammonia. — This salt is in considerable favour for hardening. Its action is very similar to that of the potassium salt. Fol says that it penetrates somewhat more rapidly, and hardens some- what more slowly. It should be employed in somewhat stronger solutions, up to 5 per cent. 59. Neutral Chromate of Ammonia is preferred by some. It is used in the same strength as the bichromate. Klein has recommended it for intestine, which it hardens, in 5 per cent, solution, in twenty -four hours. 60. Bichromate of Calcium. — Sonnenbkodt (Arch, mih: Anat., Ixxii, 1908, p. 416) fixes ovaries of Oallus in 20 parts of 2 per cent. sol. of calcium bichromate with 10 of 2 per cent. sol. of sublimate and 1 of acetic acid. 61. Bichromates and Alcohol. — -Mixtures of bichromate of potash or ammonia witli alcohol may be employed, and have a more rapid action than the aqueous solution. Thus Hamilton takes for hardening brain a mixture of 1 part methylated spirits with 3 parts of solution of Miiller ; see also Kultschitzkt's Mixture, ante, § 65). Preparations should be kept in the dark during the process of hardening in these mixtures. 62. Sulphurous Acid. — ^Waddington {Journ. Boy. Mic. Soc, 1883, p. 185) uses a saturated solution of sulphurous acid in alcohol for fixing infusoria. Oveeton (Zeit. wiss. Mik., vii, 1890, p. 9) uses the vapours of an aqueous solution for fixing algse. CHAPTER V. FIXING AND HARDENING AGENTS — CHLORIDES, ORGANIC ACIDS, AND OTHERS. Chlorides. 63. Bichloride of Mercury (Corrosive Sublimate).— Corrosive sublimate is soluble in about 16 parts of cold and 3 of boiling distilled water. It is more soluble in alcohol (1:3) or in ether (1:4) than in water. Its solubility in all these menstrua is augmented by the addition of hydrochloric acid, ammonious chloride, or camphor. With sodium chloride it forms a more easily soluble double salt ; hence sea-water may dissolve over 15 per cent. The simple aqueous solutions should always be made with distilled — not spring — water. The HgClg in them is partly split up by hydrolysis into CI, H, and (HgC])2, or HgClOH (see Chem. Centralb., 1904, i, p. 571 ; the statements of Mann [Methods, pp. 22, 77] are incorrect). These solutions should give an acid reaction with litmus paper, whilst those made with strong sodium chloride solution are neutral. For fixing, corrosive sublimate may be used pure ; but in most cases a finer fixation will be obtained if it be acidified with acetic acid, say about 1 per cent, of the glacial acid. I find that a saturated solution in 5 per cent, glacial acetic acid is a very good formula for marine animals ; for others I should take the acid weaker. Kaiser's solution consists of 10 grms. sublimate, 3 c.c. glacial acetic acid, and 300 c.c. distilled water (from Zeit. wiss. Mik., xi, p. 378). Van Beneden has used a saturated solution in 25 per cent, acetic acid, and Lo Bianco {Mitth. Zool. Stat. Neapel, ix, 1890, p. 443) a mixture of 2 parts saturated solution with 1 part of 49 per cent, acetic acid. It is sometimes advisable to take the most concentrated solution obtainable. For some very contractile forms (coral polypes, Planaria), a concentrated solution in warm or even boiling water should be employed. For Arthropoda alcoholic solutions are frequently indicated. Delicate objects, however, may require treatment with weak solutions. CHAPTER V. 45 Objects should in all cases be removed from the fixing bath as soon as fixed, that is, as soon as they are seen to have become opaque throughout, which may be in a few minutes or even seconds. Wash out with water or alcohol. Alcoholis almost always prefer- able. Alcohol of about 70 per cent, may be taken, and (Mayer,' Intern. Monatsschr. Anat. Phys., iv, 1887, p. 43) a little tincture of iodine may be added to the liquid, either alcohol or water, used for washing, enough to make it of a good port-wine colour, and the mixture be changed until it no longer becomes discoloured by the objects. Apathy [Mihrotechnik, p. 148) takes a 0-5 per cent, solution of iodine in strong alcohol, leaves the objects in it (suspended) until they have become of about the colour of the solution, and then washes for twenty-four hours in pure alcohol. In obstinate oases solution of iodine in iodide of potassium {e.g. Lttgol's) may be taken. Mayer {Zeit. wiss. Mile, xiv, 1897, p. 28) makes it by dissolving 5 grms. of iodide of potassium in 5 o.c. of distilled water and mixing this with a solution of 0-5 grm. of iodine in 46 o.o. of 90 per cent, alcohol, but seldom uses the mixture concentrated, merely adding as much of it as is required to the alcohol or water containing the objects. The important point is, that the iodine and iodide be employed together. The iodine may be washed out in obstinate cases with magnesia water. Similarly Apathy (Mitfh. Zool. Stat. Neapel, xii, 1897, pp. 729, 730). It has been objected to this process that iodine in potassic iodide precipitates corrosive sublimate instead of dissolving it. That is true, but the precipitate is soluble in excess of the precipitant. The iodide of potassium process should be employed with care, for the iodide may partly redissolve the precipitated compounds formed by the subUmate with the albuminoids, etc., of the tissues, and it may be well not to begin adding the iodine till the objects have been brought into fairly strong alcohol, 70 or 80 per cent. It is important that the subhmate be thoroughly removed from the tissues, otherwise they become brittle, and will not stain so well. They will also become brittle if they are kept long in alcohol. It may happen that if the extraction of the excess of siiblimate from the tissues in bulk has been insufficient, crystals may form in the sections after they have been mounted in balsam. This may easily be prevented by treating the sections themselves with tincture of iodine for a quarter of an hour before mounting. Some workers hold that this does away with the necessity of treating the tissues in bulk with iodine, which is frequently a very long process. Thus, Mann {Zeit. iviss. Mit, xi, 1894, p. 479) prefers treating the sections rather than the tissues in. bulk, on the ground that the iodine makes them soft, so that they shrink on coming into paraffin. Schaper 46 FIXING AND HARDENING AGENTS. {Anal. Anz., xiii, 1897, p. 463), however, has shown that neglect to extract the sublimate from the tissues in bulk may give birth to serious artifacts, which appear to arise during the imbedding process. So also LoYEZ (Arch. Anat. Micr., viii, 1905, p. 71). Heidenhain (Zeit. wiss..Mih., XXV, 1909, p. 398) removes the iodine from sections by means of sodium thiosulphate. You may stain in any way you like. Carmine stains are peculiarly brilliant after sublimate. The solutions must not be touched with iron or steel, as these produce precipitates that may hurt the preparations. To manipu- late the objects, wood, glass or platinum may be used ; for dissecting them, hedgehog spines, or quill pens, or cactus spines. When properly employed, sublimate is for general work un- doubtedly a most useful fixing agent. It is applicable to most classes of objects. It is perhaps less applicable, in the pure form, to Arthropods, as it possesses no great power of penetrating chitin. For cytological work it is, according to my experience, not to be trusted, and only to be recommended where more precise fixing agents are counter-indicated by reason of their lack of penetration, or the like. Amongst other defects it has that of frequently causing very serious shrinkage of cells. 64. Sublimate with Salt. — A solution foutaiuing 5 grms. sublimate, 0-5 grm. sodium chloride, and 100 c.o. water has been quoted as " solution of GrAULE." A -J per ceut. aqueous solution of sodiuui chloride saturated whilst hot with sublimate was mucli recommended by Heidenhain {Festsohrifi f. KoelUker, 1892, p. 109). The addition of sodium chloride allows a stronger solution to bo obtained than can be made with pure water, and also, it is stated, enhances the penetration of the sublimate. But the fixation -precipitates (§ 29) formed by the double salt are (according to Spuler, Encyl. mile. TeehniJc, p. 1274) for the most part soluble in water, thus giving rise to imperfect preservation. Concentrated {i.e. over 20 per cent.) solution in sea-water is recom- mended for some marine animals. Stoelznbe {Zeii. wiss. Mikr., xxiii, 1906, p. 25) reoommeuds sat;urated solution of subUmate in sugar solution of 4-^ per cent., as isotonic (for warm-blooded animals). Liquid of Lang (Zool. Anz'eiger, 1878, i, p. 14).— For Planaria.— Distilled water Chloride of sodium Acetic acid Bichloride of mercury (Alum, in some cases 100 parts. . 6 to 10 „ . 6 to 8 „ . 3 to 12 „ . ipart.) 65. Alcoholic Solutions.— ApAthy {Mikrotechnik, p. Ill) recom- CHAPTER V. 47 mends a solution of 3 to 4 grms. of sublimate and 0-5 grm. sodium chloride in 100 c.c. of 50 per cent, alcohol for general pur- poses. Ohlmaci-ibe (Journ. Exper. Medicine, ii, 6, 1897, p. 671) takes — Absolute alcohol . . . . .80 parts. Cliloroform . . . . . . 15 ,, Glacial acetic acid . . . . . 5 ,, Sublimate to saturation (about 20 per cent.). " Ordinary pieces " of tissue are sufficiently fixed in fifteen to thirty minutes. Entire human cerebral hemispheres, subdivided by Meynert's section, take eighteen to twenty-four hours. For Kquids containing a much higher proportion of acetic acid, see Acetic Alcohol. 66. Acetone Solution. — Held {Arch. Anat. Phys., Anat. Abth., 1897, p. 227) fixes nerve-tissue in a 1 per cent, solution of sublimate in 40 per cent, acetone, and washes out through increasingly con- centrated grades of acetone. 67. Phenol Solution. — -Pappenheim {Arch. Path. Anat, clvii, 1899, p. 23) shakes up carbolic acid with aqueous sublimate solution and filters. 68. Ciaeeio {Arch. Ital. Anat. Bmhr., vi, 1907, p. 486) has an irrational mixture of sublimate, iodine, and formol. 69. Mercuro-nitric Mixtures. — Frenzbl {Arch. mile. Anat., xxvi, 1885, p. 232) recommends a half -saturated solution of sublimate in 80 per cent, alcohol, to which is added nitric acid in the proportion of 1 drop to 1 or 2 c.c. Objects of the size of a pea to be fixed in it for five or ten minutes, then hardened in the same sublimate alcohol without the acid, and finally in 90 per cent, alcohol. It is said that the nitric acid renders after-treatment with iodine un- necessary. Gilson's Mixture (Gilson, in litt. 1895). Nitric acid of 46° strength (this would be sp. gr. 1-456, or 80 per •cent., nearly) .... 15 c.c. Glacial acetic acid . . . . 4 „ Corrosive sublimate ... 20 grms. 60 per cent, alcohol . . .100 c.c. Distilled water .... 880 „ When required fm marine animals add a few crystals of iodine, which will prevent the formation of precipitates of sea salts. If in any case the preparations should show a granular precipitate, this may be removed by washing with water containing a little tincture of iodine. 48 FIXING AND HAitDENING AGENTS. I find that it affords in general a faithful and delicate fixation, and gives to tissues an excellent consistency. Objects may remain in it for a considerable time without hurt. It has a high degree of penetration. A treatment for a few days with it will serve to remove the albumen from the ova of Batrachians. This liquid may be recommended to beginners, as it is very easy to work with. For some objects, as I found, the proportion of sublimate may be in- creased with advantage. KosTANECKi and Siedlecki {Arch. mih. Anat., xlviii, 1896, p. 181) take a mixture of saturated sublimate solution and 3 per cent, nitric acid in equal parts, or a mixture of equal parts of sublimate solution, 3 per cent, nitric acid, and absolute alcohol, fix for twenty-four hours, and wash out in iodine-alcohol. Petrunkewitsch (Zool. Jahrb. Abth. Morph., xiv, 1901, p. 576) takes water 300, absolute alcohol 200, glacial acetic acid 90, nitric acid 10, and sublimate to saturation. Both this and Gilson's have been much used lately. 70. Picro-sublimate Mixtures. — Rabl's (Zeit. iviss. Mik., xi, 1894, p. 165). — Sublimate, saturated solution in water, 1 vol. ; a similar solution of picric acid, 1 vol. ; distilled water, 2 vols. Embryos may be left in it for twelve hours, washed for two hours in water, and brought into weak alcohol. 0. vom Rath {Anat. Anz., xi, 1895, p. 268) takes cold saturated solution of picric acid, 1 part ; hot saturated solution of sublimate, 1 part ; glacial acetic acid, ^ to 1 per cent. Also the same with the addition of 10 per cent, of 2 per cent, osmic acid solution. 71. Osmio-sublimate Mixtures. — Mann's {Zeit. wiss. Mik., xi, 1894, p. 481) consists of a freshly prepared mixture of equal parts of 1 per cent, osmic acid solution and saturated solution of sublimate in normal salt solution (for nerve-centres). See "Mann-Kopsch Method," § 693. 72. Chromo-sublimate.— Lo Bianco {Mitth. Zool. Stat. 'Neapel, ix, 3, 1890, p. 443). — Concentrated sublimate solution, 100 parts ; 1 per cent, chromic acid, 50 parts. Mann {Verh. Awit. Ges., 12, 1898, p. 39) takes for nerve-cells equal parts of 5 per cent, sublimate and 5 per cent, chromic acid. 73. Sublimate and Bichromate.— Zenker's Mixture {Miinchener med. Wochenschr., xxiv., 1894, p. 534 ; quoted from Mercier, Zeit. wiss. Mik., xi, 4, 1894, p. 471).— Five per cent, of sublimate and 5 per cent, of glacial acetic acid dissolved in solution of Muller. CHAPTER V. 49 Fix for several hours or overnight, wash out with water, treat the tissues in bulk, or the sections with alcohol containing tincture of iodine. Refer to § 684, See also Eetteree,' Journ. Anat. Phys., xxxiii, 1897, p. 463, and xxxvii, 1901, p. 480. If the objects be allowed to remain too long in the fluid there may be formed precipitates, which it is very difficult to remove. Spuler (Encycl. mik Technik, 1st ed., p. 1280) says that they may be avoided by removing the objects as soon as penetrated, and com- pleting the hardening in liquid of Muller. I recommend this method. Helly [Zeit. wiss. Mik, xx, 1904, p. 413) omits the acetic acid, and adds, immediately before use, 5 per cent, of formol. This is a splendid fixative for vertebrate material. Fix overnight, wash out in running water for several hours. See footnote to § 684. Maximow (ib., xxvi, 1909, p. 179) adds 10 per cent, of formol and sometimes 10 per cent, of osmic acid of 2 per cent, (fix in the dark). Fol {Quart. Journ. Mie. 8ci., 1895, p. 287) takes equal parts of saturated solution of sublimate in normal salt solution, and of liquid of MuUer, or 5 per cent, solution of bichromate. HOTEE (Arch. Milcr. Anat., liv, 1899, p. 97) takes 1 part 5 per cent, sublimate and 2 of 3 per cent, bichromate. KOHN {ib., Lxx, 1907, p. 273) takes 5 parts 5 per cent, sublimate, 15 parts 3J per cent, bichromate, and 1 part acetic acid. 74. Sublamin (Ethylendiamin Sulphate of Mercury) is recommended in 5 per cent, solution by KLiNGMtJLLER and Veiel {Zeit. wiss. Milcr., xxi, 1904, p. 58). 75. Platinum Chloride. — -The substance used and intended by the authors who have recommended this reagent is not the true platinic chloride, or tetrachloride, PtCl4, but the compound HaPtClg, that is, platinochloric, or hydro-chloro-platinic acid, by custom called platinum chloride. It occurs as brown-red crystals, easily soluble in water and very deliquescent. For this reason it had better be stocked in the form of a 10 per cent, solution, kept in the dark (weak solutions — 0-5 per cent. — may be kept in the light). It appears that some authors have stated that they were using platinous chloride, PtCla, but that is not possible, as this salt is not soluble in water. Rabl {Morph. Jahrb., x, 1884, p. 216) employed an aqueous solution of 1 : 300. The objects remained in it for twenty-four hours, and were then washed out with water. Well-washed preparations give good chromatin stains with the " basic " tar colours ; but I find, as do others, that plasma-staining with the " acid " colours is 50 FIXING AND HARDENING AGENTS. rendered extremely difficult. It causes a certain shrinkage of chromatin. ' It is now almost always employed in the form of mixtures. For these see §§ 44, 45, 49, 76," as well as the mixtures given under " Picric Acid" and "Formol." 76. Rabl {Zeit. iviss. Mikr., xi, 1894, p. 165) takes for embryos of vertebrates, and also for other objects, 1 vol. of 1 per cent, platinum chloride, 1 of saturated sublimate, and 2 of water. Lenhossek {Arch. mikr. Anat., li, 1898, p. 220) takes 20 parts of 1 per cent, platinum chloride, 20 of 5 per cent, subhmate, and 1 of acetic acid. 77. Palladium Chloride (Schulze, Arcli. mile. Anat., iii, 1867, p. 477). — ^Used by Sohulze as a hardening agent in a 1 : 800 solution, acidified with hydrochloric acid. Cattaneo has used it in solutions of 1 : 300, 1 : 600, or 1 : 800 strength, for from one to two minutes, for Infusoria. Frenkel (Anat. Am., viii, 1893, p. 538) recommends for connective tissue a mixture of 15 parts 1 per cent, palladium chloride, 5 parts 2 per cent, osmic acid, and a few drops of acetic acid. 78. Iridium Chloride (Eisen, Zeit. wiss. Mik., xiv, 1897, p. 196). — Solution of I or J per cent., acidified with 1 per cent, of glacial acetic acid. With the ovotestis of the snail, I have obtained about the worst fixation I have ever seen, but with the testis of Triton much better results. 79. Osmium Chloride (Eisen, Journ. of Morph., xvii, 1900). — Solution of J to ,J(f per cent. From specimens I have seen I should say it is 80. Perchloride of Iron (Fol, Zeit. wiss. Zool., xxxviii, 1883, p. 491, and Lehrb. d. vergl. mih. Anat., p. 102). — ^Fol recommends 1 vol. of- Tinot. Ferri PercMor. B.P. diluted with 5 to 10 vols, of 70 per cent, alcohol. The tincture diluted with 3 to 4 vols, of either alcohol or water has been recommended for fixing meduUated nerve by Plainer ( Zeit. wiss. Mile, vi, 1889, p. 187). 81. Iron Alum. — Strong {Journ. oomp. Neur., xiii, 1903, p. 296) fixes (and decalcifies) heads of young Aoanthias in 9 parts of 5 per cent, solution of iron alum with 1 of formol, for about two weeks. 82. Chloride of Zine is sometimes used for hardening brain (see Part II). GiLSON {La Oelhde, vi, 1890, p. 122) has used it as a fixative for the silk glands of Lepidoptera, as follows : Glacial acetic acid .... Nitric acid of 46° (or 80 per cent, nearly) Alcohol of 80 per cent. Distilled water .... Dry chloride of zinc 5 c.c. 5 >> . 100 n . 300 t> 20 grms. CHAPTER V. 51 83. Iodine. — Kent (Manual of the Infusoria, 1881, p. 114) uses it for fixing Infusoria. Prepare a saturated solution of potassic iodide in distilled water, saturate this solution with iodine, filter, and dilute to a brown-sherry colour. A very small portion only of the fluid is to be added to that containing the Infusoria. Or you may use Lugol's solution : Water 100 parts. Iodide of potassium . . . . 6 ,, Iodine ....... 4 ,, Or for sm.all marine animals, a solution of iodine in sea-water. Personally I have found it very useful for the examination of sperma- tozoa. See also under Goodrich's lodine-Bouin method. Very smaU objects may be instantaneously fixed by means of vapour of Iodine. Crystals of iodine may be heated in a test-tube tiU the vapours are given off ; then on inclining the tube the heavy vapours may be made to flow over the objects arranged on a slide. The slide should then be warmed to about 40° C. for one to three minutes in order to evaporate the iodine from the objects, which may then be mounted or otherwise treated as desired (Ovekton, Zeit. wiss. Mile., vii, 1890, p. 14). Organic Acids, and other Agents. 84. Acetic Acid. — A substance most injurious to the finer elements of the cytoplasm ; in some cases it is indicated for a study of the nuclear elements. Flemming, who has made a special investigation of its action on nudei, finds (Zellsubstanz, etc., p. 380) that the best strength is from 0'2 to 1 per cent. Strengths of 5 per cent, and more bring out the nuclein structures clearly at first, but after a time cause them to swell and become pale, which is not the case with the weaker strengths {ibid., p. 103). The strong acid is, however, a valuable fixative of certain objects, which it kills with the utmost rapidity, and leaves fixed in a state of extension. The modus operandi of Van Beneden is as follows : — Pour glacial acetic acid in liberal quantity over the organisms, leave them until they are penetrated by it — ^which should be in five or six minutes, as the strong acid is a highly penetrating reagent — and wash out in frequent changes of alcohol of gradually increasing strength. Some persons begin with 30 per cent, alcohol, but this appears to me rather weak, and I think 70 per cent, or at least 60 per cent, should be preferred. Other energetic reagents inay be combined with the glacial acetic acid if desired. Dr. Lindsay Johnson (in liU.) has found that one of the best fixatives for retina is a mixture of equal parts glacial acetic acid and 2 per cent, osmio acid. S. Lo Bianco adds to his " concen- trated " (49 per cent.) acid one-tenth of a 1 per cent, solution of chromic acid. He finds that even this small proportion of chromic 4—2 52 FIXING AND HARDENING AGENTS. acid serves to counteract in a mai-ked degree the softening action of tlio acetic acid. Acetic acid, used alone, is only a fixative for a limited time. If its action be prolonged, it becomes a swelling agent. Its function in mixtures is, besides that of hilling, the valuable one of counteracting the shrinking action of the ingredients with which it is combined, and by its sweUing action enhancing the penetration of the mixture ; whilst by clarifying tissues it adds to the optical difierentiation of their elements. The proportions in which it should enter into mixtures in general seem to me to be from 0-5 per cent, to 5 per cent, of the glacial acid ; higher strengths, such as 25 per cent, to 100 per cent., being only indicated in cases in which the highest possible penetration is the chief consideration. Throughout this work, wherever acetic acid is mentioned, it is the glacial acid that is meant unless the contrary is stated. All liquids containing a large proportion of this acid {e.g., §§ 85, 86) should only be allowed to act for a very short time. 85. Acetic Alcohol (Carnoy, La Cellule, iii, 1886, p. 6 ; and ibid., 1887, p. 276; v. Beneden et Neyt, Bull. Ac. Sd. Belg., xiv, 1887, p. 218 ; Zachaeias, Anat. Anz., iii, 1888, pp. 24 — 27 ; v. Gehuch- TEN, ibid., 8, p. 227). — Carnoy has given two formulae for this important reagent. The first is — Glacial acetic acid .... 1 part. Absolute alcohol . . . . 3 parts. The second is — Glacial acetic acid . . . .1 part. Absolute alcohol . ... 6 parts. Chloroform ^ . . . . 3 „ The addition of chloroform is said to render the action of the mixture more rapid. V. Beneden and Neyt take equal volumes of glacial acid and absolute alcohol. Zachakias takes — Glacial acetic acid .... 1 part. Absolute alcohol . . . .4 parts. Osmic acid a few drops. Acetic alcohol is one of the most penetrating and qxiickly acting fixatives known. It preserves both nuclei and cytoplasm, and admits of staining in any way that may be preferred. It was employed by all of the authors quoted for the ova of Ascaris — proverbially one of the most dif&cult objects to fix, — but I have found that it is applicable to many other objects. Wasb out with alcohol, and avoid aqueous liquids as far as possible in the after- treatment. CHAPTER V. 53 86. Acetic Alcohol with Sublimate. — Cabnoy and Lebrun (La Cellule, xiii, 1, 1887, p. 68, due to Gilson). Absolute alcohol ...... 1 vol. Glacial acetic acid . . . . . 1 „ Chloroform . . . . . . 1 „ Sublimate to saturation. (The mixture does not keep long, forming ethyl acetate, which precipitates). Isolated ova of Ascaris, even though furnished with a shell, are fixed in twenty-five to thirty seconds. Entire oviducts take about ten minutes. The liquid is therefore one of the most penetrating and rapidly acting of any, if not the most. Wash out with alcohol until all traces of odour of the acetic acid have disappeared (I myself wash out with alcohol containing tincture of iodine). I consider this a very fine reagent. For Ohlmacher's mixture see § 65. G. S. Sansom's Carnoy Modification. — Absolute alcohol . . . . .65 c.c. Glac. acetic acid . . . . . 5 ,, Chloroform . . . . . . 30 ,, Corr. subl. to saturation. Leave ten minutes to half an hour ; wash in iodine absolute, then absolute. (Personal communication.) Eminently suitable for study of vertebrate material. I have seen some really brilliant results obtained by the use of this fluid (§ 590). 87. Trlchlor-aeetie Acid (Holmgren, Anat. Hefte, xviii, 1901, H. 2). — ^Five per cent, solution in water. Fix (nerve-ceUs) for eight to twenty-four hours, wash out with alcohol. See also Heidenhain, Zeit. wise. Mikr., xxii, 1905, p. 321, and xxv, 1909, p. 405, who makes a mixture of 6 per cent. subUmatc solution with 2 per cent, of trichlor- acetic and 1 per cent, of acetic acid, which he calls " Subtriessig." 88. Trichlor-acetic Fluid for Batrachia (Ghampy, Arch. d. Zool. Exper. et G6n., t. lii, 1913).— Carbolic acid cryst. in sat. aq. sol. . .15 parts. Formol, 40 per cent. . . . . 4 „ Trichlor-acetic acid, 20 per cent. . . 1-5 ,, Outside of tissue often bad, inner parts better. 89. Salicylic Acid (Heidenhain, Arch. mik. Anat., liv, 1899, p. 186). — Saturated solution in one-third alcohol. A trial has given me simply atrocious results. 90. Chloride and Acetate of Copper (Ripart et Petit's Liquid, Carnoy, La Biologie Cellulaire, p. 94). — Camphor water (not saturated) . . 75 grms. Distilled water . . . . . 75 „ Crystallised acetic acid .... 1 grm. Acetate of copper 0-30 „ Chloride of copper 0-30 „ 54 FIXING AND HARDENING AGENTS. This is a very moderate and delicate fixative, extremely useful for objects that are to be studied in as fresh a state as possible in. aqueous media. Objects fixed in it stain instantaneously and perfectly with methyl green. Osmic acid may be added to the liquid to increase the fixing action. For cytological researches a valuable medium. 91. Nitrate of Copper (Gilson, from Geldekd, La Cellule, xxv, 1909, p. 12). — Nitrate of copper 200, formol 500, sea-water 200. Seven parts of this solution to be diluted with 100 of sea-water. For Crustacea. 92. Acetate of Uranium (Sciienk, Mitth. Embryol. Inst. Wien, 1882, p. 95 ; cf. GiLSON, La Cellule, i, 1885, p. 141) lias a mild fixing action, and a liigh degree of penetration, and may be combined with methyl green. Feiedenthal (Sitzh. Oes. Nat. Freunde Berlin, 1907, p. 209) recom- mends equal parts of saturated solution of the acetate and trichlor-aoetio acid of 50 per cent. 93. Picric Acid. — Picric acid in aqueous solution should be em- ployed in the form of a strong solution whenever it is desired to make sections or other preparations of tissues with the elements in situ, as weak solutions macerate ; but for dissociation preparations or the fixation of isolated cells, weak solutions may be taken. Flemming finds that the fixation of nuclear figures is equally good with strong or weak solutions. The saturated solution is the one most employed. (One part of picric acid dissolves in about 86 parts of water at 15° C. ; in hot water it is very much more soluble.) Objects should remain in it for from a few seconds to twenty-four hours, according to their size. For Infusoria one to at most two minutes will sufiice, whilst objects of a thickness of several miUimetres require several hours. Picric acid should always he washed mil with alcohol, that of 70 per ^ cent, being mostly indicated. Staining should be performed by means of alcoholic solutions, or if with aqueous, then with such as are themselves weak hardening agents, such as hsemalum, carmalum, methyl green. Washing out is facilitated by heat, the extraction being about twice as rapid at 40° 0. as at the normal temperature (Fol). It has been found by Jelinek {Zeit. wiss. Mile., xi, 1894, p. 242) that the extraction is greatly quickened by the addition of a base to the wash-alcohol. He recommends carbonate of lithia. 'A few drops of a saturated solution of the salt in water are added to the alcohol ; a precipitate is formed. The objects are put into the turbid alcohol, which becomes clear and yellow in proportion as the ])icrin is extracted. Further quantities of carbonate are added from time to time until the colour has been entirely extracted. CHAPTER V. 55 Tissues fixed in picric acid can be perfectly stained in any stain. It is seldom necessary to remove the picric acid by washing out before staining. Paracarmine, Boraxcarmine, or Heemacalcium may be recommended for entire objects. The most important property of picric acid is its great penetration. This renders it peculiarly suitable for the preparation of chitinous structures. 94. Picric Alcohol (Gage, Proc. Amer. Soe. Mici:, 1890, p. 120). — Alcohol (95 per cent.), 250 parts ; water, 250 parts ; picric acid, 1 part. 95. Picro-acetie Acid.— Boveei {Zellenstudien, l, 1887, p. 11) dilutes a concentrated aqueous solution of picric acid with two volumes of water and adds 1 per cent, of acetic acid. According to my experience, the resu^lts are miserable. Zimmek's mixture (from Deegenek, Zool. Jahrb., Abth. Morph., xxvii, 1909, p. 634). — Saturated aqueous solution of picric acid, 10 parts ; absolute alcohol, 9 ; acetic acid, 1. 96. Picro-sulphuric Acid (Kleinenbeeg, Quart. Journ. Mic. 8ci., April, 1879, p. 208 ; Mayer, Mitt. Zool. Stat. Neapel, ii, 1880, p. 2). — Matee takes distilled water, 100 vols. ; sulphuric acid, 2 vols. ; picric acid, as much as will dissolve. Liquid of Kleinenbeeg is made by diluting the concentrated picro- sulphuric acid prepared as above with three times its volume of water. I hold that the concentrated solution is generally preferable. This particularly applies to marine organisms. Wash out with successive alcohols, beginning with 70 per cent., never with water. Warm alcohol extracts the acid much more quiolsly than cold, without which weeks may be required to fully remove the acid from chitinous ' structures. This hquid may still be useful for Arthropoda, on account of its great power of penetrating chitin ; and for some embryological purposes. For a fuller account see early editions. 97. Picro-nitric Acid (Mayer, Mitth. Zool. Stat. Neapel, 1881, p.5)- Water 100 vols. Nitric acid (of 25 per cent. NgOg) . . 5 „ Picric acid, as much as will dissolve. Properties of this fluid similar to those of picro-sulphuric acid, with the advantage of avoiding the formation of gypsum crystals, and the disadvantage that it is much more difficult to soak out of the tissues. Mayer states that with eggs containing a large amount of yolk material, like those of Palinurus, it gives better results than nitric, picric, or picro-sulphuric acid. I myself consider it distinctly superior to picro-sulphuric for most things. See Hill's fluid, § 586. 56 FIXING AND HARDENING AGENTS. 98. Picro-hydrochloric Acid (Matee, ibid.). — Water 100 vols. Hydrochloric acid (of 25 per cent. HCl) . 8 „ Picric acid, as much as will dissolve. 99. Picro-chromic Acid (Fol, LeJirh., p. 100). — Picric acid, sol. sat. in water . . .10 vols. 1 per cent, chromic acid solution . . 25 „ • Water .... . . 65 „ I have seen Pol's formula, with the addition of a trace of acetic acid, quoted as " liquid of Hae.nsel." Lo Bianco takes equal parts of picro -sulphuric acid and chromic acid of 1 per cent. Eawitz [Leitfaden, 1895, p. 24) takes 1 part of picro-nitric acid, and 4 parts 1 per cent, chromic acid. Wash out in 70 per cent, alcohol. 100. Picro-osmic Acid. — ^Plbmming (Zells. Kern u. Zellfh., p. 381) has experimented with mixtures made by substituting picric for chromic acid in the chromo-osmic mixtures (§ 42), and finds the results identical, so far as regards the fixation of nuclei. The fixation of cytoplasm is in my preparations decidedly inferior. 0. VOM Rath (Anat. Ann., xi, 1895, p. 289) adds to 200 c.c. of saturated aqueous solution of picric acid, 12 c.c. of 2 per cent, solution of osmic acid, and 2 c.c. of glacial acetic acid. Eawitz (Leitfaden, p. 24) takes picro-nitrio acid, 6 vols. ; 2 per cent. osmic acid, 1 vol. Fix for half to three hours. Transfer direct to 70 per cent, alcohol. 101. Picro-platinic and Picro-platin-osmic Mixtures. — 0. vom Rath (Uoc. cit, last §, pp. 282, 285) makes a picro-platinic mixture with 200 c.c. saturated aqueous solution of picric acid, 1 grm . of platinic chloride (dissolved in 10 c.c. of water), and 2 c.c. of glacial acetic acid. The picro-platin-osmic mixture, which is, in my opinion, much superior, is made by adding to the foregoing 26 c.jo. of 2 per cent, osmic acid. Other Picric Mixtures. See §§ 70 and 110 to 112. Other Fixing and Hardening Agents. 102. Alcohol. — For fixing only two grades of alcohol should be employed — very weak, or -absolute. Absolute alcohol ranks as a fixing agent because it kills and hardens with such rapidity that structures have hardly time to get deformed in the process ; very weak, because it possesses a sufficiently energetic coagulating action ' and yet contains enough water to have but a feeble dehydrating action. The intermediate grades do not reaUse these conditions, and therefore should not be employed alone for fixing. But they may be very useful in combination with other fixing agents by CHAPTER V. 57 enhancing their penetrating power ; 70 per cent, is a good grade for this purpose. TaUe for diluting alcohol (after Gat-Lussac).— To use this table, find in the upper horizontal row of figures the percentage of the alcohol that it is desired to dilute, and in the vertical row to the left the percentage of the alcohol it is desired to arrive at. Then follow out the vertical and horizontal rows headed respectively by these figures, and the figure printed at the point of intersection of the two rows will show how many volumes of water must be taken to reduce one hundred volumes of the original alcohol to the required grade. Weaker Original Grade. grade required. 90 p. 100. 85 p. 100. 80 p. 100. 75 p. 100. 70 ■ p. 100. 65 p. 100. 60 p. 100. 55 p. 100. 50 p. 100. p. 100. 85 6-56 80 13-79 6-83 75 21-89 14-48 7-20 70 31-05 23-14 15-35 7-64 65 41-53 33-03 24-66 16-37 8-15 60 53-65 44-48 35-44 26-47 17-58 8-76 55 67-87 57-90 48-07 38-32 28-63 19-02 9-47 50 84-71 73-90 63 04 52-43 41-73 31-25 20-47 10-35 45 105-34 93-30 81-38 69-54 57-78 46-09 34-46 22-90 11-41 40 130-80 117-34 104-01 90-76 77-58 64-48 51-43 38-46 25-55 35 163-28 148-01 132-88 117-82 102-84 87-93 73-08 58-31 43-59 30 206-22 188-57 171-05 153-61 136-04 118-94 101-71 84-54 67-45 Alcohol is an easily oxidisdble substance. Chromic acid, for instance, easily oxidises it, first into aldehyde, and then into acetic acid. It foUows that alcohol should not be combined in mixtures with oxidising agents of notable energy. Further, alcohol is a reducing agent, and therefore should not be combined with easily reducible substances. These remarks particularly apply to chromic acid. See §§ 38, 39, 48. ¥ov fixing, alcohol is a very third-class reagent, only to be used alone where better ones cannot be conveniently employed, though it enters as a useful ingredient into many mixtures, in which it serves to enhance the power of penetration. For hardening it is an im- 58 FIXING AND HARDENING AGENTS. portant one. 90 to 95 per cent, is the most generally useful strength. Weaker alcohol, down to 70 per cent., is often indicated. Absolute alcohol is seldom advisable. You ought to begin with weak, and proceed gradually to stronger, alcohol. Large quantities of alcohol should be taken. The alcohol should be frequently changed, or the tissue should be suspended near the top of it (§ 34). Many weeks may be necessary for hardening large specimens. Small pieces of permeable tissue, such as mucous membrane, may be sufficiently hardened in twenty-four hours. 103. Absolute Alcohol. — This is sometimes valuable on account of its great penetrating power. Mayer finds that boiling absolute alcohol is often the only means of killing certain Arthropoda rapidly enough to avoid maceration. It is important to employ for fixing a very large proportion of alcohol. Alum-carmine is a good stain for small specimens so fixed. For preservation, the object should be put into a weaker alcohol, 90 per cent, or less. As to the supposed superiority of absolute alcohol over ordinary strong alcohol, see last § ; and amongst authors upholding its superiority, see besides Kanvibr, Mayer {Mitth. Zool. Stat. Neapel, ii, 1880, p. 7) ; Bkuel {Zool. Jahrb., Abth. Morph., x, 1897, p. 569) ; and van Eees {ibid., iii, 1888, p. 10). Absolute alcohol is a product that it is almost impossible to preserve in use, on account of the rapidity with which it hydrates on exposure to air. Pol recommends that a little quicklime be kept in it. This absorbs part at least of the moisture drawn by it from the air. Ranvier prepares a sufficiently " absolute " alcohol as follows : — Strong (95 per cent.) alcohol is treated with calcined oupric sulphate, with which it is shaken up and allowed to remain for a day or two. It is then decanted and treated with fresh oupric sulphate," and the operation is repeated until the fresh oupric sulphate no longer becomes conspicuously blue on contact with the alcohol ; or until, on a drop of the alcohol being mixed with a drop of turpentine, no particles of water can be seen in it under the microscope. The oupric sulphate is prepared by calcining common blue vitriol in a porcelain . capsule over a flame until it becomes white, and then reducing it to powder (see Proo. Acad. Nat. Sci. Philad., 1884, p. 27 ; Journ. Boy. Mio. Soe., 1884, pp. 322 and 984). Test for the presence of water (Yvon, 0. B. Acad. Sci., 1897, p. 1181). — Add coarsely powdered calcium carbide ; the merest trace of water will cause an evolution of acetylene gas, and on agitation the alcohol will become turbid. 104. One-third Alcohol.— The grade of weak alcohol that is generally held to be most useful for fixing is one-third alcohol, or Ranvier's Alcohol. It consists of two parts of water and one part CHAPTER V. 59 of alcohol of 90 per cent, (and not of absolute alcohol). See the Traite Technique of Eanvier, p. 241, et passim. Objects may be left for twenty-four hours in this alcohol ; not more, unless there be no reason for avoiding maceration, which will generally occur after that time. You may conveniently stain with picro-carmine, alum-carmine, or methyl green. This reagent is a very mild fixative. Its hardening action is so slight that it is not at all indicated for the fixing of objects that are intended to be sectioned. Its chief use is for extemporaneous and dissociation preparations. 105. Pyridin. — ^Pyridin has been recommended as a hardening agent (by A. DE Souza). It hardens, dehydrates, and clears at the same time. It is said to harden qiiickly, and to give particularly good results with brain. See Comptes Bendus hehd. de la 8oc. de Biologie, 8 ser., t. iv, 1887, p. 622. This substance is strongly alkaline, and, either pure or diluted with water, dissolves many albumens and fats. It causes considerable shrinkage of nuclei (not so much of cytoplasm). It is now in much use in certain neuro-flbril stains, see Bielsciiowskt and Kam6n. It is soluble in water and in alcohol. Pure, it will harden and dehydrate small brains in a week. 106. Acetone is said to harden very rapidly. It precipitates lipins, and may yet prove an important reagent. Scholz (Zeit. wiss. Mikr., xxii, 1905, p. 415) fixes small objects in warm acetone for half an hour to an hour and brings them direct, or through alcohol and ether, into celloidin. Similarly Fuss {Arch. path. Anat., clxxxv, 1906, p. 5), using it cold, and Lintwarevv {ibid., ccvi, 1911, p. 36) for erythrocytes, in which it preserves the haemoglobin. 107. Lucidol. — This is a proprietary name applied to peroxide of benzol (CeHs — 00)202. It is a white powder insoluble in water, but soluble in acetone and pyridin ; heated, the white powder first of. all melts and then explodes. The introduction of " Lucidol " is -due to Ehelich, who suggested it to Szecsi. "Lucidol" must share with its solvents, acetone and pyridin, their defects as fixing agents. Acetone, especially tends to tear cells to pieces, owing to its attraction for water. So far, there have been very few references to the use of " Lucidol " in histological literature. Langeron (C R. Soc. de Biol., Ixxvi, 1914) claims that " Lucidol" is especially convenient for work on blood, fseces and cell smears, and two of SzECSi's methods arc given below. It sliould be pointed out that the Lucidol-acetone solution gives a very active penetration, and unlike Carnoy's or Schaud inn's fluids, does not dissolve away lipins. 60 FIXING AND HARDENING AGENTS. In all probability some method of mordanting the fixed material in either K2Cr207 or CrOa dissolved in a suitable solvent might help to " set " the lipins, and so avoid their dissolution when the material or smears are brought into alcohols. SzECSi (Deutsche med. WocJi., 1913) uses two solutions : one consists of 10 grms. of "Lucidor" to 100 c.c. of acetone, the other 12 grms. of " Lucidol " to 100 c.c. of pyridin. Both solutions are used for smears, or for pieces of tissue. After fixation, the latter are washed out in a mixture of acetone 3 parts, and xylol or toluol 2 parts ; and then transferred to methyl alcohol or pure xylol according to whether one is dealing with smears, or tissue for embedding in wax. For tissues proceed as follows : — Fix small pieces at room tem- perature in the acetone solution for about four or six hours, or in the pyridin solution for ten to twelve hours. In each case transfer to the above-mentioned acetone xylol mixture for several hours, bub not longer than ten. Clear in pure xylol or toluol, embed in paraffin wax. Sections can be stained as desirable. For the treatment of smears, etc., see under " Blood." 108. Formaldehyde, Formic Aldehyde, Methyl Aldehyde (Formol, Formalin, Formalose).-— Formaldehyde is the chemical name of the gaseous compound HCOH, obtained by the oxidation of methyl- alcohol. " Formol," " Formahn," and " Formalose " are com- mercial names for the saturated (40 per cent.) solution of i>his in distilled water. This quickly loses in strength through contact with air, and laboratory solutions rarely contain more than 38 per cent, of formaldehyde. Much confusion has been caused by indiscriminate use of the terms " formaldehyde " and " formol." The proper way is evidently either to state the strengths of solutions in terms of formaldehyde, and say so ; or to say " formol — or formalin — with so many volumes of water." The majority of writers seem to state in terms of formol. Solutions of formaldehyde sometimes decompose partially or entirely, with formation of a white deposit of paraformaldehyde. Fish says that to avoid this the solution should be kept in darkened bottles in the cool, or, according to some, it suffices to add glycerin to them. The solutions almost always have an acid reaction, due to the presence of formic acid ; but that is, as a rule, rather an advantage. But some observers hold that neutral or feebly alkaline solutions fix better than acid ones. Solutions may be neutralised by saturation CHAPTER V. 61 with magnesium or sodium carbonate ; some workers use lithium carbonate, but this should not be used for BouiN mixtures : it will generally suifice to make them up with spring water. It was said above that formaldehyde possesses certain hardening and .preserving qualities. It hardens gelatine, for instance, and certain albuminoids ; but others, on the contrary, are not hardened by it, but sometimes even rendered more soluble than they are naturally. For some theoretical considerations concerning its action on tissues, see F. Blum, in Anat. Anz., xi, 1896, p. 718 ; Benedecenti, in Arch. Anat. u. Phys., Abth, 1897, p. 219 ; Gekota, in Intern. Monatschr. Anat., xiii, 1896, p. 108 ; Zeit. wiss. Mik., xiii, p. 311 ; Sjobeing in Anat. Anz., xvii, 1900, p. 274 ; and Blum^ in Encycl. mik. Technik., p. 393. It seems to be generally admitted that this action consists in the formation of methylene compounds with the substances of the tissues. I find that, used pure, it is far from a first-class fixative. For it over-fixes and shrinks some things, and swells and vacuolates others. But notwithstanding this it is frequently very convenient on account of its compatibility with the most various stains. It has a high degree of penetration, and is a valuable ingredient in many mixtures. It is a powerful reducing agent, and therefore incompatible with such reagents as chromic acid or osmic acid and the like, ivhich it very rapidly decomposes. ¥oT fixing I find that a strength of about 4 per cent. (1 vol. formol to 9 of water, or to 8 of water if the formol has been long kept) is generally about right ; and this is the strength used by most writers. For cytological purposes a fixation of at least two days seems indi- cated : this applies especially to gonads which are notoriously difficult to preserve in formol. The strengths used in Cajal's and Da Fano's formol silver nitrate Golgi apparatus methods, generally give fine results for tissues other than genital. For these, injection fixation may be indicated. Seealso§141. Mayer takes 1 of formol to 9 of sea-water, for marine animals. Few workers use much stronger solutions. Only one (Hoyek, Anat. Am., ix, 1894, p. 236. Erganzungsheft) seems to have used concentrated solutions, I think this exaggerated, for I have found enormous over-fixation with solutions of 1 to 2 vols, of water. Wash out with alcohol (of 50 per cent, or more), not water. For hardening, the same strengths may be taken. Hardening is more rapid than with alcohol. For prolonged hardening, consider- able volumes of liquid should be taken, and the liquid should be 62 FIXING AND HARDENING AGENTS. renewed from time to time ; for the formaldehyde fixes itself on the tissues with which it comes in contact, deserting the solution, which thus becomes progressively weaker. The specimens should be suspended in the liquid or otherwise isolated from contact with the containing vessel. The hardening obtained is gentle and to.ugh, giving an elastic and not a brittle consistency. It varies greatly with different tissues. Mucin is not precipitated and remains trans- parent. Fat is not dissolved (see §§ 768 et seq.). Micro-organisms retain their specific staining reactions. Formaldehyde is said to harden celloidin as well as gelatin, and to be useful for celloidin- imbedding (Blum, Anat. Anz., xi, 1896, p. 724). • Several of the following mixtures are irrational, becoming reduced more or less quickly, but may give good results all the same. 109. Alcoholic Formol (Lavdowsky, Anat. Hefte, iy, 1894, p. 361). — Water 40 parts, 95 per cent, alcohol 20, formol 6, acetic acid 1 ; or water 30, alcohol 15, formol 5, acetic acid 1. GrTJi-LAND {Zeit. wiss. Mikr., xvii, 1900, p. 222) takes (for blood) 1 part formol and 9 parts of alcohol. Bles (Trans. Boy. Soc. Edinburgh, xli, 1905, p. 792) takes 7 of formol, 90 of alcohol of 70 per cent., and 3 of acetic acid. Telltesniczkt (Enoycl. mikr. Techn., i, p. 472) takes 5 of formol, 100 of alcohol of 70 per cent., and 5 of acetic acid. 110. Picro-Formol. — P. Bouin (Phenomenes cytologiques anormaux dans L'Histogenese, etc., Nancy, 1897, p. 19) recommends — • Picric acid, saturated aqueous sol. . . 75 parts. Formol 25 „ Acetic acid . . . . . . 5 „ Wash out with alcohol, first of 50 per cent., then 70 per cent, till the picric acid is mostly removed. I consider this to be for most purposes the most valuable fixative yet made known. I have satisfied myself that the proportions are exactly what they should be and cannot be changed without hurt. It is rather a strong fixative, and should not be allowed to act for more than 18 hours. If a weaker mixture be desired, dilute the whole, with water. The penetration is great, the fixation equable, delicate detail well pre- served, staining quahties admirable, especially with iron-hematoxyUn and Saiirefuchsin. See also " Cytology " sections, § 656, under heading of " Chromosomes and Urea," and Garnier, Bibl. Anat., V, 1898, p. 279. The formulse of Gkap (State Hosp. Bull. New York, 1897 ; Journ. Boy. Mic. Soc, 1898, p. 492) are in my view too weak. MOEEAUX (Bibl. AtMt., 1910, p. 265) takes 15 parts formol, 85 of trichlor-acetic acid of 3 per cent., and picric acid to saturation. CHAPTER Y. 63 111. Picro-platinie Formol (M. aud P. Bouin, Bibl. Anat., 1898, I. 2, p. 2).— Platinum cMoride, 1 per cent. sol. . . 20 parts. Picric acid, saturated sol. . . . . 20 „ Formol 10 „ Formic or acetic acid . . . 5 „ The mixture does not keep more than a day or two, and it is probably inferior. BouiN also (Arch. Biol., xvii, 1900, p. 211) simply substitutes formol for the osmic acid in Hermann's mixtrire, § 45. 112. Sublimate Formol (M. and P. Bouin, Iog. oit). — ^A similar mixture, in which sublimate of 1 per cent, is substituted for the platinum chloride. Another formula of the same authors {Areh. Biol., xvii, 1900, p. 211) is 1 part of formol to 3 of saturated aqueous sublimate. Rinse with water and bring into alcohol of 70 per cent. SptTLER {Eneycl. mik. Teehnilc, 1st ed., p. 1280) adds to sublimate of 3 per cent, or more 1 per cent, of glacial acetic acid and 10 per cent, of formol. Mann {Verh. Anat. Ges., 1898, p. 39) takes for nerve-cells 2| grms. sublimate, 1 grm. picric acid, 5 o.c. formol, and 100 c.c. water, or {Methods, etc., p. 97) for all tissues 2J grms. sublimate, 20 c.c. formol, and 80 c.c. water. Branca (Journ. Anat. et Phys., xxxv, 1899, p. 767) adds 10 parts of formol and 1 of acetic acid to 60 parts of saturated solution of picric acid in saturated aqueous sublimate. NowAK (Anat. Anz., xx, 1901, p. 244) takes 30 parts of saturated sublimate, 30 of 1 per cent, chromic acid, 27 of water, 3 of acetic acid, and 10 of formalin. 113. Formol-Miiller. — This is the name given by Orth {Bed. hlin. Wochenschr., 1896, No. 13) to a mixture of 1 part of formol with 10 of liquid of Miiller (§ 53). It should be freshly made up. Fix for three hours in the stove, or twelve at normal temperature, wash out with running water. Much used, especially for nervous tissues. MoELLER [Zeit. wiss. Zool., Ixvi, 1899, p. 85) takes 1 vol. of formol and 4 of 3 per cent, bichromate (for the intestine of mammals). Held {Abk. Sachs. Ges. Wiss., xxxi, 1909, p. 196) takes 3 per cent, sol. of bichromate with 4 per cent, of formol and 5 per cent, of acetic acid (for inner ear). See also Morel and Bassal, Journ. Anat. Phys., xlv, 1909, p. 632, and Helly and Maximow formulae. Look up section on " Mitochondria," especially paragraphs on Regaud and Schridde, §§ 685 to 687. 114. Chromic Acid Formol. — Lo Bianco fixes marine animals for half to one hour in 10 parts of 1 per cent, chromic acid with 1 of formol and 9 of sea-water, and passes into graded alcohols. 64 FIXING AND HARDENING AGENTS. Maechoux (from Peeez, Aroh. Zool. Exper., r, 1910, p. 11) takes 11 parts 1 per cent, chromic acid, 1 of acetic acid, 4 of water, and 16 of formol (added just before using). These mixtures are neither so good nor rehable as Bouin's picro- formol. 115. Copper Forniol.— Nelis {Bull. Acad. Se. Belg., 1899 (1900), p. 726) fixes spinal ganglia for twenty-four hours in 1 litre of 7 per cent, formol with 5 o.c. of acetic acid, 20 grms. of cupric sulphate, and sub- limate to saturation. Stappees {La Cellule, xxv, 1909, p. 366) used (for Sympoda) a mixture of GtIlson's : 100 parts of formol of 5 per cent, with 2 of nitrate of copper. Strong {Journ. Comp. Neur., xiii, 1903, p. 296) fixes the head of Aeanthias by injecting a mixture of equal parts of formol and 6 per cent, solution of bichromate of copper. 116. Nitric Acid Formol. — Wilhelmi {Fauna u. Flora Golf. Neapel, xxxii, 1909, p. 15) fixes. Triclads in Apathy's mixture of equal parts of 6 per cent, nitric acid and 6 per cent, formol, and brings them direct into strong alcohol. 117. Acetone Formol. — Bing and Elleemann {Arch. Anat. Phys., Phys. AbtJi., 1901, p. 260) fix medullated nerves in 9 parts of acetone with 1 of formol. CHAPTER VI. 118. Introduction. — De-alcoholisation agents are liquids employed ;for the purpose of getting rid of the alcohol which has been employed for dehydrating tissues (§ 3), and facilitating the penetration of the paraffin used for imbedding, or the balsam or other resinous medium in which preparations are, in most cases, finally mounted. Hence all of them must be capable of expelling alcohol from tissues, and must be at the same time solvents of Canada balsam and the other resinous mounting media. The majority of them are essential oils. Clearing agents are liquids whose function it is to make micro- scopic preparations transparent by penetrating amongst the highly refracting elements of which the tissues are composed, the clearing liquids themselves having an index of refraction superior, or equal, or, at all events, not greatly inferior, to that of the tissues to be cleared. Hence all clearing agents are liquids of high index of refraction. The majority of de-alcoholisation agents being also hquids of high refraction, it follows that they serve at the same time for de-alcohoU- sation and for clearing ; and in consequence it has come about that de-alcohohsation agents are generally spoken of as clearing agents. But that practice is not strictly correct, for not all clearing agents are solvents of the resius, and not all de-alcohoUsing agents can serve as clearers. I shall, however, stiU'in many cases continue to use the term " clearing " to signify " de-alcohohsing," for the sake of brevity. Neelsen and Schiefperdeckee (Arch. Anat. Phys., 1882, p. 206) examined a large series of ethereal oils (prepared by Schimmel & Co., Leipzig), with the object of finding a not too expensive substance that should combine the properties of clearing quickly alcohol preparations, not dissolving out anihn colours, clearing celloidin without dissolving it, and not evaporating too quickly. Of these, the following three fulfil the conditions -.—Cedar-wood, Origanum, Sandal-wood. 66 DE-ALCOHOLISATION AND CLEARING AGENTS. To these should be added the others recommended in the following paragraphs. See also the paper of Jordan {Zeit. wiss. Mik., xv, 1898, p. 50) as to the behaviour of some essential oils towards celloidin. 119. The Practice of De-alcoholisation or Clearing. — The old plan was to take the object out of the alcohol and float it on the surface of the de-alcoholising or clearing medium in a watch-glass. This plan was faulty, because the alcohol escapes from the surface of the object into the air quicker (in most instances) than the de-alcoho- lising or clearing agent can get into it ; hence the object must shrink. To avoid this cause of shrinkage, the operation is now generally done by the method suggested by Mayer and Giesbrecht, which consists in putting the clearing mediimi under the alcohol containing the object, as described § 5. The objects should not be considered to be perfectly penetrated by the clearing medium until the wavy refraction-lines caused by the mixture of the two liquids at their surface have ceased to form, and they should not be mounted or imbedded untU they have first been soaked for some time in a fresh quantity of clearing medium, to remove any alcohol that has got into the first bath. The penetration of all clearing media may be hastened by using them warm. It frequently happens that the essential oil with which objects are being treated in a watch-glass or on a slide becomes cloudy after a short time, and fails to clear the tissues. This is owing to a com- bination between the essential oil and moisture, derived, I think, rather from the air than from the objects themselves. The cloudi- ness can usually be removed by warming (as pointed out by Hatchett » Jackson, Zool. Anzeig., 1889, p. 630), but in certain moist states of the atmosphere it may persist, notwithstanding continued warming. It is for this reason that I advise that clearing be done, whenever possible, in shallow corked lubes, under which conditions the pheno- menon rarely occurs. In any case, be careful not to breathe on the liquid. 120. Choice of a De-aJcoholisation or Clearing Agent.— I advise the beginner to keep on his table the following :— Oil of cedar, for general use and for preparing objects for imbedding in paraflSn ; clove oil, for making minute dissections in (§ 122), and for much work with safranin, etc. ; oil of bergamot, which will clear from 90 per cent, alcohol, and which does not extract coal-tar colours ; carbolic acid, for rapidly clearing very imperfectly dehydrated objects. For special clear ers for eelloidin sections see Chapter IX. CHAPTER YI. 67 181. Cedar Oil (Neelsen and Schiefpekdecker, loc. cit., § 118). — Clears readily tissues in 95 per cent, alcohol without shrinkage ; does not extract anilin colours. Celloidin sections are cleared in five to six hours. The observer should be careful as to the quality of the cedar oil he obtains. I have examined the clearing properties of a sample, obtained from a celebrated firm, which totally failed to clear absolute alcohol objects after many days. Cedar oil is very penetrating, and for this and other reasons is, in my experience, the very best of all media for preparing objects for paraffin imbedding. I find it to be less hurtful to cells than any other medium known to me. Tissues may remain in it for any length of time without hurt. If it should become milky through keeping, filter. 122. Clove Oil. — Samples of clove oil of very different shades of colour are met with in commerce. It is frequently recommended that only the paler sorts should be employed in histology. Doubt- less it is, in general, best to use a pale oil, provided it be pure ; but it is not always easy to obtain a hght-coloured oil that is pure. Clove oil passes very readily from yeUow to brown with age, so that in choosing a colourless sample you run great risk of obtaining an adulterated sample, for clove oil is one of the most adulterated substances in cornmerce. Clove oil does not easily spread 'itself over the surface of a shde, but has a tendency to form very convex drops. This property makes it a very convenient medium for making minute dissections in. It also has the property of making tissues that have lain in it for some time very brittle. This brittleness is also sometimes very helpful in minute dissections. These quahties may be counteracted if desired by mixing the clove oil with bergamot oil. This is one of the most useful of clearers. According to Beheens {Tabellen, 3rd ed., 1898, p. 33), it will clear from alcohol of 74 per cent. It has a high index of refraction, and clears objects more than balsam mounting media. It dissolves celloidin (or collodion), and therefore should not be used for clearing sections cut in that medium without special precautions. New clove oil washes out basic tar colours more quickly than old. 123. Cinnamon (or Cassia) Oil greatly resembles clove oil, but is in general thinner, and is more liighly retractive. An excellent medium, whiQh I particularly recommend. fi — 2 68 DE-ALCOHOLISATION AND CLEARING AGENTS. 184. Oil of Bergamot (Schieffeedeckeb, Arch. Anat. Phys., 1882 [Anat. Abth.], p. 206). — Clears 95 per cent, alcohol preparations and celloidin preparations quickly, and does not extract anilin colours. Bergamot oil is, I believe, the least refractive of these essences, having a lower index than even oil of turpentine. SucHANNEK (Zeit. wiss. Mik., vii, 1890, p. 158) says that bleached, colourless bergamot oil will not take up much water, whereas a green oil will take up as much as 10 per cent. Van DEE Steicht {Arch, de Biol., xii, 1892, p. 741) says that bergamot oil will, with time, dissolve out the fatty granules of certain ova. 125. Oil of Origanum (Neelsen and Schieffeedeckeb, Arch. Anat. Phys., 1882, p. 204). — Ninety-five per cent, alcohol prepara- tions are cleared quickly, and so are celloidin sections, without solution of the celloidin. Anilin colours are somewhat extracted. ■ For work with celloidin sections care should be taken to obtain 01. Origani Cretici (" Spanisches Hopfenol "), not 01. Orig. Gallici (v. GiESON ; see Zeit. wiss. Mik., iv, 1887, p. 482). Specimens of origanum oil vary greatly in their action on celloidin.sections. Sqxjiee, in his Methods and Formulce, etc., p. 81, says that origa- num oil (meaning the commercial product) is nothing but oil of white thyme more or less adulterated (see next §), and that the product sold as 01. Origani Cretici is probably oil of marjoram. 126. Oil of Thyme.— Fish {Proc. Amer. Mic. Soc, 1893 ; Zeit. wiss. Mik., xi, p. 503), following Bumpus, says that for most of the purposes for which origanum oil has been recommended, oil of thyme wiU do just as well if not better. The red oil is just as efficient ^ as the white for clearing. Schimmel & Co., in their Keport of October, 1895, p. 69, state that in France white oil of thyme is adulterated with oil of turpentine to the extent of as much as 50 per cent. 127. Oil of Gaultheria. — Used by Unna (Monatschr. pralct. Derm., Ergdnmrvgsh, 1885, p. 53) for thinning balsam. The artificial oil, methyl salicylate, is recommended by Gubgtjen {Comp. Bend. Soc. Biol., V, 1898, p. 285) both as a de-alcoholisation and clearing agent and as a solvent of parafan. The refractive index is 1-53. It is, unfor- tunately, very sensitive to water. 128. Sandal-wood Oil (Neelsen and Schiefferdeoker, loo. oit). — Very useful, but its high price is prohibitive. 129. Oil of Cajeput.— Now much used. I have used it myself and found it to clear well, but to be rather thin. Caenoy and Lebeun CHAPTER VI. 69 {La Cellule, xiii, 1897, p. 71) have found it useful for clearing celloidin sections. It dissolves celloidin very slowly and clears without shrinkage. 130. Oil of Turpentine. — Generally used for dissolving out the paraffin from sections; but many other reagents, such as xylol and benzol, are preferable for this purpose. If used for alcohol objects, it causes considerable shrinkage, and alters the structure of cells more than any other clearing agent known to me. Turpentine has, I beUeve, the lowest index of refraction of all the usual clearing agents except bergamot oil ; it clears objects less than balsam. 131. Terpinol (liquid, from Schimmel & Co.) is recommended by Mayer, Zeit. wiss. Mikr., xxvi, 1910, p. 523. Clears from alcohol of 90 per cent., or even 80 per cent. One part xylol and 4 terpinol has been much used lately. 132. Carbolic Acid. — Best used in concentrated solution in alcohol. Clears instantaneously, even very watery preparations. This is a very good medium, but it is better avoided for preparations of soft parts which it is intended to mount in balsam, as they generally shrink by exosmosis when placed in the latter medium. It is, however, a good medium for celloidin sections. Gage's Mixture (Proc. Amer. Soc. Micr., 1890, p. 120). — Carbolic acid crystals melted, 40 c.c. ; oil of turpentine, 60 c.c. 133. Creosote. — Much the same properties as carbolic acid. Beech-wood creosote is the sort that should be preferred for many purposes, — amongst others, for clearing celloidin sections, for which it is a very good medium. 134. Anilin Oil. — Common aniUn oil will readily clear sections from. 70 per cent, alcohol, and with certain precautions (for which see the paper of Suchannek quoted below) objects may be cleared from watery media without the intervention of alcohol at aU. This renders it valuable in certain cases as a medium for preparing for parafi&n imbedding. For ordinary work the usual commercial anilin will suffice ; and it is immaterial whether it be colourless or have become brown through oxidation. For difficult work it is well to use a perfectly anhydrous oil. For directions for preparing this see Suchannek, Zeit. wiss. Mik., vii, 1890, p. 156, or the third edition of this work. Anilin is chiefly used for clearing celloidin sections. It ought however to be soaked out before mounting by something else (chloroform or xylol for instance for some hours), as if not removed it will brown both the tissues and the mounting medium. 70 DE-ALCOHOLISATION AND CLEARING AGENTS. 135. Xylol, Benzol, Toluol, Chloroform. — Too volatile to be recommendable as clearing agents in which it is desired to examine specimens, but very useful for preparing paraffin sections for balsam. Of the three first-mentioned liquids, benzol is the most volatile, then toluol, and xylol is the least volatile, in the proportion of 4 : 5 : 9 (Squire, Methods and Formulce, p. 20). Chloroform is injurious to some deUcate stains, but is in other respects an excellent de-alcohoUsa- tion agent, as it will take up a good deal of water, if any be left in the preparations. I consider it too volatile to be safe to use before balsam. Xylol is the best of these in that respect. Both xylol and toluol are liable to become acid if kept in only partially filled vessels. CHAPTER VII. IMBEDDING METHODS^— INTRODUCTION. 136. Imbedding Methods.— The processes known as Imbedding Methods are employed for a twofold end. Firstly, they enable us to surroimd an object, too small or too delicate to be firmly held by the fingers or by any instrument, with some plastic substance that will support it on all sides with firmness but without injurious pressure, so that by cutting sections through the composite body thus formed the included object may be cut into sufficiently thin slices without distortion. Secondly, they enable us to fiU out with the imbedding mass the natural cavities of the object, so that their lining membranes or other structures contained in them may be duly cut in situ ; and, further, they enable us not only to surround with the supporting mass each individual organ or part of any organ that may be present in the interior of the object, but also to fill with it each separate cell or other anatomical element, thus giving to the tissues a consistency they could not otherwise possess, and ensuring that in the thin slices cut from the mass all the minutest details of structure will precisely retain their natural relations of position. These ends are usually attained in one of two ways. Either the object to be imbedded is saturated by soaking with some material that is liquid while warm and solid when cold, which is the principle of the processes here called Fusion Imbedding Methods ; or the object is saturated with some substance which whilst in solution is sufficiently fluid to penetrate the object to be imbedded, whilst, after the evaporation or removal by other means of its solvent, it acquires and imparts to the imbedded object sufficient firmness for the purpose of cutting. The methods founded on this principle are here called Evaporation Imbedding Methods. In any of these processes the material used for imbedding is technically termed an " imbedding mass." There are two chief methods of imbedding — the paraffin method and the celloidin or collodion method. The paraffin method is the one in most use ; for it is the more rapid, requiring only hours where the celloidin process requires days or weeks ; and it is the one which the most readily affords very thin 72 IMBEDDING METHODS. sections. But this only applies to fairly small objects : with objects of much over half an inch in diameter you cannot easily get with paraffin much thinner sections than you can with celloidin ; and if you try to cut in paraffin objects of still greater size, say an inch and upwards, it will frequently happen that you will not get perfect sections at all, blocks of paraffin of this size having a tendency to split under the impact of the knife. This defect is, however, much reduced by the employment of a softer paraffin than is usual. In this way Strasser [Zeit. wiss. Mik., ix, 1892, p. 7) has obtained series of frontal sections 30 /n thick through the entire human brain, in paraffin blocks measuring 10 X 15 cm. And Mayer, with the Tetrander microtome, has obtained series of only 7-5 jj. with a surface of 4^ X 3 cm. For very large objects celloidin is safer, because it does not spht, and presents advantages for the manipulation of the sections ob- tained. For all classes of objects it has the advantages of affording a transparent mass (which facilitates orientation of the object) and of producing less shrinkage than paraffin (paraffin unavoidably shrinks on cooling to at least 12 per cent.). It is for these two reasons that celloidin is so frequently preferred by embryologists — even for small objects. Aqueous masses, such as gum or gelatin, may render great service in cases in which it is desired to avoid dehydrating tissues, and to apply chemical tests to them. 137. Imbedding Manipulations.— Imbedding in a melted mass, such as parafiln, is performed in one of the following ways. A httle tray or box or thimble is made out of paper, some melted mass is • poured into it, and the object placed in the midst of it. Or, the paper tray being placed on cork, the object may be fixed in position in it whilst empty by means of pins and the tray filled with melted ' mass at one pour. The pins are removed when the mass is cold. In either case, when the mass is cold the paper is removed from it before cutting. To make paper trays proceed as follows. Take a piece of stout paper or thin cardboard, of the shape of the annexed figure (Fig. 1) ; thin (foreign) post-cards do very well indeed. Fold it along the lines a a' and b b', then along c c' and d d', taking care to fold always the same way. Then make the folds A A', B B', C C, D D', still folding the same way. To do this you apply A c against A a, and pinch out the fine A A', and so on for the remaining angles. This done, you have an imperfect tray with dogs' ears at the angles. To CHAPTER VII. 73 finish it, turn the dogs' ears round against the ends of the box, turn down outside the projecting flaps that remain, and pinch them down. A well-made post-card tray will last through several imbeddings, and will generally work better after having been used than when new. Another method of folding the paper (Mayee) is described in the Grundziige, Lee and Mayer, 4th ed., p. 77. GriESBEECHT now makcs trays of photographic films, which, being transparent, facilitate orientation under the dissecting microscope. 0/ h A' |a b! Jf _ ^L - /■' 1 / 1 ] \ ^' ^ 1 1- 1 1 Fig. 2. D y Fig. 1. To make paper thimbles, take a good cork, twist a strip of paper several times round it so as to make a projecting collar, and stick a pin through the bottom of the paper into the cork. For work with fluid masses, such as celloidin, the cork may be loaded at the bottom by means of a nail or piece of lead, to prevent it from floating when the whole is thrown into spirit or other liquor for hardening (Fig. 2). Or you may use short lengths of solid lead rod instead of cork. . Leuckhart's Imbedding Boxes are made of two pieces of type- metal (Fig. 3). Bach of these pieces has the form of a carpenter's " square " with the end of the shorter arm triangularly enlarged outwards. The box is constructed by placing the two pieces to- gether 0*1 a plate of glass which has been wetted with glycerine and gently warmed. The area of the box will vary according to the 74 IMBEDDING METHODS. yy / Fig. 3. N position given to the pieces, but the height can be varied only by- using different sets of pieces. Two sets will be sufficient for most work, one set of 1 centimetre in height, and one of 2 centimetres, each being 8 centi- metres in length, and 3 in breadth. To make the box paraffin-tight, so that it will hold the melted paraffin long enough in the liquid state to permit of the objects being carefully orientated in it, Mayer {Mitth. Zool. Stat. Neapel, iv, 1883, p. 429) first smears the glass plate with glycerine, then arranges the metal " squares," and then fills the box with col- lodion, which is poured out again immedi- ately. As the ether evaporates, a thin layer of collodion remains behind, which suffices to keep the paraffin from running out. Even without the collodion, the mere cooling of the paraffin by the metal will generally suffice to keep it in long enough for orientation, if it is not in a superheated state when it is poured in. In such a coUodionised box the paraffin may be kept in a liquid state by warming now and then over a spirit lamp, and small objects be placed in any desired position under the microscope (Journ. Roy. Mic. Soc. [N.S.], ii, p. 880). A fighter form of " squares," made of brass, and devised by Andres, Giesbrecht, and Mayer, is described loc. cit. (See Journ. Roy. Mic. Soc, 1883, p. 913.) A more complicated sort is described by Wilson in Zeit. mss. Mile, xxvii, 1910, p. 228, for use with » imbedded threads to serve as orientation guides. See " Orientation." Feankl (Zeit. wiss. MiJc., xiii, 1897, p. 438) builds up boxes with rectangular blocks of glass, which may be found convenient, but are more expensive than the metal squares. Sblenka has described and figured another sort of apparatus having the same object. It consists of a glass tube, through which a stream of warm water may be passed and changed for cold as desired, the object being placed in a depression in the middle of the tube (see Zool. Anz., 1885, p. 419). A simple modification of this apparatus, which any one may make for himself, is described by Andkews in Amer. Natural, 1887, p. 101 ; and a more complicated imbedding and orienting box, seldom necessary, is described by Jordan in Zeit. wiss. Mik., xvi, 1899, p. 32. To imbed in a watch-glass, the object, previously saturated with paraffin, is put into a (preferably very concave) watch-glass con- CHAPTER VII. 75 taining molten paraffin. After this has been solidified by cooling (see next chapter), a block containing the object is cut out of it, and mounted on the object-holder of the microtome (this is, of course, applicable to other masses, such as celloidin). For imbedding very small objects in this way certain precautions may be necessary in order not to lose them. Samtbb {Zeit. wiss. Mik., xi, 1894, p. 469) saturates small unstained objects with parafln that has previously been strongly coloured with alkanna extract, and then imbeds them in pure paraffin. Eiiumbler {ibid., xii, 1895, p. 312, and xiii, 1896, p. 303) stains previously the objects themselves with eosin dissolved in strong alcohol, and removes the stain from the sections with weak alcohol. See also ibid., xiii, p. 200, a paper by Schtdlowski ; and in Zeit. wiss. Zool., Iviii, 1897, p. 144, a process of Bokgebt. BoEGERT (Zeit. wiss. Zool., Iviii, 1897, p. 144) allows paraffin to solidify in a watch-glass, bores a hole in it, and places the objects in the hole with a little benzol, and puts the whole for a short time into a stove. A watch-glass provided at the bottom with a groove or trough, in which small objects may be made to collect, is described by LsriivEE, Journ. App. Mic., v, 1902, p. 280 (see Journ. Roy. Mic. Soc, 1903, p. 233). LAtrxERBOEN (Zeit. wiss. Zool., lix, 1895, p. 170) brings the objectg through chloroform into paraffin in a small glass tube, and after cooling breaks the tube and so obtains a cylinder of paraffin with the objects ready for cutting. HoTEE (Arch. mik. Anat., liv, 1899, p. 98) performs all the operations in a glass cylinder (5 centimetres long and 7 millimetres wide), open at both ends, but having a piece of moist parchment paper tied over one of the openings. It is then not necessary to break the cylinder ; by removing the parchment paper the paraffin can be pushed out of it in the shape of a cylinder containing the objects imbedded at one end of it. Mater (Zeit. wiss. Mikr., xxiv, 1907, p. 130) takes the gelatin capsules used by chemists ; after cooling in water the gelatin swells and is easily removed. Mbves (Arch, mikr., Anat., Ixxx, Abth. ii, 1912, p. 85) employs wedge- shaped capsules made by Gf. Pohl, Schonbaum, Bez, Dantzig. CHAPTER VIII. IMBEDDING METHODS — PARAFFIN AND OTHER FUSION MASSES. 138. Saturation with a Solvent. — The first stage of the parafiin method consists in the saturation of the object with some substance which is a solvent of paraffin. The process is sometimes called " clearing," since many of the substances used for infiltration are also " clearing " agents. The process of saturation should be carefully performed with well-dehydrated objects in the manner described in § 119. Saturation liquids being liquids that are, on the one hand, miscible with alcohol, and on the other hand good solvents of parafiin, are not quite as numerous as could be wished. According to Geaefe (Chem. Centralb., 1906, p. 874), at 20° C. petroleum ether (1 c.c.) dissolves 200 mg. of paraffin, chloroform 246, benzol 285, carbon tetrachloride 317. And according to Apathy, at 20° C. benzol dissolves 8 parts per cent., chloroform 10, toluol 10, xylol 12, oil of turpentine 8, cedar oil 4 to 6, bergamot oil 0-5 to 3, creosote and clove oil hardly any. Acetone, according to Mayer, dissolves hardly any. Turpentine I do not recommend, because in my experience it is of all others the clearing agent that is the most hurtful to delicate structures. Clove oil mixes very imperfectly with parafiin, and quickly renders tissues brittle. Oil of bergamot mixes still more imperfectly with parafiin. Benzol has been recommended by Brass {Zeit. wiss. Mile, ii, 1885, p. 301), and is now much used. Toluol (or toluen) has been recommended by Holl {Zool. Anz.', 1885, p. 223). Xylol is said by M. Heidenhain {Kern, und Protoplasm, p. 114) to be a cause of shrinkage in cells. So it is, if you use it to de- alcoJiolise the specimens. But used after oil of cedar, or the like, it is very good, as it is one of the best of solvents of parafiin. Chloroform is deficient in penetrating power, so that it requires an excessive length of time for clearing objects of any size ; and it must be very thoroughly got rid of by evaporation in the paraffin CHAPTER Yin. 11 bath, or by successive baths of paraffin, as if the least trace of it remains in the paraffin used for cutting it will make it soft. The process of removal requires a very long time, in some cases days. It ought therefore to be reserved for small and easily penetrable objects. Naphtha has been recommended by Websteu {Journ. Anat. and Physiol, XXV, 1891, p. 278). Field and Martin (Zeit. wiss. MiJc., xi, 1894, p. 10) recommend a light petroleum known as " petroleum-setlier." It is highly volatile, and thus a cause of shrinkage. Sulphide of carbon has been recommended by Heidenhain {Zeit. wiss. Mih., xviii, 1901, p. 166) as being a very powerful solvent of parafl&n. Most workers have found it to be much too disagreeable and dangerous a reagent for ordinary work, and not necessary even for delicate work. See under "Teeth" and "Chitin." Carbon tetrachloride has been recommended by Plecnik (op. cit., xix, 1903, p. 328) and Pranter (ihid., p. 329) on the ground of not dissolving out osmium-blackened fats. Mater finds it no better than benzol. As a general thesis, the best of all these are cedar oil, benzol, and chloroform. Cedar-wood oil is, according to my continued experience, for the reasons stated by me in Zool. Anz., 1885, p. 563, for general work the very best clearing agent for paraffin imbedding. It penetrates rapidly, preserves delicate structure better than any clearing agent known to me, does not make tissues brittle, even though they may be kept for weeks or months in it, and has the great advantage that if it be not entirely removed from the tissues in the paraffin bath it will not seriously impair the cutting consistency of the mass ; indeed, I fancy it sometimes improves it by rendering it less brittle. 139. The Paxaffin Bath.— The objects having been duly saturated with a solvent, the next step is to substitute melted paraffin for the saturating medium. Some authors lay great stress on the necessity of making the passage from the saturating agent to the paraffin as gradual as possible, by means of successive baths of mixtures of solvent and paraffin kept melted at a low temperature, say 35° C. With oil of cedar, at all events, this is n'ot necessary. I simply put the objects into melted paraffin kept just at its melting-point, and keep them there till they are thoroughly saturated ; the paraffin being changed once or twice for fresh only if the objects are sufficiently voluminous to have brought over with them a notable quantity of clearing agent. If the objects have been for a very long time— months or 78 IMBEDDING METHODS. years — in the cedar oil, so that this has become thick, I remove it partially or entirely by soaking in xylol (thirty minutes to several hours) before putting into the paraffin. But with fresh oil of cedar I find no advantage in doing so. Giesbeecht's method (Zool. Anz., 1881, p. 484), is as foUows : — Objects to be imbedded are saturated with chloroform, and the chloroform and objects are gradually warmed up to the melting-point of the paraffin employed, and during the warming small pieces of paraffin are by degrees added to the chloroform. So soon as it is seen that no more bubbles are given off from the objects, the addition of paraffin may cease, for that is a sign that the paraffin has entirely displaced the chloroform in the objects. This displacement having been a gradual one, the risk of shrinkage of the tissues is reduced to a minimum. Mayer (Grundziige, Lee and Mayer, 1910, p. 84) first saturates the objects with benzol, and then adds to the benzol some small pieces of paraffin, and lets them dissolve in the cold. After several hours (up to eighteen) the whole is brought in an open vessel on to the cold water-bath, the bath is then warmed gradually so as to attain a temperature of 60° C. in about two hours, and as fast as the benzol evaporates melted paraffin is added to it. Lastly, the paraffin is changed once before the definitive imbedding. He rarely leaves objects overnight in the water-bath. ApIthy {Mikrotechnik, pp. 149, 150) first clears with oil of cedar, then brings the objects (by the process described § 119) into a solution of paraffin in chloroform saturated at the temperature of the laboratory. The objects remain in the chloroform-paraffin solution for from one to three hours, without warming, until all the cedar oil is soaked out of them. The whole is then warmed on the water- bath or oven to a few degrees above the melting-point of the paraffin intended to be used for imbedding, and the object is brought into a mixture of equal parts of paraffin and chloroform, being suspended therein near the top on a bridge made of hardened filter paper (or in a special apparatus to the same end, not yet described). It remains in this mixture, at the temperature of the oven, for one to three hours, and lastly is brought (still on the paper bridge or in the apparatus) into pure paraffin, where it'remains for half an hour to two hours. Denne (inlitt., 1907) points out that the objects ought at first to be at the bottom of the mixture. For this mixture is not a true solution, and the lower section of the contents of the tube is com- paratively free from paraffin while the upper part is nearly pure CHAPTER VIII. 79 paraffin. He moves the holder up in the tube at intervals, and the infiltration proceeds gradually with the minimum risk of shrinkage. Lastly, he removes the objects, on the holder, to the top of a tube of pure paraffin. The practice of giving suocessive baths first of soft and then of hard paraffin, which has been frequently advised, appears to me entirely illusory. It is important to keep the paraffin dry — that is, protected from vapour of water during the bath. It is still more important to keep it as nearly as possible at melting- point. If it be heated for some time to a point much over its normal melting-point, the melting-point will rise, and you will end by having a harder paraffin than you set out with. And as regards the preser- vation of tissues, of course, the less they are heated the better. Overheating, as well as prolonged heating, tends, amongst other things, to make tissues brittle. The duration of the bath must, of course, vary according to the size and nature of the object. An embryo of 2 to 3 miUimetres in thickness ought to be thoroughly saturated after an hour's bath, or often less. Many workers habitually give much longer baths, I think often longer than necessary. But some objects, such as ova of Crustacea, may require three or four days (Hbidecke, Jena. Zeit., xxxviii, 1904, p. 506 ; Mayee, Grundziige, Lee and Mayeb, 1910, p. 85 ; Bbinkmann, Mitth. Zool. Stat. Neapel, xvi, 1903, p. 367, three to five days for uterus of Selachians ; Mullee, Arch, mihr. Anat., IxLx, 1906, p. 3, for lungs of mammals ; Poso, Esperienze miorotechniche, Napoli, 1910, p. 29, five to twelve days for uterus and placenta of Homo). I take as a guide, generally, the length of time the object has taken to clear in the cedar oil, assuming that the warm melted paraffin ought to penetrate at least as quickly as the coU oil ; and then allowing somewhat longer, say as much again, in order to be on the right side. 140. Water-toaths and Ovens. — It is important that the paraffin should not be exposed to a moist atmosphere whilst it is in the liquid state. If a water-bath be used for keeping it at the required tempera- ture provision should be made for protecting the paraffin from the steam of the heated water. A very convenient apparatus for this purpose is that of Paul Mayer, or " Naples water-bath," which will be found described at p. 146 of Journ. Boy. Mic. Soo., 1883, or Caepentek's The Microscope, p. 452. An extremely simple stove, which any one can make for himself, is described in Oentralbl. Bakt, xlv, 1907, p. 191 (see Journ. Boy. Mic. Soc, 1908, p. 109). For others, see the price-lists of the instrument makers, 80 IMBEDDING METHODS. especially Jung, and Gkublek and Hollboen ; and the descriptions in the technical journals. 141. Imbedding in Vacuo. — ^There are objects which, on account of their consistency or their size, cannot be penetrated by paraffin in the ordinary way, even after hours or days jn the bath. For such objects the method of imbedding under a vacuum (strictly, under diminished atmospheric pressure) renders the greatest service. It not only ensures complete penetration in a very short time — a few minutes — but it has the further advantage of preventing any falling in of the tissues, such as may easily happen with objects possessing internal cavities if it be attempted to imbed them in the ordinary way. It is realised by means of any arrangement that will allow of keeping paraffin melted under a vacuum. That of Hoffmann is described and figured at p. 230 of Zool. Anz., 1884. In this arrangement the vacuum is produced by means of a pneumatic water aspiration pump, the vessel containing the paraffin being placed in a desiccator heated by a water-bath and furnished with a tube that brings it into communication with the suction apparatus. Feancotte (Bull. 8oc. Belg. Mic, 1884, p. 45) produces ttie requisite vacuum by the condensation of steam. Pol [Lejirb., p. 121) employs the vacuum apparatus of HofEmann, but simplifies the arrangement for containing the paraffin. The paraffin is contained in a stout test-tube furnished with a rubber stopper traversed by a tube that puts it into communication with the pump. The lower end of the test-tube dips into a water-bath. You pump out the air once or twice, wait a few minutes, then turn out the object with the paraffin (which by this time will have become abnormally hard), and re-imbed in fresh paraffin. See also Pringlb, in Journ. Path, and Bacteriol., 1892, p. 117, or Journ. Boy. Mic. Soc, 1892, p. 893 ; Kolster, in Zeit. wiss. Mile., xviii, 1901, p. 170 ; Berg, Zeit. wiss. Mik., xxvi, 1909, p. 209 ; Fuhrmann, ibid., xxi, 1904, p. 462 ; Kolmer and Wolff, ibid., xix, 1902, p. 148 ; Gemmill, Journ. Boy. Mic. Soc, 1911, p. 26. 142. Imbedding and Orientation.— ^s soon as the objects are thoroughly saturated with paraffin they should be imbedded by one of the methods given above (§ 137), and the paraffin cooled as described next §. But it may be desirable to have the object fixed in the cooled paraffin in a precisely arranged position, and, above all, in a precisely marked position. Very small objects may be oriented as follows : — The object is removed from the melted paraffin, and placed on a cylinder of sohd paraffin. A needle or piece of stout iron wire is now heated in the flame of a lamp, and with it a hole is incited in the end of the cyKnder ; the specimen is pushed into the melted paraffin, and placed in any desired position. The advantages of the method lie in the quickness and certainty with which it can be performed. CHAPTER VIII. 81 In using the needle it is important to melt as little paraffin as possible at one time, in order that that which is melted may cool again as rapidly as possible. Kekr {Qua/rt. Joum. Micr. Sc, xlv, 1901, p. 4) employs an electrically heated needle. The method of Patten {Zeit. wiss. Mile, xi, 1894, p. 13) is useful when one desires to orient large numbers of small objects. You get some writing paper of the sort that is made with two sets of raised parallel lines running at right angles to each other, ("linen cloth paper "). Small strips are cut from this, and at suitable intervals along them small drops of a mixture of collodion and clove oil, of about the consistency of thick honey, are arranged close together along one of the ribs that run lengthwise. The objects to be imbedded are cleared in clove oil or oil of bergamot. They are taken one by one on the point of a knife, and after the excess of oil has been drawn off, are transferred each to a drop of the collodion mixture, in which they will stay in any required position. When half a dozen or more objects have been oriented in reference to the cross Unes {which are to be parallel to the section planes) the whole thing is placed in turpentine. This washes out the clove oil and fixes the objects very firmly to the paper. The paper with the attached objects is now passed through the bath of paraffin and imbedded in the usual way. After coohng on water the block is trimmed and the paper peeled off, leaving the objects in the paraffin close to the under-surface of the block. This surface is now seen to be marked by the orienting lines of the ribbed paper, and also by any record numbers which may before imbedding have been written with a soft pencil on the paper. Knowen {Journ. Morpji., xvi, 1900, p. 507) takes smooth paper and engraves parallel Unes on it with a needle, and takes xylol instead of turpentine. A somewhat more complicated form of this process has been described by WooDWOKTH, Bull. Mus. Gomp. Zool., xxxviii, vol. xxv, 1893, p. 46. A similar process has also been described by Field and Martin in Zeii. wiss. Mih., xi, 1894, p. 11, small strips of gelatin being used instead of paper. Mater also (Orundziige, Lee and Matek, 1910, p. 89) takes strips of photographic gelatin, and lets the collodion set in benzol. Hoffmann (Zeit. wiss. Mile., xv, 1899, p. 312, and xvii, 1901, p. 443) takes, instead of the ribbed paper, glass sUps ruled with a diamond, and completely imbeds the objects in large drops of clove oil collodion (equal parts), allowed to stand for twenty-four hours in an open vessel. The drops are caused to set in xylol. See also Samtek, ibid., xiii, 1897, p. 441 ; Jordan, ihid., xvi, 1899, p. 33 ; and Peter, Verh. Anat. Oes., xiii Vers., 1899, p. 134. Entz (Arch. Protistenk., xv, 1909, p. 98) orients in clove oil collodion on a cover-glass coated with paraffin, and puts the whole into chloroform in which the mixture sets into a sheet which can be detached. Denne (Journ. Appl. Mic, iii, 1902, p. 888) imbeds on disks of paper held at the bottom of glass tubes containing the paraffin by bent wires, 82 IMBEDDING METHODS. by means of which a cylinder of paraffin containing the object may be lifted out as soon as cool. Wilson (Zeit. wiss. Mik., xvii, 1900, p. 169) makes orientation lines by imbedding alongside the objects strands of osmium-blackened nerve- flbres. See also a further development by Wilson, ibid., xxvii, 1910, pp. 228 and 231. 143. Cooling the Mass. — Whatever method of imbedding and orientation in the molten paraffin has been employed, the important point now to be attended to is that the paraffin be cooled rapidly. The object of this is to prevent crystallisation of the paraffin (which may happen if it be allowed to cool slowly) and to get as homogeneous a mass as possible. If the definitive imbedding has been done in a watch-glass, hold it on the top of cold water until all the paraffin has solidified, and then let it sink to the bottom. When thoroughly cool, cut out blocks containing the objects. If the watch-glass has been smeared with a drop of a mixture of equal parts of glycerinfe and water before putting the paraffin into it, the solidified paraffin will generally detach itself in a single cake and float up in a few minutes, or hours at any rate. Do not attempt to remove it entire by warming the bottom of the watch-glass. Similarly with the paper trays or metal imbedding boxes. Or you may put them to cool on a cold slab of metal or stone. Selenka cools the mass by passing a stream of cool water through the imbedding tube described above (§ 137). Mater cools the mass in the paraffin-tight moulds (§ 137) by passing cold water through a special movable water-bath, which allows of the arrangement of the objects by transmitted light under a dissecting microscope, see Mitth. Zool. Stat. Neapel, iv, 1883, p. 429 ; Intern. Monatssehr. Anat. Hist., iv, 1887, p. 39. A complicated apparatus for the same purpose is described by Meissnbe {Zeit. wiss. Mik., xviii, 1902, p. 286). SimUarly, Hahn, ibid., xxv, 1908, p. 184, and Kappebs, ibid., xxiv, 1907, p. 254. See also Faekas, ibid., XXX, 1913, p. 168, for experiments on cooling methods. The paraffin blocks with the objects are now mounted on the carrier of the microtome in position for cutting, and pared to the proper shape (next §). If any bubbles or cavities or opaque spots be present, prick with a heated needle till all is smooth and homo- geneous. The same should be done if any cavities present them- selves in the course of cutting. In bad cases, re-imbed. 144. Shape and Orientation of the Block of Mass to be cut.— These differ accordingly as the cutting is done with a slanting knife or a square-set knife (see next §). In the first case, the block is best trimmed to a three-sided^prism, and orientated as in Fig. i, so that CHAPTER VIII. 83 the knife enters it at the angle a and leaves it at the angle c. When the section is cut it will adhere to the knife only by the angle c, and can thus most readily be removed by means of a brush or needle. The object itself should come to lie in the block close to the line b c, so that the knife at first cuts only paraffin, and that if the section begins to roll it may be caught and held down by a brush or section- stretcher before the object itself is reached. For the square-set knife the block is best trimmed to a four-sided prism, and orientated as in the first case, so that the knife first touches one angle, if only isolated sections are to be cut. But if ribbons (§ 148) are to be cut, the block must be orientated with one of its sides parallel to the knife-edge, and the opposite side must be strictly parallel to this one. An object which is not approximately isodiametrical but gives a section which is wider in one direction than another should be orientated end on, that is, so as to present its narrowest diameter to the knife-edge : for it is in this position that it will offer the least resistance to the blade, and tend the least to make the edge bend away or dig into it. This is specially impor- tant with longikidinal sections of worms, Amphioxus, embryos of vertebrates, and the like. Most especially with a square-set knife should the narrowest diameter of the object be presented to the knife ; and only when the object is particularly hard, or otherwise diflBcult to cut, should it be turned so as not to let the whole of that diameter be attacked at once by the knife, but only a corner of it. And as far as possible arrange that the hardest part of an object be the last to be touched by the knife. For Noack's simple apparatus for accurately orientating small blocks, see Zeit. wiss. Mik., xv, 1899, p. 438, or Journ. Roy. Mic. Sac, 132, 1899, p. 550. For Eternod's machine for trimming blocks to true cubes, see Zeit. iviss. Mik., XV, p. 421, and for that of Schaffek, ibid., xvi, 1900, p. 417. 145. Knife Position. — The position to be given to the knife may be considered under two heads, viz., its slant and its tilt. By the slant of the knife is meant the angle that its edge mak s with the line of section : that is, with the line along which it is drawn through the object (or along which the object moves across it in the case of microtomes with fixed knives). The position is 6—2 Fig. 4. 84 IMBEDDING METHODS. transverse when the edge makes an angle of 90° with the line of section, or the knife in that case is said to be set square. It is oblique or slanting when it makes a smaller angle with that line. The difference between the effect of the two positions is that the obKque position afEords a more acute-angled wedge than the transverse one. It does so for the following reasons : — Neglecting for the moment the distinction between the cutting-facets and the surfaces of the blade (which are distinct usually because they are not ground to the same angle),* it is clear that the knife itself is a wedge, the angle of which depends on the relation between the height of its base and the distance from the base to the edge. With the same base the angle becomes more acute the greater the distance from edge to base. Now by slanting the knife we can effect what is equivalent to an increase in the distance from edge to base ; for we can thus increase the distance between the point of the edge which first touches the object, and the point of the back (strictly, of the back edge of the under cutting-facet) which last leaves it. When the knife is set transversely, the line along which any point of it traverses the object is the shortest possible from edge to base of the wedge, and the effective angle of wedge is the least acute obtainable ■\\ith that knife. But if it is set as obliquely as possible, the line along which any point of it traverses the object traverses the knife from heel to toe, that is, along the greatest possible distance from edge to base, and therefore affords practically a much more acute- angled wedge than in the first case ; and so on, of course, for inter- mediate positions. (See the stereometrical constructions of these relations by Schiefferdecker, op. cit., p. 115 ; and also with more instructive figures. Apathy, " Ueber die Bedeutung des Messer- halters in der Mikrotomie," in Sitzber. med.-naturw. Section d. Siebenburgischen Museumvereins, Bd. xix. Heft 7, p. 1 (Kolozsvar, 1897, A. K. Ajtai). Por honing knives see Ssobolew, Zeit. wise. Mik., xxvi, 1909, p. 65 ; Lendvai, iUd., p. 203 ; Punck, ibid., xxvii, 1910, p. 75. Very large objects are best cut with the slanting knife, and so are all objects of very heterogeneous consistency, such as tissues * The edge of a microtome knife is composed of two plane surfaces — the upper and lower cutting-facets, which meet one another at an acute angle, the cutting-edge, and posteriorly join on to the upper and lower surfaces of the blade (see some good figures of diflerently shaped knives in Behkens, Kossel und Schiefferdecker, Das MiJcroslcop., p. 115, et seq. ; and in Apathy's paper quoted below). It will be seen that the two facets together form a wedge welded on to the blade by the base. CHAPTER VIII. 85 that contain much chitin or much muscular tissue ; and better with a slowly working sliding microtome than with a quick-working Rocker or the like. Soft masses such as gelatin or celloidin cut wet, can only be cut with the slanting knife. The slanting position causes less compression of sections than the transverse one. It has the defect of producing rolling in paraffin sections more easily than the transverse position. The latter is the proper position for putting ribbons of sections from paraffin. By the tilt of the knife is meant the angle that a plane passing through its back and- edge makes with the plane of section : or, practically, the gseater or less degree of elevation of the back above the edge (it is not to be confounded with the inclination of the long axis of the knife to the horizon ; any accidental inchnation that this may have is a matter of no moment). The question of the proper tilt to be given to the knife under different circumstances has been investigated by Apathy, foe. cit. supra. He concludes — (1) The knife should always be tilted some- what more than enough to bring the back of the under cutting-facet clear of the object. (2) It should- in general be less tilted for ha,rd and brittle objects than for soft ones ; therefore, cceteris paribus, less for paraffin than for celloidin. (3) The extent of useful tilt varies between 0° and 16° or occasionally 20°. (4) Excessive tilt causes rifts (longitudinal) in the paraffin, also furrows that in bad cases split up the section into narrow ribbons. It also makes sections roll. Also it may cause the knife not to bite, thus causing sections to be missed. Or it may give an undulatory surface to the sections, owing to vibrations set up in the knife, which may be heard as a deep humming tone. Further, I would add, excessive tilt may cause the knife to act as a scraper, carrying away portions of tissue bodily from their places. Excessive tilt may often be recognised by the knife giving out a short metallic sound just as it leaves the object. For knives with plane under-surfaces it is seldom advisable to give less than 10° tilt. Knives with concave under- surfaces, on the contrary, may require to be placed almost horizontal. Jung's knife-holders give mostly a tilt of about 9°, which is only enough for cutting ribbons with hard paraffin. A knife with too little tilt mU often cut a second section, or frag- ments of one, without the object being raised, showing that during the first cut the object was pressed down by the knife, and recovered itself afterwards. This fault is denoted by the ringing tone given out by the knife on passing back over the object before the latter is raised. Such a knife gives out a dull rattling "sound whilst 86 IMBEDDING METHODS. cutting. Too little tilt causes folding or puckering of sections, and does not allow of the cutting of the thinnest possible sections,^ as the edge does not bite enough. It is thus frequently a cause of sections being missed,, or coming off thicker at one end than the other. A slanting knife should have more tilt given to it than a square- set one. Ribbon section-cutting (§ 148) requires a relatively hard paraffin and less tilt. With celloidin it is very important to avoid insufficient tilt, as the elastic celloidin yields before an insufficiently tilted knife and is not cut. The tilt of the knife is given to a certain extent by the knife- holder sold with the microtome. With plane-concave knives it can be regulated to a certain extent by simply turning the blade over. It is more accurately regulated by means of mechanical contrivances, of which the most simple are the horseshoe-shaped wedges of Neumayer (see Jung's price-list). A pair of these, each ground to the same angle, is taken, and one of them placed (thin end towards the operator) under, and the other (thick end towards the operator) over, the clamping-arm of the knife-holder. Three pairs, having different degrees of pitch, are supphed, and are sufficient for most work. Other contrivances to the same end consist of knife-holders that permit of rotating the knife on its long axis, and, though more costly, will be found a great convenience where much section- cutting has to be done. For these see Jung's price-Hst, and various recent papers in Zeit. iviss. Mik., also that of Apathy, in the paper quoted above (very complicated), and especially the description of the two latest of Jung, viz., his model I and model n, by Mayee and ScHOEBEL, in Zeit. urns. Mile, xvi, 1899, p. 29 (see figure of model I in Journ. Roy. Mic. Soc, 132, 1899, p. 546). Also Carpenter's The Microscope, p._463. 146. Cutting and Section-stretching.— Paraffin sections are cut dry, — that is, with a knife not moistened with alcohol or other liquid. By this means better sections are obtained, but a difficulty generally arises owing to the tendency of sections so cut to curl up on the blade of the knife. It is sometimes difficult by any means to unroll a thin section that has curled. To prevent sections from rolling, the following points should be attended to. First and foremost, the paraffin must not be too hard, see § 151. If, after cutting has begun, the paraffin be found to be too hard, it may be softened by placing a lamp near the imbedded object. But then, the paraffin being warmed most on the side nearest the lamp. CHAPTER VIII. 87 becomes softer ou that side, and the sections have a tendency to become compressed and puckered-in on that side. If, on the contrary, the paraffin be found too soft, it may be hardened by exposing it to the cooling influence of a lump of ice. It is often sufficient to moderate the temperature of the room by opening or closing the window, stirring the fire, setting up a screen, or the hke. For other devices for warming or cooling the paraffin see Held, Arch. Anal. Phys., Anat. Abth., 1897, p. 345 ; van Walsem, Zeit. wiss. Mik., xi, 1894, p. 218 ; Lendenfeld, ibid., xviii, 1901, p. 18 ; Krause, ibid., XXV, 1908, p. 299 ; Foot and Steobell, Biol. Bull. Wood's Hole, ix, 1906, p. 281. Secondly, the knife should be set square, for the oblique position encourages rolling, and the more the knife is oblique the more do the sections roll. Thirdly, it is better to cut ribbons than disconnected sections ; ribbons of sections will often cut flat, when the same mass will only give rolled sections if cut disconnectedly. Eolling may often be lessened or suppressed by cutting the sections thinner. Mechanical means may be employed. The simplest of these is as follows : During the cutting the edge of the section that begins to curl is caught and held down on the blade of the knife by means of a small camel-hair brush with a flat point, or by a small spatula made by running a piece of paper on to the back of a scalpel. Or, which is much better, the section is held down by means of an instrument called a " section-stretcher." This consists essentially of a little metallic roller suspended over the object to be cut in such a way as to rest on its free surface with a pressure that can be delicately regulated so as to be sufflcient to keep the section flat without in any way hindering the knife from ghding beneath it. See the descriptions of various forms of section-stretchers, Zool. Anzeig., vol. vi, 1883, p. 100 (Schtjltze) ; Mitth. Zool. Stat. Neapel, iv, 1883, p. 429 (Mayeb, Andres, and GtIesbrecht) ; Arch. mik. Anat., ' xxiii, 1884, p. 537 (Decker) ; Bull. Soo. Belg. Mic, x, 1883, p. 55 (Pran- cotte) ; The Microscope, February, 1884 (Gage and Smith) ; Whit- man's Meth. in Mic. Anat., 1885, p. 91 ; Zeit. wiss. Mile, iv, 1887, p. 218 (Strassee) ; ibid., x, 1893, p. 157 (Born). The best are those of Mayer and Bom. I find that Mayee's, beautifully made by Jung, works admirably and is most valuable. Another plan is to allow the sections to roll, but to control the rolling. To this end, the block of parafiin is pared to the shape of a 88 IMBEDDING METHODS. wedge five or six times as long as broad, the object being contained in the broad part, and the edge turned towards the knife (see Fig. 4). The sections are allowed to roll and come off as coils, the section of the object lying in the outermost coil, which wiU be found to be a very open one — indeed, very nearly flat. Lay the coil on a slide with this end downwards, warm gently, and the part containing the object will unroll completely and lie quite flat. AsiLE {Glandole duodenali, Napoh, 1903, p. 51) and Vastarini- Cresi {Mm. Zool. Ital, 1906, p. 164) lay a strip of wet filter-paper on the block. A defect opposite to that of the rolling of sections is the comp-ession and the crumpling or puckering of sections, indicating that the paraffin has been compressed by the knife instead of being merely cut true by it. Such sections, besides showing creases or folds, have a smaller area than that of the block from which they are cut. This is a bad fault, for the compression may obhterate important cavities or efiace important limits between ceU-layers, etc. It may be caused by a badly cutting knife, and is very easily caused by the paraffin being too soft. To prevent it, correct the knife or cool the paraffin, or re-imbed in harder parafiin. Very large sections tend to form folds on the knife, and are difl&cult to remove from it. Mater {Orundziige, Lee and Matee, p. 94) gets tliem to wrap themselves round a glass or gelatin tube laid on tte block just in front of the knife-edge and rolled forwards as it progresses. When cut, the section is rolled off on to the surface of water. 147. Cutting Brittle Objects (Collodionisation). — Some objects are by nature so brittle that they break or crumble before the knife, or furnish sections so friable that it is impossible to mount them in the ordinary way. Ova are frequently in this case. A remedy for this state of things consists in covering the exposed surface of the object just before cutting each section with a thin layer of collodion, which serves to hold together the loose parts ; and will enable the operator to cut sections considerably thinner than can be obtained in the usual way. The primitive form of the process was to place a drop of collodion on the free surface of each section just before tutting it. But this practice has two defects ; the quantity of collodion employed sensibly softens the parafiin, and the thick layer of collodion when dry causes the sections to roll. Mark {Amer. Natural, 1885, p. 628 ; cf. Journ. Roy. Mic. Soc, 1885, p. 738) gives the following directions : " Have ready a little very fluid collodion in a small bottle, through CHAPTER VIH. 89 the cork of which passes a small camel-hair brush, which just dips into the collodion with its tip. The collodion should be of such a consistency that when applied in a thin layer to a surface of paraf&n it dries in two or three seconds without leaving a shiny surface. It must be diluted with ether as soon as it begins to show signs of doing so. " Take the brush out of the collodion, wipe it against the neck of the bottle, so as to have it merely moist with collodion, and quickly pass it over the free surface of the preparation. Care must be taken not to let the collodion touch the vertical surfaces of the paraffin, especially not the one which is turned towards the operator, as that will probably cause the section to become stuck to the edge or under- surface of the knife. As soon as the collodion is dry, which ought to be in two or three seconds, cut the section, withdraw the knife, and pass the collodion brush over the newly exposed surface of the paraffin. Whilst this last layer of collodion is drying, take up the section from the knife and place it with the collodionised surface downwards on a shde prepared with fixative of Schaellibaum. Then cut the second section, and repeat the manipulations just described in the same order." Henki^g {Zeit. wiss. Mih., iii, 1886, p. 478) takes instead of collodion a solution of paraffin in absolute alcohol. For extremely brittle objects, such as ova of Phalangida, be recom- mends a thin (light yellow) solution of shellac in absolute alcobol. Heider {Embryonalentw. v. HydropMlus„ 1889, p. 12 ; cf. Zeit. wiss. Mih., viti, 1892, p. 509) employs a solution made by mixing a solution of gum mastic in ether, of a syrupy consistency, witb an equal volume of collodion, and diluting the mixture with ether until quite thin and liquid. Eabl [ihid., xi, 2, 1894, p. 170) employs superheated paraffin (of about 100° C. j. This has the advantage of filling up any cavities there may be in the objects, and also of preventing the sections from rolling. A compli- cated development of this process is described by Lbndenfeld in Zeit. wiss. Mih., xviii, 1901, p. 18. ApItht (Mihrotechnik, p. 183) employs a 1 -per cent, solution of celloidin, aUows the sections to roU, and unrolls them by the water- process (§ 149). Jordan (Zeit. wiss. Mih.) adds 6 drops of oil of cedar to 15 c.c. of the solution of celloidin, and finds that roUing is prevented. 148. Ribbon Section-cutting. — If a series of paraffin sections be cut in succession and not removed from the knife one by one as cut, but allowed to lie undisturbed on the blade, it not imfrequently happens that they adhere to one another by the edges so as to form a chain or ribbon which may be taken up and transferred to a slide 90 IMBEDDING METHODS. without breaking up, thus greatly lightening the labour of mounting a series. For the production of a ribbon, the paraffin must be of a melting-point having the right relation to the temperature of the laboratory, see § 151. Secondly, the knife should be set square. Thirdly, the block of paraffin should be trimmed so as to present a straight edge parallel to the knife-edge ; and the opposite edge should also be parallel to this. It is by no means necessary to have recourse to special mechanical contrivances, as in the so-called ribbon microtomes ; the Thoma microtome is sufficient. But the automatic microtomes, and amongst them the Cambridge Rocking Microtome and the Minot, are certainly most advantageous for this purpose. If the paraf3S.n is very hard, it is necessary for sections of 10 ix, and advisable for thinner ones, to coat the block with softer paraffin. To do this, take paraffin of about 40° C. melting-point, melt it, heat it to about 80° on the water-bath, dip the block into it for an instant, and rapidly turn it over so that the fluid paraffin may run down away from the top part as much as possible. Allow it to cool, and pare away again the soft paraffin from the two sides that are not to be arranged parallel to the knife. Or, as I frequently prefer, simply plaster a wall of soft paraffin (superheated) on to the fore and aft faces of the block with a small spatula. Large blocks may have two coatings given them. It sometimes happens that the ribbon becomes electrified during the cutting, and twists and curls about in the air in a most fantastic and undesirable manner. It may be got flat by warming slightly. 149. Section Flattening. — The sections having been obtained may be cleared and mounted at once if they are quite perfect, that is, neither rolled nor creased nor compressed. But should they in the least degree show any of these defects, they must first be unrolled or smoothed, or expanded to their proper dimensions. The most efficacious plan is combined treatment with fluid and heat. The sections are either floated on to the surface of warm water or warm alcohol contained in a suitable dish, which causes them to flatten out perfectly, and are then transferred to a slide, by floating them into position, or otherwise. Or the slide has a layer of water spread over it, the sections are laid on the water, and the slide is heated (to somewhat below the melting-point of the paraffin) until the sections flatten out, which happens in a few seconds. A special water-bath for flattening sections is described by Nowak in Zeit: wiss. Mile, xii, 1896, p. 447. CHAPTER VIII. 91 150. Clearing and Mounting. — The sections having been .duly smoothed by one of these processes, and duly fixed to the slide (Chapter X), unless it is desired to keep them loose, all that now remains is to get rid of the paraffin and mount or stain as the case may be. Many solvents have been recommended for this purpose : — Turpentine, warm turpentine, a mixture of i parts of essence of turpentine with 1 of creasote, creasote, a mixture of turpentine and oil of cloves, benzin, toluol, xylol, thin solution of Canada balsam in xylol (only applicable to very thin sections), hot absolute alcohol, naphtha, or any other paraffin oil of low boihng-point. Of these xylol and toluol are generally in most respects the best. Benzol and chloroform are too volatile for safe manipulation. If the sUde be warmed to the melting-point of the paraffin, a few seconds will suffice to remove the paraffin if the slide be plunged into a tube of xylol or toluol. For thin sections, 10 to 15 /x, it is not necessary to warm at all. The sections may be mounted direct from the xylol, or the shde may be brought into a tube of alcohol to remove the solvent for staining. Paraffin sections can be stained without removal of the paraffin, so tliat after-treatment with alcohol can be suppressed, but this is only- very exceptionally advantageous. 151. Pure Paraflta,. — It is now almost universally admitted that pure paraffin is superior for ordinary work to any of the many mixtures with wax and the like that used to be recommended. Paraffin varies enormously in hardness according to the temperature of its surroundings. It should therefore be taken of a melting- point suitable to the temperature of the laboratory. A paraffin melting at 50° C. or a little harder, is that which in my experience gives the best results so long as the temperature of the laboratory is between 15° and 17° C. For higher temperatures a harder paraffin is required, and for lower temperatures a softer one. Many workers of undoubted competence prefer masses somewhat harder than this ; so, for instance, Heidenhain (58°), Apathy (55°), Rabl (56°), Mayer (58° to 60° in summer ; in winter about 56°, but never less than 50°). Mayer points out that at Naples the tempera- ture during five months of the summer and autumn is over 22° C. in the laboratory, sometimes over 30°. Temperatures such as these are seldom realised in the British Isles, and, whilst I quite admit that such hard paraffin may have its raison d'Stre for Naples, I hold that for that very reason it is in general unnecessarily hard for cooler chmates. My recommendation of a relatively soft paraffin refers to work with the Thoma sliding microtome. Microtomes with fixed knives, 92 IMBEDDING METHODS. such^s the Cambridge, the Minot, or the Eeinhold-Giltay, will give good results with much harder paraffin, and, in fact, require such. Stout knives of hard steel will take a harder paraffin than thin ones of soft steel ; but the latter may be preferable for soft masses. For thin sections a harder paraffin is required than for thick ones. Hard objects require a harder paraffin than soft ones. Brass {Zeit. wiss. MiL, ii, 1885, p. 300) recommends paraffin that has been kept for some years, as it has less tendency to crystalUse than new paraffin. Paraffin of various melting-points is easily found in commerce. Intermediate sorts may be made by mixing hard and soft paraffin. I find that 2 parts of paraffin melting at 50° with 1 of paraffin melting at 36° C. give a mass melting at 48° C, and a mixture of 1 part of that melting at 53° with 1 part of that melting at 45° gives a mass melting at 50° C. According to E. Burchardt {Jena Zeit. Naturw., xxxiv, 1900, p. 719) mixtures of paraffins of different melting-points give better results than an unmixed paraffin of the same melting-point as the mixture. He recommends 10 parts of 40° paraffin + 1 of 45° -f 1 of 52° + 1 of 58° + 6 of 60°. For methods for ascertaining melting-points see Kissling, Chem. Centralb. ii, 1901, p. 507. 152. Overheated Paraffin. — Spee {Zeit. wiss. Mile, ii, 1885, p. 8) takes paraffin of about 50° C. melting-point and heats it in a porcelain capsule by means of a lamp until it has become brownish-yellow, and after oooHng shows an unctuous or soapy surface on being cut. This mass may be obtained ready prepared from Griibler. The object of this preparation is to make the mass stickier, in view of cutting ribbons. Van Walsem (Verh. Ahad. Wetenseh. Amsterdam, 1899, p. 132) still recommends the addition of 5 per cent, of yellow wax to paraffin of 52° to 57° melting-point (for large sections of central nervous system). Johnston {Journ. Appl. Micr., vi, 1903, p. 2662) adds 1 per cent, of india-rubber in very small pieces, dissolved by heating to 100° G. for twenty-four hours, or several days to 60° C. Clear with xylol. For very brittle objects. 153. Soap Masses. — ^These have never been much used, and are now entirely discarded. But see early editions, or Polzam {Morpli. Jahrb., iii, 1877, p. 558) ; Kadyi (Zool. Anz., 1879, vol. ii, p. 477) ; Dollkbn {Zeit. wiss. Mile., xiv, 1897, p. 32). Gelatin Masses. 154. Gelatin Imbedding is a method that has the advantage of being applicable to tissues that have not been in the least degree dehydrated. CHAPTER VIII. 93 The modus operandi is, on the whole, the same as for other fusion masses, with the difference that the objects are prepared by satura- tion with water instead of alcohol or a clearing agent. After the cooling of the mass it may sometimes be cut at once, but it is generally necessary to harden it. This may be done by treatment for a few minutes with absolute alcohol (Kaiser), or for a few days with 90 per cent, alcohol (Klebs) or chromic acid (Klebs) or formaldehyde (Nicolas), or it may be frozen (Sollas). The mass can be removed from the sections by means of warm water. 155. Glycerin Gelatin, Klebs' {Arch. mik. Anat., v, 1869, p. 165). — A concentrated solution of isinglass mixed with half its volume of glycerin. Kaiser's (Bot Oentralb., i, 1880, p. 25). — One part by weight of gelatin is left for about two hours in 6 parts by weight of water ; 7 parts of glycerin are added, and for every 100 grras. of the mixture 1 grm. of concentrated carbolic acid. The whole is warmed for ten to fifteen minutes, stirring all the while, until the whole of the flakes produced by the carbolic acid have disappeared. GtERLACh's (Unters. a. d. Anat. Inst. Erlangen, 1884 ; Journ. Boy. Mie. Soc, 1885, p. 541). — ^Take gelatin, 40 grms. ; saturated solution of arsenious acid, 200 c.c. ; glycerin, 120 c.c. Clarify with white of egg. The objects to be prepared for imbedding by a bath of one-third glycerin. Apathy {Mitth. Z. Stat. Neapel, xii, 1897, p. 718, and Zeit. wiss. Mihr., xxix, 1913, p. 472) soaks small objects first in glycerin and water (equal parts) and then for at least twenty -four hours at 40° C. in a solution of 1 part of gelatin in 3 of glycerin and 6 of water. They are then arranged in some of this in an imbedding box:, and the whole is warmed (over calcium chloride) in a stove at 45° to 60° C. until the mass has evaporated down to one-half, losing 5 of its 6 volumes of water (as I understand — ^the description is not clear). Blocks are then cut out and hardened in absolute alcohol (suspended therein) for several days (one day per millimetre of thickness), cleared in terpinol (one day per millimetre), and cut with a knife wetted with the same. Said to give sections of 3 fi, without the least shrinkage. Brunotti's Gold Gelatin Mass (Journ. de Botan., vi, 1892, p. 194). —Twenty grms. gelatin dissolved with heat in 200 c.c. distilled water, and 30 to 40 c.c. of glacial acetic acid with 1 grm. corrosive sublimate added after filtering. Objects are prepared by soaking in some of the mass diluted with 2 to 3 volumes of water, then imbedded in the undiluted mass. The mass is then hardened in spirit or bichromate of potash, picric acid, or the like. No heat at all is required in this process. Nicolas's Method (Bibliogr. Anat, Paris, 3 annee, 1896, p. 274).— Preparations are first soaked for one or two days iu a 3 to 94 IMBEDDING METHODS. 4 per cent, aqueous solution of gelatin kept at 25° C, then for the same time in a 10 per cent, solution, and then for two or three days more in a 20 to 25 per cent, solution containing 8 to 10 per cent, of glycerin and kept at 35° C. They are then imbedded in some of the same mass in paper trays, and a,s soon as the gelatin has set are thrown into a mixture of formol 1 part, water 7. After a few days therein the gelatin has become hard and insoluble, and may be cut or preserved for months in weak formol solution, or dilute alcohol or glycerin, or even in pure water. Sections must be very gradually passed through successive alcohols for dehydration, as they curl up very easily. They, however, flatten out at once on being brought from absolute alcohol into cresylol, and may then be mounted in balsam. To mount in glycerin is of course easy. BuEZYNSKi [Polu. Arch. Biol. Med. Wiss., i, 1901, p. 39) finds that alkaline formol hardens gelatin better than acid. Gaskell (Journ. Path. Bad., July, 1912, p. 58) soaks in pure gelatin, melted s.a., for two to five hours at 37° C, and hardens the mass in vapour of formol, for three or more days. To cut, he freezes. He mounts in glycerin jelly, to avoid dehydration and shrinkage. CHAPTER IX. COLLODION (CELLOIDIN) AKD OTHER IMBEDDING METHODS. 156. Introduction. — Collodion (or celloidiia) masses do not require the employment of heat. They do not require that the objects should be cleared before imbedding, and that is an advantage in the case of very large objects. They are more or less transparent, which facilitates orientation. And they are specially indicated for very large objects, for the soaking in collodion, being quite inoffensive to the most delicate elements, may be prolonged if necessary for weeks. Lastly, the mass being quite transparent after moimting, it is not necessary to remove it from the sections before staining and mounting them ; it may remain, and fulfil the function of an admirable support to the tissues, holding in their places brittle or detached elements that without that help would fall to pieces and be lost. ■ There are disadvantages. One is that the process is a very long one ; as usually practised, it requires some three days for the imbedding of an object that can be imbedded in parafiin in an hour. Another is that it is impossible to obtain with celloidin sections qiiite so thin as those furnished by parafSn. In the older celloidin method the mass is cut wet, before clearing. I strongly recommend the more recently introduced practice of clearing before cutting, and cutting dry as described in §§ 168 — 170. 157. Collodion, Celloidin, Parlodion and Photoxylin. — The collodion method is due to Duval (Journ. de I'Anat., 1879, p. 185). Celloidin, recommended later on by Merkel and Schieefer- DECKER (Arch. Anat. Phys., 1882, p. 200), is merely a patent collo- dion. It may be obtained from almost all of the usual dealers in histological reagents. It is sent out in the form of tablets. These tablets may, if desired, be dissolved at once in ether, or a mixture of ether and alcohol, to make a collodion of any desired strength. But it is better, as recommended by ApIthy, to cut them up into thin shavings, which should be allowed to dry in the air until they become yellow, transparent, and of a horny consistency, and that these be then dissolved in alcohol and ether (sulphuric, free from acid). The solutions thus prepared are free from the excess of water that is present in the undried celloidin, and give after hardening a mass 96 COLLODION AND OTHER IMBEDDING METHODS. that is more transparent and of a better consistency for cutting {Zeit. wiss. Mit, vi, 1889, p. 164). Imbedding masses of excellent quality can be prepared with ordinary coUodion, but celloidin furnishes more readily solutions of known concentration. Otherwise there is but Uttle to choose between the two, and therefore in this work the terms collodion and celloidin are used indifferently. According to Unna {Monatschr. p. Bermtdol., xxx, 1900, pp. 422 and 476 ; Zeit. wiss. Mik., xviii, 1901, p. 32) a more inelastic, and therefore better, mass is obtained by adding to celloidin 2 per cent, of oil of turpentine, stearate of soda, or (best of all) castor oil. Celloidin with this addition has been put on the market under the name of " Celloi- dinum inelasticum," by the Chemisehe Fabrik vorm. E. Sohering, in Berlin. Photoxylim, (Ketsinskt, Vikchow'S ArcMv; cviii, 1887, p. 217 ; BussE, Zeit. wiss. Mik., ix, 1892, p. 47) is a dry substance, of the aspect of cotton-wool, and chemically nearly related to ceUoidin. It can be obtained from GtKUBlee. It gives a clear solution in a mixture of equal parts of ether and absolute alcohol, and should be used in exactly the same way as ceUoidin. It has the advantage of aflording a mass which after hardening in 85 per cent, alcohol remains perfectly trans- parent. Some writers say that it gives a better consistency, but others deny this (Apathy, e.g.) TscHEKNrscHBFF {Zeit. wiss. Mik., xvii, 1900, p. 449) recommends Golloxylin (10 grms. dissolved in 10 grms. of eugenol or clove oil, with the addition of 60 c.o. of ether and 1 of absolute alcohol). The Older Celloidin Method. 158. Preparation of Objects. — The objects must first be very thoroughly dehydrated with absolute alcohol. They are then soaked till thoroughly penetrated in ether, or, which is better, in a mixture of ether and absolute alcohol. Duval (Joe. cit.) takes for this purpose a mixture of ten parts of ether to one of alcohol ; Schieffer- DECKER (and the majorit}?^ of workers) a mixture of equal parts of ether and alcohol ; Tubby (in Nature, November 17th, 1892, p. 51) advises a mixture of four parts of ether and one of alcohol. Fish advises acetone, see next §. Mann {Methods, etc., p. 172) takes equal parts of ether and methyl alcohol. So also Pavlow, Zeit. iviss. Mikr., xxi, 1904, p. 15. This stage may be omitted if the objects are of a sufficiently permeable nature, and they may be brought direct from alcohol into the collodion bath. 159. The Collodion Bath.— The secret of success here is to infiltrate the objects first with thin solutions, then with the definitive thick CHAPTER IX. 97 one. (A thin solution may be taken to mean one containing from 4 to 6 per cent, of celloidin [dried as described in § 157] ; a thick solution, one containing 10 to 12 per cent.) If collodion be taken, the thin solutions may be made by diluting it with ether. If photoxyHn or celloidin be taken, the solutions are made in a mixture of ether and absolute alcohol in equal parts. The dried celloidin shavings dissolve very slowly in the mixture. Elschnig (Zeit. ims. Mikr., x, 1893, p. 443) has found that solution is obtained much quicker if the shavings be first allowed to swell up for twenty-four hours in the necessary quantity of absolute alcohol, and the ether be added afterwards. BussE {op. dt., ix, 1892, p. 47) gives the following proportions for the successive baths : — No. 1, 10 parts by weight of photoxyHn or perfectly dried celloidin to 150 parts of the ether and alcohol mix- ture ; No. 2, 10 parts of photoxyUn or celloidin to 105 of the mixture ; No. 3, 10 parts to 80 of the mixture (already-used solution may be employed for the first bath). I generally use only two solutions : one weak one, and one strong one corresponding approximately to Busse's No. 2. His No. 3 is so thick that excessive time is required to obtain penetration by it. Mann {Methods, p. 172) uses solutions of 2^ and 10 per cent. ApIthy (Behrens, Tabellm., 1898, p. 82) takes 2 per cent, and 4 per cent, for the first baths, 8 per cent, for the last. Myers {Arch. Anat. Phys., Anat. Ahth., 1902, p. 370) takes 1^ per cent., 6 per cent., and 16 per cent. See also Neumatek, Zeit. wiss. Mik., xxv, 1908, p. 38 ; De Vbcohi, ihid., xxiii, 1906, p. 312 ; and Fuhrmann, Z&it. wiss. Zool, Ixxviii, 1905, p. 524. Fish {Journ. Appl. Mioroscop., ii, 1899, p. 323) first infiltrates with acetone (which he says may be used as a fixing and dehydrating agent at the same time), then with a 4 per cent, solution of pyroxylin (gun- cotton) in acetone, and, lastly, in an 8 per cent, acetone solution of the same. See for other solutions §§ 170 and 171. The objects ought to remain in the first bath until very thoroughly penetrated ; — days, even for small objects, — weeks or months for large ones (human embryos of from six to twelve weeks, for instance). When the object is duly penetrated by the thin solution, or solutions, if more than one have been employed, it should be brought into the thickest one. This may be done (as first described in this work, 1st ed., 1885, p. 194) by" allowing the thin solution to con- centrate slowly (the stopper of the containing vessel being raised, for instance, by means of a piece of paper placed under it), and making up *;he loss from evaporation with thick solution. M. '' 98 COLLODION AND OTHER IMBEDDING METHODS. ApIthy (Milcrotechnik, p. 121) holds that it is preferable to transfer to fresh thick solution, as he finds that a better consistency after hardening is thus obtained. 160. Imbedding.— The objects must now, if it has not been done before, be imbedded— that is, arranged in position in the thick coUodion in the receptacle in which they are to be hardened. For the usual manipulations see § 137. If paper thimbles be taken for imbedding, the bottoms should be made of soft wood in preference to cork. See § 165. They should be prepared for the reception of the object by pouring into them a drop of collodion, which is allowed to dry. The object of this is to prevent bubbles coming up through the wood or cork and lodging in the mass. Watch-glasses, deep porcelain water-colour moulds, and the like, also make convenient imbedding receptacles. Care should be taken to have them perfectly dry. It not infrequently happens that during these manipulations bubbles make their appearance in the mass. Before proceeding with the hardening these should be got rid of by exposing the whole for an hour or two to the vapour of ether in a desiccator or other well-closed vessel. Care should be taken that the ether (which may be poured on the bottom of the vessel) does not wet the mass (BussE, Zeit. wiss. Mik., viii, 1892, p. 467). 161. Orientation. — CeUoidin being more or less transparent, it is seldom necessary to resort to sj)ecial aids to orientation. ApIthy (Zeit. wiss. Mik., v, 1888, p. 47) arranges objects on a small rectangular plate of gelatin, placed on the bottom of the imbedding-recipient. The gelatin is turned out with the mass after hardening, and cut with it. The edges of the gelatin form good ' orientation lines. Halle and Boen {Zeit. wiss. Mik., xii, 1896, p. 364) use plates of hardened white of egg, in which a shallow furrow for the reception of the objects has been cut by means of a special instrument. See also § 142. For the complicated method of Eycleshymer (Amer. Nat., xxvi, 1892, p. 354) see previoics editions. See also the article " Rekonstruction " in the Encycl. mik. Technik. 162. Hardening, Preliminary.— The objects being imbedded, the treatment should be as follows : — The receptacles or supports are set with the mass under a glass shade, allowing of just enough com- munication with the air to set up a slow evaporation. Or porcelain moulds or small dishes may be covered with a lightly fitting cover. CHAPTER IX. 99 As soon as the added thick collodion (of which only just enough to cover the object should have been taken) has so far sunk down that the object begins to lie dry, fresh thick solution is added, and the whole is left as before. (If the first layer of collodion has become too dry, it should be moistened with a drop of ether before adding the fresh collodion.) Provision should be again made for slow evaporation, either in one of the ways above indicated, or — which is perhaps better — by setting the objects under a hermetically fitting beU-jar, which is Ufted for a few seconds only once or twice a day. I have frequently found it advantageous to set the objects under a bell-jar, together with a dish containing alcohol, so that the evapora- tion is gone through in an atmosphere of alcohol. This is especially indicated for very large objects. The whole process of adding fresh collodion and placing the objects under the required conditions of evaporation is repeated every few hours for, if need be, two or three days. When the mass has attained a consistency such that the ball of a finger {not the nail) no longer leaves an impress on it, it should be scooped out of the dish or mould, or have the paper removed if it has been imbedded in paper, and be submitted to the next stage of the hardening process. (If the mass is found to be not quite hard enough to come away safely, it should be put for a day or two into weak alcohol, 30 to 70 per cent.) 163. Hardening, Definitive. — Several methods are available for the definitive hardening process. One of these is the chloroform method, due to Viallanes (Rech. sur I'Hist. et le Dev. des Insectes, 1883, p. 129). It consists in bringing the objects into chloroform. In some cases a few hours' immersion is sufficient to give the requisite consistence. In no case have my specimens required more than three days. The collodion frequently becomes opaque on being put into the chloro- form, but regains its transparency after a time. Small objects may be hardened by chloroform without preliminary hardening by evaporation. All that is necessary is to expose the mass to the air for a few seconds until a membrane has formed on it, and then bring it into chloroform. If the mass is in a test-tube this may be filled up with chloroform and left for two or three days if need be. By this time the collodion mass will be considerably hardened, and also somewhat shrunk, so that it can be shaken out of the tube. It is then brought into fresh chloroform in a larger vessel, where it remains for a few more days until it is ready for cutting. But sufficient hardening is sometimes obtained in a few hours. 7—2 100 COLLODION AND OTHER IMBEDDING METHODS. Good chloroform is a necessity. The above processes are excellent, but I regard them as primitive forms of the chloroform method. I now almost always harden in vapour of chloroform. All that is necessary is to put the liquid mass (after having removed bubbles as directed in § 160), with its recipient, into a desiccator on the bottom of which a few drops of chloroform have been poured. The action is very rapid, and the final con- sistency of the mass at least equal to that obtained by alcohol hardening. The more commonly employed hardening method is the alcohol method. The objects are thrown into alcohol and left there until they have attained the right consistency (one day to several weeks). The bottle or other vessel containing the alcohol ought not to be tightly closed, but should be left at least partly open. The strength of the alcohol is a point on which the practice of different writers differs greatly. Busse (Zeit. f. wiss. Mikr., ix, 1, 1892, p. 49) has found, as I also have done, that alcohol of about 85 per cent, is the best, both as rega/rds the cutting consistency and the transparency of the mass. (Care must be taken to keep masses hardened in this grade of alcohol moist while cutting, as they dry by evaporation very quickly.) Some workers use lower grades, 70 to 80 per cent., or even lower. ApIthy (Microtechnik, p. 185) mentions " glycerin-alcohol," but without giving details. Blum (Anat. Anz., xi, 1896, p. 724) mentions " weak spirit with formol added to it," saying that formol hardens celloidin. Lastly, the mass may be frozen. After preliminary hardening by alcohol, it is soaked for a few hours in water, in order to get rid of the* greater part of the alcohol (the alcohol should not be removed entirely, or the mass may freeze too hard). It is then dipped for a few moments into gum mucilage in order to make it adhere to the freezing plate, and is frozen. If the mass have frozen too hard, cut with a knife warmed with warm water. Floeman {Zeit. wiss. Mik., vi, 1889, p. 184) recommends that the definitive hardening should be done without the aid of alcohol or chloro- form, by simply cutting out the blocks, turning them over, and carefully continuing the evaporation process in the way described above. I described this process myself in the first edition of this work. I doubt whether it is possible in this way to carry the hardening much beyond the point attained by the chloroform or alcohol method without incurring a very undesirable degree of shrinkage. 164. Preservation. — The hardened blocks of collodion may be preserved till wanted in weak alcohol (70 per cent.), or dry, by dipping CHAPTER IX. 101 them into melted paraifin (ApAthy, Zeit. wiss. Mikr., v, 1888, p. 45), or, after rinsing with water, in glycerine- jelly, which may be removed with warm water before cutting (Apathy, MiUh. Zool. Stat. Neapel, xii, 1897, p. 372). Reference numbers may be written with a soft lead pencil on the bottom of the paper trays, or with a yellow oil pencil on the bottom of the watch-glasses in which the objects are imbedded. On removal of the paper from the collodion after hardening, the numbers will be found impressed on the collodion. 165. Cutting. — If the object has not been stained before imbedding, it may form so transparent a mass with the oollodion that the arrange- ment of the object and sections in the right position may be rendered very diflcult. It is, therefore, well to stain the oollodion lightly, just enough to make its outlines visible in the sections. This may be done by adding picric acid or other suitable coloiiring matter dissolved in alcohol to the collodion used for imbedding, or to the oil used for clearing. To fix a collodion block to the microtome take a piece of soft wood, or, for very small objects, pith, of a size and shape adapted to fit the holder of the microtome. Cover it with a layer of collodion, which you allow to dry. Take the block of collodion or the infil- trated and hardened but not imbedded object, and cut a slice off the bottom, so as to get a clean surface. Wet this surface first with absolute alcohol, then with ether (or allow it to dry) ; place one drop of very thick collodion on the prepared wood or pith and press down tightly on to it the wetted or dried surface of the block or object. Then throw the whole into weak (70 per cent.) alcohol for a few hours, or even less, or, better, into chloroform, or vapour of chloroform, for a few minutes, in order that the joint may harden. Lindsay Johnson prefers a mixture of beeswax, 1 part ; rosin, 2 parts. To use it you must get the block of celloidin perfectly dry at the bottom, then warm the object-holder slightly, if possible over a flame ; drop on to it a few drops of melted cement, and press on to it the block of collodion, which will be firmly fixed as soon as the cement is cool — that is, in a few seconds. For objects of any considerable size it is best not to use cork for mounting on the microtome, if the object-holder be a vice ; for cork bends under the pressure of the holder, and the elastic collodion bends with it, deforming the object. If the object-holder be of the cylinder type, a good cork may be used ; but even then, I think, wood is safer. Gage has recommended bits of glass cylinders. Jelinek (Zeit. wiss. MiL, xi., 1^94; p. 237) recommends a sort of 102 COLLODION AND OTHER IMBEDDING METHODS. vulcanite known as " Stabilit," which is manufactured for electrical insulation purposes. It is supplied in suitable blocks by Jung, and by Geubler. Wood is liable to swell in alcohol so that it no longer fits into the object-holder. Babcock (Joum. R. Micr. Soc, 1901, p. 339) uses a block of hard paraffin, with the surface corrugated. Sections (from such masses as have not been cleared before cutting) are cut with a knife kept abundantly wetted with alcohol (of 50 to 85 or even 96 per cent.). ApIthy recommends that the knife be smeared with yellow vaseline ; it cuts better, is protected from the alcohol, and the mobility of the alcohol on the blade is lessened. The knife is set in as oblique a position as possible. Very brittle sections may be coUodionised as explained § 147. The sections are either brought into alcohol (of 50 to 85 or 95 per cent.) as fast as they are made, or if it be desired to mount them in series, they are treated according to one of the methods described below, in Chapter X. Masses that have been cleared before cutting with cedar oil or the like may be cut dry, § 170. 166. Staining. — The sections may now be stained as desired, either loose, or mounted in series on slides or on paper as described in Chapter X. It is not in general necessary, nor indeed desirable, to remove the mass before staining, as it usually either remains colourless, or gives up the stain on treatment with alcohol. But if it be desired, the mass may be removed by treating the sections with absolute alcohol or ether.' 167. Clearing and Mounting. — You may mount in glycerin without ' removing the mass, which remains as clear as glass in that medium. You may mount in balsam, also, without removing the mass, which does no harm, and serves the useful purpose of holding the parts of the sections together during the manipulations. Dehydrate in alcohol of 95 or 96 per cent, (not absolute, as this attacks the collodion). Nikifoeow (Zeit. wiss. MiL, viii, 1891, p. 189) recom- mends a mixture of equal parts of alcohol and chloroform. Clear with a substance that does not dissolve collodion. The clearing agents most recommended are origanum oil {01. Origan. Cretici, it is said, should be taken, not 01. Orig. Gallici ; but see as to this reagent the remarks in § 125), bergamot oil (said to make sections shrink somewhat), oil of sandal- wood, lavender oil, oil of cedar- wood (safe and gives excellent results, but acts rather slowly), chloroform, xylol, or benzol (may. make sections shrink if not well dehydrated), CHAPTER IX. 103 or Dunham's mixture of 3 or 4 parts of white oil of thyme with 1 part of oil of cloves. (As to oil of thyme, see also §§ 125, 126.) Fish (Proc. Amer. Mik. Soc, 1893) advises a mixture of 1 part of red oil of thyme with 3 parts of castor oil, the latter being added in order to counteract the volatility of the thyme oil. But later (June, 1895), writing to me. Dr. Fish says he has substituted the white oil of thyme for the red, and finds it an advantage in orien- tating. See also § 126, and under " Euparal." Some specimens of clove oil dissolve collodion very slowly, and may be used, but I would not be understood to recommend it. The action of origanum oil varies much, according to tbe samples ; some sorts do not clear the collodion, others dissolve it, others pucker it. Minot {Zeit. wiss. Mile, iii, 1886, p. 175) says that Dunham's mixture " clarifies the sections very readily, and softens the ceUoidin just enough to prevent the puckering which is, so annoying with thyme alone." Carbolic acid has been recommended. Weigeet [Zeit. wiss. Mik., iii, 1886," p. 480) finds that a mixture of 3 parts of xylol with I'part of carbolic acid (anhydrous) clears well. But it must not be used with the basic aniUn stains, as it discolours them. For these anilin oil may be used with xylol in the place of carboUo acid. Anilin oU clears well (it wiU clear from 70 per cent, alcohol), but unless thoroughly removed the preparation becomes yellowish-brown, see § 134. See van Gieson, Amer. Mon. Mic. Journ., 1887, p. 49, or Jowm. Boy. Mic. Soc, 1887, p. 619, for a review of these clearing agents. Beech-wood creasote has been recommended (by M. Plesch). Etoleshtmer (Amer. Nat, xxvi, 1892, p. 354) advises a mixture of equal parts of bergamot oil, cedar oil, and carbolic acid. For oil of cajeput see § 129 ; and for this and other clearers see also Jordan, Zeit. wiss. Mik., xv, 1898, p. 51, who recommends, amongst other things, oil of Linaloa, which remains colourless. . The Newer CeUoidin Method, 168. The New Method, by Clearing before Cutting.— This process is due, I believe^ in the first instance to E. Meyer (Biol. Centralb., X, 1890, p. 508), who' advised soaking blocks before cutting for twenty-four hours in glycerin. Bumpus (Amer. Anat, xxvi, 1892, p. 80) advises clearing the mass, after hardening in chloroform, with white oil of thyme or other suitable clearing agent. See § 167. The knife is wetted with the clearing oil, and the same oil is employed for covering the exposed surface of the object after each cut. Similar recommendations are made by Eycleshymer {op. cit., pp. 354, 563), carbolic acid, or glycerin, or the mixture given § 167, being suggested for clearing ; and Gilson has for a long time past 104 COLLODION AND OTHER IMBEDDING METHODS. adopted the practice of clearing before cutting with cedar oil, as described in the next §. Fish {loc. cit., § 167) also advocates the practice of clearing in the mass, recommending the clearing mixture there given. Similarly Gage, Trans. Amer. Mih. Soc, xvii, 1896, p. 361. All the authors above quoted cut in the wet way, that is to say, with a knife wetted with the clearing liquid. 169. Gilson's Rapid Process (communicated April, 1892). — The object is dehydrated, soaked in ether, and brought into a test-tube with collodion or thin celloidin solution. The tube is dipped into a bath of melted parafiin, and the collodion allowed to boil (which it does at a very low temperature) until it has become of a sjnrupy consistence. (It should be boiled down to about one-third of its volume.) The mass is then turned out, mounted on a block of hardened celloidin, and the whole hardened in chloroform or in a mixture of chloroform and cedar oil for about an hour. It is then cleared in cedar oil (if hardened in pure chloroform : special clearing will not be necessary if it has been hardened in the mixture). It may now be fixed in the microtome and cut, using cedar oil to wet the knife, and cover the exposed surface of the object after each cut. This process is very much more rapid than the old process : small objects can be duly infiltrated in an hour, where days would be required by the old process. As collodion boils at a very low temperature, very little heat is required, and there is no risk of the tissues suffering on that head. 170. The Dry Cutting Method. — I recommend the following as a further improvement. Infiltrate with collodion or celloidin either by Gilson's process, or by soaking in the cold in the usual way, § 159. Imbed as usual. Harden in vapour of chloroform for from one hour (generally sufficient for small objects) to overnight. This is 'done by putting the object (definitively imbedded in the final thick solution, but without any preliminary hardening in the air) into a Steinach's sieve-dish or into a desiccator, on the bottom of which a teaspoonfrd of chloroform has been poured. (The objects may remain for months in the chloroform vapour if desired.) As soon as the mass has attained sufficient superficial hardness, it is, of course, well to turn it out of its recipient, and turn it over from time to time, in order that it may be equally exposed on all sides to the action of the vapour. When fairly hard throw it into Gilson's mixture. This should be at first a mixture of 1 part of chloroform with 1 or 2 parts of cedar oil. From time to time more cedar oil CHAPTER IX. 105 should be added, so as to bring the mixture up gradually to nearly pure cedar oil. As soon as the object is cleared throughout, the mass may be exposed to the air, and the rest of the chloroform will evaporate gradually. The block may now either be moimted on the holder of the microtome, § 165, and cut at once, or may be preserved indefinitely without change in a stoppered bottle. Cut dry, the cut surface will not dry injuriously under several hours. The cutting quality of the mass is often improved by allowing it to evaporate in the air for some hours. The hardening may be done at once in the chloroform and cedar oil mixture, instead of the chloroform vapour, but I find the latter preferable. And clearing may be done in pure cedar oil instead of the mixture, but then it will be very slow, whereas in the mixture it is extremely rapid. Stepanow {Zeit. wiss. Mik., xvii, 1900, p. 185) soaks and imbeds in a solution of ceUoidin in a mixture of equal parts of ether and clove oil, hardens in alcohol or vapour of chloroform, or in benzol, and cuts either wet or dry. See also TscHEENiscHErF, ibid., p. 449. Jordan, ibid., p. 193, imbeds in a mixture of 5 parts of 8 per cent. ceUoidin solution with 1 of oil of cedar, hardens first in vapour of chloroform and then in a mixture of 5 parts of chloroform with 1 of oil of cedar, and cuts wet or dry. 171, Double Imbedding in Collodion and ParaflSn.^-This is some- times employed for objects of which it is desired to have very thin sections, and which are too brittle to give good sections by the plain parafiin process. Kultschitzky's Method (Zeit. wiss. Mik., iv, 1887, p. ^8). — After the collodion bath, the object is soaked in oil of origanum (Oleum Origani vulg.). It is then brought into a mixture of origanum oil and paraffin heated to not more than 40° C, and lastly into a bath of pure paraffin. The mass may be preserved in the dry state, and may be cut dry. Etder [Queen's Mier. Bull., 1887, p. 43 ; Journ. Boy. Micr. Soc, 1888, p. 512) modified the process by substituting chloroform for the origanum oil. Ide (La Cellule, vii, 1891, p. 347, and viii, 1, 1892, p. 114) imbeds in coUodion in a tube by G-ilson's process (§ 169) ; the collodion is boiled for forty minutes, then brought for fifteen minutes (this is for small objects) into chloroform heated to 30° C. containing J part of parafiin dissolved in it, then for ten minutes into pure melted paraffin. Field and MARTm'(Bull. Soc. Zool. de France, 1894, p. 48) make a solution of dried ceUoidin in a mixture of equal parts of absolute alcohol and toluene, of about the consistency of clove oil. This solution is 106 COLLODION AND OTHER IMBEDDING METHODS. saturated witi paraffin, added in shavings at a temperature not exceed- ing 20° to 23° C. The tissues are prepared by soaking- in some of the , mixture of alcohol and toluene, and are then penetrated with the celloidin -paraffin solution. The mass is hardened in a saturated solution of paraffin in chloroform or in toluene, and is finally imbedded in pure paraffin in the usual way. Stbpanow imbeds in paraffin after clearing with benzol, last §. JOKDAN, after imbedding as in last §, passes through a bath of paraffin dissolved in chloroform into pure paraffin. WiLHELMi (Fauna Flora Oolf. Neapel, xxxii, 1909, p. 17), following Apathy, imbeds in celloidin, hardens in chloroform, then adds benzol to the chloroform, and passes through pure benzol (half an hour to an hour) into paraffin, and cuts dry. Similarly, Beeckneb, Zeit. wiss. Mih., xxv, 1908, p. 29. Sterling {Jena Zeit, 1909, p. 253) soaks for two or three days in equal parts of clove oil and collodion, puts for a couple of hours (until clear) into xylol, and imbeds in paraffin. See also Dahlgeen, Journ. Appl. Microsc, 1898, p. 97 ; SabusSow, Mitth. Zool. Stat. Neapel, xii, 1896, p. 353 ; Meteb, ibid., xiv, 1901, p. 295 ; MiTEOPHANOW, Arch. Zool. Fxpir. (3), 3, 1896, p. 617 ; Fedeeici, Anat. Anz., xxi, 1907, p. 602 ; Boedagb, Bull. 8ei. France Belg., xxxix, 1905, p. 385 ; Gandolfi, Zeit. wiss. Mile, xxv, 1909, p. 421 ; Matee, ibid., xxiv, 1907, p. 132. Other Cold Masses. 172. Lead-Gum Imbedding Method of J. Salkind {C. R. Soc. de Biol., t. Ixxix, 1916, No. 16). — The principle of this method is that- an aqueous solution of gum treated by acetate of lead, when exposed to the action of ammonia, is transformed into a gel, sufficiently stiff to allow of thin sections being cut. (1) •Dissolve a quantity of gum of cherry (white for preference) in double its weight of aq. dest. After filtration, add to the solution one-third its volume of the liquid subacetate of lead (extract of Saturne), to which has been added 5 per cent, of glacial acetic acid. This gives a kind of thin collodion-like solution, in which you place the pieces at room temperature, to be imbedded, after a fixation, for which see below. (2) Leave about twelve ho\irs for pieces about a millimetre in thickness : larger pieces must be left longer. After the correct period has elapsed, you let the lead gum solution evaporate in the air tiU the solution reaches the consistency of a thick celloidin solution. (3) Arrange the pieces to be imbedded in a paper box (or on a piece of paper), in a large drop of the thick solution. Expose to strong ammonia vapour for about five minutes till the block hardens to the consistency of cartilage. CHAPTER IX. 107 (4) Trim the block, and fasten it on to the plate of a microtome by means of some of the thick lead-gum (hardened afterwards in ammonia vapour). Cut sections with an oblique knife, the block being moistened with a solution 1 per cent, sodium chloride in aq. dest. The sections are placed in the same solution, in which they must not stay more than one hour. (5) Sticking the sections to the sUde is done by a modification of Olt's method, § 182. Cover the slide with albumen, then with gelatin, arrange the sections, press down with a cloth, and harden in formol vapour. See also J. A. Murray, below. (6) The lead-gum is then dissolved away in 5 per cent, acetic acid. After washing you stain and mount in any way desired. Neither gum arable (acacia), plum, nor apricot give quite such good results as cherry gum. Salkind recommends two fixatives to precede this method of imbedding. A. Formol, acetic acid, sub-acetate of lead, 1 part each. Aq. dest., 5 parts. B. Formol 10 c.c. Acetone . . . . . 30 „ Water 40 „ Citric acid , . . . 5 to 10 grms. Saturated with Sudan III. After A, it is not necessary to wash out. After B, and the majority of such fixatives as bichromate especially, you must wash out in running water. J. A. Murray (Beport of Itnper. Cancer Bureau, 1919) fixes cartilage in 10 per cent, formol-salt solution for at least twenty-four hours. After Salkind's lead-gum imbedding, cuts sections 10 — 15 /i thick with sliding microtome. Transfers sections for from ten minutes to one hour in 1 per cent. NaCl solution. Special slides prepared beforehand by coating in 1 per cent, gelatin and allowing to dry. The prepared sUde is immersed in the salt solution (NaCl), sections arranged with a smooth-pointed glass rod, superfluous liquid drained ofl, and a wetted cigarette paper carefully lowered over the sections. Firm pressure with several layers of filter paper makes the sections adhere to the sUde. Withdraw the cigarette paper and expose to formol vapour for a few minutes. Transfer to 10 per cent, formol five minutes, then treat in the 5 per cent, acetic to remove the lead-gum. Stain, • 173. Joliet's Gum and Glycerin Method {Arch. Zool. Exp6r. et Gen., X, 1882, p. xliii). — Pure gum arable dissolved in water to the consistency of a thick syrup. Pour a little of the solution into a watch-glass, and add from 6 to 10 drops of pure glycerin. In the winter or in rainy weather less glycerin should be taken than in the summer or dry weather. The object is imbedded in the mass in the watch-glass and the whole 108 COLLODION AND OTHER IMBEDDING METHODS. left to dry for from one to four days. When it has assumed a cartila- ginous consistency, a block containing the object is out out, turned over, and allowed to dry again until wanted for use. A stove, or the sun, may be employed for drying, but it is best to dry slowly at the normal temperature. 174. Strickeb's Gum Method (Hdb. d. Gewebel, p. xxiv). — A concen- trated solution of gum arable. The object is imbedded in the gum in a paper case. The whole is thrown into alcohol, and after two or three days may be cut. The alcohol should be of about 80 per cent. (Matee). I have seen masses of sufficiently good consistency prepared by this simple method. 175. Hyatt's Shellac Method, see Am. M. Mic. Journ., i, 1880, p. 8 ; Journ. Boy. Mic. 8oc., iii, 1880, p. 320. For sections through hard chitinous organs consisting of several pieces, such as stings and oviposi- tors, retaining all the parts in their natural positions. 176. Beunotti's Cold Gelatin Mass has been given, § 156. Masses for Grinding Sections* 111. G. VON Koch's Copal Method {Zool. Anz., i, 1878, p. 36).— Small pieces of the object are stained in bulk and dehydrated with alcohol. A thin solution of copal in chloroform is prepared by triturating small fragments of copal in a mortar with fine sand, pouring on chloroform to the powder thus obtained and filtering. The objects are brought into a capsule filled with the copal solution. The solution is now slowly evaporated by gently heating the capsule on a tile by means of a common night-light placed beneath it. As soon as the solution is so far concentrated as to draw out into threads that are brittle after cooling, the objects are removed from the capsule and placed to dry for a few days on the tile in order that they may more quickly become hard. When they have attained such a degree of hardness that they cannot be indented by a finger- nail, sections are cut from them by means of a fine saw. The sections are rubbed down even and smooth on one side with a hone, and cemented, with this side downwards, to a slide, by means either of Canada balsam or copal solution. The slide is put away for a few days more on the warmed tile. As soon as the cement is perfectly hard the sections are rubbed down on a grindstone, and then on a hone, to the requisite thinfiess and polish, washed with water, and mounted in balsam. The process may be varied by imbedding the objects unstained, * For the manipulations of section-grinding, see Carpenter's The Microscope. CHAPTER IX. 109 removing the o pal from tie sections by soaking in chloroform, decalcifying them if necessary, and then staining. It is sometimes a good plan, after removing the copal, to cement a section to a slide by means of hard Canada balsam, then decalcify cautiously the exposed half of the specimen, wash, and stain it. This method was invented in order to enable the hard and soft parts of corals to be studied in their natural relations, and is valuable for this and similar purposes. 178. Ehrenbaum's Colophonium and Wax Method {Zeit. wiss. Mik., 1884, p. 414). — Ehrenbaum recommends a mass consisting of 10 parts of colophonium to 1 of wax. The addition of wax makes the mass less brittle. Sections are obtained by grinding in the usual way. The mass is removed from them by means of turpentine followed by chloroform. 179. Johnstone-Lavis and Vosmaer's Balsam Method (Joum. Roy. Mic. Soc, 1887, p. 200).— Alcohol material is carefully and gradually saturated, first with benzol, and then with thin and thick solution of benzol-balsam. It is then dried for a day in the air and for several days more in a hot-air bath. When hard it is ground in the usual way. 180. Weil's Canada Balsam Method, see Zeit. wiss. Mile., v, 1888, p. 200. 181. Giesbkecht's Shellac Method. — ^For hard parts only, spines of Echinus, shell, etc., see Morph. Jajirb., vi, 1880, p. 95, or the abstract in Lee und Matek, Cfrundmge. Congelation Masses. 182. The Methods of Freezing. — For the requisite manipulations and means of producing the requisite degree of cold, see Carpenter's The Microscope (ether spray) ; Johne, Zeit. wiss. Mik., xiv, 1897, p. 370 (Uquid carbonic acid) ; Wolff, ibid., xxv, 1908, p. 175 (ethyl chloride) ; Krause, ibid., p. 289 (solid carbonic acid) ; Jung, Verh. Ges. Naturf. Aertze, Ixix, 1898, p. 129 (ethyl chloride) ; Brissy, C. R. Soc. Biol, Ixii, 1907, p. 1115 (liquid air). Fregh tissues may be, and are, frequently frozen without being included in any mass. But the formation of ice crystals frequently causes tearing of delicate elements, and it is better to infiltrate the tissues with a mass that does not crystallise in the freezing mixture, but becomes simply hard and tough, such as one of those given below. When sections have been obtained, it is difficult to manipulate them. Olt {Zeit. wiss. Mik., xxiii, 1906, p. 327) puts them into a 1 per cent, solution of gelatin, brings them therein on to a slide, no COLLODION AND OTHER IMBEDDING METHODS. hardens for an hour in vapour of formaldehyde, and soaks for a few minutes in formol of 10 per cent. AifiTSCHKOW (tW., xxvii, 1910, p. 73) puts them into alcohol of 50 per cent., gets them on to a sUde prepared with Mayer's albumen, presses down with paper, puts into alcohol of 98 per cent., and thence through lower grades into water. 183, Gum and Syrup Masses. — Hamilton {Journ. of Anat. and Phys., xii, 1878, p. 254) soaked tissues in sjnrup made with double refined sugar, 2 ounces ; water, 1 fluid ounce ; then washed the superfluous syrup from the surface, and put into ordinary gum mucilage for an hour or so, and then imbedded in the freezing microtome with mucilage in the usual way. Cole (Methods of Microscopical Research, 1884, p. xxxix) takes gum mucilage (B. P.), 5 parts ; syrup, 3 parts. (For brain and spinal cord, retina, and all tissues liable to come in pieces put 4 parts of syrup to 5 of gum.) Add 5 grains of pure carbolic acid to each ounce of the medium. (G-um mucilage [B. P.] is made by dissolving 4 ounces of picked gum acacia in 6 ounces of water. The syrup is made by^ dissolving 1 pound of loaf sugar in 1 pint of water and boiling.) The freezing is conducted as follows : — The gum and syrup is removed from the outside of ths object by means of a cloth ; the spray is set going and a little gum mucilage painted on the freezing plate ; the object is placed on this and surrounded with gum muci- lage ; it is thus saturated with gum and syrup, but surrounded when being frozen with mucilage only. This combination prevents the sections from curling up on the one hand, or splintering from being too hard frozen on the other. Should freezing have been carried too far, wait for a few seconds. Webb (The Microscope, ix, 1890, p. 344 ; Journ. Roy, Mic. Soc, 1890, p. 113) takes thick solution of dextrin in solution of carbolic acid in water (1 in 40). 184. Gelatin (Sollas, Quart. Journ. Mic. Soe., xsiv, 1884, pp. 163, 164). Gum Gelatin (Jacobs, Amer. Natural., 1885, p. 734). White of Egg (EOLLETT, DenskseJir. math, naturw. Kl. Tc. Acad. Wiss. Wien, 1885 ; Zeit. wiss. Mik., 1886, p. 92). — Small portions of tissue brought in the white of a fresMy laid egg on to the freezing stage, frozen and cut. Oil of Aniseed (Kuhne, Centralh. f. Bakteriol., xii, 1892, p. 28 ; Journ. Boy. Mic. Soc, 1892, p. 706 ; V. A. Moore, Amer. Man. Mic. Journ., 1894, p. 373 ; Journ. Boy. Mic. Soc, 1895, p. 247). Anethol (anise camphor) (Stepanow, Zeit. wiss. Mik., xvii, 1900, p. 181). For details of these see previous editions. For Dollkbn's method of solidifying formol by means of resorcin, see Zeit. wiss. Mik., xiv, 1, 1897, p. 33. CHAPTEE X. SERIAL SECTION MOUNTING. 185. Choice of a Method. — I recommend the following : — For general work with paraffin sections, the combined water and albumen method, § 188. For very delicate work, the water method. For collodion sections, the albumen method ; for large collodion sections, GrRAHAM Kerr's seems the most convenient. Methods for Paraffin Sections. 186. The Water or Desiccation Method. — Gaule {Arch. Anat. Phys., Phys. Abth., 1881, p. 156) ; Suchannek {Zeit. wiss. Mik., vii, 1891, p. 464) ; Gulland (Journ. Anat. and Phys., xxvi, 1891, p. 56) ; Schieiterdecker (Zeit. wiss. Mik, ix, 1892, p. 202) ; Heidenhain (Kern, und Protoplasma, p. 114) ; Nusbaum (Anat. Am., xii, 2, 1896, p. 52) ; Mayer in the GrundzUge, Lee und Mayer, 1898, p. 113 ; De Groot [Zeit. wiss. Mik., xv, 1898, p. 62), and others. — The principle' of this method is that the sections are made to adhere to the slide without the intervention of any cementing substance, being brought into intimate contact with the glass by being slowly drawn down by the evaporation of a layer of water on which they are floated. It is now practised, with unessential variations, as follows : (a) For sections that are large and not numerous. The sections are flattened out on water by one or other of the processes described in § 149. The slide is then drained and put away to dry until every trace of water has completely evaporated away from under the sections. This drying may be performed at the temperature of the laboratory, in which case many hours will be necessary (to be safe it will generally be necessary to leave the sections overnight). Or it may be performed in a stove or on a water-bath at a temperature a few degrees below the melting-point of the paraffim (best not above 40° C), in which case fixation wiU be much more rapid, large thin sections being often sufficiently fixed in an hour, though thick ones will require half a dozen hours or more. The paraffin must not be allowed to melt before the sections are perfectly dry; the sections are sure to become detached if it does. Perfectly dry sections have a 112 SERIAL SECTION MOUNTING. certain briUiant transparent look that is easily recognisable. As soon as dry the paraflftn may be removed, and they may be further treated as desired. To remove the paraffin all that is requisite is to put the slide into a tube of xylol or other good solvent, which in a few seconds, or minutes at most, removes the paraffin perfectly. Most workers first melt the paraffin, but I find this is not necessary. (6) For series of numerous small sections. Clean a slide perfectly, so that water will spread on it without any tendency to run into drops (see below). Breathe on it, and with a brush draw on it a streak of water as wide as the sections and a little longer than the first row of sections that it is intended to mount. With a dry brush arrange the first row of sections (which may be either loose ones or a length of a. ribbon) on this streak. Breathe on the sHde again, draw on it another streak of water under the first one and arrange the next row of sections on it, and so on until the slide is fuU. Then breathe on the sHde again, and with the brush add a drop of water at each end of each row of sections, so as to enable them to expand freely ; then warm the slide so as to flatten out the sections, taking care not to melt the paraffin. Some persons do this by holding it over a small flame for a few seconds. I prefer to lay it on a slab of thick glass, warmed, watching the flattening of the sections through a lens if necessary. As soon as they are perfectly flat, draw off the excess of water from one corner of the mount with a dry brush, and put aside to dry as before (a). In order to succeed in this method it is absolutely essential that the sections be perfectly expanded and come into close contact with the slide at all points. And to ensure this it is necessary that the slide should be perfectly /ree/rom grease, so that the water may wet it equaUy everywhere. The test for this is, firstly, to breathe on the slide ; the moisture from the breath should condense on it evenly all over, and disappear evenly. Secondly, streaks of water drawn on it with a brush should not run. To obtain a slide that will fulfil these conditions, clean it well in the usual way, place a drop of water on it and rub it in thoroughly with a damp cloth and try the tests. If this does not suffice, take a turn of a corner of the cloth round a finger and rub it with a piece of chalk, then damp the cloth and rub the sUde with it, finishing up with a clean part of the cloth and clean water (De Gkoot, loc. cit., supra). If after performing this operation twice the slide still refuses to take the water thoroughly it should be rejected as incorrigible ; for there are apparently some sorts of glass that can never be got to wet properly. Mayer finds carbonate of magnesia or soda useful. CHAPTER X. 113 GuDERNATSCH {Zeit. wiss. Mikr., xxiv, 1908, p. 358) washes tke slide well with potash soap, and arranges the sections on it whilst still wet. Helly {ibid., 1906, p. 330) passes it two or three times over the flame of a Bunsen burner. Tap water seems preferable to distilled water ; it seems to spread better and give a stronger adhesion. Nusbaum adds a trace of gum arabic (1 or 2 drops of mucilage to a glass of water) ; Apatht {Micro- technik, p. 126) adds 1 per cent, of Mayer's albumen {§ 187) ; and Henneguy [Lemons sur la Cellule, 1896, p. 62) takes a 1 : 5,000 solution of gelatin, with a trace of bichromate of potash, added just before using, and dries the slides exposed to light. Similarly, Buechakdt (Jena Zeit, xxxiv, 1900, p. 719). Some workers have used alcohol (50 or 70 per cent. ) instead of water ; but this I believe to be now generally abandoned. This is the most elegant method of any, as there is nothing on the slide except the sections that can stain, or appear as dirt in the mount. Tissues do not suffer from the drying, provided the material has been properly imbedded. Sections stick so fast by this method that they wiU stand watery or other fluids for weeks, so long as they are not alkaline. When successfully performed it is quite safe, provided that the sections are of a suitable nature. They must be such as to afford a sufficient continuous surface, everywhere iu con- tact with the slide. Sections of parenchymatous organs stick well ; sections of thin-waUed tubular organs stick badly. Sections of chitinous organs are very unsafe. The larger and thinner sections are, the better do they stick, and vice versa. Sections from chromic or osmic material adhere less well than sections from alcohol or sublimate material. By taking a staining solution instead of pure water for expanding, the sections can be got to stain at the same time, and so be brought into balsam without passing through alcohol ; see Matek, Mitth. Zool. Stat. Neapel, xii, 1896, p. 320 ; Schmokl, Path.-hist. Untersuchungsmethoden, 1897, p. 38 ; Smith, Journ. Anat. PTiys., xxxiv, 1899, p; 151. 187. Mayer's Albumen {Mitth. Zool. Stat. Neapel, iv, 1883 ; Internat. Monatschr.f. Anat., iv, 1887, p. 42).— White of egg, 50 c.c. ; glycerin, 50 c.c. ; salicylate of soda, 1 grm. Shake them well together, and filter into a clean bottle. The filtering may take days or a week, but the preparation does not spoil meanwhile. Francotte shakes up the albumen with a few drops of acetic acid before adding the other ingredients, and finds the filtering greatly quickened. So do I. Be careful with the acid. A very thin layer of the mixture is spread on a slide with a fine brush and well rubbed in with the finger (I prefer a small rubber 114 SERIAL SECTION MOUNTING. " squeegee ")• The sections are laid on it and pressed down lightly with a brush (if they will bear it). The shde may then be warmed for some minutes on a water-bath, and the paraffin removed with a solvent. It is not necessary to warm the slide at all ; the paraffin can be removed in the cold if desired by putting the slide into toluol, xylol, or the hke. But the slide must, in any case, be treated with alcohol after removal of the paraffin, in order to get rid of the glycerin, which will cause cloudiness if not perfectly removed. This method allows of the staining of sections on the sUde with perfect safety, both with alcoholic and aqueous stains, provided they be not alkaline. According to my experience, the albumen method is absolutely safe, provided that alkaline fluids be avoided in the after-treatment. It has the defect that certain plasma stains (not chromatin stains) colour the albumen very strongly, and cannot be removed from it, and that sections are not expanded by it. It sometimes happens that the mixture after it has stood for some time becomes turbid, and at last coagulates, passing into a caseous state ; or it may undergo a hyaline coagulation, drying up like amber. But up to the very last it does not in general lose its adhesive properties. I have, however, found it to do so, after keeping for five or six years, so that, to be on the safe side, it may be well to make it up fresh every six months. Heidenhain (Zeit. wiss. Mikr., xxii, 1905, p. 331) makes it up with 1 grm. of blood albumen dissolved in 25 c.c. of water, and an equal volume of 50 per cent, alcohol. 188. The Albumen and Water Method (Henneguy, Journ. dff I'Anat. et de la Physiol., 1891, p. 398). — A drop of water is spread on a slide painted with Mayer's white-of-egg mixture, the sections are arranged on it, the whole is warmed (not to the melting-point of the paraffin) until the sections flatten out ; the water is then evaporated off at a temperature of about 40° C, and as soon as it has sufficiently disappeared, which at that temperature will be in about ten to fifteen minutes, the slide is further treated as described last §. This is a most valuable method. It is quicker than the water method, and, for difficult material, safer. See also Ohlmacheb, Journ. Amer. Med. Assoc, April, 1893. The so-called " Japanese " method, attributed to Ikeda by Eeinke {Zeit wiss. Mih., xii, 1895, p. 21), is merely that of Henneguy. Mann {Armt. Anz., viii, 1893, p. 442) shakes up white of egg with CHAPTER X. 115 water, coats slides with it and dries them. He flattens sections on water at 40° C, lifts them out on a prepared slide, and dries for five minutes at 36° C. 189. Garlic-water. — Hollande (Arch. d'Anat. Mior., xiii, 1911, p. 171) gives the following as more adhesive than albumen : — 50 grms. of crushed and chopped garlic are rubbed up with 80 c.c. of chloroform- water (Codex, A.C.) and filtered after twenty-four hours. Use as albumen. 190. SchIllibaum's Collodion (Arch. mikr. Anat., xxii, 1883, p. 565). ^Onepart of collodion shaken up with 3 — 4 parts of clove or lavender oil. Use as albumen. Sections can be treated with alcohol (not absolute) and divers staining fluids. I do not find it safe for this. Rabl, however (Zeit. wiss. Mik., xi, 1894, p. 170), finds that it is if you take 2 parts of coUodion to 3 of clove oil, and make up fresh every four or five days. 191. Obeegia's Method for Paraffin or Celloidin Sections (Neuro- logisches Centmlb., ix, 1890, p. 295 ; G-ulland, Journ. of Path., February, 1893). — Slides, or glass plates of any size, are coated with a solution made of — Syrupy solution of powdered candy-sugar made with boiling distilled water . . . .30 c.c. 95 per cent, alcohol . . . . . 20 „ Transparent syrupy solution of pure dextrin made by boiling with distilled water . . . 10 „ They are dried slowly for two or three days until the surface is just sticky to the moist finger. Paraffin sections are arranged and heated for a few minutes to a temperature slightly above the melting- point of the paraffin. The parafiin is removed by some solvent, and this in turn by absolute alcohol. The alcohol is poured off, and the sections are covered with solution of celloidin. The plates are left to evaporate for ten minutes in a horizontal position, then brought into water, in which the sheet of celloidin with the sections soon becomes detached, and may be further treated as desired, e.g., as in Weigert's process, § 198. The evaporation must not be artificially hastened. Dimmer (Zeit. wiss. Mik., xvi, 1899, p. 44) coats the slides with a solution of about 16 parts of gelatin in 300 of warm water, and dries them (two days), and proceeds in other respects as above. A good method for large sections, equally applicable to paraffin sections, to celloidin sections, and to sections of material that has not been imbedded at all. For Blochman's modification of Weigert's process, by means of which large sections can be preserved unmounted, see Zeit. wiss. Mik., xiv, 1897, p. 189. 8 2 116 SERIAL SECTION MOUNTING. 192. Strasser's Collodion Paper Method (ibid., iii, 1886, p. 346). — This is an extremely complicated modification of Weigert's method for celloidin sections, and is only adapted for use with Strasser's automatic ribbon-microtome. See Zeit. wiss. Mile, iii, 1886, p. 346 ; vi, 1889, p. 154; vii, 1890, pp. 290 and 304; ix, 1892, p. 8 ; xii, 1895, p. 154; and xiv, 1897, p. 39 ; also Schoenemann, ibid., xix, 1903, p. 333 ; Strasser, ibid., p. 337 ; and Ruppricht, ibid., xxviii, 1912, p. 281. Methods for Watery Sections. 193. Fol's Gelatin (Fol, LeJirb., p. 132).— Four grammes of gelatin are dissolved in 20 c.c. of glacial acetic acid by heating on a water-bafch. and agitation. To 5 c.c. of the solution add 70 c.c. of 70 per cent, alcohol and 1 to 2 c.c. of 5 per cent, aqueous solution of chrome-alum. Pour the mixture on to the slide and allow it to dry. In a few hours the gelatin passes into the insoluble state. It retains, however, the property of swelhng and becoming somewhat sticky in presence of water. The slide may then be immersed in water containing the sections ; these can be slid into their places, and the whole lifted out ; the sections will be found to be fixed. This method is specially intended for sections made under water, large celloidin sections amongst others. Similarly, Euppeicht, he. cit., last §, with the needless com- plication of a seriation on Strasser's coUodionised paper. Steassee (he. dt., last §) also employs a dry gelatin film which he makes sticky by means of carbol-xylol. Methods for Celloidin Sections. 194. The Albumen Method. — I find that celloidin sections may be mounted on Mayer's albumen, and have the celloidin removed, if desired, by putting them into ether-alcohol. Care must be taken* to press them down very thoroughly on to the albumen ; and it is well not to have them too wet. Similarly, Joedan (Zeit. wiss. Mih., xv, 1898, p. 54), and Aegu- TiNSKY {ibid., xvii, 1900, p. 37). See also Jordan, ibid., 192 — 194 ; Dantschakofp, ibid., xxv, 1908, p. 35 ; Maximo w, ihid., xxvi, 1909, p. 184 ; Anitschkow, ibid., xxvii, 1910, p. 68 ; Webee, ihid., xxix, 1912, p. 186 ; Rubaschkin, Anat. Anz., xxxi, 1907, p. 30. Weber paints over the series on the albumen with a layer of thin collodion, and puts into alcohol of 50 per cent., then into a mixture of equal parts of chloroform and absolute alcohol. After staining, pure absolute alcohol must be avoided. 195. SuMMEEs' Ether Method (Amer. Mon. Mic. Journ., 1887, p. 73). — Place* the sections in 95 per cent, alcohol for a minute or CHAPTER X. 117 two, arrange on the slide, and then pour over the sections sulphuric ether vapour, from a bottle partly full of liquid ether. The coUoidin will immediately soften and become perfectly transparent. Place the slide in 80 per cent, alcohol, or even directly in 95 per cent, if desired. I have not myself found this method safe. Instead of pouring the ether vapour over the slide, it may, of course, be treated with ether vapour in apreparation glass or similar arrangement, which I think preferable. Gage {Proc. Amer. Soc. Mic, 1892, p. 82) advises that the slide be one that has been previously coated with a 0-5 per cent, solution of white of egg and dried ; the collodion adheres much more strongly to an albuminised surface. AuBURTiN {Anat. Anz., xiii, 1897, p. 90) arranges on a clean slide, dehydrates the sections with blotting-paper and treatment with absolute alcohol, then drops on to them a mixture of alcohol and ether which dissolves out the oeUoidin from the sections, then allows the thin coUodion thus formed to evaporate into a thin sheet on the slide. Then 70 per cent, alcohol and other desired reagents. Similarly, Maier (Muneh. med. WocJienschr., Ivii, 1910, No. 12 ; Zeit. wiss. Mik., xxvii, 1910, p. 385), but adding a treatment for ten to fifteen minutes with sulphide of carbon. See also Mtees, Arch. Anat. Phys., Anat. Abth., 1902, p. 371 (com- plicated). 196. ApIthy's Oil of Bergamot Method {Mitth. Zool. Stat. Neapel, 1887, p. 742 ; Zeit. wiss. Mik., v, 1888, pp. 46 and 360, and vi, 1889, p. 167). — Cut with a knife smeared with yellow vaseline and wetted with 95 per cent, alcohol. Float the sections, as cut, on bergamot oil (must be green, must mix perfectly with 90 per cent, alcohol, and must not smell of turpentine), or on carbolxyol {Mikrotechnih, p. 176). The sections flatten themselves out on the surface of the oil, and are then transferred to a slide which (ApAthy, Mikrotechnik, pp. 127 and 176) has been previously coUodionised and dried. If the sections are to be stained, the slide after removal of the bergamot oil, by a cigarette paper, is exposed for a few minutes to the vapour of a mixture of ether and alcohol, then brought into 90 per cent, alcohol, and after a quarter of an hour therein may be stained in any fluid that contains 70 per cent, alcohol or more. If it be desired to stain in a watery fluid, care must have been taken when arranging the sections to let the celloidin of each section overlap that of its neighbours at the edges, so that the ether vapour may fuse them all into one continuous plate. This will become detached from the slide in watery fluids, and may then be treated as a single section. Terpinol may be taken instead of bergamot oil. 118 SERIAL SECTION MOUNTING. 197. Apathy's Series-on-the-Knife Method {Zeit. wiss. Mik, vi, 1888, p. 168). — The knife is well smeared with yellow vaseline, rubbed evenly on, and is wetted with alcohol of 70 to 90 per cent. As fast as the sections are cut they are drawn with a needle or small brush to a dry part of the blade, and there arranged in rows, the celloidin of each section overlapping or at least touching that of its neighbours. When a series (or several series, if you like) has been thus completed, the sections are dried by laying blotting-paper on them, and. the series is painted over with some of the thinnest celloidin solution used for imbedding, is allowed to evaporate for five minutes in the air, and the knife is then removed and brought for half an hour into 70 per cent, alcohol. This hardens the celloidin around the sections into- a continuous lamella, which can be easily detached by means of a scalpel, and stained, or further treated as desired. 198. Weigert's Collodion Method {Zeit. iviss. Mikr., 1885, p. 490). — Shdes, or larger plates of glass, are prepared by coating them with collodion in a thin layer, as photographers do, and allowing them to dry (they may be kept thus in stock). Sections (cut wet with alcohol) are got on to one of these (by a roundabout process, not essential), and arranged in order, and gently pressed down with paper. Now remove with blotting-paper any excess of alcohol that may remain on or around the sections, pour collodion over them, and get it to spread in an even layer. As soon as this layer is dry at the surface you may write any necessary indications on it with a small brush charged with methylen blue (the colour will remain fast throughout all subsequent manipulations). The plate may now be either put away till wanted in 80 per cent, alcohol, or may be brought into a staining fluid. The watery fluid causes the double sheet of collodion to become detached from the glass, holding the sections fast between its folds. It is then easy to stain, wash, dehydrate, and mount in the usual way, merely talcing care not to use alcohol of more than 90 to 96 per cent, for dehydra- tion. Weigert recommends for clearing the mixture of xylol and carbolic acid (§ 167). The series should be cut into the desired lengths for mounting whilst in the alcohol. A good method for iarge and thick sections. For Blochman's modification see § 191. Steassbe takes gummed paper instead of the glass plates used in this process. See the papers quoted § 192. CHAPTER X. 119 See also Wintersteinee (Zeit. wiss. Mik., x, 1893, p. 316) and Kubo (Arch. mik. Anat, Ixx, 1907, p. 173). 199. Obrbgia's Method.— Slides are prepared as directed (§ 191), the sections are arranged on them and covered with celloidin or photoxylin and evaporated as described, § 191. For Dimmer's modification see also § 191. 200. CoUodion Film Method.*— Graham Kerr (in litt, 1908) seriates on Kodak films. A film has the emulsion removed by hot water. The sections are arranged on a dry film, and the application of a drop of absolute alcohol and ether (or an atmosphere of alcohol and ether) suffices to weld them into a mass with the film. The sheet may then be stained and moimted, or rolled up and stored in cedar oil. Other Methods for CeUoidin Sections.— See §§ 193 (Fol) and 182 (Olt). * The late Dr. S. G. Scott used mica sheets, upon which he stuck paraffin sections. These could be distributed to a class of students by simply cutting out pieces of mica supporting the sections. CHAPTEE XI.* STAININa. 801. Dyes. — In practice, the staining materials are usually of the nature of neutral salts. But in the so-called " basic " dyes it is the base or the cation that consists of the complex organic compound possessing colour ; in the " acidic " dyes it is the acid or anion that is the staining agent. In the former case the colour-base is com- bined with a simple acid, generally hydrochloric or sulphuric, but sometimes acetic. In the latter case the colour-acid is combined with an inorganic base, usually sodium. For example, the dye caUed fuchsin is the hydrochloride of the base rosanihne, and its staining properties are clearly due to the latter. Acid-fuchsin, on the other hand, is the sodium salt of a sulphonic acid derived from fuchsin, a,nd its coloured constituent is present as the acid. The free colour-base or colour-acid is in most cases insoluble in water, although it may exist therein in the colloidal state. These are frequently colourless in themselves. It follows that the addition of acids to formulae for " acidic " dyes or bases to " basic " dyes is devoid of a rational foundation. Although the terms " acidic " and " basic " serve to indicate an important difference between dye-salts, their careless use may lead to unwarranted conclusions. Thus, if a cell-constituent takes up a basic dye, it does not follow that this body has the chemical nature of an acid. It may have, but substances other, than acids are stained by basic dyes, as we shall see below. A " neutral " dye-salt may clearly also be formed by combination between a colour-base and a colour-acid. These compounds are for the most part insoluble in water, although soluble in alcohol. Being of high molecular dimensions, they have the properties of colloids, amongst others, that of forming permanent coUoidal solutions in the presence of excess of either component. Moreover, the compo- sition of the dye that is precipitated varies according to the relative proportion of the two reagents in the solution. It is, therefore, not* a simple salt, but rather a " colloidal-complex " or " adsorption- compound." These dyes have limited use, more especially in * W. M. Bayliss. CHAPTER XL 121 distinguishing between various kinds of leucoc5d;es in blood (see Ehrlich and Lazarus, Die Anaemie, Wien, 1898). Since the acidic dyes are salts of fairly strong sulphonic acids with strong bases, they are electrolj^tically dissociated in solution to a large extent. Thus their solutions contain coloured anions, colourless cations (usually Na), together with undissociated salt. They are only hydroljiiically dissociated to a negligible degree, if at all. The basic dyes, on the other hand, are salts of weak bases (amino- or imino-derivatives) with strong acids. Althougli electro- Ijrtically dissociated as salts, so that their solutions contain coloured cations and undissociated salt together with colourless anions, they also undergo hydrolytic dissociation to a notable degree. Thus they contain free colour base and free colourless acid in addition to their ions. It is important to remember that commercial samples of dyes contain mineral salts, as a rule, sodium chloride or sulphate, some- times as much as 30 per cent, or more. This must be borne in mind in statements as to their properties. For instance, it is often said that the Congo-red dyes are direct dyes for cotton fibre. This is only the case in the presence of salts,' as will be shown presently. Details of the chemical composition of different dyes are beyond the scope of this book. The reader is referred to Cain and Thorpe's Synthetic Dyestuffs, 1913. 202. The Nature of the Staining Process. — Prom what has been said in the preceding paragraph it will be realised that a solution of a dye is a complex system from a physico-chemical standpoint. Moreover, the structures to be stained are present as separate phases, sohd or hquid, of a heterogeneoiis system. It is clear, therefore, that the properties of boundary surfaces must be taken into consideration, in addition to differences of chemical composition and of coUoidal state. Much discussion has taken place with respect to the process of dyeing, and various theories of its nature as being essentially chemical or essentially physical, in the sense of adsorption, mechanical or electrical, or in the sense of solid solution, involving partition between the solution and the tissue elements according to relative solubility of the dye therein, have been advocated. It is probable that all these factors play their part in varying proportion and that no one theory alone can explain all the facts. We shall be in a better position to appreciate the complexity of the conditions present if we examine, to begin with, the case of a pure substance, cellulose, in relation to pure solutions of an acidic and a basic dye respectively. 122 STAINING. We take, then, a piece of the purest analytical filter paper, wash it with distilled water to remove possible traces of acid, and place it in a dilute solution of Congo red, freed from foreign salts. It is scarcely stained at all. Add next a very small amount of a neutral salt, say, sodium chloride. The paper is deeply stained. How are these facts to be explained ? In view of the chemical inertness of cellulose, it seems unlikely that a chemical combination occurs between the dye and the paper under the influence of a neutral salt at ordinary temperatures. . Moreover, the same behaviour is shown by such different substances as charcoal, silk, alumina, silica, and so on. The process must be one of adsorption or deposition of the dye on the surface by some means. In other words, it must be associated with the decrease of surface energy of some kind. In the absence of foreign electrolytes, adsorption may be due to decrease of surface energy of the ordinary kind, shown as surface tension. This is confirmed by the fact that the dye, in the absence of electrolytes, can be washed out again by water. But since the degree of staining is very small, there must be some influence at work restricting the mechanical adsorption. There is, indeed, another property of the boundary surfaces between phases which demands attention hBre. This is the electrical charge, nearly always present. If we test paper in water, we find that it has a negative charge. Similarly, by appropriate means, we find that the dye itseK has a negative charge. Whether this is that of the coloured anion or of complex aggregates of these ions with undissociated salt is not certain, but, according to Freundlich, " acidic " dyes are adsorbed as a whole. In the present case it is immaterial, because the material to be adsorbed has a negative charge in either case. This being so, there are repellent forces acting between the dye and the paper. Or, if we take the point of view of energetics, the adsorption of electro-negative dye would increase the negative charge on the paper, with an increase of free energy, which is contrary to the Second Law of Thermo- dynamics. Suppose, however, that we have also present the ions into which a neutral salt dissociates. The cations, being positively charged, are deposited on the surface of the paper, decreasing or annulling its negative charge and reducing the free energy. There is now little or no obstacle to the adsorption of the dye. From the work of Peerin we know that an ion may be adsorbed on an oppositely charged surface to so great an extent that the charge on this surface may actually be reversed in sign. This occurs, as it appears, when surface energy of some kind other than electrical is diminished by the presence of such ions, and is found mainly with CHAPTER XI. 123 plurivalent or organic ions. But since colloids are readily precipi- tated by such ions, owing to neutralisation of their charges, it may happen that a dye such as Congo red is neutralised and precipitated before it has attached itself to the adsorbing surface by virtue of its charge. Since the amount adsorbed is dependent on the magnitude of the electric charge, we have an explanation of the fact that alcohol diminishes the effect of electrolytes. The charge on a surface is proportional to the dielectric constant of the liquid phase in which it is immersed, and the dielectric constant of alcohol is less than that of water. It is a well-known fact that colloids of the suspensoid class, such as gold and coagulated egg white, are much more sensitive to the action of electrolytes than are those of the emulsoid class, in which the colloidal particles themselves contain water and differ from the medium in which they are suspended merely by the smaller quantity of water which they contain. Such are gum arable, starch, colloidal silica, and proteins in general, gelatin, raw egg white, and so on. Faraday showed that colloidal gold could be protected from precipitation by salt if a trace of gelatin was added. This is explained by the adsorption of a coating of gelatin over the gold particles, which are thereby converted into the emulsoid variety so far as their surfaces are concerned. We find similar phenomena in the staining of paper by Congo red. It is protected from the dye even in the presence of salts. But the conditions are made more complex by the possibility of using in the experiment either an electro-positive or electro-negative protein. A trace of acid or alkali respectively has this effect on proteins, by the production, of dissociated salts. Now the former are more powerfully adsorbed by the negative paper than the latter are, while at the same time they reduce, instead of increasing, the electric charge. Actually the latter effect preponderates, so that the presence of electro- positive protein increases the depth of staining. Turning now to the basic dyes, we find that the paper is the more deeply stained the lower the concentration of salt present. Accord- ing to Freundlich, it is the colour base that is chiefly adsorbed in this case. As was pointed out above, these dyes are hydro- lytically dissociated, so that free base is present. This free base, being insoluble, is in the colloidal state, and, like colloidal bases in general, has a positive charge, due to electrolytic dissociation of the surface of the particles. See Hardy in Van Bemmelen Gedenkboek, p. 188. Thus, not only are the coloured ions, in this case the 124 STAINim. cations, strongly adsorbed by the negative paper, but the free base is also. Since foreign electrolytes diminish the charge on the paper, their effect on staining by basic dyes is naturally of the opposite kind to that described in the case of the acidic dyes. The effect of alcohol is in the same direction as that of electrolytes, since it also decreases the electric charge and, therefore, the amount of dye adsorbed. Facts of the kind referred to in the preceding statements have given rise to an " Electrical Theory of Dyeing," which probably accounts for a larger number of them than any other single theory is able to do. For further particulars of theories and facts relating to dyeing and staining, the reader is referred to Alfred Fischer's Fixirung, Farhung und Bau des Protophsmas, Jena, 1899 ; Pelet- Jolivet's Theorie des Farbeprozesses, Dresden, 1910 ; First Report on Colloid Chemistry, Brit. Ass,, 1917 ; Gee and Harrison, Trans. Faraday Sac, vol. vi, 1910 ; Harrison, Journ. Soc. Dyers and Colourists, December, 1911 ; Bayliss, Biochem. Journ., vol. i, 1906, p. 175. The reducing properties, in the chemical sense, of cell constituents play an important part in certain special staining reactions, particu- larly those with metallic salts. Osmic acid (osmium tetroxide) is reduced to a lower oxide or perhaps to the metallic state by un- saturated fats. Gold and silver salts are reduced by nervous tissue under appropriate conditions and deposited in the colloidal state. AU the various colours of coUoidal gold in different degrees of dispersion may be seen in tissues prepared by the gold impregna- tion methods. Details of these methods will be found in other chapters of this book. Effect of Heat. — Since surface tension is decreased by rise of temperature, it would naturally be expected that, if this factor is concerned in the process of staining, the depth of the stain would be lessened at a higher temperature. This is actually the case with simple substances like cellulose up to 50° C. or thereabouts. At first sight this would seem to be at variance with the frequent practice of fixing stains by heating the preparation. But the temperatures used are much higher than those referred to, and, in point of fact, if filter paper is stained in a Congo red solution at 100° C, the dye is firmly fixed, and cannot readily be removed by washing. It is difiicult to say what actually happens here. Chemical combina- tion suggests itself, but the nature of the compound formed is not easy to conceive. The temperature at which a material possesses the maximum electric charge, as shown by Gee and Harrison, also plays a part in the phenomena. CHAPTER XI. 125 Chemical combination obviously occurs in some cases when substances stained by adsorption are heated to 100° C. Thus Bayliss (Proc. Roy. Soc., B., vol. Ixxxiv, 1911, p. 83) showed that various insoluble hydroxides, such as that of aluminium, are stained blue by the free acid of Congo red, which is its own colour. When heated to 100° C, combination takes place with the formation of the usual red colour of the salts of this dye. Silk behaves in the same way, and even crystals of leucine, doubtless to be explained by the formation of salts with the fairly strong acid. Conditions similar to this, however, do not arise in histological staining, where dye salts are used. It is difficult, moreover, to see how^ the conditions for reaction by double decomposition could arise in the cell, since acids and bases sufficiently strong to displace hydrochloric acid or sodium .hydroxide are required. Reactions with precipitation might occur, but these would give rise to the appearance of new solid structures in the cell. . There is no evidence that such precipitates are produced in a simple staining process, although they are undoubtedly, formed by fixing agents (Hardy, Journ. of Physiology, vol. xxiv, p. 158). The following experiment by Martin Heidenhain is sometimes given as evidence of the formation of salts of dyes with proteins. A solution of Congo red, as is well known, turns blue when made acid with acetic acid, owing to the separation of the free acid. If such a blue solution is added to an acidulated solution of serum albumin, a red solution is obtained. Since this is the characteristic colour of the salts of Congo red, it is natural to interpret it as a salt of the dye acid with the protein base. But the fact that it exists in a solution sufficiently acid to decompose the sodium salt of the dye shows that the colour acid is more firmly combined with protein than with sodium, a view that it is difficult or impossible to hold. Moreover, it appears that even 5 per cent, sulphuric acid is unable to split off the acid from some of these protein " compounds." It is clear that the phenomena must have a different interpretation. It may be that the free colour acid exists in two forms, a true and a pseudo- acid — ^the former of a red colour and ionised; the latter, blue, in- soluble and non-ionised. When adsorbed by protein, for some reason or other, the acid may be for the most part in the former condition. See the work of Wo. Ostwald on Congo-rubin (Koll. Ghem. Beihefte, B. X, 1919). But further evidence is required. 203. Removal of Dyes. — When a stain is fixed by adsorption of the ordinary, non-electrical type, it can be removed by frequent washing with distilled water. This is very difficult if the dye is held 126 STAINING. by electrical forces. The reason is, in all probability, that the dye can only be set free by reversing the sign of the charge on the surface. This cannot be done by pure water alone. It can be done, however, by acid or alkali in the appropriate case. For example, if an acidic dye has been fixed on a negative surface by the aid of cations, which convert the charge to a po?itive one, OH' ions, provided by alkali, are powerful enough to change the sign of the charge back again to negative and thus free the dye, whereas H' ions from an acid only increase the positive charge and fix the dye more firmly. Hence the statement that acidic dyes are fast to acids. A basic dj^e, adsorbed by a negative surface, is removed by acids and intensified by allcalies. A corresponding explanation holds. Thus, H' ions from acids make the surface more positive, hence the dye is released. OH' ions make it more negative, hence the dye is held faster. In all cases, if the acid or alkali is strong enough, any dye-salt adsorbed is decomposed, somei;imes with change of colour. The process of " differentiation " by alcohol or other agent, to be referred to below, is an application of these facts. Alcohol removes a " basic " dye because it reduces the negative charge of the tissue elements and thus releases part of the positively charged constituent of the dye adsorbed. 204. "Specific" Stains. — Certain tissue elements and cell- constituents have the property of staining deeply with particular dyes. That of nervous structures with methylen blue and of mitochondria with dyes containing di-ethyl-safranin, such as Janus- green, may be given as examples. The property may be shown either by their taking up the stain from a dilute solution more rapidly than other structures present do (" progressive " staining), or by their holding on to it more tightly when excess of general stain is washed away by appropriate treatment. This latter process is sometimes known as " differentiation " or as " regressive " staining. It is natural to interpret this behaviour as due to a chemical combination of a special kind, as did Ehrlich in his well-known theory of " chemo-receptors," according to which certain " side- chains " of protoplasmic molecules have special affinities for parti- cular groups in the dye molecules. "While this may be the case in isolated instances, there are many facts which show that it cannot be accepted as a general law. It is difficult to see what purely chemical relationship can exist between complex, substituted, diazo-sulphonates, as a large- number of these specific dyes are, and the chemical components of cells. Moreover, although methylen CHAPTER XL 127 blue and other thiazines are specific vital stains for nervous tissue, certain safranin azo-dyes — diazin-green, for example — which have no chemical relationship to the former, are also vital nerve stains ; while similar compounds of the safranin series itself have no such property. See Michaelis, Ghemie der Farbstoffe, 1902, p. 104. We must remember also that the conception of large protoplasmic molecules with side-chains in the chemical sense, the so-called " biogens," is becoming more and more discredited. See Hopkins, Address to Physiology Section, Brit. Assoc, 1913. We have already seen how complex are the physical factors that intervene in such a simple case as the staining of paper, and to these may be added questions of solid solution, distribution between phases, diffusibility, and so forth. Indeed, it would seem that each individual case of specific staining requires investigation by itself. Evans (Amer. Journ. of Physiol., vol. xxxvii., p. 255) in an investigation of " macro- phages " comes to the conclusion that chemo-receptors are not responsible for vital staining with the benzidine and related series of dyes. Alfred Fischer {op. cit., pp. 107—150) gives interesting cases of staining differentially particles of the same substance by different dyes. Particles can be made to take up either dye, according to their size, the order in which the different dyes are applied and the degree of differentiation by removal of stain. 205. Some Applications of the Theory of Staining.— It is of interest to see how some histological facts are explained in the theory sketched above. Most of the structures in the living ceU have negative electric charges, probably on account of the slightly alkaline nature of the surrounding fluids. This fact accounts for the ease with which tissues in general are stained by basic dyes. It is remark- able that haemoglobin is one of the few constituents that have a positive charge. Accordingly, it is stained by acidic dyes, such as eosin or acid fuchsin. The effect of electrolytes is shown in the experiments of Mayr (Hofmeister's Beitrage, vol. vii., p. 560). He finds that the affinity of Nissl bodies for basic dyes is abolished by previous treatment with neutral salts. 206. Objects of Staining.— Most constituents of cells are, in their natural state, either colourless or only faintly coloured. Thus they are only visible if their refractive indices differ from those of the media in which they are immersed. Such, for example, are fatty globules and the granules of many secreting cells. But, as seen thus, it is not an easy matter to judge of their true forms. This is 128 STAINING. greatly facilitated by staining them either more deeply than or of a different colour from their surroundings. If colourless glass heads, although they are easily seen by refraction, could only be observed from the direction of a line through the hole in the centre, the recognition of their true form would be difficult. Immersing them in a medium of the same refractive index as themselves would render them invisible. But if they were made of coloured glass and im- mersed in such a medium, they would be readily detected and their shape recognised. The chief object of histological staining is then to cause certain constituents of the cells to take on a different intensity of tint from others. This may be done in various ways, as will be seen later. It is usual to distinguish two kinds of selective staining, histological and cytological selection. In the former an entire tissue or group of tissue elements is prominently stained, the elements of other kinds present remaining colourless or being differently stained, as in the impregnation of nerve endings by the silver and gold reduction methods. In the latter the stain is taken up or retained by some constituent element of the cell, such as the chromatin of the nucleus or an element of the cytoplasm. The nuclear stains are of importance in marking out the contours and relations of the tissues making up regions or organs as a whole and are thus of special value to the embryologist and morphologist. At one time, it was thought to be possible to distinguish between " basophilous " and " acidophilous " tissue elements, according to their affinity for basic or acidic dyes. Ehrlich (Dm Bois Reymond's Archiv., 1879, p. 571) thought that the basic dyes have a special affinity for the chromatin of nuclei and the acidic dyes for the cytoplasm and intercellular substances. But we have already seen that the same substance may take up either kind of dye, according to the conditions present. Most staining processes are undertaken on cells which have been acted on by fixing reagents or by the so- called " mordants," and these may reverse the natural behaviour to dyes. Ehrlich's statement only applies in fact to cover-glass preparations fixed and dried by heat, without the action of reagents. The acidic colours, orange and acid fuchsin, although they stain cytoplasm, may give good chromatin differentiation when used as regressive stains. Methylen blue is basic, but stains nerves. The widely used carmin and haematoxylin are both acidic dyes, but in combination with alum they give nuclear stains. Other instances might be given, but these wiU suffice. CHAPTER XI. 129 807. Intra-vitam Staining.— It is clear that unless the cell-membrane of a living cell is permeable to a dye, no constituent of the cell can be stained by it. Most dyes appear to be more or less toxic if they enter the cell. But, while alive, the latter is to a large extent protected, since the dye does not obtain entrance. A living Amoeba is stained by very few dyes. Neutral red, however, passes through the membrane and stains various structures, while having no apparent effect on the activities of the organism. The auricle of the frog's heart can also be stained with this dye, while continuing its normal contractions. Used in this way, the dye is appUed in very dilute solution. Since neutral red is a very sensitive indicator just about the neutral point, the fact of its permeability and non-toxicity makes it a valuable test for the presence of acid or alkali within the ceU. When a dye enters a living cell, it usually stains various granules and structures contained therein, while at the same time it is uni- formly diffused through the liquid phase of the protoplasm. If the process of staining is conditioned by phenomena at boundary sur- faces, simple undifferentiated protoplasm in the living state should be incapable of staining, and this seems to be the general experience. As regards the question of permeability to a given dye, unless the cell is able to show that it is still ahve by movement or by con- tractility, it is clearly a matter of difficulty to be certain that, when a particular dye enters, it does so during life or only after it has destroyed the normal properties of the cell-membrane. The nucleus itself seems to be very resistant to dyes while alive, and it_has been stated that the appearance of stain in it is a sure indication of death. BoLLES Lee made a large number of observations and came to the conclusion that most of the " intra-vitam " stains are either due to ■ mere diffusion through the hquid protoplasm or that the stained constituents were not really living, being food particles or products of cell activity. At the same time, many of the methods which come under this heading are of much value. Methylen blue may be injected into the living animal and frequently gives very successful staining of nervous structures, owing to the fact of its being conveyed into intimate contact with the cells by means of the blood vessels. The various methods of preserving the stain in the structures to which it was localised during life obviously depend on the adequacy of the means used to fix and maintain these structures and to retain the properties owing to which the stain was taken up. This is by no means a simple matter. Mott describes in living nerve cells a 130 STAINING. number of minute particles which stain on the outside with methy- len blue. In fixed cells, as is well known, these particles aggregate together to form the " Nissl granules." Michaelis found similar granules in hver cells. As the cells die, the stain leaves the granules and passes into the nucleus. The behaviour of the living nucleus to methyl green has given rise to some discussion. It appears that no uni-cellular organism in which the nucleus was stained has been observed to move, whereas the chlorophyll grains may take up the stain while the cell is normally motile. No convincing case of staining of the living nucleus has in fact been described. The question as to whether the cell elements which stain during life are to be described as Uving or not is scarcely putting the problem from the right point of view. If a dye obtains contact with the interfaces between constituents of a cell, it will in all probability be deposited there to a degree depending on the various properties of the interface described previously. This may occur independently of the fact as to whether one or both of the phases is living. Apart, however, from these questions, it must be conceded that these so-called " vital stains " are frequently very useful. According to BoLLES Lee's experience, methylen blue is the most generally useful of them. It has (with Bismarck brown, Congo red, and neutral red) the valuable point that it is sufficiently soluble in saline solutions, and may therefore be employed with marine organisms by simply adding it to sea-water. The others are not thus soluble to a practical extent, but BoLLES Lee finds that gentian and dahlia become so if a trace of chloral hydrate — 0'25 per cent, is ample enough — be added to the saline solution. Any of these reagents may be rubbed up with serum, or other " indiflerent " liquid. Methylen blue may be fixed in the tissues, and permanent preparations ' made, by one or other of the methods described in Chap. XVI. Bismarck brown stains may be fixed with 0-2 per cent, chromic acid or with sublimate solution (Mateb), or 1 per cent, osmic acid (Loisel, Journ. de TAnat. et de la Phys., 1898, No. 2, p. 212^a work that contains a good deal of information on the subject of intra-vitam stains), and the preparations may be stained with safranin, care being taken not to expose them too long to the action of alcohol. For the study of cell- granules, neutral red is perhaps the best. Fisci-IEL {Unters. ueb. vitale Faerbungen, Leipzig, 1908) finds that alizarin is specific for nerves. Add excess of alizarin to boiling water, boil and filter, and add 1 vol. of the filtrate to the water containing the organisms (Cladocera). The stain takes several hours. For sulphorhodamin, which is selective for many organs (kidney, liver, uterus, skin, lymph-glands, etc.), see Andeeew, in Virchow's Arch., ociv, 1911, p. 447. The details of the various methods used for intra-vital staining CHAPTER XL 131 and the fixation of the results are described in other p'arts of this book. The reader may be referred to the work of Goldman {Unters. iteber die Sekretion des Organismus im Lichte der " vitalen Farhung" Laupp : Tubingen, 1912) for certain aspects of the problem. 208. Dead Cells. — Changes take place in cells in consequence of which they cease to respond to external stimuli and are said to be " dead." Their staining reactions are naturally different in this state both from those during life and from those after their structures have been " fixed " b)'^ treatment with some appropriate solution. Although it seems likely that valuable information would be obtained from the investigation of their behaviour in the dead but unfixed state, very little work appears to have been done. Dogiel obtained various differentiations in nerve cells by observations at various stages after death. 209. Fixed Tissues. — The majority of staining methods are under- taken on tissues that have been*fixed and hardened by reagents. It is sufficient to mention here that some of these reagents merely serve to coagulate or precipitate the constituents of cells without marked changes in their chemical nature, although their physical state is more or less altered. Alcohol is one of these agents. Other fixing fluids, of which those containing chromic acid are represen- tatives, produce what seem to be compounds of cell proteins with the reagent. In this latter case, we have what is known as a " mordant " present. The distinction is often made between " substantive " and " adjective " staining. These expressions are really based on a particular theory of the staining process and are somewhat difficult to justify logically. When a substance takes up a dye without the necessity of the presence of any additional reagent, the staining is said to be " substantive." When a mordant is required, it is " adjective." The only true case of the former process is when an electro-negative surface adsorbs a basic dye. Such a surface is practically unstained by an " acidic " dye, when pure. The presence of an electrolyte is necessary. But sodium chloride may suffice, and the question arises whether this should be called a mordant. Mordants are usually understood to be agents which form insoluble compounds with dyes and in this way cause their fixation in places from which otherwise they might be washed out by the subsequent treatment with dehydrating agents, etc. Such a substance may clearly be either already present in the fixed preparation when the dye is added, or it may be added together with or subsequently to 9—2 132 STAINING. the staining agent. It will readily be understood that the pro- duction of an insoluble dye salt renders solution in water more diffi- cult, but this alone is not enough. The compound must also be firmly attached to the surface. Thus, the barium salts of colour acids are iasoluble, but barium salts cannot be used as mordants for acidic dyes ; the compound formed is readily washed off mechani- cally. • The insoluble compounds between a dye and a mordant are known as " lakes." But the chemical nature of these substances is by no means clear — especially when they are produced in situ in stained tissues. In fact, they do not behave as simple compounds of the dye and the mordant. They resist the action of strong acids and bases iq moderately strong solutions. So that the stains obtained by this method are characterised by durability and " fastness." On the whole, we must conclude that there are factors still un- known, but probably related to the electrical properties of the surface, which play an important part in these reactions with mordants. A simple chemical theory does not account for all the phenomena. Changes in the properties of the surface by the action of the mordant are concerned. In this connection, we may call to mind how frequently multivalent cations are used as mordants. Alum is one of the commonest of these. A further mention may here be made of the " progressive " and " regressive " methods, between which there is a more definite distinction than that between " substantive " and " adjective " staining. A preparation may be placed in a very dilute solution of a dye and the action stopped when the elements with the greatest " affinity " for the dye have taken it up. The staining of nuclei , with dilute haematoxylin-alum may be mentioned. If the action is prolonged, various other constituents of the cell, besides the nucleus, take on the colour. As a rule, no great differentiation is obtained by this method. Better results are given by the " regressive " method, in which a general overstaining is followed by a partial decoloration, in which certain elements retain the stain, owing to special chemical or physical properties, after it has been removed from the rest. Thus safranin stains the whole section of a deep red colour ; acting on it with alcohol removes the stain from all but the chromatin and the nucleoli. This action of alcohol may be explained, as already pointed out, by its effect on the magnitude of the electric charge, since its dielectric constant is lower than that of water. Other d^jfferentiating agents are also used. Iron-alum, in the iron- hsematoxylin method, serves both as preliminary mordant and as CHAPTER XI. 133 differentiating agent. This double action is not easy to explain and confirms what was said above as to the complexity of the process. 810. Metachromasy. — There are a few dyes, mostly of the basic aniline series, which stain certain eleme^ats in the colour of the ordinary solutions of the dye,- other elements in that of the free colour-base. Safranin stains nuclei red ; mucin and the ground substance of cartilage, orange. Methyl violet stains " amyloid," and mucin red. Although in a few cases this behaviour may be due to the dj-^e being really a mixture of two dyes, as in the case of iodine green, there is no doubt that this is not the explanation of genuine cases. According to Michaelis, the appearance of the colour of the base is not due to the alkalinity of the elements in question. The fact that the red stain given to mucin by thionin can be changed into blue by alcohol and back to red by water shows that the change is not one involving great alterations of chemical structure, and a tautomeric one is naturally suggested. It would appear that the change is one by which an amino-group becomes freed from its combination with the mineral acid of the salt. In the case of thionin, the acid is supposed to change its connection to the nitrogen which unites the two benzene rings. What are the conditions which regulate the change from one form to the other is unknown. A similar difficulty is met with in the case of iodine, which is brown in solution in alcohol, violet in chloroform. We must, however, not overlook the changes in colour shown by substances in the colloidal state merely in consequence of a decrease in their degree of dispersion or increase in size of particles. Gold is a notable case. It riiay be red, violet, blue or green. All of these tints are met with in its use as a histological reagent. Whether similar phenomena may occur in the adsorption of dyes is uncertain, but cannot be dismissed without further evidence. 211. The Use of Stains in Practice. — Stains for special purposes are described in other pages of this book. It will be obvious from the contents of the present chapter that caution must be exercised in making deductions as to chemical composition from behaviour to dyes. In general, the dyes of the anilin series, although of great value for particular purposes, are apt to be less permanent than the old logwood and carmine stains. The Canada balsam used must be as pure as possible if the stain is to last for any length of time. 134 STAINING. The most distinctive processes, involving the use of mordants and regressive differentiation, can only be undertaken on sections. Staining in bulk is useful when the general anatomy is the object of study. For cytological work it is of little value. As remarJced, the main object ofstainmg is to obtain better information of the appearance of the structures present in the cell. The fact, hov;ever, that this appearance is not necessarily that of the living state should never be allowed to escape remembrance. Without special investigation of the case, it is not permissible to draw conclusions as to the chemical nature of a cell constituent from its behaviour to dyes. 213. Choice of a Stain. — Mr. Bolles Lee believes that the follow- ing may be recommended to the beginner for general work : — For sections, Mayer's hcemalum ; or, for chromosmium objects more especially, Benda's or HEiDENHAm's.iron hsematoxylin. For staining in toto Grenacher's alcoholic borax-carmine, or Mayer's carmalum, or hsemalum, unless the object be so impermeable as to require a very highly alcohohsed stain, in which case take Mayer's paracarmine, or for chromic acid objects Mayer's hcema- calcium. Foi fresh tissues or small entire objects, methyl green, if it is not important to have permanent preparations ; if it is, take carmalum or alum-carmine (but both of these may give precipitates with marine animals). CHAPTER XII. CARMINE AND •COCHINEAL STAINS. 813. Carmine.— Carmine is by no means merely carminic acid with at most certain impurities. According to the analysis of LiEBERMANN (Ber. d. Chem. Ges., Jahrg. 18, 1886, pp. 1969—1975) it is a very peculiar alumina-Ume-protein compound of carminic acid, a true chemical compound from which at all events aluminium and calcium can no more be absent than sodium from salt. It results from the researches of Mayer {Mitth. Zool. Stat. Neapel, x, 1892, p. 480) that in the processes of histological staining (not of industrial dyeing) the active factors of the compound are, besides the carminic acid, always the alumina, and in some cases the lime. The other bases are inactive ; the nitrogenous matters, so far as they have any influence at all, are an obstacle, as it is they that give rise to the well- known putrefaction of the solutions. " This being so, it follows that carminic acid may, if desired, be taken as the basis of staining solutions instead of carmine. Staining solutions thus prepared do not give essentially better stains than those made with carmine ; but have the advantage of being of more constant composition. For carmine is a product which varies greatly from sample to sample. Carminic acid of sufficient purity is furnished by Grubler and HoLLBORN (or C. A. F. Kahlbaum, in Berlin). It is soluble in water and weak alcohol (that of 70 per cent, only dissolves less than 3 per cent.). It cannot be used alone for staining, as it only gives in this way a weak and diffuse stain. 214. Cochineal. — According to Mayer (Mitth. Zool. Stat. Neapel, X, 1892, p. 496), the active principle of extract or tincture of cochineal (as used in histology) is not free carminic acid, but carminic acid chemically combined with a base which is not lime, but some alkali. The watery extract made with alum, or cochineal-alum carmine (§ 216), owes its staining power to the formation of carminate of alumina (last §). The tincture made with pure alcohol, on the other hand, contains only the above-mentioned carminate of some alkali. This carminate alone stains weakly and diffusely (like carminic acid alone). But if in the tissues treated with it it meets with lime salts, 136 CARMINE AND COCHINEAL STAINS. alumina or magnesia salts, or even metallic salts capable of com- bining with it and forming insoluble coloured precipitates in the tissues, then a strong and selective stain may result. And if the necessary salts be added to the tincture itself, there results a solution containing the necessary elements for affording a strong and selective stain with all classes of objects. Hence Mayer's new formula, § 236. 215. General Remarks. — Carmine stains are chiefly used for staining entire objects, or tissues in bulk. In most cases this can be done more satisfactorily by means of carmine than by means of any other known agent. For most hsemateiin solutions have a disastrous tendency to overstain ; and the tar-colours are generally inappUcable to staining in bulk. Grenacher's alcoholic borax-carmine may be recommended to the beginner as being the easiest of these stains to work with : or para- carmine, for objects which require a highly alcoholic solution. Carmalum, or one of the alum-carmines, is also an easy and safe reagent. Overstains may in all cases be washed out with weak HCl (e.g. 0-1 per cent.). Alum-solution will often suffice, or, according to Henneguy (Journ. de VAnat. et de la Physiol., xxvii, 1891, p. 400), permanganate of potash. All carmine stains, with the exception of aceto-carmine, are permanent in balsam. The alum-carmines are fairly permanent in glycerin. None of the acid stains, nor any of Grenacher's fluids, should be used with calcareous structures that it is wished to preserve, unless they be taken in a state of extreme dilution. A. Aqueous Carmine Stains. a. Acid. 216. Alum-carmine (Grenacher, Arch. mik. Anat., xvi, 1879, p. 465). — An aqueous solution (of 1 to 5 per cent, strength, or any other strength that may be preferred) of common or ammonia alum is boiled for ten to twenty minutes with J to 1 per cent, of powdered carmine. (It is perhaps the safer plan to take the alum solution highly concentrated in the first instance, and after boiling the carmine in it dilute to the desired strength.) When cool, filter. This stain must be avoided in the case of calcareous structures that it is wished to preserve. TizzoNi (Bull. 8c. Med. Bologna, 1884, p. 259), Pisenti (Oaze. degli Os2}elnU, No. 24 ; Zeit. wiss. Mile, ii, 1885, p. 378), and Grieb (Mem. CHAPTER XII. 137 Soc. Ital. Sci., t. vi, No. 9, 1887 ; Zeit. wiss. Mik., vii, 1, 1890, p. 47) have given modifications of Grenadier's formula wMch do not appear to me rational. Mater {ibid., xiv, 1897, p. 29) makes a stronger stain by taking 2 grms. carmine, 5 grms. alum, and 100 o.c. water, and boiling for an hour. Alum-carmine is an excellent stain. It is particularly to be recommended to the beginner, as it is easy to work with ; it is hardly possible to overstain with it. Its chief defect is that it is not very penetrating, and therefore unsuitable for staining objects of consider- able size in bulk. 217. Acetic Acid Alum-Carmine (Henneguy, in Traite des Meth. Techn., Lee et Henneguy, 1887, p. 88). — Excess of carmine is boiled in saturated solution of potash alum. After cooUng add 10 per cent, of glacial acetic acid, and leave to settle for some days, then filter. For staining, enough of the solution is added to distilled water to give it a deep rose tint. In order to ensure rapid diffusion it is well to bring the tissues into the stain direct from alcohol. Stain for twenty-four to forty-eight hours, and wash for an hour or two in distilled water. Mount in balsam. You can mount in glycerin, but the preparations do not keep so well. The advantage of this carmine is that it has much greater power of penetration than the non-acidified alum-carmine. 218. Cochineal Alum-Carmine (Paetsch, Arch. mik. Anat., xiv, 1877, p. 180). — Powdered cochineal is boiled for some time in a 5 per cent, solution of alum, the decoction filtered, and a little salicylix; acid added to preserve it from mould. Another method of preparation has been given by Czokoe [ibid., xviii, 1880, p. 413). — Mayer finds that Partsch's is the more rational, the proportion of alum in it being exactly right, whilst in Czokor's it is insufficient. Partsch's fluid also keeps better. Eabl {Zeit. wiss. Mik., xi, 2, 1894, p. 168) takes 25 grms. each of cochineal and alum, 800 c.c. of water, and boils down to 600 c.c. He prefers this because it is not so purely nuclear a stain as the others. These solutions give a stain that is practically identical with that of alum-carmine made from carmine, with perhaps even more dehcate differentiations. Eawitz (Zeit. wiss. Mik., xxv, 1909, p. 392) takes cochineal 4 grms., nitrate of aluminium (or ammonio-sulphate of cobalt) 4 grms., water 100 c.c, and glycerin 100 c.c. Only for sections. 219. Mayer's Caxmalum (Mitth. Zool. Stat. Neapel, x, 1892, p. 489).— Carminic acid, 1 grm. ; alum, 10 grms. ; distilled water. 138 CARMINE AND COCHINEAL STAINS. 200 c.c. Dissolve with heat (if necessary). Decant or filter. Add some antiseptic, either 1 c.c. formol, or 0-1 per cent, salicylic acid, or 0-5 per cent, salicylate of soda. The solution will then keep. It stains well in bulk, even osmium objects. If washed out with distilled water only, the plasma will remain somewhat stained. If this be not desired, wash out carefully with alum solution, or, in difficult cases with weak acid, followed in either case with water. The general effect is that of an alum-carmine stain. A weaker solution may be made by taking from three to five times as much alum and five times as much water, and dissolving in the cold. With either solution the objects to be stained should not have an alkaline reaction. Rawitz {Anat. Anz., xv, 1899, p. 438) takes 2 grms. carminio acid, 20 grms. ammonia-alum, 150 c.c. water, and 150 c.c. glycerin. A strongly staining solution, which is said to heep well. Only for sections. All solutions prepared with alum tend to precipitate. Carmalum made up with 500 c.c. of water instead of 200, and with glycerin or 10 per cent, of formol or pyroligneous acid added, keeps well. 220. Mater's Aqueous Aluminium-Chloride Solution (Mitth. Zool. Stat. Neapel, x, 1902, p. 490). — Carminio acid, 1 grm. ; chloride of aluminium, 3 grms. ; water, 200 c.c. Add an antiseptic, as for carmalum. Use as carmalum. The stain is of a blue-violet colour, very powerful, and elective, but not so purely nuclear as carmalum. It is recom- mended only as a substitute for carmalum in cases in which the latter is counter-indicated on account of the alum in it or the like. 221. Alum-Carmine and Picric Acid. — Alum-carmine objects may be double-stained with picric acid. Legal (Marph. Jahrb., viii, p. 353) combines the two stains by mixing 10 vols, of alum-carmine ' with 1 of saturated picric acid solution. I find this very recommend- able. Aceto-Carmine (Acetic Acid Carmine) Schneider {Zool. Anzeig., 1880, p. 254). — ^To boiling acetic acid of 45 per cent, strength add carmine until no more will dissolve, and filter. (Forty-five per cent, acetic acid is, according to Schneider, the strength that dissolves the largest proportion of carmine.) To use the solution you may either dilute it to 1 per cent, strength, and use the dilute solution for slow staining ; or a drop of the concen- trated solution may be added to a fresh preparation under the cover- glass. If you use the concentrated solution it fixes and stains at the same time, and hence may render service for the study of fresh objects. It is very penetrating. The stain is a pure nuclear one. Unfortunately the preparations cannot be preserved, and for this and other reasons the stain is of very restricted appliodbility . CHAPTER XII. 139 A similar stain has been prepared with formic acid by Pianese (see Zeit. wiss. Mik., x, 4, 1894, p. 502). For Bukchardt's pyroligneous-aoid carmines see Aroh. mile. Anat., liii, 1898, p. 232 ; and Jena Zeit Naturw., xxxiv, 1900, p. 720. 228. Iron Carmine. — I recommend trial of the following, which I have already published in the Traite des Meth. Techniques, Lee et Henneguy, 1902. Sections (I have not tried material in bulk) are mordanted (a few hours will suffice) in sulphate of iron (Benda's liquor feni, as for iron haematoxylin), washed, and stained for an hour or so in 0-5 per cent, solution of carminic acid in alcohol of 50 per cent. Wash in alcohol of 50 pe? cent. ; no differentiation is necessary. When successful, an almost pure chromatin stain, quite as sharp as iron haematoxylin, but somewhat weak. Iron Carmine. — ^Pfbiffee von Wellhbim {Zeit. wiss. Mik., xv, 1898, p. 123) mordants for six to twelve hours in a very weak solution of chloride of iron in 50 per cent, alcohol, washes in 50 per cent, alcohol, and stains as above. Overstains may be corrected with 0-1 to 0'5 per cent. HCl alcohol. I find this good, but not so good as the last. Iron Carmine (Zachabias, Zool. Anz., 1894, p. 62). — Stain for several hours in an aceto-carmine (made by boiling 1 grm. of carmine with 150 to 200 c.c. of acetic acid of 30 per cent., for twenty minutes, and filtering). Einse the objects with dilute acetic acid, and bring them (taking care not to touch them with metallic instruments) into a 1 per cent, solution of ammoniated citrate of iron. Leave them, for as much as two or three hours if need be, till thoroughly penetrated and blackened (with sections this happens in a few minutes). Wash for several hours in distilled water. A chromatin and plasma stain. HoUande's Chlorcarmin Staining Method (0. B. Soc. Biol., 1916, Ixxix, p. 662, and Jour. Boy. Micr. Soo., 1920). — Place 5 c.c. pure hydrochloric acid in a porcelain dish ; add little by little 14 grms. powdered carmine, stirring constantly to m^ke a homogeneous doughy mass. Allow to digest for twenty-four hours : add 250 c.c. aq. dest., bring to the boil, and keep boiling for half an hour. Filter ; make up to 180 c.c. with aq. dest., and then add enough 75 per cent, alcohol to make a- total volume of 200 c.c. Stain sections or pieces of tissue for two to twenty-four hours. Rinse in aq. dest. or 30 per cent, alcohol ; immerse in 3 percent, iron alum solution, in which the sections become black, and are then slowly decolourised; when differentiation is com- plete, rinse in a 1 per cent, pyridin solution, and wash under the tap for ten to fifteen minutes. Counterstain and mount as desired. This is a very intense stain suitable for mitocliondria and cell granules. 223. Iron Carmalum (de Groot, Zeit. wiss. Mik., xx, 1903, p. 21). — Dissolve 0-1 grm. of ferric alum in 20 c.c. distilled water and add 1 grm. carminic acid. Dissolve, add 180 c.c. of water, warm, add 5 grms. potash alum, dissolve, cool, filter, and add 2 drops of hydrochloric acid. To be used as carmalum, and said to give a stronger stain. 140 CARMINE AND COCHINEAL STAINS. 224. Iron Cochineal (Spulbr, Enoydlopcedie d. mik. Technik, 1903, p. 153, and 1910, p. 240).— Stain for forty-eiglit hours in a stove, in extract of cochineal (made in a highly complicated way), wash with water, put into solution of ferric alum of f per cent, strength for twenty- four hours or more. If the stain is not sufficiently intense, the whole process may be repeated. i Peter {Zeit. wiss. Mik., xxi, 1904, p. 314) stains material in bulk for forty-eight hours (sections eighteen to twenty-four) in an incubator, in a similar extract, acidifled with HCl, treats with iron-alum of 2J per cent, for one hour to one day (sections half to two minutes), then alcohol, xylol, paraffin, or balsam. Chromatin black, protoplasm grey, yolk granules red. Hansen {ibid., xxii, 1906, p. 85) stains sections or entire objects in a solution of 5 to 10 grms. cochineal, 8 grms. ferric alum, 250 c.c. water, and 25 c.c. sulphuric acid of 10 per cent., boiled for fifteen to twenty minutes. /3. So-called "Neutral" and Alkaline. 225. Ammonia-Carmine. — Best made by the method of Eanviee. Make a simple solution of carmine in water with a slight excess of ammonia, and expose it to the air in a deep crystallising dish until it is entirely dried up. It should be allowed to putrefy if possible. Dissolve the dry deposit in pure water, and filter. Van Wijhe {Vers. Akad., Amsterdam, viii, Deel, p. 507) takes an old strong solution of carmine in ammonia (or boils carmine with ammonia and peroxide of hydrogen), then precipitates it by adding alcohol to excess, washes the precipitate with alcohol, and dries it. 226. Soda-Carmine appears to be still used by some for central nervous system (see Cuccati, Zeit. wiss. Mik., iv, 1887, p. 50). It can be obtained from GtEUbler & Hollborn (Natron-Carmin). 226a. Orth's Lithium-Carmine (see early editions) macerates strongly, and is superfluous. For that of Best, see Zeit. wiss. Mik., xxiii, 1906, , p. 322. 227. Magnesia-Carmine (Mater, Zeit. wiss. Mik., xiv, 1897, p. 23). — Take 1 grm. carmine, 0-1 grm. magnesia usta, and 50 c.c. distilled water, boil for five minutes, filter, and add 3 drops of formol. This is the stock solution. A weak solution may be made by boiling 0-1 grm. carmine for half an hour in 50 c.c. of magnesia water (made by leaving 0-1 grm. of magnesia usta in contact with 100 c.c. of spring water for a week with frequent agitation, and decanting when required for use). Said to be less injurious to tissues than the other. alkaline carmines. 228. As to Picro-Carmine. — The term " picro-carmine " is commonly used to denote a whole tribe of solutions in which carmine, ammonia, and picric acid exist uncomhined in haphazard proportions. These solutions do not contain a double salt of picric and carminic acid and ammonia, or ficro-oarminate of ammonia. They are always alkaline, and frequently injurious to tissues. The CHAPTER XII. 141 raison d'etre of picro-carmine does not lie in its capacity of affording a double stain, but in that the picric acid in it is supposed to neutral- ise the ammonia, which it only does imperfectly. See Mayer in Zeit. wiss. MiL, xiv, 1897, p. 18. 229. RANviEK's'Plcro-Carmine, Original Formula {TraiU, p. 100). — To a saturated solution of picric acid add carmine (dissolved in ammonia) to saturation. Evaporate down to one-fifth the original volume in a drying oven, and separate by filtration the precipitate that forms in the liquid when cool. Evaporate the mother liquid to dryness, and you will obtain the picro-carmine in the form of a crystalline powder of the colour of red ochre. It ought to dissolve completely in distilled water; a 1 per cent, solution is best for use. For slow staining, dilute solutions may advantageously have 1 or 2 per cent, of chloral hydrate added to them. Overstains may be washed out with hydrochloric acid, say 0-5 per cent, in water, alcohol, or glycerin. Preparations should be mounted in balsam, or if in glycerin, this should be acidulated with 1 per cent, of acetic acid, or better, formic acid. Eanviee's Newer Formula does not give a more constant product (see previous editions). 230. Van Wijhe dissolves 0-5 per cent, of the dry ammonia-carmine, § 225, in a 1 per cent, solution of neutral picrate of ammonia, boils until the vapour ceases to blue reddened litmus paper, and adds 1 per cent, of chloral hydrate. Gives an almost neutral preparation. 231. Mater's Picro-Magnesia Carmine (Zeit. wiss. Mik., xiv, 1897, p. 25) is relatively constant and innocuous to tissues. It consists of 1 vol. of the stoch solution of magnesia-carmine (§ 227), and 10 vols, of a 0-6 per cent, solution of picrate of magnesia, or of equal parts of the weah solution and the picrate solution. The picrate may be obtained from GrEUBLEK & HoLLBOEN, or the solution may be made by heating 0-25 grm. of carbonate of magnesia in 200 c.c. of 0-5 per cent, solution of picric acid, allowing to settle, and filtering. De Geoot's pioro-magnesia carmine {ibid., xxix, 1912, p. 184) contains ammonia, which is bad, and seems to me superfluous. 232. Other Formulae for Picro-Carmine and Other Aqueous Carmines (Acid and Alkaline). — ^I have tried most of them, and found no real advantage in any of them (see previous editions). B. Alcoholic Carmine Stains. 233. Alcoholic Borax-Carmine (Grenacher, Arch. mik. Anaf., xvi, 1879, pp. 466 et seq.). — Make a concentrated solution of carmine in borax solution (2 to 3 per cent, carmine to 4 per cent, borax) by boiling for half an hour or more (or allowing it to stand, with occasional stirring, for two or three days) ; dilute it with about an 142 CARMINE AND COCHINEAL STAINS. equal volume of 70 per cent, alcohol, allow it to stand some time and filter. Preparations should remain in the stain until they are thoroughly penetrated (for days if necessary), and then be brought (without first ivashing out) into alcohol of 70 per cent, acidulated with 4 to 6 drops of hydrochloric acid to each 100 c.c. of alcohol. They are left in this until they have taken on a bright transparent look (which may require days), and may then be washed or hardened in neutral alcohol. Four drops of HCl is generally enough. Three drops I find not quite sufficient. For dehcate objects, and for very impermeable objects, it may be well to increase the proportion of alcohol in the stain ; it may conveniently be raised to about 50 per cent. It should not exceed 60 per cent, in any case (Mayee). This stain used to be the most popular of any for staining in bulk. It is easy to use, and gives a most splendid coloration. But it is not so penetrating as is commonly supposed, and has the defect of sometimes forming precipitates in the cavities of bulky objects which cannot be removed by washing out. And the fluid is alkaline, and therefore may not be suitable for certain delicate work. 334. Mayee's Paracarmine [Mitth. Zool. Stat. Neqpel, x, 3, 1892, p. 491). — Carminic acid, 1 grm. ; chloride of aluminium, 0-5 grm. ; chloride of calcium, 4 grms. ; 70 per cent, alcohol, 100 c.c. Dissolve cold or warm, allow to settle, and filter. Objects to be stained should not have an alkaline reaction, nor contain any considerable amount of carbonate of lime (spicules or skeletal parts of corals, etc.) which would give rise to precipitates. Wash out sections or objects intended to be sectioned, with pure 70 per cent, alcohol. Objects intended to be mounted whole may be washed out with a weak solution of aluminium chloride in alcohol, or if this be not sufficient, with 5 per cent, common acetic acid (or 2-5 per cent, glacial acetic acid) in alcohol. This may also be done with section material, if it is desired to obtain a more purely nuclear stain. For staining bulky objects with large cavities, such as Salpa, the solution should be diluted (with alcohol) ; and as this may cause precipitates to form during the staining, especially if the objects are not very clean, it is advisable to slightly acidify the dilute solutions. Instead of calcium chloride, which is very hygroscopic, strontium chloride may be taken. Paracarmine is less hurtful to delicate tissues than borax carmine ; it is more highly alcoholic, therefore more penetrating ; and has less CHAPTER XII. 143 tendency to form precipitates in the interior of objects. But, in my hands, it does not give quite so fine a stain. 234a. Alcoholic Hydrochloric-Acid Carmine. — Geenacher's receipt {Arch. f. Mik. Anat., xvi, 1879, p. 468) is troublesome. That of Mater {Mitth. Zool. Stat. Neapel, iv, 1883, p. 521 ; Intern. Monatsschr. f. Anat, etc., 1897, p. 43) is better : Carmine 4 grms. ; water, 15 c.c. ; hydro- chloric acid, 30 drops. Boil till the carmine is dissolved, add 95 c.c. of 85 per cent, alcohol, and neutralise by adding ammonia until the carmine begins to precipitate. If it be desired to dilute the solution, it should be done with alcohol not water, and alcohol of 80 to 90 per cent, should be taken for washing out. A very powerful stain, which I have found useful. If it be desired to have a purely nuclear stain, the alcohol must be very slightly acidulated with HCl. For a oompUcated receipt of Loewenthal see Zeit. wiss. Mik., xix, 1902, p. 56. 235. Alcoholic Cochineal, Mayer's Old Formula {Mitth. Zool. Stat. Neapel, ii, 1881, p. 14). — Cochineal in coarse powder is macerated for several days in alcohol of 70 per cent. For each gramme of the cochineal there is required 8 to 10 c.c. of the alcohol. Stir frequently. Filter. The objects to be stained must previously be saturated with alcohol of 70 per cent., and alcohol of the same strength must be used for washing out or for diluting the staining solution. The washing out must be repeated with fresh alcohol until the latter takes up no more colour. Warm alcohol acts more rapidly than cold. Overstaining seldom happens ; it may be corrected by means of 70 per cent, alcohol, containing -^ per cent, hydrochloric or 1 per cent, acetic acid. Small objects and thin sections may be stained in a few minutes ; larger animals require hours or days. A nuclear stain, slightly afEecting protoplasm. The colour varies with the reaction of the tissues, and the presence or absence of certain salts in them. Crustacea with thick chitinous integuments are generally stained red, most other organisms blue. The stain is also often of difEerent colours in different tissue elements of the same preparation. Glands or their secretion often stain grey-green. Acids lighten the stain and make it yellowish-red. Caustic alkalies turn it to a deep purple. All acids must be carefully washed out from the objects before staining, or a diffuse stain will result. The stain is permanent in oil of cloves and balsam. Very penetrating and especially useful for Arthropoda. 144 CARMINE AND COCHINEAL STAINS. It has over the new fluid (next §) the advantage of being more highly alcoholic ; and it does not contain free acid, so that it can be used with calcareous structures which it is wished to preserve — which the new fluid cannot. For specimens of Pluteus, for instance, I find it excellent. But it only gives good results with such objects as contain the necessary salts, § 212. 236. Matee's Alcoholic Cochineal, New Formula (Mitth. Zool. Stat. Neapel, x, 1892, p. 498). — Cochineal, 6 grms. ; chloride of calcium, 5 grms. ; chloride of aluminium, 0-5 grm. ; nitric acid of 1-20 sp. gr., 8 drops ; 50 per cent, alcohol, 100 c.c. Powder the cochineal and rub up with the salts, add the alcohol and acid, heat to boiling-point, leave to cool, leave for some days standing with frequent agitation, filter. Use as the old tincture, the objects being prepared and washed out with 50 per cent, alcohol. Mayer only recommends it as a succedaneum of paracarmine. Since this fluid contains in itself all the necessary salts (§ 212), it gives good results with all classes of objects. CHAPTER XIII. HjEMATEIN (HiEMATOXYLIN) STAINS. 237. Introduction. — Hcematoxylin is a dye extracted from log- wood. It is a substance that oxidises very readily, thus becoming converted into hcematdn, or, as often happens, into other more highly oxidised products. It appears to be now thoroughly well established (see Nietzki, Chemie der organischen Farbstoffe, Berlin, Springer, 1889, pp. 215—217, and Mayer, Mitth. Zool. Stat. Neapel, X, 1891, p. 170) that the colouring agent in solutions of log- wood or hsematoxyHn is not the hsematoxyUn itself, but hsematein formed in them (or, in some cases, one of the higher oxidation products). Hsematein is an acid body, a " colour acid " (§§ 201, 206). Sub- stantively employed, it is a very weak plasma stain. But combined with appropriate mordants it becomes basic, and can be made to give a powerful nuclear stain, or at the same time a nuclear and a selective plasma stain. The mordants employed in histology are aluminium, chrome, iron, copper, and (rarely) vanadium and molybdenum. Aluminium and iron are the mordants most em- ployed, the former furnishing lakes used for progressive staining of material in bulk, the latter forming in most cases in the tissues a lake that requires differentiation, and is only applicable to the staining of sections. The presence of a sufficient amount of hsematein in staining solutions was formerly brought about by aUowing solutions of hsematoxyHn to oxidate spontaneously by exposure to air. The change thus brought about in the solutions is known as " ripening," and until it has taken place the solutions are not fit to use for staining. It was discovered by Mayer and Unna independently (see Mayer in Mitth. Zool. Stat. Neapel, x, 1891, pp. 170—186 ; Unna in Zeit. wiss. Mih., viii, 1892, p. 483) that nothing is easier than to bring about this change artificially ; all that is necessary being, for in- stance, to add'to a solution of hsematoxylin containing alum a little neutralised solution of peroxide of hydrogen or other powerful 10 146 HMMATEIN (HEMATOXYLIN) STAINS. oxidising agent.* The solution becomes almost instantaneously dark blue, " ripe " and fit for staining. Other methods of " ripen- ing," or of preparing haematein separately, are given further on, and constibute a great progress. For under the old practice of leaving staining solutions to " ripen " by the action of the air, it is necessary to wait for a long time before the reaction is obtained. During all this time, it may be weeks or months, there is no means, except repeated trial, of ascertaining whether the solution at any moment contains sufficient hsematein to afford a good stain. And here a second difficulty arises : the oxidising process continuing, the solutions become "over-ripe"; the hsematein, through further oxidation, passes over into colourless compounds, and the solutions begin to precipitate. They are therefore, in reality, a mixture in constantly varying proportions of " unripe," " ripe," and " over- ripe " constituents (the first and last being useless for staining purposes), and, in consequence, their staining power is very in- constant. Logically, therefore, as concluded by Maybe, not hsematoxylin, but hcematein, should be taken in the first instance for making the staining solution. But this is not always indicated ; for such solutions may easily over-oxidise, either in the bottle or on contact with the tissues. So that it is always preferable to start from hsematoxylin. In this case, it should not be done by dissolving the hsematoxylin straight away in the other ingredients of the staining solution. The solutions should be made up from a strong stock solution made by dissolving hsematoxyhn crystals in absolute alcohol : one in ten is a good proportion. This solution should be kept for a long time— months, , at least, a year if possible ; it gradually becomes of a vinous red, and shoidd not be used till it has become quite dark. It has then become to a great extent oxidised into hsematein, and the staining solutions made up from it will be at once fairly ripe. Hsematein (or hsematoxylin) affords a stronger stain than carmine, and gives better results with tissues fixed in osmic or chromic mixtures. The alum solutions are indicated for staining in bulk, iron hsematoxylin for sections. 238. Hsematoxylin is found in commerce in the form of crystals, either colourless or browned by oxidation, easily soluble in either water, glycerin, or alcohol. * Ee-invented lately (Zeit. wiss. Mile, xxix, 1912, p. 69) by Piazza who adds to Boelimer's solution about 20 per cent., to Delafleld's about 7 per cent., to Ehrlioh's about 12 per cent, of peroxide of hydrogen. CHAPTER XIII. 147 239. Hsematein is found in commerce as a brown powder, entirely, though with difficulty, soluble in distilled water and in alcohol, giving a yellowish-brown solution, which remains^ clear on addition of acetic acid. Alkalies dissolve it with a blue-violet tint. (See also last edition.) 240. Iron Haematoxylin, Generalities. — This method is due to Benda (Verh. Phys. Ges., 1885—1886, Nos. 12, 13, 14 ; Arch. Anat. Phys., 1886, p. 562 ; third ed. of this work, p. 365). The method was independently worked out about the same time by M. Heidenhain. The method is almost universally practised in the form given by Heidenhain, not on account of any essential difference between the two, for there is none, but chiefly because Heidenhain has given more precise instructions concerning the process. After carefully comparing Heidenhain's process with Benda's later process (next §), I find that the two give an absolutely identical stain ; that is to say, that if you mordant in Benda's liquor ferri (next §), and differentiate in the same, you wiU get exactly the same efEect as by mordanting in ferric alum and differentiating in the same. But you may vary the results somewhat by varying the differentia- tion. Benda has pointed out (Verb. Anat. Ges., xv, 1901, p. 156) that you may differentiate either by an agent which simply dissolves the lake — such as acetic or hydrochloric acid ; or by an oxidising agent, such as chromic acid, or the liqitor ferri or the ferric alum. The former, he thinks, are the best for the demonstration of nuclear structures, the latter for cytoplasmic structures. For these he greatly recommends Weigeet's borax-ferricyanide mixture, as being the easiest and safest to employ. For myself, I find that differentiation in the iron salt (§ 241 or § 242) is sufficient for almost all purposes. Acetic acid of 30 per cent, acts much too quickly to be safe, and causes swelling of the tissues. Van GtIESOn's picro-saurefuchsin has been recommended as a differentiation fluid by Benda (Deutsch. med. Wochenschr., 1898, No. 30). I find it gives very delicate differentiations, but acts very slowly, requiring nearly as many hours as the iron alum solution does minutes. The addition of the saurefuchsin to the picric acid is, I find, not necessary, and may prove an injurious complication. In these processes haematoxylin is generally used for the stain, not hamatein, the iron salt oxidising it into hasmatein, or into a higher oxidation product. I have obtained some good stains with 10—2 148 HMMATEIN (HMMATOXYLIN) STAINS. haematein, but also some very bad ones ; presumably the solutions easily over-oxidise on contact with the iron salt. The hsematoxylin is generally dissolved in water. I frequently prefer alcohol, of 50 per cent., as less injurious to tissues. The method is a regressive one. It has been proposed to stain progressively, which I have tried, and had extremely bad results. The differentiation requires to be carefully timed. For this reason the method is only applicable to sections, which should be thin, best not over 10 /u. Iron hsematoxylin is one of the most important of stains. It enables us to stain elements which cannot be selectively stained in any other way. The stain is very powerful, and of a certain optical quality that is peculiarly suited to the employment of high powers ; it will allow of the use of deeper eye-pieces than other stains. It will take effect on any material, and "is quite permanent. Further details as to the characters of the stain are given in § 242. 241. Benda's later Iron Hsematoxylin {Verb. d. Anat. Ges., vii, 1, 1893, p. 161). — Sections are mordanted for twenty-four hours in liquor ferri sulphurici oxidati, P.G.,* diluted with one or two volumes of water. They are then well washed, first with distilled water, then with tap water, and are brought into a 1 per cent, solution of hsematoxylin in water, in which they remain till they have become thoroughly black. They are then washed and differentiated. The differentiation may be done either in 30 per cent, acetic acid, in which case the progress of the decoloration must be watched ; or in a weaker acid, which will not require watching ; or in the sulphate solution strongly diluted with water. I find that if the iron solution be taken for the differentiation, it should be taken extremely diluted (of a very pale straw-colour, about 1 : 30 of water), and the progress of the differentiation watched ; as if it be only diluted about tenfold, for instance, the decoloration is extremely rapid. See also last §. I also find that Benda's mordant is unnecessarily, sometimes harmfully, strong, and that the liquor ferri may be diluted tenfold with advantage. The duration of the bath in the mordalit is also for most purposes excessive as directed by Benda. I find that three to six hours in the solution diluted tenfold is generally sufficient with favourable material. * This preparation consists of sulphate of iron, 80 parts ; water, 40 ; sulphuric acid, 15 ; and nitric acid, 18, and contains 10 per cent, of Fe. Doubtless the ferri persulphatis liquor B. P. will do instead ; the point is, to have a per-salt, and not a proto-salt. CHAPTER XIII. 149 842. Heidenhain's Iron Hsematoxylin (M. Heidenhain, " Uber Kern und Protoplasma," in Festschr. fur Kolliker, 1892, p. 118). — Sections are treated from half an hour to at most two or three hours with a 1-5 to 4 per cent, solution of ferric alum (ammonio-ferric sulphate). By this is always meant in histology the double salt of ammonium and sesquioxide of iron (NH4)2Fe2 (804)4, in clear violet crystals ; the double salt of the protoxide, or salt of Mohr in green crystals, will not serve. If the crystals have become yellow and opaque, they have gone bad, and should be rejected. They ought to be kept in a stoppered bottle, and the solution should be made in the cold {Arch. mik. Anat., xKii, 1894, pp. 431, 435). The sections are then washed with water and stained for half an hour in an aqueous solution (of about 0-5 per cent.) of hsematoxylin. They are then rinsed with water, and again treated with the iron solution, which slowly washes out the stain. The progress of the differentia- tion ought to be controlled under the microscope. The sections should to this end be removed from time to time from the alum solution, and put into tap-water whilst they are being examined. This is favourable to the stain. As soon as a satisfactory differentia- tion has been obtained, the preparations are washed for at least a quarter of an hour in running water; but not more than an hour, and mounted. The results differ according to the duration of the treatment with the iron and the stain. If the baths have been of short duration, viz. not more than half an hour in the iron and as much in the stain, blue preparations will be obtained. These show a very intense and highly differentiated stain of nuclear structures, cylioplasmic structures being pale. If the baths in the iron and in the stain have been prolonged (twelve to eighteen hours), and the subsequent differentiation in the second iron bath also duly pre longed, black preparations 'will result. These show chromosomes stained, central corpuscles stained intensely black, cytoplasm some- times colourless, sometimes grey, in which case achromatic spindle- fibres and cell-plates are stained, connective-tissue fibres black, red blood-corpuscles black, micro-organisms sharply stained, striated muscle very finely shown. Later {Zdt. miss. Mik., xiii, 1896, p. 186) Heidenhain gives further instructions for the employment of this stain in the study of central corpuscles. All alcohol should be removed from the tissues * by means of distilled water before bringing them into the mordant. * Why ? I find my iron-alum solution, as well as the Uquor ferri sulph. oxid., last §, mix clear with alcohol without the least precipitate forming. 150 HMMATEIN (HEMATOXYLIN) STAINS. Tliis should be a 2^ per cent, solution of ferric alum, not weaker. Leave the sections therein (fixed to slides by the water method, § 186) for six to twelve hours, or at least not less than three. Keep the slides upright in the mordant, not lying flat. Wash out well with water before staining. Stain in a " ripened " hsematoxyhn solution, i.e. one that has stood for four weeks [of course, if you make it up with the ripened brown alcoholic solution recommended § 237 sub. fin., this will be superfluous]. Stain from twenty-four to thirty-six hours. Use the same staining solution over and over again until it becomes spoilt ; for the solution after having been used gives a more energetic stain, owing to its containing a trace of iron brought over by the sections. Differentiate in a 2-J- per cent, solu- tion of ferric alum. Rinse for ten minutes in running water, clear with xylol, not with any essential oil, and mount in xylol-balsam. See also under " Centrosomes," and " Chromosomes," etc. BiELASZEWics {Bull. Acad. Oracovie, 1909, 2 seri6, p. 152) difEerentiates with very weak solution of calcium chloride ; G-trARNiERi (Mon. Zool. Ital., xvii, 1906, p. 44) with, saturated solution of picric acid. GuEwiTSCH {Zeit. wiss. Mile, xviii, 1902, p. 291) floods sections on the slide with mordant, warms on a water-bath tiU bubbles are given ofE or the mordant becomes turbid, then stains with the hsematoxylin in the same way. The whole process takes about ten minutes. Held {Arch. Anat. Phys., Anat. Abth., 1897, p. 277) adds to the staining bath a very little of the iron-alum solution until a scarcely perceptible precipitate is produced. A dangerous practice. I find it is not even safe to add a little of an over-used bath {supra). rEANCOTTE {Arch. Zool, ExpSr., vi, 1898, p. 200) mordants with tartrate of iron, Malloet {Journ. Exper. Med., v. 1900, p. 15) with chloride. 243. Iron Hsematoxylin (Butsohli, Unters. iiher mikroshopische Schaume u. das Protoplasma, etc., 1892, p. 80). — -Sections treated with a weak brown aqueous solution of ferric acetate, washed with water, and stained in 0-5 per cent, aqueous solution of hsematoxylin. A stain of extraordinary intensity, used by Biitschli for sections, 1 /i in thickness, of Protozoa. 244. Weigert's Iron Hsematoxylin Mixture {Zeit wiss. Mih., xxi, 1904, p. 1). — -Mix 1 part of a 1 per cent, solution of hsematoxylin in alcohol of 96 per cent, with 1 of a solution containing 4 o.c. of liq. ferri sesg[uichlor., 1 c.c. of officinal hydrochloric acid (sp. gr. 1-124) and 95 of water. The mixture may be kept for some days (until it begins to smeU of ether), but is best used fresh. Stain sections for a few minutes ; no differentiation is necessary. For an earlier process of Weigert's {Allg. Zeit. Psyohiatr., 1894, p. 245) see last edition. MoEEL and Bassal {Journ. Anat. Phys., xlv, 1909, p. 632) stain in hulk in Weigert's mixture with the addition of 1 c.c. of 4 per cent, solution of acetate of copper. CHAPTER XIII. 151 245. Janssens' Iron Hsematoxylin ( " H6matoxyline noire"; La Oellule, xiv, 1897, p. 207). — ^A similar mixture to that of Delafield, ferric alum being taken instead of ammonia alum, the rest as in Delafield's. A progressive stain, nuclear : for yeast cells. 24.6. Hansen's Iron Hsematoxylin {Zeit. wiss. Mik., xxii, 1905, p. 55). — ^A solution of 10 grms. ferric alum in 150 c.c. water is added to a solution of 1-6 grm. hsematoxylin in 75 c.c. water, the mixture heated to boiling-point and cooled without access of air. Filter before use. To get a pure nuclear stain, add dilute sulphuric acid. 247. Aluminium Hsematein (Alum Hsematoxylin) Generalities.— The mordant and dye are generally combined in a single staining bath, giving a 'progressive stain. The stain is in different tones of blue or red according to the composition of the staining solution. Neutral or alkaUne solutions give a blue stain ; acid solutions give a red one. In order to get a blue stain in preparations that have come out red through the acidity of the staining bath, it is a common practice to treat them with weak ammonia, in the belief that the blue colour is restored by neutralisation of the acid that is the cause of the redness. According to Mayer, the ammonia acts, not by neutralising the acid, but by precipitating the alumina, which carries down the hsematein with it (if no alumina were present the colour would be purple, not blue). The same result can generally be obtained by merely washing out with common tap-water, which is usually sufficiently alkaline, and can be obtained with certainty by treatment with bicarbonate of soda or acetate of soda or potash. And this is the preferable course, as ammonia is certainly a dangerous thing to treat delicate tissues with. See Scott's tap-water substi- tute, § 676. Of course this is a different question from that of neutralising with an alkali tissues that have been treated with an acid to correct over-staining. Here the neutralisation may be indicated in the interest of the preservation of the stain. Squire {Methods, p. 22) finds that sections can be blued in a few seconds by treatment with a 1 : 1000 solution of bicarbonate of soda in distilled water. Mayer holds that acetate of potash is the most inoffensive reagent to take ; a strength of 0-5 to 1 per cent, may be taken. Several of these solutions have a great tendency to over-stain. Over-stains may be corrected by washing out with weak acids {e.g. 0-1 to 0-2 or even 0-5 per cent, of hydrochloric acid, or with oxalic or tartaric acid), but this is not favourable to the permanence of the stain. Carnoy {La Cellule, xii, 2, 1897, p. 215) recommends iodised water. If acids be used, it is well to neutrahse afterwards with ammonia or bicarbonate of soda (0-1 per cent.). 152 HMMATEIN (HEMATOXYLIN) STAINS. Bicarbonate of soda may be used for neutralisation with 70 per cent, alcohol as the vehicle (von Wistinghausen, Mitth. Zool. Stat. Neapel, x, 1891, p. 41). Over-staining may be avoided by staining very slowly in dilute solutions. The purest chromatin stains are obtained by staining for a short time (sublimate sections half an hour, 'say) in solutions of medium strength, such as hsemalum diluted ten to twenty-fold with water. The stain obtained either with very strong solutions, or with the slow stain of the dilute solutions, is at the same time a plasma-stain, which of course may or may not be desired. Mayer says that very dilute solutions will give a pure nuclear stain if they have been diluted with alum-solution, or have been acidified. Chrom- osmium material will not yield a pure chromatin stain unless it is very fresh ; it is consequently next to impossible to obtain the reaction with paraffin sections of such material ; they constantly give a plaslna-stain in addition to the chromatin stain, which is not the case with sublimate material. The stain is fairly permanent in balsam, but is very liable to fade a little, and may fade a great deal. If acids have been used after staining, great care should be taken to wash them out thoroughly before mounting. In aqueous media the stain cannot be relied on to keep (this refers to the old solutions : Mayer finds that his hsematein preparations have kept well for at least some months in glycerin, if not acid, and, with certain precautions, in balsam). Turpentine-balsam should not be used. Formulae §§ 248 to 259 give aqueous solutions, and §§ 260 to 263 alcoholic ones. 248. Mayer's Hsemalum, Newer Formula (Zeit. wiss. Mih., xx, 1903, p. 409). — Hcematoxylin, 1 grm. ; water, 1 Htre. Dissolve, and add 0-2 grm. of iodate of sodium (NalOj) and 50 grms. of alum, dissolve and filter. This is an amended formula. The original one {Mitth. Zool. Stat, i Neapel, x, 1891, p. 172) was : 1 grm. of hcematein (or the ammonia salt, §§ 238, 239) dissolved with heat in 50 o.c. of 90^per cent, alcohol, and added to a solution of 50 grms. of alum in a litre of distilled water. This solution does not keep very well, but may be made more stable by adding 50 grms. of chloral hydrate and 1 grm. of citric (or acetic) acid. It stains equally well, either at first, or later. Concentrated, it stains sometimes almost instantaneously, or in any case very rapidly. (Spring water or tap-water containing lime must not be used for diluting ; perhaps weak solution of alum in distilled water is the CHAPTER XIII. 153 best means of all.) After staining, sections may be washed out either with distilled or common water. It is admirable for staining in bulk. Large objects will, however, require twenty-four hours' staining, and should be washed out for the same time (this should be done with 1 per cent, alum solution if a sharp nuclear stain be desired). All alum must be carefully washed out of the tissues before mounting in balsam ; and it is well to blue the stain with tap- water or otherwise, § 257. The stain is generally a nuclear one ; in any case such may be obtained by washing out with alum-solution. Mayer's preparations have kept well in glycerin (care being taken not to have it acid), also in balsam. If oil of bergamot be used for clearing, it must be thoroughly removed by means of oil of turpen- tine before mounting, and oil of cloves is dangerous. It is best (Mayer, in litt.) to use only xylol, benzol, or chloroform, and to mount in xylol-balsam or chloroform-balsam or benzol-balsam. Hsemalum may be mixed with alum-carmine, Saurefuchsin, or the Uke, to make a double staining mixture ; but it seems preferable to use the solutions in succession. 249. Mayer's Acid Hsemalum {Miuh. Zool. Stat. Neapel, x, 1891, p. 174). — This is heemalum with 2 per cent, glacial acetic acid (or 4 per cent, common acetic acid). To be used as the last, washing out with ordinary water in order to obtain a blue-violet tint of stain. The solution keeps better. 250. Unna's Half-ripe Constant Stock Solution (Zeit. wiss. Mik., viii, 1892, p. 483). Haematoxylin ...... 1 Alum . Alcohol Water Sublimed sulphur 10 100 200 2 If the sulphui be added to the hssmatoxylin solution only when the latter has become somewhat strongly blue, i.e. after two or three days' time, the stage of oxidation attained by the solution wiU be fixed for some time by the sulphur, and according to Unna the solution will remain "constant" in staining power. Mateb (Mitth. Zool. Stat. Neapel, xii, 1896, p. 309) finds that the sulphur process does not preserve the solutions for long, whilst glycerin does. See below, " GLTOHiEMALUM." 251. Matek's Glychsemalum (Mitth. Zool. Stat. Neapel, xii, 1896, p. 310). — ^Hsematein (or hsemateate of ammonia), 0-4 grm. (to be rubbed up in a few drops of glycerin) ; alum, 5 grms. ; glycerin, 30 ; distilled water, 70. The stain is not purely rmclear, but may be made so by washing out with alum solution or a weak acid. The solution keeps admirably. 154 EMMATEIN (HEMATOXYLIN) STAINS. Eawitz {Leitfaden, 2iid ed., p. 63) takes 1 grm. haematein, 6 grms. ammonia alum, 200 grms. each of water and glycerin. Or {Zeit. wiss. Mile., xxv, 1909, p. 391) 1 grm. hsematein, 10 grms. of nitrate of aluminium, 250 grms. each of water and glycerin. 252. Hansen's Solution (^ooZ. J.m^., 1895, p. 158). — See fourth edition. 253. Harris's Solution (Micr. Bull, xv, 1898, p. 47 ; Journ. App. Mic, iii, p. 777). — ^Alum-hsematoxylin solution ripened by addition of mercuric oxide. Mater (Ch-undziige, 1901, p. 171) finds the formula " gives too much hsematein." 254. Bohmer's Hsematoxylin {Arch. mik. Anat., iv, 1868, p. 345 ; Aerzt. Intelligenzbl, Baiern., 1865, p. 382). — Make (a) a solution of hsematox. cryst. 1 part, alcohol (absolute) 12 parts, and (b) alum 1 part, water 240. For staining, add two or three drops of A to a watch-glassful of b. The alcoholic solution of hsematoxylin ought to be old and darh (§ 237). A. Gr. HORNTOLD {Trans. Manch. Micr. 8oc., 1915) prepares : — Solution A: hsematoxylin, 0-7 grm. ; absolute alcohol, 20 c.o. Solution B : alum, 0-35 grm. ; aq. dest., 60 c.c. Mix A and B, expose to light in window for three or four days, then add 20 drops of tincture of iodine. Stain sections five to ten minutes till red-brown. Differentiate in 70 per cent, alcohol, to which add a few drops of acetic acid. The sections then turn blue. Good stain after osmic fixatives. 255. Delafield's Heematoxylin {Zeit. wiss. Mik., ii, 1885, p. 288 ; frequently attributed erroneously to Geenacher or Prudden). — To 400 c.c. of saturated solution of ammonia-alum (that is about 1 to 11 of water) add 4 grms. of hsematox. cryst. dissolved in 25 c.c. of strong alcohol. Leave it exposed to the light and air in an un- stoppered bottle for three or four days. Filter, and add 100 c.c. of glycerin and 100 c.c. of methylic alcohol (CH^O). Allow the solution to stand (uncorked) until the colour is sufficiently dark, then filter. This solution keeps for years. It is well to allow it to ripen for at least two months before using it. For staining, enough of the solution should be added to pure water to make a very dilute stain. It is an extremely powerful stain. It is still much used. I find that when well ripened— tor years rather than months — it is quite a first-class stain. BtJTSCi-iLi {Unters. iib. milcroscopische Schaume u. das Protoplasma, etc., 1892) recommends, under the name of "acid hsematoxylin," solution of Delafield very strongly diluted, and with enough acetic acid added to it to give it a decidedly red tint. This gives a sharper and more differentiated nuclear stain than the usual solution. Martinotti {Zeit. wiss. Mik., xxvii, 1910, p. 31) makes it up with 0-2 per cent, of hmmatein, and less alum (2 per cent.). CHAPTER Xin. 155 256. Ehrlich's Acid Hsematoxylin {Zeit. iviss. MiL, ] 886, p. 150).— Water 100 c.c, absolute alcohol 100, glycerin 100, glacial acetic acid 10, hsematoxylin 2 grms., alum in excess. -Let the mixture ripen in the light (with occasional admission of air) until it acquires a dark red colour. It will then keep, with constant power, for years, if kept in a well-stoppered bottle. It is very appropriate for staining in bulk, as overstaining does not occur. I find it excellent. Mann (ibid., xi, 1895, p. 487) makes up this stain with an equal quantity of haematein instead of hsematoxylin. Mayer (Grundziige, Lee and Mayer, 1st ed., p. 154) finds that this is too much and makes the mixture overstain ; 0-4 grm. of haematein is quite enough. For proper method of using Ehrlich's hsematoxylin, see Scott, §669. 257. Burchakdt's Pyroligneous Acid Hsematoxylin {Arch. miJc. Anat, liii, 1898, p. 232) would seem to be superfluous at least. 258. Unna's Oxidised Hsematoxylin (from Martinotti, Zeit. wiss. Mik., xxvii, 1910, p. 31). — Hsematoxylin 0-5, alum 2, water 60, alcohol 10, glycerin 20, peroxide of hydrogen solution 10, carbonate of soda 0-05. Martinotti, he. cit., makes it up with hcematein (0-2 grm.). 259. ApIthy's Haematein Mixture I A (Mitth. Zool. Stat. Neapel, xii, 1897, p. 712). — Make (a) a solution of 9 per cent, alum, 3 per cent, glacial acetic acid, and 0-1 per cent, salicylic acid in water, and (b) a 1 per cent, solution of hsematoxylin in 70 per cent, alcohol, preserved for six to eight weeks in a bottle not quite full. Mix 1 part of A with"l of B and 1 of .glycerin. Stains either sections or material in bulk. Apathy uses it for staining neuro-fibrils. 260. Kleinenbekg's Hsematoxylin (Quart. Journ. Micr. 8oi., Ixxiv, 1879, p. 208). — Highly irrational and very inconstant in its composition and its effects ; see early editions ; also the criticism of Mayer {Mitth. Zool. Stat. Neapel, x, 1891, p. 174), and that of Squiee in his Methods and Formulw, p. 26, and the alternative formulas of Squire {loo. cit.) and of VON Wistinghausen (Mitth. Zool. Stat. Neapel, x, 1891, p. 41). 261. Mayer's Hsemacalcium (Mitth. Zool. Stat. Neapel, x, 1891, p. 182). — Hsematein (or hsemateate of ammonia, §§ 238, 239), 1 grm. ; chloride of aluminium, 1 grm. ; chloride of calcium, 50 grms. ; glacial acetic acid, 10 c.c. (or common acetic acid, 20 c.c.) ; 70 per cent, alcohol, 600 c.c. Eub up finely together the first two ingre- dients, add the acid and alcohol, dissolve either cold or with heat ; lastly add the chloride of calcium. 156 HMMATEIN (HEMATOXYLIN) STAINS. If the objects stain in too red a tone they may be treated with a solution (of about 2 per cent.) of chloride of aluminium in 70 per cent, alcohol, or with a 0-5 to 1 per cent, solution of acetate of soda or potash in absolute alcohol ; but washing with neutral alcohol will generally suffice. With certain objects this solution does not penetrate well. This may be remedied by acidifying the solution, or, which is better, by leaving the objects for some time before staining in acid alcohol. Anyway objects ought not to have an alkaUne reaction. If these precautions be taken, it will not be necessary to use acid for washing out. The solution is not recommended as giving as good results as hsemalum, and Mayer recommends it merely as a substitute for Kleinenberg's, in cases in which an alcoholic hsematein stain seems indicated, as being easy to prepare, and constant in its effects. 862. Mayer's Hsemastrontium {Grundziige, Lee and Mayee, 1910, p. 166). — 1 grm. hsematein, 1 grm. aluminium chloride, 50 grms. strontium chloride, 600 c.c. alcohol of 70 per cent., and (if- desired) 0-25 grm. citric acid. Prepare and use as hsemacalcium. ' 263. De Gboot's Alcoholic Hsemalum {Zeit. wiss. Mik., xxix, 1912, p. 182). — ^Mix 20 CO. of glycerin with. 240 of alcohol of 70 per cent. Take 4 c.c. of the mixture, 2 c.c. of hydrogen peroxide, and 0-5 grm. of hsematoxylin, and dissolve with lieat. Add 60 c.c. of the mixture, 4 grms. of calcium chloride, and 2 grms. of sodium bromide. Dissolve, add 3 grms. of alum, heat and add 100 c.c. of the mixture. When the alum is dissolved add 0-2 grm. of ferri-cyanide of potassium ; dissolve and add 3 grms. more of alum and the rest of the mixture. Said to stain almost as weU as hsemalum. Wash out with alcohol of 70 per cent. 264. Other Alumina-HsBmatein Solutions. — ^A large number of suppressed receipts wiU be found given in the earlier editions. 265. E. Heidenhain's Chrome Hsematoxylin (AreJi. mile. Anat., xxiv, 1884, p. 468, and xxvii, 1886, p. 383). — -Stain for twelve to twenty- four hours in a J per cent, solution of hsematoxylin in distilled water. Soak for the same time in a 0-5 per cent, solution of neutral chromate of potash. Wash out the excess of chromate with water. Objects that have been fixed in corrosive sublimate ought to be very carefully washed out with iodine, or the Uke, as neutral hsematoxyhn forms a black precipitate with any excess of subhmate that may remain in the tissues. See Toeniek, in ArcTi. mile. Anat, 1886,. p. 181. The process is adapted to staining in bulTc. You can decolour the objects to any extent by prolonging the soaking in the chromate. Bichromate will do instead of the neutral chromate. 266. Apathy's Modlflcation of Heidenhain's Process {Zeit. wiss. Mik., V, 1888, p. 47). — ^This is an alcohoUo method. Stain in a 1 per CHAPTER XIII. 157 cent, solution of hsematoxylin in 70 or 80 per cent, alcohol. Diflerentiate sections of 10 to 16 ;*, half the time of staining, sections of 25 to 40 fi, twice the time of staining, in 1 per cent, solution of bichromate of potash in 70 to 80 per cent, alcohol, and wash out in alcohol of 70 per cent. AU these processes should be done in the dark. For ceUoidin series of sections, Ap&thy (ihid., 1889, p. 170) stains in the hsematoxylin solution as above for ten minutes ; then removes the excess of hsematoxylin fluid from, the sections by means of blotting- paper, and brings the series for five to ten minutes into 70 per cent, alcohol containing only a few drops of a strong (5 per cent.) solution of bichromate. 267. Schultze's Chrome Hsematoxylin [Zeit. wiss. Mih., xxi, 1904, p. 5). — ^The tissues to be fixed for twelve or more hours in a bichromate or chromic acid solution, preferably an osmium-bichromate mixture or liquid of Flemming, then to be washed out for twenty-four hours in 50 per cent, alcohol in the dark and stained for twenty-four hours or more in 0-5 per cent, hsematoxylin in alcohol of 70 per cent., then washed out in alcohol of 80 per cent. 268. Hansen's Chrome Hsematoxylin (ibid., xxii, 1905, p. 64). — Ten grms. of chrome alum boiled in 250 o.c. of water till green, and 1 grm. hsematoxylin (dissolved in 15 c.c. of water) added ; to the mixture when cold add 5 c.o. of sulphuric acid of 10 per cent, and (drop by drop) a solution of 0-55 grm. of bichromate of potash in 20 c.o. of water. Filter before use. Wash out with water free from air. 269. Vanadium Hsematoxylin (Heidenhain, Encyolop. mik. Tecjinik., 1903, p. 618). — ^Add 60 c.c. of a 6 per cent, solution of hsematoxylin to a 0-25 per cent, solution of vanadate of ammonium (quantity not stated ; should be 30 c.c, see Cohn in Anat. Eefte, xv, 1895, p. 302). The mixture to be used after three or four days ; it will not keep over eight days. To be used with sections of sublimate material. A strong plasma stain for spedal purposes, especially mucus glands. 270. Benda's Copper Hsematoxylin {Aroji. mik. Anat., xxx, 1887, p. 49). — See fourth edition. According to my experience, not to be compared with iron hsematoxylin, and superfiuous. 271. Malloey's Phospho-molybdic Acid Hsematoxylin (Anat. Anz., 1891, p. 375). — One part 10 per cent, phospho-molybdic acid solution, 1 part hsematoxylin, 100 parts water, and 6 to 10 parts chloral hydrate. Let the solution ripen for a week in sunlight, and filter. Chiefly for central nervous system. Sections should be stained for from ten minutes to one hour, and washed out in two or three changes of 40 to 50 per cent, alcohol. It is necessary that the solution should be saturated with hsematoxylin in order to obtain the best results ; if a good stain be not obtained at once, more hsematoxylin must be added. Water must never be used for diluting it. 158 HMMATEIN {HEMATOXYLIN) STAINS. See also Eibbert {Oentralb. allg. Path., vii, 1896, p. 427 ; Zeit. wiss. Mik., XV, 1898, p. 93), Patellani (Mon. Zool. Ital, xiii, 1902, p. 6), and GrOLOViN (Zeit. wiss. Mik., xix, 1902, p. 184). Sakgent (Anat. Ann., xv, 1898, p. 214) quotes this stain, preceded by mordanting for twenty-four hours in 5 per cent, sulphate of copper, as Kenton's. KoDis (Aroli. mik. Anat., lix, 1901, p. 211) takes hsematoxylin, 1 part ; molybdic anhydride, 1-5 ; water, 100 ; H2O2, 0-5, or a crystal of HgO. Police (Arch. Zool. Napoli, iv, 1909, p. 300) takes 0-35 grm. hsema- toxylin, 10 drops phospho -molybdic acid of 10 per cent., 10 grms. chloral hydrate, and 100 grms. alcohol of 70 per cent. 272. Mallory's Phospho-tungstic Hsematoxylin {Journ. Exp. Med., V, 1900, p. 19 ; Zeit. iviss. Mik., xviii, 1901, p. 178) : HBematoxylin . . . . . . 0-1 . Water 80-0 10 per cent, solution of (Merck's) phospho- tungstic acid ...... 20-0 Peroxide of hydrogen (U.S. Ph.) . . .0-2 (Dissolve the heematoxylin, add the acid, then the peroxide.) Stain sections two to twenty-four hours, wash out with water. A poly- chromic stain, nuclei blue, intercellular substances pink. I consider this a fine stain. 273. DoNAGGio's Tin Hsematoxylin (Ann. Nevrol. Napoli, xxii, 1904, p. 192). — A 1 per cent, solution of hsematoxylin is poured slowly into an equal volume of 20 per cent, solution of pink-salt (ammonio -chloride of tin). Keep in the dark. 274. Osmium Hsematoxylin. — Schultze (Zeit. wiss. Mik., xxvii, 1910, p. 465) treats tissues for twenty-four hours or more with osmic acid of 1 per cent., washes well with water, and puts for a couple of days into ripened 0-5 per cent, solution of hsematoxylin in alcohol of 35 to 50 per cent. "Wash out for a day or more with alcohol of 70 per cent. Intense plasma stain. CHAPTER XIV. NUCLEAR STAINS WITH COAL-TAE DYES. 275. Introduction. — Very few coal-tar dyes give a precise nuclear or chromatin stain by the progressive method (§ 209). Two of them — methyl green and Bismarck brown — are pre-eminAitly progressive chromatin stains. Many of the others — for instance, safranin, .gentian, and especially dahha — may be made to give a progressive nuclear stain with fresh tissues by combining them with acetic acid ; but in general are not so suitable for this kind of work as the two colours first named. Again, very few coal-tar dyes give a pure plasmatic stain (one leaving nuclei unaffected). The majority give a diffuse stain, which in some few cases becomes by the application of the regressive method (§ 209) a most precise and splendid chromatin stain. But plasma staining is generally done by the progressive method. The basic anilin dyes were at one time greatly in vogue for the staining of chromatin in researches on the structure of nuclei. They have been Httle used for that purpose since the working out of the iron hsematoxylin process, which gives a more energetic stain. But they may still be useful as a means of controlling the iron hsematoxylin process, which frequently stains all sorts of things besides chromatin, which does not occur with the best -tar colour stains. The acid and neutral anihn dyes afford some of our best plasma stains. I recommend — for staining nuclei of fresh tissues, methyl green ; for staining nuclei of fixed tissues by the regressive method, safranin for a red stain, and gentian violet or Thionin for a blue one ; as a plasma stain for sections, Saurefuchsin ; for entire objects, picric acid. A. Progressive Stains. 876. Methyl Green. — This is the most common in commerce of the " aniUn " greens. It appears to go by the synonyms of Methyl- anilin green, OrUwpulver, Vert Lumiere, Lichtgrun ; these two last are in reality the name of another colour. When first studied by Calberla, in 1874 (Morphol. Jahrh., iii, 1887, p. 625), it went by the 160 NUCLEAR STAINS WITH COAL-TAR DYES. name of Vert en cristaux. It is commonly met with in commerce under the name of more costly greens, especially under that of iodine green. It is important not to confuse it with the latter, nor with aldehyde green (Vert d'Eusebe), nor with the phenylated rosanihns, Paris green, and Vert d'Alcali, or Veridine. Methyl green is the chloromethylate of zinc and penta-methyl rosanihn-violet. It is obtained by the action of methyl chloride on methyl violet. The commercial dye always contains unconverted- methyl violet as a consequence of defective purification. It is sometimes adulterated with anilin blue (soluble blue). It is also sometimes adufterated with a green bye-product of the manufacture — the chloride of nona-methyl-para-leukaniUn. See Benbdikt and Knecht's Chemistry of the Coal-tar Colours. For tests for purity see Mayer, Mitth. Zool. Stat. Neapel, xii, 1896, p. 312, and Fischer, Fixirung, Farbung, u. Bau des Protoplasmas, p. 89. Methyl green is extremely sensitive to alkahes. It is therefore important to use it only in acidified solutions and to use only acid, or at least perfectly neutral fluids for washing and mounting. This is an extremely important histological reagent. Its chief .use is as a chromatin stain ion fresh, unfixed tissues. For this purpose it should be used in the form of a strong aqueous solution containing a httle acetic acid (about 1 per cent, in general). The solutions must always be acid. If the tissues have been previously fixed with acetic acid you will not get a chromatin stain. The same applies to fixation with acetic acid sublimate : whilst pure sublimate will allow of a chromatin stain (Burckhardt, La Cellule, xii, 1897, p. 364). You may wash out with water (best acidulated) and mount in some acid aqueous medium containing a little of the methyl green ii} solution. The mounting medium, if aqueous, must be acidulated. Employed in this way, with /resA unfixed tissues, methyl green is a pure chromatin stain, in the sense of being a precise colour reagent for chromatin. For in the nucleus it stains nothing but chromosomes or chromatin elements ; it does not stain plasmatic nucleoli (unless indeed these contain chromatin), nor caryoplasm, nor achromatic filaments. Outside the nucleus it stains some kinds of cytoplasm and some kinds of formed material, espec'ially glandular secretions (silk, for instance, and mucin). The chromatin elements are in- variably stained of a bright green (with the exception of the nuclein of the head of some spermatozoa), whilst extra-nuclear structures are in general stained in tones of blue or violet. But this meta- chromatic reaction is probably due to the methyl-violet impurity, and is not obtained with a chemically pure methyl green. CHAPTER XIV. 161 Staining is instantaneous; overstaining never occurs. The solution is very penetrating, kills cells instantly without swelling or other change of form, and preserves their forms for at least some hours, so that it may be considered as a dehcate fixative. It may be combined without precipitating with divers fixing or preserving agents. Osmic acid (of 0-1 to 1 per cent.) may be added to it, or it may be combined with solution of Eipaet and Petit (this is an excellent medium for washing out and mounting in). Alcoholic solutions may also be used for staining. They also should be acidulated with acetic acid. The stain does not keep easily. It is difficult to mount it satis- factorily in balsam, because the colour does not resist alcohol sufficiently (unless this be charged with the colour). The resistance of the colour to alcohol is, however (at all events if it be used in the Ehrlich-Biondi combination), considerably increased by treating the sections for a few minutes with tincture of iodine before staining (M. Heidenhain). Of preparations mounted with excess of colour in the usual aqueous media, I find the most fortunate only survive for a few months. Dr. Henneguy, however, writes to me that it keeps well in Beun's glucose medium. It was first pointed out, I believe, by Heschl {Wiener med. Wochensehr., 2, 1879), that methyl green is a reagent for amyloid degeneration. His observations were confirmed by Cursohmann {Virojiow's Arch., vol. Ixxix, 1880, p. 556), who showed that it colours amyloid substance of an intense violet ; but this, as pointed out by Squike (Methods and Formulas, etc., Churchill, 1892, p. 37), is un- doubtedly due to its containing methyl violet as an impurity. 277. Bismarck Brown (Manchester Brown, Fhenylen Brown, Vesuvin, La Ph6nicienne). — A fairly pure nuclear stain that will work either with fresh tissues or with such as have been hardened in chromic acid, or otherwise. The colour is not very easily soluble in water. You may boil it in water, and filter after a day or two (Weigeet, in Arch. miJc. Anat., XV, 1878, p. 258). You may add a little acetic or osmic acid to the solution. Maysel (ibid., xviii, 1880, pp. 237, 250) dissolves the colour in acetic acid (this solution does not give a permanent stain). Alcoholic solutions may also be used, e.g. saturated aqueous solution diluted with one-third volume, of 90 per cent, alcoholic ; or Cal- beela's glycerin-and-alcohol mixture or dilute glycerin (say of 40 per cent, to 50 per cent.) may ve!ry advantageously be employed. The watery solutions must be frequently filtered (but then much u. 11 162 NUCLEAR STAINS WITH COAL-TAR DYES. of the colour is retained on the filter). The addition to them of carbolic acid has been recommended (vide Journ. Roy. Mic. Soc, 1886, p. 908). Bismarck brown stains rapidly, but never over- staias. The stain is permanent both in balsam and in glycerin. This colour may be used as a chromatin stain for fresh tissues in the same way as methyl green. Heela {ArcJi. Biol, xiii, 1893, p. 423) employs for ova of Ascaris a mixture of 0-25 parts vesuvin, 0-25 malachite green, 10 of glycerin and 100 of water, and washes out with weak glycerin. The chief use of this colour is for progressive staining ; but it may be employed for staining by the regressive method (see § 289), and also for intra-vitam staining (§ 208) (for this purpose it is necessary to see that the colour employed be pure and neutral). 278. Methyl Violet (Methylanilin Violet, Anilin Violet, Paris Violet).— Grasee (Deutsche Zeit. OUrurgie, xxvii, 1888, pp. 538 — 584 ; Zeit. wiss. Mik., V, 3, 1888, p. 378) stains sections from twelve to twenty-four hours in a solution so dilute that at the end of that time the sections will have taken up all the colour from the liquid. They are then washed out for a short time in acidulated alcohol, and then in pure alcohol. The method is applicable to objects fixed in Flemming's mixture. 279. Other Progressive Stains. — ^Most of the basic tar colours used for regressive staining wiU also give by the progressive method a nuclear stain of greater or less purity if used in solutions acidified with acetic acid. Amongst these may be mentioned thionin, which need not even be acidified ; also, for fresh tissues especially, gentian violet, dahlia, and iohiidin blue. B. Regressive Stains. 280. The Practice of Regressive Staining : The Staining Bath.— . Sectiotis only, or material that is thin enough to behave like sections, such as some membranes, can be stained by this method. The solutions employed are made with alcohol, water, or anilin, or sometimes other menstrua, according to the solubility of the colour. There seems to be no special object in making them with alcohol if water will suffice, the great object being to get as strong a solution as possible. Indeed, the solutions made with strong alcohol are found not to give quite such good results as those made with water or weak alcohol. Alcohol of 50 per cent, strength, however, may be said to constitute a very generally desirable medium. The sections must be very thoroughly stained in the solu- tion. As a general rule they cannot be left too long in the staining fluid. With the powerful solutions obtained with anilin a few minutes or half an hour wiU usually suffice, but to be on the safe side CHAPTER XIV. ' 163 it is frequently well to leave the sections twelve to twenty-four hours in the fluid. Up to a certain point the more the tissues are stained the better do they resist the washing-out process, which is an advantage. Some workers, indeed, prefer weak solutions ; so Heidenhain, Encycl. mik. Technik, i, pp. 433, 434 ; but the nature of the fixiag agent should be taken into account. Material fixed in chromic or chromo-osmic mixtures gives a sharper and more selective stain than material fixed in sublimate or the like. In fact, to ensure the best results, only material fixed in chromic mixtures (or Hermann's fluid) should be employed. During the staining the tissues become overstained, that is, charged with colour in an excessive and diffuse manner. The stain must now be differentiated by removal of the excess of colour. 281. Differentiation, — This is generally done with alcohol, some- times neutral, sometimes acidulated (with HCl). The stained sections, if loose (celloidin sections), are brought into a watch-glassful of alcohol ; if mounted in series on a sUde, they are brought into a tube of alcohol (differentiation can be done by simply pouring alcohol on to the sHde, but it is better to use a tube or other bath). It is in either case well to just rinse the sections in water, or even to wash them well in it, before bringing them into alcohol. The sections in the watch-glass are seen to give up their colour to the alcohol in clouds, which are at first very rapidly formed, afterwards more slowly. The sections on the shde are seen, it -the slide be gently lifted above the surface of the alcohol, to be giving off their colour in the shape of rivers running down the glass. In a short time the formation of the clouds or of the rivers is seen to be on the point of ceasing ; the sections have become pale and somewhat transparent, and (in the case of chrom-osmium objects) have changed colour, owing to the coming into view of the general ground colour of the tissues. (Thus chrom-osmium-safranin sections turn from an opaque red to a delicate purple.) At this point the differentiation is complete, or nearly so. It is generally directed that absolute alcohol be taken for differen- tiation. This may be well in some cases, but in general 95 per cent, is found to answer perfectly well. Heidenhain (Encycl., i, p. 434) takes methyl alcohol. The hydrochloric-acid-alcohol extracts the colour much more quickly from resting nuclei than from kinetic nuclei. Therefore, washing out should be done with neutral alcohol whenever it is desired to have resting nuclei stained as weU as dividing nuclei ; the acid process serving chiefly to differentiate karyokinetic figures. 11—2 164 NUCLEAR STAINS WITH COAL-TAR DYES. The proportion of HCl with which the alcohol should be acidi- fied for the acid process should be about 1 : 1000, or less ; seldom more. The length of time necessary for differentiating to the precise degree required varies considerably with the nature of the tissues and the details of the process employed ; all that can be said is that it generally lies between thirty seconds and two minutes. The acid process is vastly more rapid than the neutral process, and therefore of course more risky. There exists also a metliod of differentiation known as substitution — one stain being made to wash out another. Thus methylen blue and gentian violet are discharged from tissues by aqueous solution of vesuvin or of eosin ; fuchsin is discharged from tissues by aqueous solution of methylen blue. The second stain " substitutes " itself for the first in the general " ground " of the tissues, leaving, if the operation has been successfully carried out, the nuclei stained with the first stain, the second forming a " contrast " stain. In the paper of Resegotti in Zeit. wiss. Mik., V, 1888, p. 320, it is stated as a very general rule that colours that do not give a nuclear stain by the regressive method wiU wash out those that do. But Resegotti used the second colour in alcoholic solution ; so that it remains uncertain how far the differentiation should be attributed to the second colour itself, and how far to the alcohol used as a vehicle. The same remark applies to Benda's Safranin-and- Lichtgriin process. 282. Clearing. — After due differentiation, the extraction of the colour may be stopped by putting the sections into water ; but the general practice is to clear and mount them at once. You may clear with clove oil or anilin, which will extract some more colour from the tissues. Or you may clear with an agent that does not attack the stain (cedar oil, bergamot oil, xylol, toluol, etc. ; t see the chapter on Clearing Agents). If you have used neutral alcohol for washing out, you had perhaps better clear with clove oil, as neutral alcohol does not always, if the staining have been very prolonged, extract the colour perfectly from extra-nuclear parts. But if you have not stained very long, and if you have used acidulated alcohol for washing out, clove oil is not necessary, and it may be better not to use it, as it somewhat impairs the brilliancy of the stain. A special property of clove oil is that it helps to differentiate karyoldnetic figures, as it decolours resting nuclei more rapidly than those in division. Some colours are much more sensitive to the action of clove oil than others ; and much depends on the quality of this much- adulterated essence. New clove oil extracts the colour more quickly than old, and anilin than clove oil. CHAPTER XIV. 165 Series of sections on slides are conveniently cleared by pouring the clearing agent over them. After clearing you may either mount at once in damar or balsam,- or stop the extraction of the colour, if clove oil have been used, by putting the sections into some medium that does not affect the stain (xylol, cedar oil, etc.). Chloroform should be avoided, either as a clearer or as the menstruum for the mounting medium. 283. General Results. — The results depend in great measure on the previous treatment of the tissues. If you have given them a prolonged fixation in Flemming's stron,g chromo-aceto-osmic mixture, and have differentiated after staining with acid alcohol and cleared with clove oil, you will get, with some special exceptions, nothing stained but nucleoli and the chromatin of dividing nuclei, that of resting nuclei remaining unstained. If you have given a hghter fixation, with Flemming's weak mixture or some other fixing agent not specially inimical to staining, and have differentiated after staining with neutral alcohol, you will get the chromatin of resting nuclei stained as well. Either process may also stain mucin, the ground-substance of connective tissues (especially cartilage), the bodies of Nissl in nerve-cells, and the yolk of ova. 284. Hennegut's Permanganate Method (Journ. de I'Anat, et de la PJiysiol., xxvii, 1891, p. 397). — Sections are treated for five minutes with 1 per cent, solution of permanganate of potassium. They are then washed with water and stained (for about half the time that would have been taken if they had not been mordanted with the permanganate) in safranin, rubin, gentian violet, vesuvin, or the like, and are difEerentiated with alcohol, followed by clove oil in the usual way. The mordanting action of the permanganate is so energetic that if it has been overmuch prolonged before staining with safranin, or, still more, with rubin, it becomes almost impossible to differentiate the sections properly ; it may be necessary to leave them for a month or more in clove oil. 285. Ohlmachek's Formaldehyde Process {Medical News, February 16th, 1895). — Ohlmacher states that formaldehyde is a powerful mordant for tar colours. Tissues may either be mordanted separately by treatment for a short time (one minute is enough for cover-glass preparations) with a 2 per cent, to 4 per cent, formalin solution ; or the formalin may be combined with the stain. One grm. of fuchsin or methylen blue dissolved in 10 c.c. of absolute alcohol may be added to 100 c.c. of 4 per cent, formahn solution. Sections are said to stain in half a minute and to resist alcohol much more than is the case with those treated by the usual solutions. 286. Safranin. — One of the most important of these stains, on account of its power, brilliancy, and permanence in balsam, and the 166 NUCLEAR STAINS WITH COAL-TAR DYES. divers degrees of electivity that it displays for the nuclei and other constituent elements of different tissues. - The great secret of staining with safranin is to get a good safranin. In ordering it, from Griibler & HoUborn or elsewhere, it is well to specify whether you want it for staining nuclei or for staining elastic fibres, or for what other purpose you may require it. There are presumably at least a score of sorts of safranin in the market, differing to a considerable extent in colour, weight, solubility, and histological action. Some are easily soluble in water and not so in alcohol, some the reverse, and some freely soluble in both. The brand I have been using for a long time, which gives good results, is the " Safranin " of Grubler & Co. Staining. — The majority of safranins are not sufficiently soluble in water, so that solutions in other menstrua must be employed. Pfitzner {Morph. Jahrb., vi, p. 478, and vii, p. 291) advised a solution of safranin 1 part, absolute alcohol 100 parts, and water 200 parts, the last to be added only after a few days. Flemming (Arch. mile. Anat., xix, 1881, p. 317) used a concen- trated solution in absolute alcohol, diluted with about one-half of water. Babes {ibid., 1883, p. 356) used (a) a mixture of equal parts of concentrated alcoholic solution and concentrated aqueous solution (this is very much to be recommended), or (b) a concentrated or supersaturated aqueous solution made with the aid of heat. Some people still employ simple aqueous solutions. The anilin solution of Babes {Zeit. wiss. Mih., iv, 1887, p. 470) consists of water 100 parts, anilin oil 2 parts, and an excess of safranin. The mixture should be warmed to from 60° to 80° C, and filtered through a wet filter. This solution will keep for a month or two. Zwaardemaker {ibid., iv, 1887, p. 212) makes a mixture of about equal parts of alcoholic safranin solution and anilin water (saturated solution of anibn oil in water ; — to make it, shake up anilin oil with water, and filter). This, I find, will keep for many months, perhaps indefinitely. I myself use equal parts of saturated solution in anilin water and saturated solution in absolute alcohol. Differentiation. — ^For general directions see §§ 281 and 282. Flemming's acid differentiation {Zeit. wiss. Mile., i, 1884, p. 350). — Differentiate, until hardly any more colour comes away, in alcohol acidulated with about 0-5 per cent, of hydrochloric acid, followed by pure alcohol and clove oil. (You may use the HOI in watery GHAPTER XIV. 167 solution if you prefer it.) Or you may use a lower strength, viz. 0-1 per cent, at most (see Arch. mik. Anat., xxxvii, 1891, p. 249) ; and this I find is generally preferable. Objects are supposed to have been well fixed — twelve hours at least— in the strong chromo-aceto-osmic mixture, and stained for some hours. In this way you get kinetic chromatin and nucleoli alone stained. PoDWYSSOZKi {Beitr. z. Path. Anat., i, 1886, p. 289) differentiates (for from a few seconds to two minutes) in a strongly alcoholic solution of picric acid, followed by pure alcohol. Same results (except that the stain will be brownish instead of pure red). Babes recommends treatment with iodine, according to the method of Gram (see next section). This process has also been recommended by Prenant {Int. Monatsschr. Anat., etc., iv, 1887, p. 368). It has been shown by Ohlmachek (Journ. Amer. Med. Assoc, vol. xx. No. 5, February 4tli, 1893, p. Ill) that if tissues be treated with iodiue or picric acid after staining with safranin, there may be produced in the tissue elements a precipitate of a dark red substance of a crystalline nature, but of lanceolate, semilunar, falciform, or navioellar forms. The precipitate is formed both in normal and pathological tissue, readily in carcinomatous tissues ; and Ohlmacher concludes that many of the bodies that have been described as " coccidia," " sporozoa," or other " parasites " of carcinoma are nothing but particles of this precipitatfe. See also the differentiation process of Maetinotti and Resegotti {Zeit wiss. Mik., iv, 1887, p. 328) for aloohol-flxed material, and of Gaebini {Zeit. wiss. Mik., v, 2, 1888, p. 170). In preparations made with chromo-aceto-osmic acid, safranin stains, besides nuclei, elastic fibres, the cell bodies of certain horny epithelia, and the contents of certain gland-cells (mucin, under certain imperfectly ascertained conditions). The stain is perfectly permanent. 287. Gentian Violet may be used in aqueous solution, or as directed for safranin. In some cases it may be useful to employ the method devised by Gram for the differentiation of bacteria in tissues {Fortschr. d. Medicin., ii, 1884, No. 6 ; British Med. Journ., September 6th, 1884, p. 486 ; Journ. Roy. Mic. Soc. [N.S.], iv, 1884, p. 817). In this the sections are treated, after staining, with a solution composed of— Iodine . . • • • • 1 grm. Iodide of potassium .... 2 grms. Water 300 „ 168 NUCLEAR STAINS WITH COAL-TAR DYES. for two or ttree minutes, until they become black. They are then differentiated with neutral alcohol, until they turn grey, and are then finally differentiated with clove oil. By this process, in resting nuclei the nucleoli alone are stained, or the chromatin if stained is pale ; in dividing nuclei the chromatin is stained with great intensity, being nearly black in the equatorial stage. Gentian violet is an exceedingly powerful stain, quite as precise as safranin. The stain keeps well. It is more or less dichroic, possibly owing to the fact that the dye is not a pure substance, but a mixture of " Krystallviolett " and methyl violet. HEEMANisr {Arch. mile. Anal., xxxiv, 1889, p. 58) first stains for twenty- four hours or more in safranin, differentiates incompletely with, alcohol, then stains for three to five minutes in the anilin-water gentian solution, treats with the iodine solution for one to three hours, and finally diilerentiates with absolute alcohol. 288. Thionin. — The hydrochloride of thionin, or violet of Lauth, is a colour chemically nearly allied to methylen blue. Its action is so selective from the first that it may almost be considered to be a progressive stain. If you stain for only a short time (a few minutes) in a concentrated aqueous solution, hardly anything but the chro- matin will be found to be stained. If the staining be prolonged, plasmatic elements will begin to take up the colour. After a short stain no special differentiation is required ; all that is necessary is to rinse with water, dehydrate, and mount. After a strong stain you differentiate with alcohol in the usual way, with this advantage, that the stain is so highly resistant to alcohol that there is no risk what- ever of over-shooting the mark ; the stain will not be more extracted in an hour than that of gentian or dahlia is in a minute, so that the process may be controlled under the microscope if desired. For this reason I think this stain may be useful to beginners, but I myself prefer gentian. It is a very powerful stain. Thionin is a specific stain for mucin, q. v. Some observers have found the stain to fade. Wolff {Zeit. wiss. Mik., xv, 1899, p. 312) says that, to avoid this, preparations should be mounted in a little solid coIo' phonium or balsam melted over a flame. Felizat and Beanca (Journ. Anat. PJiys., xxxiv, 1898, p. 590) mount without a cover. Hennegtjt (in litt.) clears with acetone. King [Anat. Becord, iv, 1910, p. 236) stains with a saturated solution in carbolic acid of 1 per cent., and finds the stain permanent. Nicolle's "thionine ph6niqu4e " consists of 1 part of saturated solution in alcohol of 60 per cent., and 6 parts of 2 per cent, aqueous solution of carbolic acid. CHAPTER XIV. 169 289. Other Regressive Stains. — The following may be useful : — Dahlia, according to Flemming {Arch. mih. Anat., xix, 1881, p. 317), best used in aqueous solution, either neutral or acidified with acetic acid, and differentiated with neutral alcohol. A pure blue stain, which keeps well. See also Schuberg, in Zeit. wiss. Zool., Ixxiv, 1903, p. 7, and Ixxxvii, 1907, p. 557. Victoria Blue (Victoriablau) (Lustgarten, Med. Jdhrh. k. Ges. d. Aerzte zu Wien, 1886, pp. 285— 291).— This dye ("Victoriablau 4 A ") has a special affinity for elastic fibres. For this object Lust- garten recommends an alcoholic solution of the dye diluted with 2 to 4 parts of water. Fixation in chrom-osmium, or at least in a chromic mixture, is, I believe, a necessary condition to this reaction. And you must stain for a long time. Victoria has also a special affinity for mucus-cells, from which it is not washed out by alcohol, and for cartilage. This stain keeps very well. With Toluidin Blue I have had some superb stams of chromatin, unfortunately accompanied by a difEuse staining of cytoplasm. Mann {Zeit. wiss. Mih., xi, 1894, p. 489) states that he has had good results by staining with it after eosin. See further, as to the micro -chemical properties of this dye, Hakeis, The Philadelpjiia Medical Journal, May 14th, 1898. It much resembles methylen blue. Metznek (Nagel's Eandb. Phys., ii, 1907, p. 915) mordants sections, before staining, for three-quarters of an hour in iron alum. Magdula Red (Naphthalin Red, Rose de Naphthaline). Fuchsin (meaning the basic fuchsins, a series of Eosanilin salts having very similar reactions, and found in commerce imder the names of Fuchsin, Anilin Eed, Eubin, Eosein, Magenta, Solfeeino, Coeallin). — Geasee (Deutsche Zeit. Ohirurgie, xxvii, 1888, pp. 538 — 584 ; Zeit. wiss. Mih., v, 1888, p. 378) stains for twelve to twenty-four hours in a dilute aqueous solution, washes out for a short time in alcohol, stains for a few minutes in aqueous solution of methylen blue, and dehydrates with alcohol. A double stain. Chromatin and nucleoli red, all the rest blue. Ziehl's Carbolic Fuchsin {Zeit. wiss. Mile, vii, 1890, p. 39) consists of fuchsin 1 grm., acid, carbol. crist. 5 grms., alcohol 10 grms., aq. dest. 100 grms. The stain is differentiated with alcohol followed by clove oil. Kresofuchsin (Eothig, Arch. mih. Anat, Ivi, 1900, p. 364). — Its aqueous solution is red and stains mucus, cartilage, keratin, and nuclei red, whilst its alcoholic solution is blue and stains elastin blue. See also under " Connective tissues." Bismarck Brown has this advantage, that being sufBciently resistant to alcohol it may be utihsed for staining entire objects. 170 NUCLEAR STAINS WITH COAL-TAR DYES. Kaiser {Bihliofh. Zool., H. 7, 1 Halft, 1891 ; Zeit. viiss. Mik., viii, 1891, p. 363) stains for forty-eight hours, and at a temperature of 60° C. in saturated solution of Bismarck brovra in 60 per cent, alcohol (the solution to be made in boiling alcohol), and washes out (until aU is decoloured except the karyokinetic figures) in 60 per cent, alcohol, containing 2 per cent, hydrochloric acid or 3 per cent, acetic acid. Methyl Violet. See ante, § 278. Benzoazurin (Martin, Zeit. wiss. Mile., vi, 3, 1889, p. 193). — Stain for an hour or so in dilute aqueous solution, and wash out with HCl alcohol. Methylen Blue. Nigrosin (Errbra, Proc.-Verb. Soc. Beige de Mik., 1881, p. 134) gives a good stain which resists alcohol weU. Methyl Green is sometimes useful in certain mixtures (see next chapter). CHAPTER XV. PLASMA STAINS* WITH COAL-TAR DYES. 290. Introduction. — By a plasma stain is meant one that stains the extra-nuclear parts of cells and the formed material of tissues, or one of these. The plasma stains described in this chapter are for the most part those obtained by means of " acid " dyes (§ 201) ; but some of them are obtained by means of " neutral " dyes (§ 201), and a few by " basic "dyes. The mode of staining is generally progressive, almost always so when acid colours, used substantively (§ 205), are employed. But the regressive method, with differentiation, is sometimes made use of, especially when a mordant has been used with the dye. In some processes, e.g., Flemming's orange method, a basic and an acid dye (or vice versa) being employed in succession, there is formed in the tissues a neutral colour (§ 201) which effects the desired stain. These may be considered as adjective stains, the first colour serving as a mordant for the second. Not any two dyes taken at haphazard will behave in this way : they must be such as to form by combination a suitable neutral lake (cf. § 201). The basic dye may be made the primary stain, as in Flemming's process : or the contrary. In such stains as Reinke's orange method, or the EhrHch-Biondi mixture, and many others, one or more neutral colours are formed in the mixture and stain progressively. Excepting Biebrich scarlet, I am not acquainted with any plasma stain that is thoroughly satisfactory for delicate work. In addition to Biebrich scarlet, I recommend for sections Saurefuchsin, either alone or in the form of Ehdich-Biondi mixture, or Ehrlich's triacid : for material in bulk, picric acid (but only for rough work). 291. Saurefuchsin (Acid Fuchsin, Fuchsin S, Acid Rubin, Rubin S, Saurenibin, Acid Magenta, Magenta S).— -The chemical descrip- * This chapter includes only such stains as are used in ordinary work on tissues in bulk or sections, stains for special purposes being treated under " Nervous tissue," " Blood," etc. It includes some double or triple stains that affect nuclei as well as plasma, but in different hues. 172 PLASMA STAINS WITH GOAL-TAR DYES. tion of this acid colour has been given (§ 201) : it must not be confounded with basic fuchsin, as seems to have been done by some writers. This dje is highly soluble in water, less so in alcohol. I use a 0-5 per cent, solution in water and allow it to act on sections for a few minutes in the case of easily stainable material, or twenty-four hours or more for chrom-osmium material. The stain is fast to neutral alcohol. It is very sensitive to alkalies, so that overstains can easily be removed by washing for a few minutes in tap-water. Acids strengthen the stain, so that it is frequently useful to treat sections after staining for a few seconds with acidulated water. A good stain should show the reticulum of c3i}oplasm, together with nuclear spindles and asters, stained red, and connective tissue strongly brought out. It may be advisable to acidify the staining bath very slightly. Successful stains are admirably sharp. 292. Pyronin. — A basic dye, red, only used (as far as I can find) in mixtures. Pappenheim {Arch. Path. Anat., clxvi, 1901, p. 427) takes 2 parts 1 per cent, solution of methyl green and 1 part 1 per cent, solution of pyronin, stains sections for five minutes, rinses, and differentiates in a solution of resorcin or hydroquinon in absolute alcohol. According to Corti and Ferrara, Mon. zool. Ital., xvi, 1905, p. 319, this mixture generally stains chromatin green and cytoplasm red, but in Memming or Hermann material the reverse. It seems to me a coarse plasma stain, but likely to be sometimes useful. Unna's carbol-pyronin-methyl green modification {Encycl. Mik. Tech., 1910, ii, p. 412 : I am indebted for the formula to Dr. Gaudlitz) is as follows : Stain for five to ten minutes at 30° to 40° C. ' in methyl green 0-15 parts, pyronin 0-25, alcohol 2-5, glycerin 20, and carbolic acid of 0-5 per cent, to make up 100 volumes. Cool rapidly, rinse, dehydrate, and pass through bergamot oil, or xylol or benzol (not clove-oil), into balsam. Brings out bacteria (red) in organic liquids. The mixture may be had from Griibler & HoUborn. 293. Orange G.— This is the benzenazo-beta-naphthol-disul-- phonate of soda. As indicated by its chemical description, this is an " acid " colour. It is easily soluble in water, less so in alcohol. Use as directed for Saurefuchsin. Almost, if not quite, as precise a stain as Saure- fuchsin. It does not overstain, but may wash out other dyes. 294. Saurefuchsin and Orange G.— I have had good results by mixing the aqueous solutions of these two dyes, but unfortunately CHAPTER XV. 173 have not noted the proportions. Squieb (Methods and Formulce, p. 42) takes 1 grm. Sauiefuchsin, 6 grms. Orange G in 60 c.c. of alcohol and 240 c.c. of water. See also under " Connective tissues." 295. Ehblich-Biondi Mixture (or Ehrlich-Biondi-Heidenhain Mixture) (Pfluger's Arch., xUii, 1888, p. 40). To 100 c.c. saturated aqueous solution of orange add with con- tinual agitation 20 c.c. saturated aqueous solution of Saurefuchsin (Acid Fuchsin) and 50 c.c. of a hke solution of methyl green. (According to Krause (Arch. mik. Anat, xlii, 1893, p. 59), 100 parts of water will dissolve about 20 of Saurefuclisin (Eubin S), 8 of orange G and 8 of methyl green.) The solutions must be absolutely saturated, which only happens after several days. Dilute the mixture with 60 to 100 volumes of water. The dilute solution ought to redden if acetic acid be added to it ; and if a drop be placed on blotting-paper it should form a spot bluish green in the centre, orange at the periphery. If the orange zone is surrounded by a broader red zone, the mixture contains too much fuchsin. According to M. Heidenhain (" Ueber Kern u. Protoplasma," in Festschr.f. KolUker, 1892, p. 115) the orange to be used should be " Orange G," the Acid Fuchsin or Saurefuchsin should be " Eubin S " (" Eubin " is a synonym of Fuchsin) and the methyl green should be " Methylgriin 00." And it is ahsolutely necessary that these ingredients be those prepared imder those names by the Actimfabrikfilr Anilin-fabrikation in Berlin. They can be obtained from Griibler & Hollborn, either separately, or as a mixture of the three dyes in powder (which I do not recommend). The strong solutions directed to be taken readily precipitate on being mixed. To avoid this it is recommended by Squire {Methods and FormulcB, etc., p. 37) to dilute them before mixing. Other proportions for the mixture have been recommended by Keause {loe. eit. supra), viz. 4 c.c. of the Saurefuchsin solution, 7 of the orange G and 8 of the methyl green ; the mixture to be diluted 60 to 100-fold with water. Thome (Areh. mik. Anat, lii, 1898, p. 820) gives the proportions 2:5:8, and dilutes 100-fold. Stain sections (N.B. sections only) for six to twenty-four hours. Dehydrate with alcohol, clear with xylol, and moimt in xylol balsam. In the intention of the observers who have elaborated this stain it is a progressive stain, and not a regressive one. It does not require any differentiation, and the sections should be got thro.ugh the alcohol into xylol as quickly as possible in order to avoid any extrac- 174 PLASMA STAINS WITH GOAL-TAR DYES. tion of the metliyl green, which easily comes away in the alcohol. Druner {Jena Zeit., xxix, 1894, p. 276) stains for ten minutes in the concentrated solution, treats for one minute with alcohol con- taining 0-1 per cent, of hydrochloric acid, and then with neutral alcohol. The best results are obtained with sublimate material ; chrom- osmium material, and the like, give a much inferior stain. Prepara- tions made with the usual mixture, as given above, are liable to fade ; by acidifying the mixture a stronger and more sharply selective stain is obtained, which does not fade. But too much acid must not be added, as this would cause a staining of the interfilar substances. According to the Encycl. mik. Technih, you may add 15 to 24 drops of 0-2 per cent, acetic acid to 100 c.c. of the diluted solution. Another process of acidification is given by M. Heidenhain (Ueher Kern und Protoplasma, p. 116) ; for this.sse fourth edition. See also ISKAEL {PraUihum Path. Eist., 2 Aufl., Berlin, 1893, p. 69) ; Trambusti (Bicerche Lab. Anal. Boma, v, 1896, p. 82 ; Zeit. wiss. Mik., xiii, 1896, p. 357) ; and Thome {op. eit. supra). Eisen {Proe. Calif. Acad. (3), i, 1897, p. 8) acidifles with oxalic acid. After acidification the solution must not be filtered, and if it has been kept for some time a little more acid must be added. Before staining (M. Heidenhain, loc. cit.), sections should be treated for a couple of hours with 0*1 per cent, acetic acid, then for ten to fifteen minutes with officinal tincture of iodine, and be rinsed with alcohol before bringing into the stain. The treatment with acid is necessary in order to ensure having the sections acid on mounting in balsam. The primary object of the iodine is to remove any subhmate from the preparations, but it also is said to enhance the power of staining of the chromatin with methyl green, and to produce a more selective staining of protoplasmic elements. The stain is a very fine one when successful. But it is very capricious. The correct result should be a precise chromatin stain combined with a precise stain of the plastin element of cytoplasm by the Saurefuchsin. Now the least defect or excess of acidity causes the plasma stain of the Saurefuchsin to become a diffuse one, instead of being sharply limited to the plastin element. It is difficult to dehydrate the sections without losing the methyl green. For this reason the stain will only work with very thin sections ; to be quite sure of good results, the sections should be of not more than 3 IX in thickness, and if they are over 6 the desired results are almost hopeless. The stain keeps very badly. I admit that the CHAPTER XV. 175 method has its raison d'Stre for the very special objects for which it was imagined — for the researches on cell-granulations for which Ehrlich employed the three colours, or for the researches on the plastin element of c3rtoplasm for which Martin Heidenhain em- ployed the mixture ; for the study of gland cells ; and for similar objects. But to recommend it, as has been done, as a general stain for ordinary work, is nothing but mischievous exaggeration. For it is far from having the qualities that should be possessed by a normal section stain. Workers have at length found this out, and it is now but little used except for the special purposes above indicated. 296. Ehrlich's " Tiiacid " Mixture. — This name would seem to indicate that the mixture contains three " acid " colours, which is not the case, methyl green being a strongly " basic " colour. Ehrlich explains in a letter to Mayer (see also Ehrlich and Lazarus, Die Ancemie, 1898, p. 26) that it is so called " because in it all the three basic groups of the methyl green are combined with the acid dye- stufEs." A very pretty conundrum ! The latest receipt {op. cit., p. 28) is as follows : Prepare separately saturated solutions of orange G-, Saurefuchsin, and methyl green, and let them clarify by settling. Then mix, in the order given, using the same measure-glass, 13 to 14 c.c. of the orange, 6 to 7 of the Saurefuchsin, 15 of distilled water, 15 of alcohol, 12^ of the methyl green, 10 of alcohol, and 10 of glycerin. After adding the methyl green, shake well, but do not filter. The mixture keeps well. I find its qualities and defects to be much those of the Ehrlich-Biondi mixture. The stain seems more powerful but less delicate, and the methyl green in it appears to have more resistance to alcohol, so that it is better adapted for ordinary work. Mater {Orundeiige, Lee and Mayer, p. 197) has simplified the foimtda thus : Take 1 g. methyl green, 2 g. orange, 3 g. Saurefuchsin, and dissolve in a mixture of 45 c.c. water, 10 c.c. glycerin, and 20 c.c. alcohol of 90 per cent. Morel and Doleris {0. B. Soc. Biol., liv, 1902, p. 1255) mjg; 1 vol. of the SDlution with one of 8 per cent, formalin and add 0-1 per cent, of acetic acid, and state that thus the methyl green is better fixed in the nuclei. 297. Pianese's Saurefuchsin-malachlte Green (from M^tjllek, Arch. Zellforsoh., viii, 1912, p. 4) consists of 0-5 grm. malachite green, 0-1 grm. Saurefuchsin, and 0-01 grm. Martins yellow in 150 c.c. water and 50 c.c. alcohol. Stain for twenty-four hours, difEerentiate with alcohol, containing 1 to 2 drops of HOI per 200 c.c. 176 PLASMA STAINS WITH COAL-TAR DYES. 298. Picric Acid. — Picric acid gives useful plasma stains after carmine and lisematoxylin. The modus operandi consists merely in adding picric acid to the alcohols employed for dehydrating the objects. Picric acid has considerable power of washing cut other anilin stains ; and in combination with hydrochloric acid it very greatly enhances the power with which this acid washes out carmine stains. It should, therefore, not be added to the acidulated alcohol taken for differentiating boraz-carmine stains, or the like, but only to the neutral alcohol used afterwards. It has the great quahty that it can be used for staining entire objects, and is much indicated for such objects as small Arthropods or Nematodes, mounted whole. It can in some cases be employed by dissolving it in the solution of another dye (see Picro-carmine, Legal's alum-carmine, § 221, etc.) ; or (for sections) by dissolving it in the xylol or chloroform used for clearing. Though picric acid is a useful ground stain, it is at most a rough one, being very diffuse. It stains, however, horn, chitin, muscle and eryi}hrocytes, with special energy. According to Feohlich {Zeit. wiss. Mile., xxvii, 1910, p. 349) picraminic acid (from Griibler & Hollborn) has some advantages over picric acid. 299. Van Gibson's Picro-Saurefuchsin (from Zeit. wiss. Mik., xiii, 1896, p. 344). — To a saturated aqueous solution of picric acid are added a few drops of saturated aqueous solution of Saurefuchsin, until the mixture has become garnet-red. Or [Trans. Amer. Micr. Soc, xix, 1898, p. 105) to 100 parts of the picric acid solution add 5 parts of 1 per cent, solution of Saurefuchsin. After staining - (sections only), rinse with water, dehydrate, and clear in oil of origanum. Ohlmachee (Journ. Exper. Med., ii, 1897, p. 675) adds 0-5 per cent, of Saurefuchsin to a saturated solution of picric acid which has been diluted with an equal quantity of water. He uses this after previous staining with gentian violet. Ramon y Cajal recommends 0-1 grm. of Saurefuchsin to 100 of saturated solution of picric acid (Schaffer, Zeit. wiss. Zbol., Ixvi, 1899, p. 236). Hansen [Anat. Anz., xv, 1898, p. 152) adds 5 c.c. of 2 per cent, solution of Saurefuchsin to 100 c.c. saturated solution of picric acid, and for staining adds to 3 c.c. of the mixture one-third of a drop of 2 per cent, acetic acid, stains for a few minutes or hours, rinses in 3 c.c. of water with 2 drops of the acidified stain added, dehydrates CHAPTER XV. 177 clears with xylol, and mounts in xylol-balsam. Connective-tissue red, elastin and all other elements yellow. Weigeet {Zeit. wiss. MiL, 1904, p. 3) adds 10 parts of 1 per cent. Saurefuchsin to 100 of saturated picric acid. See also Mollee, op. cit., xv, 1898, p. 172. This stain is generally used as a contrast stain to follow heemato- xylin. Apathy {Behrens' Tabelkn, 3rd ed., p. 129) takes for this purpose 1 grm. of Saurefuchsin in 500 c.c. of saturated solution of picrate of ammonia. WiLHELMi (Fauna Flora Golf. Neapel, xxii, 1909, p. 18) takes 0-2 grm. Saurefuchsin, 0-8 grm. picrate of ammonia, 10 grm. absolute alcohol, and 89 grm. water. E. and T. Savini (Zeit. wiss. Mile, xxvi, 1909, p. 31) use a formula due to Benda. Ninety-five volumes of saturated solution of picrate of ammonia are mixed with 5 volumes of 1 per cent, solution of Saurefuchsin. For use, two to four drops of saturated solution of picric acid are added to 10 c.c. of the mixture. This neither over- stains nor attacks the primary stain. 300. Flbmming's Orange Method (Arch. mih. Anat, xxxvii, 1891, pp. 249 and 685). — Stain sections of Flemming or Hermann material in strong alcoholic safranin solution diluted with anilin water (§ 286) ; differentiate in absolute alcohol, containing at most 0-1 per cent, of hydrochloric acid, until hardly any more colour comes away ; stain for one to three hours in gentian violet (§ 287) ; wash for a short time in distilled water ; treat with concentrated, or at least fairly strong, aqueous solution of orange G. After at most a few minutes, whilst pale violet clouds are still being given o& from the sections on agitation, bring them into absolute alcohol until hardly any more colour comes away, clear in clove or Bergamot oil, and mount in damar or balsam before the last pale clouds of colour have ceased to come away. The orange must be orange G. Winiwarter and Sainmont (Zeit. wiss. Mih., xxv, 1908, p. 157, and Arch. Biol., xxiv, 1909, p. 15) stain for twenty -four hours in the gentian, wash out after the orange for two to three hours in 100 c.c. absolute alcohol with 3 to 4 drops of HCl, and differentiate finally with oil of cloves. This is not a triple stain in the sense of giving three different colours in the result ; it is a nuclear and plasmatic stain in mixed tones ; the orange, apparently, combines with the gentian to form a "neutral" dye, soluble in excess of the orange (§ 203) which thus differentiates the stain. See also Flemmincs in Arch. Anat. Phys. Anat. Abth., 1897, p. 175. Never popular, this clumsy and uncertain process is now little used. 301. Eeinke's Orange Method (Arch. mih. Anat, xliv, 2, 1894, p. 262), — ^To a concentrated aqueous solution of gentian violet are added " a few props " of a like solution of orange G. The solution precipitates in part. M. 12 178 PLASMA STAINS WITH COAL-TAR DYES. owing to the formation of an imperfectly soluble " neutral " colour, but becomes almost clear again if an excess of water be added. The solution is not to be filtered, but the sections are to be stained in the mtKture made almost clear by addition of water. It is said that the " neutral " solution may be preserved for future use by adding to it one third of alcohol. After staining (sections previously stained with safranin), you difierentiate rapidly with alcohol and clear with clove oil. I have tried this process and obtained exactly the same results as with Flemming's process, and so have other workers. Arnold's Orange Method {Arch. Zellforsch, iii, 1909, p. 434).— Sections (of chrome material) are treated for five minutes with solution of equal parts of iodine and iodide of potassium in alcohol of 40 per cent., then washed and stained for four hours in saturated solution of safranin in alcohol of 75 per cent. : then washed and put for five to fifteen minutes into solution of 7 parts of methylen blue, 0-5 of carbonate of soda and 100 of water, washed, dehydrated, and treated until pale blue with solution of orange G in oil of cloves. Cytoplasmic reticulum blue on orange ground, nucleoli and centrosomes red. Instead of the safranin, basic fuchsin may be taken. 302. Bonnet's Triple Stain (Virchow's Arch., cxciii, 1908, p. 547, and elsewhere).— Stain sections (of acetic alcohol or sublimate material, not chrome or formol material) for two minutes in a solution of 0-25 parts methyl violet and 1 part pyronin in 100 of water. Wipe slide dry, and flood twice with the following : 2 per cent, aqueous solution of orange G, boiled and filtered, is added drop by drop to 100 c.c. of acetone, with agitation, until there is formed a flocculent precipitate, which redissolves on further addition of the orange. Wash rapidly in pure acetone, and pass through xylol into balsam. Chromatin violet, cytoplasm red, connective- tissue yellow, keratin violet. Not adapted for blood films. 303. Bordeaux R. — An " acid " dye, giving a general stain taking * effect both on chromatin and cytoplasm, and, I consider, a very good plasma stain. I use for chrom-osmium material a 1 per cent, solution, and stain for twelve to twenty-four hours. The stain is sufficiently fast. 304. Bordeaux R, Thionin, and Methyl Green (Geaberg, Zeit. wiss. Mile., xiii, 4, 1896, p. 460). 305.. Congo Red (Congoroth) (see Griesbach, in Zeit. wiss. Mik, iii, 1866, p. 379).— An " acid " colour. Its solution becomes blue in presence of the least trace of free acid (hence Congo is a valuable reagent for demonstrating the presence of free acid in tissues ; see the papers quoted he. cit.). A stain much of the same nature as Saurefuchsin. It is useful for staining some objects during life (see ante, § 208). Carnoy {La Cellule, xii, 1897, p. 216) has had very CHAPTER XV. 179 good results with it after hsematoxylin of Delafield. He used 0-5 per cent, solution in water. Note that this colour is not to be confounded with other Congos, as Congo yellow, or brilliant Congo. It is one of the azo dyes. 306. Congo-Corinth. — ^Also an acid dye. Heidenhain (Zeit. viiss. Mih., XX, 1903, p. 179) recommends Congo-Corinth. G- (or the allied colour Benzopurpurin 6 B) (Elberfelder Farbwerke). Sections must be made allcaline before staining, by treating them with very weak sal ammoniac or caustic soda, in alcohol. After staining, pass through absolute alcohol into xylol. Used after alum lisemotoxylin, the stain of which it does not cause to fade. 307. Benzopuipurin. — ^According to Griesbach (toe. dt, § 305), another " acid " colour very similar in its results to Congo red. See also ZscHOKKE {ibid., v, 1888, p. 466), who recommends Benzopurpurin B, and says that weak aqueous solutions should be used for staining, which is effected in a ■ few minutes, and alcohol for washing out. Deltapurpurin may be used in the same way. See last § as to the necessity of alkalising the sections, which Heiden- hain states is necessary with all dyes of this group. 308. Neutral Red (Neutralroth) (Ehelich, Allg. med. Zeit, 1894, pp. 2, 20 ; Zeit. wiss. Mik., xi, 1894, p. 250 ; Galeotti, ihid., p. 193). — A " basic " dye. The term " neutral " refers to the hue of its solution. Its neutral red tint is turned bright red by acids, yellow by alkalies. The stain in tissues is in general metachromatic, nuclei being red, cell-bodies yellow {cf. Eosin, in Deutsche med. Wochenschr., xxiv, 1898, p. 615 ; Zeit. wiss. Mik., xvi, 2, 1899, p. 238). Up to the present this colour has chiefly been employed for intra-vitam staining. Tadpoles kept for a day or two in a solu- tion of 1 : 10,000 or 100,000 absorb so considerable a quantity of the colour that all their tissues appear of a dark red. The stain is limited to cytoplasmic granules (Ehelich), and to the contents of mucus cells (Galeotti). According to Ehelich and Lazarus (Spec. Pathol, und Therapie, herausgeg. von Nothnagel, viii, 1, 1898, p. 1 ; Zeit.f. iviss. Mik., XV, 3, 1899, p. 338) it may be used for intra-vitam staining of tissues in the same way as methylen blue, by injection or immersion with contact of air. It is especially a granule stain. Similar results are recorded by Aenold {Anat. Anz., xvi, 1899, p. 568, and xxi, 1902, p. 418). See also Ehelich and Lazarus, Ancemie, i, 1898, p. 85 ; Loisel (Journ. de I'Anat. et de la Physiol., 1898, pp. 197, 210, 217) (intra-vitam staining of sponges) ; and Prowazek (Zeit. wiss. Zool., Ixii, 1897, p. 187) (intra-vitam staining of Protozoa). I myself have had very good results with it as an intra^itam stain. According to Golovine (Zeit. imss. Mik., xix, 1902, p. 176), the 12—2 180 PLASMA STAINS WITH COAL-TAR DYES. stain may be fixed in the tissues by means of sublimate, chromic acid, bichromates, picric acid, or platinum chloride, followed by molybdate of ammonium. It has also been found useful for staining, in hardened material, the corpuscles of Nissl (q. v.) in nerve-cells. S. Mayer {Lotos, Prague, 1896, No. 2) states that it also stains degenerating myelin. The solutions that have been employed for staining fixed material are strong aqueous ones, 1 per cent, to concentrated. 309. Biebrich Scarlet (British Dyes, Ltd., Huddersfield).— A. K. Gordon (British Medical Journ., 1917, p. 828) finds this an excellent acid dye which never overstaina and is not diffuse. Use in 1 per cent, solution, or as recommended by Scott (§ 676). I have used this stain a good deal and find it better than any other plasm dye that I have tried. 310. The Bosins, found in commerce under the names of Eosin, Saffrosin, Primerose Soluble, Phloxin, Bengal Rose, Erythrosin, Pyrosin B, Rose B, k I'Eau, etc., are all " acid " phthalein colours. They are not quite identical in their properties. Most of them are soluble both in alcohol and in water, but some only in alcohol (" Primerose a VAlcool "). They are all diffuse stains, formerly much used as contrast stains, less so now. Hansen (Anal. Hefte, xxvii, 1905, p. 620) adds 1 drop of acetic acid of 2 per cent, to 9 c.c. of 1 per cent, eosin, which makes the stain more selective. For Bengal Rose see Griesbach, Zool. Am., 1883, p. 172. Eosin is a specific stain for red blood-corpuscles, and also for certain granules of leucocytes (see under " Blood "). The yolk of some ova takes the stain strongly, so that it is useful in some embryological researches. 311. Ehrlich's Indulin-Aurantia-Eosin, or Acidophilous Mixture, or Mixture C, or Mixture for Eosinophilous Cells (from the formula kindly sent me by Dr. Grubler). — Indulin, aurantia, and eosin, of each 2 parts ; glycerin, 30 parts. This gives a very thick, syrupy solution. To use it, cover-glass preparations majr be floated on to it ; or sections on slides may have a few drops poured on to them, the slide being laid flat till the stain has taken effect (twenty-four hours for Flemming material). I find that with Flemming material it gives a powerful and good stain, which is much more resistant to alcohol than that of the Ehrlich-Biondi mixture, and is, therefore, much more adapted to ordinary work. The stain keeps well. ISEAiiL (Prahtik. Path. Hist, Berlin, 1893^ p. 68) gives a more complicated receipt. ■ CHAPTER XV. 181 312. Methyl Green and Eosin (Calbeela, Morph. Jahrb., iii, 1877, Heft 3, p. 625 ; List, Zeit. wiss. Mik., ii, 1885, p. 147 ; Balbiani, Ann. Microgr., Paris, vii, 1895, p. 245 ; Rhumblek, Zeit. wiss. Zool., Ixi, 1895 , p. 38). — See early editions. 313. Methylen Blue and Eosln (Chenzinsky, quoted from Zeit. wiss. Mik., xi, 2, 1894, p. 269). Methylen blue, sol. sat. in water . . .40 Eosin, 0-5 per cent, in 70 per cent, alcohol . 20 Distilled water, or glycerin . . . .40 This solution wiU only keep for about eight days. PiANESE {ibid., xi, 1894, p. 345) adds a considerable proportion o£ carbonate of lithia. See also the mixture of Bremek (Aroh. mik. Anat., xlv, 1895, p. 446). I have tried Chenzinskt's mixture as a tissue stain, without good results ; but see Eosin, Berliner klin. Wochenschr., 1898, p. 251 ; Zeit. wiss. Mik., xvi, 1899, p. 223, and xvii, 1900, p. 333. See also Laxjkent {Centralb. ally. Path., xi, 1900, p. 86 ; Zeit. wiss. Mik., xvii, 1900, p. 201). 314. Maxloet's Eosin and Methylen Blue (Journ. Med. Besearcli, January, 1904). — Sections of Zenker material (other sublimate material not so good) are stained for half to three quarters of an hour at 56° C. in 5 per cent, aqueous solution of eosin, rinsed and flooded with solution of 1 part of methylen blue, and 1 of potassium carbonate in 100 of water, diluted with about 7 parts of water. After forty minutes they are flooded (not washed) with water, and differentiated for about five minutes in alcohol of '95 per cent. Absolute alcohol, xylol, balsam. 315. Other Eosin and Methylen-blue Stains. — For some very important ones see under " Blood." 316. Light Green (Lichtgriin S. F.). — An " acid " colour, soluble in alcohol, and a good plasma stain. Benda {Verh. physiol. Ges. Berlin, December ISth, 1891, Nos. 4 u. 5) stains sections for twenty-four hours in anilin-water safranin solution, then for about half a minute in a solution of 0-5 grm. Lichtgriin or Saureviolett (G-riibler) in 200 c.c. of alcohol, dehydrates and mounts in balsam. This process gives a very elegant stain, but requires very thin sections, and there is always risk of the safranin being washed out. The Lichtgriin stain unfortunately does not keep at aU well. See also Peenant, Arch. mik. Anat., vii, 1905, p. 430, and GuiEYSSE, C.R. Soc. Biol, Ixii, 1907, p. 1212. 317. Janus Green (Michaelis, Arch. mik. Anat., Iv, 1900, p. 665). — Used in solution of 1 : 30,000 for staining certain granules (pancreas, salivary glands, etc.) in the fresh state. 318. Malachite Green (syn. Solid Green, Victoria Green, New Green, Benzoyl Green, Fast Green). — ^A basic colour, which has been used as a 182 PLASMA .STAINS WITH COAL-TAR DYES. plasma stain for the ova of Ascaris by van Beneden and Nett. These authors used it for glycerin preparations ; it can hardly be got into balsam. Flemming {Arch. mik. Anat., xix, 1881, p. 324) attributes to it a special affinity for nucleoli. 319. Iodine Green ("Hofmann's Griin "), see Geiesbacii {Zool. Am:., No. 117, vol. v, 1882, p. 406). — Stain essentially that of methyl green, but plasma often violet through the presence of a violet impurity (Mater, Mitth. Zool. Stat. Neipel, xii, 1896, p. 311 ; see also earUer editions). It is now only used by botanists. 320. Thiophen Green (Thiophengrlin), see Kea^se, Intern. Monatsschr. Anat, etc., iv, 1887, Heft 2. 321. Coerulein S., a green " acid " dye, is recommended for the staining of muscle-fibrils by M. v. Lenhossiiik (Anat. Anz., xvi, 1899, p. 339). See also Heidenhain, ihid., xx, 1901, p. 37, and Kawitz, ibid., xxi, 1902, p. 554. 322. Quinolein Blue (Cyanin, Chinolinblaii ; v. Kanviee, Traits, p. 102). — Quinolein is said by Ranvier to have the property of staining fatty matters an intense blue. It is useful for staining Infusoria, which in dilute solution it stains during Ufe. See the methods of Certes. Prom the reactions mentioned by Eanvier it would seem that his "bleu de quinol6ine" is not the preparation that usually goes under that name. See Ehrlich, in Arch. mile. Anat., xiii, 1877, p. 266. 323. Indulin and Nigrosin, — Indulin, Nigrosin; Indigen, Coupler's Blue, Fast Blue R, Fast Blue B, Blackley Blue, Guernsey Blue, Indigo substitute are the names of brands of a group of dyes, mostly"" acid," related to the base violanUin. According to Beheens the name Indulin is generally given to a bluish brand, and that of Nigrosin to a blacker one. Nigrosin, used with sublimate material, I find stains both nuclei and cytoplasm, the chromatin strongly. It will not give the stain at all with chrom-osmium material. According to Calbeela (Morph. Jahrb., iii, 1877, p. 627) the concen- trated aqueous solution of Indulin should be diluted with 6 volumes of water. Sections wiU stain in the dilute solution in five to twenty minutes. He also says that it never stains nuclei ; the remaining cell- contents and intercellular substance are stained blue. This seems to me to be, roughly, correct. 324. Safranin and Nigrosin (or Indigo-Carmine) (Eossinski, Zeit. wiss. Mik., vi, 1880, p. 61). — See early editions. 325. Picro-Nigrosin, Pfitzee {Deutsch. Botan. Geselhch., 1.883, p. 44) dissolves nigrosin in a saturated solution of picric acid in water, and uses it for fixing and staining at the same time, on the slide. See also under " Connective Tissues." 326. Anilin Blue. — Under this title are comprised various " basic " derivatives of the base rosanilin. They occur under the names CHAPTER XV. • 183 Spirit Soluble Blue (Bleu Alcool), Gentian Blue 6 B, Spirit Blue 0, Opal Blue, Bleu de Nuit, Blue Lumi^re, Parma Bleu, Bleu de Lyon. Some authors give the name Bleu de Nuit and Griindstichblau as synonyms of Bleu de Lyon. The Encycl. mik. Technih. says it is " AniUnblau B — 6 B," with many synonyms, or designations of brands, Parma blue being " AniUnblau Eor 2 R." I find it a fairly good stain, giving very good differentiations of nerve-tissue and of cartilage (as has already been pointed out by Baumgarten and by Jacoby). Maurice and Schulgin stain in bulk with it after borax- carmine, using a very dilute alcoholic solution. Baumgarten and Jacoby stain sections in a 0-2 per cent, alcoholic solution. ToNKOFF {Arch. mik. Anat., Ivi, 1900, p. 394) adds a little tincture of iodine to the solution of the dye, or mordants the sections with iodine. Skrobansky {Intern. Monatsschr. Anat., xxi, 1904, p. 20) uses it in water with picric acid. 327. Carmine Blue (Bleu Carmln Aqueux, from Mesiter, Lucius, and Brunig, at Hochst-a-M.). — Janssens (La Cellule, ix, 1893, p. 9) states that this colour possesses a special affinity for the parts of cytoplasm that are undergoing cuticular differentiation. He uses it in alcoholic solution acidifled. 328. Methyl Blue. — Under this title are comprised some other derivatives of the base rosanihn. They are " acid " colours. Here belong Methyl Blue, Cotton Blue, Water Blue (Wasserblau), Methyl Water-Blue, China Blue (Chinablau), Soluble Blue. Amongst these Water Blue (Wasserblau) possesses some useful properties. According to Mitrophanow (quoted from Zeit. wiss. Mik., V, 1888, p. 513), used in concentrated aqueous solution it gives a very good double stain with safranin. It is very resistant to alcohol. Using the Wasserblau first, and then the safranin, I have had some interesting results. The Wasserblau must be used first. With chrom-osmium material, twelve to twenty-four hours in the blue, and four or five in the safranin, may not be too much. My stains have not kept well. Mann {Methods, etc., p. 216) uses a mixtiu-e of 35 parts 1 per cent, solution of eosin, 45 of methyl blue 1 per cent., and 100 of water. He has also {Zeit. wiss. Mik., xi, 1894, p. 490) used a similar mixture for nerve-ceUs. 329. Anilin Blue-blaek. — ^A preparation cited under this name has been recommended by Bevan Lewis and others for nervous tissue. The dye used by them cannot now ba identifled. Dr. GrEtJBLEK writes me that the anilin blue-black of his list is the oxyazo colour blue-black B or Azoschwaez ; but that dye had not been discovered when Bevan 184 PLASMA STAINS WITH COAL-TAR DYES. Lewis wrote. See also Heidenhain in Zeit. wiss. Mile, xx, 1903, p. 185, and XXV, 1909, p. 407. 330. Violet B (or Methyl Violet B) (S. Mayeb, Sitzh. h. h. Ahad. wiss. Wien, iii, Abth., February, 1882). — Used in solutions of 1 grm. of the colour to 300 grms. of 0-5 per cent, salt solution, and with fresh tissues that have not been treated with any reagent whatever, this colour gives a stain so selective of the elements of the vascular system that favourable objects, such as serous membranes, appear as if injected. The preparations do not keep well ; acetate of potash is the least unsatisfactory medium for mounting them in, or a mixture of equal parts of glycerin and saturated solution of picrate of ammonia {Anat. Am., 1892, p. 221). See also under " Plasmafibrils." The allied dye, Crystal Violet, has been employed for staining sections, e.g. by Keomayer and others. Benda (Neurol. Centralb., xix, 1900, p. 792) stains in a mixture of 1 vol. saturated sol. of the dye in 70 per cent, alcohol, 1 vol. 1 per cent. sol. of hydrochloric acid in 70 per cent, alcohol, and 2 vols, of anilin water, the liquid being warmed until vapour is given off, then cooled and the sections dried with blotting-paper, treated one minute with 30 per cent, acetic acid, dehydrated with alcohol and cleared with xylol. 331. Kresyl Violet. — ^An oxyazin dye, giving metachromatic stains. Herxi-ieimbe, {Arch. mik. Anat., liii, 1899, p. 519, and liv, p. 289) stains sections of skin with Kresyl-echtviolett. Nuclei blue, plasma reddish. Similarly FiCK {Gentralh. allg. Path., xiii, 1902, p. 987 ; Zeit. wiss. Mik., XX, 1903, p. 223), staining for three or four minutes in a concentrated aqueous solution, and differentiating in alcohol until the connective tissue has become colourless. Keratohyalin violet-red to salmon- coloured. 332. Saureviolett, see § 316. 333. Benzoazurin may be made to give either a diffuse or a nuclear stain, according to Martin (see Zeit. wiss. Mik., vi, 1889, p. 193). 334. Kawitz' " Inversion " Plasma Stains. — It has been discovered by Rawitz that by means of appropriate mordants certain basic anilins, which by the usual methods of regressive staining are pure chromatin stains, may be made to aflord a pure plasma stain, thus giving an " inversion " of the usual stain. The stain, in my opinion, is a vile one. For details see fourth edition, or Rawitz (Sitzh. Gesnaturf. Freunde, Berlin, 1894, p. 174 ; Zeit. wiss. Mik., xi, 1895, p. 603 ; and hiaLeitfaden f. hist. Untersuohungen, Jena, 1895, p. 76). 335. Artificial Alizarin (Rawitz, Armt. Anz., xi, 10, 1895, p. 295). — A double stain by means of artificial Alizarin, or Alizarin-cyanin, requiring the use of special mordants supplied by the colour manu- facturers, and very complicated. See fifth edition. CHAPTER XV. ■ 185 Rawitz [Zeit wiss. Mik., 1909, pp. 393 and 395) also recommends a solution of 1 grm. of Saure-AIizarlnblau BB (or Sauregriin G) (both from Hoclist), 10 grms. ammonia alum, 100 c.c. glycerin, and 100 c.c. water. SztJTZ {ibid,, xxix, 1912, p. 289) fixes, n a mixture of 15 c.c. 1 per cent, platinum chloride, 15 c.c. formol, and 30 c.c. saturated solution of sublimate, makes paraffin sections, and stains them with Heidenhain's iron-hsematoxylin. They are then treated for five to six hours with 5 per cent, solution of aluminium acetate, rinsed, and stained for five to six hours with Benda's sulphaHzarinate of soda (given under " Mito- chondria "), and got into balsam. A red plasma stain, affecting plasma- fibrils. For intra-vitam stains with alizarin see § 208 (Pischel), and NiLSSON, Zool. Anz., XXXV, 1909, p. 196. 336. For Benda's Alizaxin Stains, see under " Centrosomes," " Mitochondria," and " Neuroglia." CHAPTER XVI. METHYLEN BLUE. 337. Methylen Blue is a " basic " dye, being the chloride or the zinc chloride double salt of tetramethylthionin. It appears that some persons have confounded it with the " acid " dye methyl blue, to which it has not, histologically, any resemblance. Commercial methylen blue sometimes contains as an impurity a small quantity of a reddish dye, which used to be taken to be methylen red. This impurity is present from the beginning in many brands of methylen blue, is frequently developed in solutions of the dye that have been long kept (so-called " ripened " solutions), and is still more frequently found in kept alkaline solutions. Accord- ing to NocHT (Centralh. Bakteriol., xxv, 1899, pp. 764 — 769 ; Zeit. wiss. MiJc., xvi, 1899, p. 225) it is not methylen red, nor methylen violet either, but a new colour, for which Nocht proposes the name " Eoth aus Methylenblau." According to MiCH^Lis (Centralb. Bakteriol., xxix, 1901, p. 763, and XXX, 1901, p. 626 ; Zeit. wiss. MiL, xviii, 1902, p. 305, and xix, 1902, p. 68) confirmed later by Nocht, Reutee, and Giemsa, this dye is Methylenazur, an oxidation-product of methylen blue, already described by Bernthsen in 1885. It is an energetic dye, of markedly metachromatic action, and to it are due the metachromatic effects of methylen blue solutions (methylen blue itself is not metachro- matic). The presence of this dye as an impurity in methylen blue is not always an undesirable factor ; on the contrary, it sometimes affords differentiations of elements of tissues or of cells that cannot be produced by any other means. Methylen blue that contains it is known as polychrome methylen blue, and is employed for staining certain cell-granules. Unna {Zeit. wiss. Mik., viii, 1892, p. 483) makes this as follows : A solution of 1 part of methylen blue and 1 of carbonate of potash in 20 of alcohol and 100 of water is evapo- rated down to 100 parts. (It may be used at once, or after diluting \vith an equal volume of anilin water, for sections, which after staining may be differentiated with glycol, creosol, or Unna's glycerin-ether mixture — all of which, as well as the polychrome methylen blue, can be obtained from Griibler & Hollborn.) Mich.5;lis CHAPTER XVI. 187 {(yp. cit.) makes it as follows : 2 gr. of medicinal methylen blue are dissolved in 200 c.c. of water, and 10 c.c. of ^ normal solution of caustic soda added. Boil for a quarter of an hour ; after cooling add 10 c.c. of -^ normal sulphuric acid, and filter. Methylenazur is isolated from methylen blue by the prolonged action of an alkaU or of silver oxide. It seems also that it is formed in certain mixtures of methylen blue with eosin (Romanowskt, Laveran, Giemsa and others), by means of the eosin, which in these mixtures acts ohemi- oally, and can be replaced by resorcin, hydroquinon, and the Hke. It is best procured from Uriibler & HoUborn, who supply it pure as " Azur I," and mixed with an equal quantity of methylen blue as "Azur II." See further as to this dye under " Stains for Blood." See also an important paper by Pkowazek {Zeit. wiss. Mikr. Tech., 31). There are several sorts of methylen blue sold, the most important being — " methylen blue, according to Ehelich " ; " methylen blue, according to Koch " ; " methylen blue BX, according to S. Mayer " ; " Methylenblau, medic, pur." The colour to be employed for intra-vitam nerve staining should be as 'pure as possible. ApIthy {Zeit. wiss. Mik., ix, 1893, p. 466) writes that the best — in fact, the only one that will give exactly the results described by him — is that of B. Mekck, of Darmstadt, described as " medicinisches Methylenblau." Dogiel {Encycl. mik. Technik., 1st edition, p. 811) has had his best results with " Methylenblau n. Ehrlich," or " BX," obtained from Griibler & HoUborn. 338. The Uses of Methylen Blue. — ^As a histological reagent it is used for sections of hardened central nervous tissue, in which it gives a specific stain of meduUated nerves. It gives more or less specific stains of the basophilous granulations of " Mastzellen " and plasma-cells, and the granules of NissL in nerve-ceUs, also mucin. It is much used — in the form of mixtures affording methylen azur — in the study of blood, blood parasites, and similar objects. For all of these see the respective sections in Part II. Further, it stains a large number of tissues intra vitam, with little or no interference with their vital functions. And last, not least, it can be made to furnish stains of nerve tissue, intercellular cement substances, lymph spaces, and the like, that are essentially identical with those furnished by a successful impregnation with gold or silver, and are obtained with greater ease and certainty ; with this difference, however, that gold stains a larger number of the nervous elements that are present in a preparation, sometimes the totality of them ; whilst methylen blue stains only a selection of them, so bringing them more prominently 188 MBTHYLBN BLVE. before the eye, and allowing them to be traced for greater distances. These two uses form the subject of this chapter. 339. Staining in toto during Life. — Small and permeable aquatic organisms may be stained during life by adding to the water in which they are confined enough methylen blue to give it a very light tint. After a time they will be found to be partially stained — that is, it will be found that certain tissues have taken up the colour, others remaining colourless. If now you put back the animals into the tinted water and wait, you will find after a further lapse of time that further groups of tissues have become stained. Thus it was found by Ehrlich (Biol. Centralb., vi, 1886, p. 214 ; Abh. h. Akad. Wiss. Berlin, February 25th, 1885) that on injection of the colour into living animals axis-cyhnders of sensory nerves stain, whilst motor nerves remain colourless. [The motor nerves, however, will also stain, though later than the sensory nerves.] It might be supposed that by continuing the staining for a sufiicient time, a point would be arrived at at which all the tissues would be found to be stained. This, however, is not the case. It is always found that the stained tissues only keep the colour that they have taken up for a short time after they have attained the maximum degree of coloration of which they are susceptible, and then begin to discharge the colour even more quickly than they took it up. According to Ehrlich this decoloration is explained as follows : methylen blue, on contact with reducing agents in alkaline solution, can be reduced to a colourless body, its " leucobase." Now living or recently dead tissue elements are, or may be, both alkaline and very greedy of oxygen, and thus act on the dye as reducing agents. The leucobase thus formed is easily reoxidised into methylen blue by oxidising substances, or acids, or even by the mere contact of air — which latter property is taken advantage of in practice. It follows that a total stain of all the tissues of a living intact organism can hardly be obtained under these conditions, but that a specific stain of one group or another of elements may be obtained in one of two ways. If the tissue to be studied be one that stains earher than the others, it may be studied during Hfe at the period at which it alone has attained the desired intensity of coloration. If it be one that stains later than the others, it may be studied at the period at which the earher stained elements have already passed their point of maximum coloration and have become sufiiciently decoloured, the later stained ones being at a point of desired intensity. Or the observer may fix the stain in either of these stages and preserve it for leisurely study by means of one of the processes given § 343. CHAPTER XVI. 189 The proper strength of the very dilute sohitions to be employed for the staining of living organisms must be made out by experiment for each object. I think the tint is practically a sufficient guide, but it may be stated that when in doubt a strength of 1 : 100,000 may be taken, and increased or diminished as occasion may seem to require. Zoja {Rendic. R. 1st. Lombardo, xxv, 1892 ; Zeit. wiss. Mik., ix, 1892, p. 208) finds that for Hydra the right strength is from 1 : 20,000 to 1 : 10,000. The stain is capricious. It is not possible to predict without trial which tissues will stain first in any organism. The stain penetrates very badly, which is no doubt one cause of its capriciousness. Gland cells generally stain early ; then, in no definable order, other epithe- lium cells, fat cells, plasma cells, "Mastzellen," blood and lymph corpuscles, elastic fibres, smooth muscle, striated muscle. There are other elements that stain in the Hving state, but not when the staining is performed by simple immersion of intact animals in a dilute staining solution in the manner we are considering. Chief amongst these are nerve-fibres and ganglion-cells, which remain unstained in the intact organism. To get these stained, it is neces- sary to isolate them sufficiently, as explained in the following sections. 340. Staining Nervous Tissue* during Life. — It was made out by Ehrlich {op. cit., last §) that by injecting a solution of methylen blue into the vessels or tissues of living animals and shortly after- wards cutting out and examining small pieces of their tissues, these will be found to be intensely stained in some of their elements (chiefly nervous). If the tissues are mounted under a cover-glass, the stain will fade in a short time ; but if the cover-glass be removed, so that oxygen can have access to the tissues, the stain will be restored, as explained last §. The chief elements stained in this way are peripheral nerves, and amongst these more especially axis- cylinders of sensory nerves. EhrKch held that the stain so obtained is a product of a vital reaction of the tissues, and that it cannot be obtained with dead material. Dogiel, however {Arch. mik. Anat., xxxv, 1890, pp. 305 et seq.), found that muscle nerves of limbs of the frog could be stained as much as from three to eight days after the limbs had been removed from the animal. He concludes, indeed, that the reaction shows that the nerves were still living at that time. But it seems more natural to conclude with Apathy {Zeit. wiss. Mik, ix, 1892, pp. 15 et seq.) that nerve-tissue can be stained after life has ceased. ApAthy * See also p. 477. 190 METHYLEN BLUE. lias directly experimented on this point, and sums up the necessary- conditions as follows : The tissue need not be living, but must be fresh ; nothing must have been extracted from it chemically, and its natural state must not have been essentially changed by physical means. For example, the tissue must not have been treated with even dilute glycerin, nor with alcohol, though a treatment for a short time with physiological salt solution is not very hurtful ; it must not have been coagulated by heat. Michailow {ibid., xxvii, 1910, p. 7) prefers tissues that have lain from one and a half to two hours after the death of the subject in Ringer's salt solution. As above explained, the primary stain obtained by injecting methylen blue, or immersing tissues in it, only lasts a very short time. In order to get it to last long enough for study, it must be re-blued by oxidation (see last §). It is therefore the usual practice to dissect out the tissues to be examined, and leave them for some time exposed to the air. This is done in order that they may take up from the air the necessary oxygen. Another consideration that justifies the practice is that by exposure to air the preparations take up a trace of ammonia, and ApAthy has experimentally established that this is an important factor in the sharpness of the stain. Ehrlich also {op. cit.) holds that an alkaline reaction of the tissues is a neces- sary condition to the stain. Apathy further holds that the stain is a regressive one, easily washed out by the surrounding liquid ; and in order to prevent this washing-out being excessively rapid, it is desirable to have it go on in presence of as little liquid as possible. 341. The Modes of Staining. — The practice of the earlier workers at this subject was (following Ehelich) to inject methylen blue into the vascular system or body-cavity of a living animal, wait a suffi- cient time, then remove the organ for further preparation and study. And there appears to have been a belief with some workers that it was essential that the stain shoftld have been brought about by injection of the colouring matter into the entire animal. It is now known that the reaction can often be equally well obtained by re- moving an organ and subjecting it to a hath of the colouring matter in the usual way. But in some cases it seenis that injection is preferable, if not necessary. 342. The Solutions employed.— The solutions used for injection are generally made in salt solution (physiological, or a little weaker) ; those for staining by immersion, either in salt solution or other " indifferent " liquid, or in pure water. The earlier workers generally took concentrated solutions. Thus Aenstein {Anat. Am., 1887, CHAPTER XYl. 191 p. 125) injected 1 c.c. of saturated {i.e. about 4 per cent.) solution into the vena cutanea magna of frogs, and removed the organ to he investigated after the lapse of an hour. Biedermaitn (Sitzb. AJcad. Wiss. Wien, Math. Nat. CI, 1888, p. 8) injected 0-5 to 1 c.c. of a nearly saturated solution in 0-6 per cent, salt solution into the thorax of era joshes, and left the animals for from two to four hours before killing them. S. Mayer {Zeit. wiss. Mile., vi, 1889, p. 423) took a strength of 1 : 300 or 400 of 0-5 per cent, salt solution. The solutions of Eetzius are of the same strength. But the tendency of more recent practice is decidedly towards the employment of weaker solutions. ApIthy [ibid., ix, 1892, pp. 25, 26 et seq.) finds that it fs not only superfluous, but positively disadvantageous, to take solutions stronger than 1 : 1000. Dogiel (Encycl. Mih. Technik., 1st ed., p. 815) recommends ^ to J per cent., or at most \ per cent. For warm-blooded animals the solution should be warmed to 36° -or 37° C, and before sending in the injection the blood-vessels should be well washed out with similarly warmed salt solution. The injected organs may be removed after twenty to thirty minutes. They should be placed on a thin layer of spun glass moistened with weak (^ to ^ per cent.) methylen blue, or simply spread out on a slide, and the whole placed in a Petri dish with a layer of the methylen blue on the bottom. The dish is best placed in a stove at 36° C, and after fifteen to thirty minutes (if the pieces are thin) or one hour to one and a half hours (if they are thick) specimens may be removed for examination or preservation ; or, without using the stove, specimens may be removed ten to twenty minutes after injection, placed on a sHde, and moistened with weak methylen blue or salt solution, and brought imder the microscope. Then as soon as the stain is sufficiently brought out (forty to sixty minutes) they may be fixed (§ 343). For staining by immersion the solutions should, if anything, be still weaker. Dogiel {Arch. mih. Anat., xxxv, 1890, p. 305) places objects in a few drops of aqueous or vitreous humour, to which are added 2 or 3 drops of a Jg to Jg per cent, solution of methylen blue in physiological (0-75 per cent.) salt solution, and exposes them therein to the air. In thin pieces of tissues the stain begins to take effect in five or ten minutes, and attains its maximum in from fifteen to twenty minutes. For thicker specimens — retina, for instance — several hours may be necessary. The reaction is quickened by putting the preparations into a stove kept at 30° to 35° C. EouGET {Compt. Rend., 1893, p. 802) employed a 0-05 per cent, solu- jiipn in 0-6 per cent, salt solution (for muscles of Batrachia). Allen 192 METHYLEN BLUE. (Quart. Journ. Micr. Sci., 1894, pp. 461, 483) take.* for embryos of the lobster a solution of 0-1 per cent, in 0-75 per cent, salt solution, and dilutes it with 15 to 20 volumes of sea- water. Seidenmann (Zeit. wiss. Mik, xvii, 1900, p. 239) takes for the choroid a solution of 0-02 per cent, in 0-5 per cent, salt solution. Lavdowsky (ibid., xii, 1895, p. 177) takes -j^ to ^ per cent, in white of egg, or serum. Similarly YouNG (ibid., xv, 1898, p. 253). MiCHAiLOW (ibid., xxvii, 1910, p. 10) takes | to g^ per cent, in Ringer's salt solution (for nerves of Mammals). ApAthy (Zeit. wiss. Mik., ix, 1892, p. 15 ; see also his Mikro- technik, p. 172) jiroceeds as follows for Hirudinea and other inverte- brates. A portion of the ventral cord is exposed, or dissected out. If it be desired to stain as many ganglion cells as possible, as well as fibres, the lateral nerves, as well as the connectives, should be cut through near a ganglion. The preparation is then treated with the stain. This is, for the demonstration chiefly of fibres in Hirudo and Pontobdella, either a 1 : 1000 solution in 0-5 to 0-75 per cent, salt solution, allowed to act for ten minutes ; or a 1 : 10,000 solution allowed to act for an hour to an hour and a half ; or a 1 : 100,000 solution allowed to act for three hours (Lumbricus requires twice these times ; Astacus and Unio require three times ; medullated nerves of vertebrates four times). For the demonstration of ganghon cells the stain is allowed to act three or four times as long. The preparations from the 1 : 1000 solution are then washed in salt solution for an hour ; those from the 1 : 10,000 solution for a quarter of an hour ; those from the 1 : 100,000 solution need not be washed at all. They are then treated with one of the ammoniacal fixing and differentiating liquids described in § 343. This is done , by pouring the liquid over them, and leaving them in it without moving them about in it for at least an hour, and by preference in the dark. The further treatment is as described in § 343. The object of the ammonia in these liquids is to differentiate the stain — to produce an artificial " secondary differentiation." It acts by washing out the absorbed colour from certain elements, others resisting longer. See also, for Hirudinea, Sanchez, in Trab. Lab. Invest. Biol. Univ. Madrid, vii, 1909, fasc. 1 — 4, or Zeit. wiss. Mik., xxvii, 1910, p. 393 (injection of solutions of 0-2, 0-1, or 0-05 per cent., with further treatment as Apathy or Bethe). 343. Fixation of the Stain. — The stain obtained by any of these methods may be fixed, and more or less permanent preparations be made by one or other of the following methods : ■ CHAPTER XVI. 193 Arnstein {Anat. Anz., 1887, p. 551) puts the tissue for half an hour into saturated aqueous solution of picrate of ammonia. S. Mayer {Zeit. wiss. Mik, vi, 1889, p. 422) preferred a mixture of equal parts of glycerin and saturated picrate of ammonia solu- tion, which served to fix the colour and mount the preparations in. This was also in principle the method of Eetzius {Intern. Monatsschr. Anat. Phys., vii, 1890, p. 328). DoGiEL {Encycl. Mik. Techn., ii, p. 105) puts for two to twenty- four hours into saturated aqueous picrate of ammonia, and then into equal parts of glycerin and the picrate solution. (Thin mem- branes, and the like, may be fixed with 1 or 2 per cent, of 2 per cent, osmic acid solution added to the picrate solution and stained with picro-carmine before putting into the glycerin mixture.) Other workers have employed saturated solution of iodine in iodide of potassium (so Abnstein) or picro-carmine (so Feist, Arch. Anat. Entvnchel., 1890, p. 116 ; cf. Zdt. wiss. Mik., vii, 1890, p. 231), the latter having the advantage of preserving the true blue of the stain if it be not allowed to act too long, and the preparation be mounted in pure glycerin. Picric acid has been used by Lavdowsky, but this after careful study is rejected by Dogiel. ApIthy {op. cit., § 342) brings preparations either into a concen- trated aqueous solution of picrate of ammonia /ree/rom picric acid, and containing 5 drops of concentrated ammonia for every 100 c.c. ; or, which is generally preferable, into a 1 to 2 per cent, freshly prepared solution of neutral carbonate of ammonia saturated with picrate. They remain in either of these solutions, preferably in the dark, for at least an hour. They are then brought into a small quantity of saturated solution of picrate of ammonia in 50 per cent, glycerin, where they remain until thoroughly saturated. They are then removed into a saturated solution of the picrate in a mixture of 2 parts 50 per cent, glycerin, 1 part cold saturated sugar solution, and 1 part similarly prepared gum-arabic solution. When thoroughly penetrated with this they are removed and mounted in the following gum-syrup medium {loc. cit., p. 37) : Picked gum-arabic 50 grms. Cane-sugar (not candied) . . . . 50 „ Distilled water . . . . • • 50 ,, Dissolve over a water-bath and add 0-05 grm. thymol. (This mounting medium sets quickly and as hard as balsam, so that no cementing of the mounts is necessary. Farrants' medium [with omission of the arsenious acid] will also do. In neither case should 13 194 METHYLEN BLUE. either ammonium picrate or methylen blue be added to the medium.) Preparations that have heen fully differentiated (§ 342) do not keep more than a few weeks ; whilst those in which the differentiation has not been carried to the point of thorough tinctorial isolation of the neuro-fibrils have kept for five or six years (ApAthy, Mitth. Zool. Stat. Neapel, xii, 1897, p. 712). Pleschko {Anat. Anz., xiii, 1897, p. 16) fixes with picrate, and then puts into 10 per cent, formol for a few days. The methods described next § are also available for material not destined to be sectioned. 344. Methods for Sections. — The preceding methods do not give preparations that wiU resist the operations necessary for imbedding in parafiin or mounting in balsam. A strong solution of platinum chloride is said to do this (see Feist, Arch. Anat. Entw., 1890, p. 116), but the preparations are not very satisfactory. For the earUer method of Parker {Zool. Anzeig., 1892, p. 375) with methylal see early editions. Later {Mitth. Zool. Stat. Neapel, xii, 1895, p. 4) he fixes the stain by dehydrating the objects in successive alcohols of 30, 50, 70, 95, and 100 per cent, strength, each containing 8 per cent, of corrosive sublimate, then brings them into a mixture of the last with an equal volume of xylol, and lastly into pure xylol. For the earlier method of Bethe (Arch. mih. Anat., xliv, 1894, p. 585), see last edition. Bethe's later method [Anat. Anz., xii, 1896, p. 438) is as follows : After staining, pieces of tissue of 2 to 3 mm. thickness are treated for ten to fifteen minutes with a concentrated aqueous solution of picrate of ammonia and then brought into a solution of 1 grm. of molybdate of ammonium, either in 20 of water, or in 10 of water and 10 of 0-5 per cent, osmic acid or 2 per cent, chromic acid ; or into a solution of phosphomolybdate of sodium in the same propor- tions, each of these solutions having added to it 1 drop of hydro- chloric acid, and if desired 1 grm. of peroxide of hydrogen. They remain in one of these solutions for three quarters to one hour (or from four hours to twelve in the osmic acid one), and are then passed through water, alcohol, xylol, balsam, or paraffin. (The objects that have been treated with one of the solutions of the sodium salt are not thoroughly resistant to alcohol, so that for them it is well to cool the alcohol to under 15° C.) Sections may be after-stained with alum carmine, or " neutral " tar colours. Slight modifications of this method are given by Dogiel {Arch. CHAPTER XVI. 195 mik. Anat., xlix, 1897, p. 772 ; liii, 1898, p. 237 ; Zeit. wiss. Zool, Ixvi, 1899, p. 361 ; and Encycl. mik. Technik, 1903, p. 825, and 1910, p. 108.) He omits the peroxide, the hydrochloric acid, and the cooling. Bethe {Zeit. wiss. Mik., xvii, 1900, p. 21) does not approve of these modifications. Further modifications of the molybdenum method have been published by Leontowitsch (Intern. Mmiatsschr. Anat., xviii, 1901, p. 142). MiCHAiLOW (Zeit. wiss. Mik., xxvii, 1910, p. 19) adds to 8 per cent, solution of molybdate 0-5 per cent, of formalin, leaves the objects in a large quantity of it (filtered) for twenty-four hours at 37° C, washes with warm water, and passes through alcohol and xylol into xylol-damar (not balsam). See also Schmidt (Arch. Ges. Phys., ciii, 1906, p. 522). Harris (Philadelphia Medical Journ., May 14th, 1898), after' staining, rinses with water, and brings into a saturated solution of either ferrocyanide or ferricyanide of potassium which has been cooled to within a few degrees of zero (a trace of osmic acid may be added to prevent maceration). They remain therein for three to twenty -four hours, and are then washed in distilled water for an hour, and are dehydrated in absolute alcohol kept at a low tempera- ture, cleared in xylol or cedar oil, and imbedded in paraffin. 345. Impregnation of Epithelia, Lymph-spaces, etc. {Dogiel, Arch. mik. Anat., xxxiii, 1889, pp. 440 et seq.). — Suitable pieces of tissue (thin membrane by preference) are brought fresh into a 4 per cent, solution of methylen blue in physiological salt solution (in the Encycl. mik. Technik, 1903, p. 827, Dogiel gives the strength of the methylen blue as ^ to 1 per cent.). After a few minutes therein they are brought into saturated solution of picrate of ammonia, soaked therein for half an hour or more, then washed in fresh picrate of ammonia solution, and examined in dilute glycerin. If it be wished only to demonstrate the outhnes of endothelium cells, the bath in the stain should be a short one, not longer than ten minutes in general ; whilst if it be desired to obtain an impregna- tion of ground-substance of tissue, so as to have a negative image of juice canals or other spaces, the staining should be prolonged to fifteen or thirty minutes. If it be desired to preserve the preparations permanently, they had better be mounted in glycerin saturated with picrate of ammonia, or (Encycl., 1910, ii, p. 110) fixed with ammonium molybdate and a trace of osmium. 13—2 196 METHYLEN BLUE. The effect is practically identical (except as regards the colour) with that of a negative impregnation with silver nitrate. S. Maybe (Zeit. wiss. Mik., vi, 1889, p. 422) stains tissues for about ten minutes in a 1 : 300 or 400 solution of methylen blue in 0-5 per cent, salt solution, rinses in salt solution, and puts up in the glycerin-picrate of ammonia mixture given § 343. The images are generally positive after injection of the colour into the vascular system ; negative after immersion of the tissues. TiMOPEJEW (Anat. Anz., xxxv, 1909, p. 296) impregnates for fifteen to twenty minutes in a solution of 1 : 300 or 400 strength, fixes with a very weak solution of ammonium picrate in salt solution, and puts up in a mixture of 50 c.c. glycerin, 50 c.c. water, and 35 c.c. saturated solution of the picrate : or fixes with ammonium molybdate of 8 per cent, and moimts in balsam. 346. Toluidin Blue' or Thionin as succedanea of methylen blue. — Harris {Pkilidelphia Med. Journ., May 14th, 1898) has found that there is no reaction of methylen blue that cannot be equally well obtained with toluidin blue or thionin. For staining pieces of tissue he takes : Toluidin blue, 0-1 per cent. sol. in physio- logical salt solution . . . . .2 parts. Ammonium chloride 0-25 per cent, in water . 1 part. Egg albiunen . . . . . . 1 „ For injections he uses 1 part of the dye to 1000 of physiological salt solution. Any of the methylen blue fixing methods may be employed and the whole technique is the same. L. Martinotti {Zeit. wiss. Mik., xxvii, 1910, p. 24) recommends a polychrome toluidin blue, made by adding 0-5 per cent, of lithium carbonate to a 1 per cent, solution of the dye and keeping till a purple-red tone appears. Or, a stock solution made of 1 grm. toluidin blue, 0-5 grm. lithium carbonate, glycerin 20 grms., alcohol 5 grms., and water 75. CHAPTER XVII. METALLIC STAINS (iMPEEGNATION METHODS). 347. The Characters of Impregnation Stains. — By impregnation is understood a mode of coloration in which a colouring matter is deposited in tissues in the form of a precipitate — ^the impregnated elements becoming in consequence opaque. By staining, on the other hand, is understood a mode of coloration in which the colouring matter is retained by the tissues as if in a state of solution, showing no visible solid particles under the microscope, the stained elements remaining in consequence transparent. But it is not right to draw a hard and fast Kne between the two Mnds of coloration. Some of the metallic salts treated of in this chapter give, besides an impregna- tion, in some cases a true stain. And some of the dyes that have been treated of in the preceding chapters give, besides a stain, a true impregnation. Methylen blue, for instance, will give in one and the same preparation an impregnation and a stain ; and in most chloride preparations the coloration is in places of the nature of a finely divided solid deposit, in others a perfectly transparent stain. 348. Negative and Positive Impregnations. — In a negative- impieg- nation intercellular substances alone are coloured, the cells them- selves remaining colourless or verj'' lightly tinted. In a positive impregnation the cells are stained and the intercellular spaces are unstained. (A directly contrary statement, made in a recent Lehrhuch, is erroneous.) Negative impregnation is generally held to he 'primary because brought about by the direct reduction of a metal in the intercellular spaces; positive impregnation to be secondary (in the case of silver nitrate at least) because it is brought about by the solution 'in the liquids of the tissues of the metallic deposit formed by a primary impregnation, and the consequent staining of the cells by the new solution of metallic salt thus formed. These secondary impregnations take place when the reduction of the metal in the primary impregnation is not sufBciently energetic (see on these points His, Schweizer Zeit. Heilk., ii. Heft 1, p. 1 ; GiEKEE, Zeit. wise. Mile., i, p. 393 ; Ranvier, TraitS, p. 107). As to the nature of the black or brown deposit or stain formed in the intercellular spaces in cases of primary impregnation see Schwalbe, Arch. mile. Anat., vi, 1870, p. 5 ; Giekke's Fdrberei zu mikroskopischen Zviecken, in vols, i and ii of Zeit. wiss. Mik. ; Joseph, Sitzb. Ahad. Wiss. 198 METALLIC STAINS {IMPREGNATION METHODS). Berlin, 1888 ; Zeit. wiss. Mik., xi, 1, 1894, p. 42 et seq. It evidently cannot consist of metallic silver, as it is soluble in hyposulphite of soda. See also Macallum, Proc. Boy. 8oe., Ixxvi, 1905, p. 217, and Achabd and RETNAtnD, O.B. Soc. Biol., Ixi, 1906, p. 43. 349. Action of Light on Solutions of Metallic Salts. — Stock solutions of metallic salts are generally kept in the dark, or at least in coloured bottles, under the belief that exposure to light reduces them. It has been pointed out in § 35 that in the case of osmic acid, not light, but dust is the reducing agent, and that solutions may be exposed to light with impunity if dust be absolutely denied access to them. I have now good evidence to the effect that the same is the case with other metallic solutions ; and the point is raised whether such solutions are not positively improved for impregnation purposes by exposure to light ! Dr. Lindsay Johnson writes me as follows : " One may (I find by experiment) state as a rule without exception that all the solutions of the chlorides and nitrates of the metals will keep indefinitely in clean white stoppered bottles in the sunlight ; and as far as osmium, uranium, gold and silver, and platinum are concerned, actually improve or ripen by a good sunning. All photographers tell me their papers salt more evenly by old well- sunned silver nitrate than by a fresh solution kept in the dark ; and I go so far as to say that this is one of the reasons why gold stains are so unsatisfactory." Apathy {Mitt. Zool. Stat. Neapel, xii, 1897, p. 722) leaves his gold solutions exposed to light, so long as there are no tissues in them. 350. State of the Tissues to be Impregnated. — The majority of stains given by dyes are only obtained with tissues that have been changed in their composition by the action of fixing and preservative reagents. With metallic impregnations the case is different ; perfectly /resA tissues — ^that is, such as are either living, or at all events have not been treated by any reagent whatever — will also impregnate with the greatest ease and precision. Indeed, some impregnations will not succeed at all with tissues that are not fresh in the sense above explained. Silver. 351. Silver Nitrate : Generalities. — The principles of its employ- ment are given by Ranvier {TraM, p. 105) as follows : Silver nitrate may be employed either in solution or in the solid state. The latter method is useful for the study of the cornea and of fibrous tissues, but is not suitable for epithelia. For the cornea. CHAPTER XVII. • 199 for instance, proceed as follows : The eye having been removed, a piece of silver nitrate is quickly rubbed over the anterior surface of the cornea, which is then detached and placed in distilled water ; it is then brushed with a camel's hair brush in order to remove the epithelium. The cornea is then exposed to the action of light. It will be found that the nitrate has traversed the epitheUum and soaked into the fibrous tissue, on the surface of which it is reduced by the light. The cells of the tissues will be found unstained. It is generally employed in solution, in the following manner : In the case of a membrane, such as the epiploon, the membrane must be stretched like a drum-head over a porcelain dish, * and washed first with distilled water, and then washed with a solution of silver nitrate. In order to obtain a powerful stain it is necessary that this part of the operation be performed in direct sunlight, or at least in a very brilliant Hght. As soon as the tissue has begun to turn of a blackish grey the membrane is removed, washed in distilled water, and mounted on a slide in some suitable examination medium. If the membrane were left in the water the cells would become detached, and would not be found in the finished preparation. If the membrane had not been stretched as directed the silver would be precipitated not only in the intercellular spaces, but in all the small folds of the surface. If the membrane had not been washed with distilled water before impregnation there would have, been formed a deposit of silver on every spot on which a portion of an albuminate was present, and these deposits might easily be mistaken for a normal structure of the tissue. It is thus that impurities in the specimen have been described as stomata of the tissue. If the solution be taken too weak — for instance, 1 : 500 or 1 : 1000, or if the Ught be not brilliant — a general instead of an interstitial stain will result ; nuclei -will be most stained, then protoplasm, and the intercellular substance will contain but very little silver. In general in a good " impregnation " the contents of the cells, and especially nuclei, are quite invisible. The tissues should be constantly agitated in the silver-bath in * The Hoggans Histological Rings wiU be found much more con- venient. They are vulcanite rings made in pairs, in which one ring just fits into the other, so as to clip and stretch pieces of membrane between them. They will be found described and figured in Journ. Boy. Mic. Soo ii, 1879, p. 357, and in Robin's Journ. de VAnat, 1879, p. 54. They may be obtained of Burge & Warren, 42, Kirby Street, Hatton Garden, London, E,C, 200 METAULIC' STAINS [{IMPREGNATiON METHODS). order to avoid the formation on their surfaces of deposits of chlorides and albuminates of silver. These impregnations only succeed mth fresh tissues. 352, Silver Nitrate : the Solutions to be employed (Ranvier).— The solutions generally employed by Ranviee vary in strength from 1 : 300 to 1 : 500. Thus 1 : 300 is used for the epiploon, pulmonary endothelium, cartilage, tendon ; whilst a strength of 1 : 500 is employed for the phrenic centre, and the epithelium of the intestine. For the endothelium of blood-vessels (by injection) solutions of 1 : 500 to 1 : 800 are taken. M. Duval (Precis, p. 229) takes solutions of 1, 2, or at most 3 per cent. V. Recklinghausen used, for the cornea, a strength of from 1 : 400 to 1 : 500 (Die Lymphgefasse, etc., Berlin, 1862, p. 5). RoBiNSKi (Arch, de Physiol, 1869, p. 451) used solutions varying between 0-1 and 0-2 per cent., which he allowed to act for thirty seconds. RouGET (Arch, de Physiol., 1873, p. 603) employed solutions as weak as 1 : 750, or even 1 : 1000, exposing the tissues to their action several times over, and washing them with water after each bath. The Hebtwigs take, for marine animals, a 1 per cent, solution (Jen. Zeit. Naturk., xvi, pp. 313 and 324). The HoGGANS (Journ. of Anat. and Physiol., xv, 1881, p. 477) take for lymphatics a 1 per cent, solution. TouRNEUX and Herrmann (Robin's Journal de I'Anat., 1876, p. 200) took for the epithelia of Invertebrates 3 : 1000, and in some cases weaker solutions, — for one hour, washing out with alcohol of 90 per cent. HoYER (Arch. mik. Anat., 1876, p. 649) takes a solution of nitrate of silver, and adds ammonia to it until the precipitate that is formed just redissolves, then dilutes the solution until it contains from 0-75 to 0-50 per cent, of the salt. This ammonio-nitrate solution has the advantage of impregnating absolutely nothing but endothelium or epithelium ; connective tissue is not affected by it. Ranvier's injection-mass for impregnating endothelium is given under " Injection." Dekhuyzen (Anat. Anz., iv, 1889, No. 25, p. 789) has applied to terrestrial animals the method of Harmer for marine animals (§ 356). For details see previous editions. Regaud (Journ. Anat. et Phys., xxx, 1894, p. 719) recommends for the study of lymphatics a process devised by Renaut, for the details of which see also previous editions. CHAPTER XVII. 201 353. Other Salts of Silver. — ^Alferow (Arch. Phys., i, 1874, p. 694) employs the piorate, lactate, acetate, and citrate, in solution of 1 : 800, and adds a smaU quantity of the acid of the salt taken (10 to 15 drops of a concentrated solution of the acid to 800 c.c. of the solution of the salt). This decomposes the precipitates formed by the action of the silver salt on the chlorides, carbonates,- and other substances existing in the tissues. Eegaud and Dubeeuil (G.B. Ass. Anat, 5 Sess. 1903, p. 122) take a fresh solution of protargol or a mixture of equal parts of 1 per cent, protargol and 1 per cent, osmic acid, thus avoiding precipitates. 354. Silver Nitrate : Reduction. — Eeduction may be effected in media other than distilled water. V. Recklinghausen washed his preparations in salt solution before exposing them to the light in distilled water (Arch. path. Anat., xix, p. 451). Physiological salt solution (0-75 per cent.) is commonly used for these washings. MiJLLER (Arch.f. path. Anat., xxxi, p. 110), after impregnation by immersion for two or three minutes in a 1 per cent, solution of nitrate of silver in the dark, adds to the solution a small quantity of 1 per cent, solution of iodide of silver (dissolved by the aid of a little iodide of potassium). After being agitated in this mixture the preparations are washed with distilled water, and exposed to the light for two days in a 1 per cent, solution of nitrate of silver (see also Gierke, in Zeit. wiss. MiL, i, 1884, p. 396). RoTJGET {Arch, de Physiol., 1873, p. 603) reduces in glycerin ; Szutz (Zeit. wiss. Mik., xxix, 1912, p. 291) in glycerin with ^q of formol. Sattlee {Arch. mik. Anat., xxi, p. 672) exposes to the light for a few minutes in water acidulated with acetic or formic acid. Than- HOFFER {Das Mikroskop, 1880) employs a 2 per cent, solution of acetic acid. Krauss brings his preparations, after washing, into a Ught red solution of permanganate of potash. Reduction takes place very quickly, even in the dark. Oppitz puts for two or three minutes into a 0-25 or 0-50 per cent, solution of chloride of tin. Jakimovitch {Journ. de VAnat., xxiii, 1888, p. 142) brings nerve preparations, as soon as they have become of a dark brown colour, into a mixture of formic acid 1 part, amyl alcohol 1 part, and water 100 parts, and exposes to the light for five to seven days, the mixture being renewed from time to time. ' Pekhuyzen {op. cit., last §) reduces in oil of cloves, after dehydra- tion. 355. Fixation. — ^Legkos (Journ. de VAnat., 1868, p. 276) washes his preparations, after reduction, in hyposulphite of soda, to prevent after- 202 METALLIC STAINS (IMPREGNATION METHODS). blackening. According to DtrvAL {Pricis, p. 230) they should be washed for a few seconds only in 2 per cent, solution and then in distilled water. GrEROTA {Arch. Anat. Phys., Phys. Abth., 1897, p. 428) reduces in a hydroquinone developing solution, followed by fixation in hyposulphite of soda, just as in photography. 356. Impregnation of Marine Animals. — On account of the chlorides that bathe the tissues of marine animals, these cannot be treated directly mth nitrate of silver. Heetwig (Jen. Zeit., xiv, 1880, p. 322) recommends fixing them with a weak solution of osmic acid, then washing with distilled water until the wash-water gives no more than an insignificant precipitate with silver nitrate, and then treating for six minutes with 1 per cent, solution of silver nitrate. Haemer (Mitth. Zool. Stat. Neapel, v, 1884, p. 445) washes them for some time (half an hour) in a 5 per cent, solution of nitrate of potash in distilled water ; they may then be treated with silver nitrate in the usual way. For some animals he recommends a 4-5 per cent, solution of sulphate of soda. 357. Double-staining Silver-stained Tissues.- The nuclei of tissues impregnated with silver may be stained with the usual reagents, provided that solutions containing free ammonia be avoided. These stains will only succeed, however, with successful negative impregna- tions, as nuclei that have been impregnated will not take the second stain. Impregnation with silver may be followed by impregnation with gold. In this case the gold generally substitutes itself for the silver in the tissues, and though the results are sharp and precise, the effect of a double stain is not produced. See hereon Geeota, loc. cit., §355. 358. Impregnation of Nerve Tissue.— .Fo/' this subject, which includes the important bichromate-and-silver method of Golgi, and the neurofibril methods of Bielschowsky and Ram 6m y Cajal, see Part II. These give important results, not only with Nervous tissue, but with various forms of Connective tissue, mitochondrial formations, etc. Gold. 359. The Characters of Gold Impregnations. — Gold chloride differs from nitrate of silver in that it generally gives positive (§ 348) im- pregnations only. It generally gives negative images only with such tissues as have first received a negative impregnation with CHAPTER XVII. 203 silver, the gold substituting itself for the silver. In order to obtain these images you first impregnate very lightly with silver ; reduce ; treat for a few minutes with a 0-5 per cent, solution of gold chloride, and reduce in acidulated distilled water. This process, however, is in but little use, and except for certain special studies on the cornea and on connective tissue, the almost exclusive function of gold chloride is the impregnation of nervous tissue, for which it exhibits a remarkable selectivity. 360. Pre-impregnation and Post-impregnation.— Gold methods may be divided into two groups : viz. pre-impregnation methods, characterised by employing perfectly fresh tissues, and post-impregna- tion methods, characterised by the employment oi fixed and hardened tissues. Both are chiefly used for nervous tissue. They give in some respects opposite results. Pre-impregnation gives nuclei unstained, cjrtoplasm rather strongly stained, axis-cylinders reddish- violet. Post-impregnation gives nuclei sharply stained, c3rfcoplasm pale, axis-cylinders black, and (when successful) showing their neurofibrils sharply distinguished from the interfibrillar substance. In ApIthy's view {Mitth. Zool. Stat. Neapel, xii, 1897, p. 718) successful gold preparations should show a true stain, not an im- pregnation (§ 347), the stain being brought about by the formation of gold oxide (AuO) which combines with the tissue elements. He advises in consequence that preparations should not be moved about more than can be helped in the reducing bath, so that the colouring oxide may not be washed away from the tissues before the stain has taken effect. 361. As to the Commercial Salts of Gold. — Squire's Methods and FormulcB, etc. (p. 43), says : " Commercial chloride of gold is not the pure chloride, AuClg, but the crystalUsed double chloride of gold and sodium, containing 50 per cent, of metalUc gold. " Commercial chloride of gold and sodium is the above crystallised double chloride mixed with an equal weight of chloride of sodium, and contains 25 per cent, of metallic gold." This, however, appears not to be the case in Germany. Dr. Geublbe, writing to Mayee (see the Orundzuge, Lee und Mayee, p. 215), says: " Aurum chloratum fuscum contains about 53 per cent. Au, the flavum about 48 per cent. ; in both of them there should be only water and hydrochloric acid besides the gold, no sodium chloride. Pure Auronatrium chloratum contains 14-7 per cent, of sodium chloride, though samples are found in commerce with much more." 204 METALLIC STAINS {IMPREGNATION METHODS). Apathy (Mitth. Zool. Stat. Neapel, xii, 1897, p. 722) formerly employed the aurum chloratum flavum, but now prefers the fuscum. A. Pre-impregnation. 362. The State of the Tissues to be Impregnated.— The once classical rule, that for researches on nerve- endings the tissues should be taken perfectly fresh, seems not to be valid for aU cases. For Deasch {Sitzb. Akad. Wiss. Wim, 1881, p. 171, and 1884, p. 516 ; and Ahhand. math.-phys. CI. K. Sack. Ges. Wiss., xiv, No. 5, 1887 ; Zeit. wiss. Mik., iv, 1887, p. 492) finds that better results are obtained with tissues that have been allowed to lie after death for twelve, twenty-four, or even forty-eight hours in a cool place. 363. Cohnhbim's Method (Virchow's Arch., Bd. xxxviii, pp. 346 — 349 ; Strieker's Handb., p. 1 100).- — Fresh pieces of cornea (or other tissue) are put into 0-5 per cent, solution of chloride of gold until thoroughly yellow, and then exposed to the light in water acidulated with acetic acid until the gold is thoroughly reduced, which happens in the course of a few days at latest. They are then mounted in acidulated glycerin. Eesults very uncertain and anything hut permanent. 364. Lowit's Method {Sitzgsber. Akad. Wim, Bd. Ixxi, 1875, p. 1). — The following directions are from Fischer's paper on the corpuscles of Meissner {Arch. mik. Anat., xii, 1875, p. 366). Small pieces oi fresh skin are put into dilute formic acid (1 volume of water to 1 of the acid of 1-12 sp.gr.), and reniain there until the epidermis peels off. They then are put for fifteen minutes into gold chloride solution (1|- to 1 per cent.), then for twenty-four hours into dilute formic acid (1 part of the acid to 1 — 3 of water), and then for twenty-four hours into undiluted formic acid. (Both of these stages are gone through in the dark.) Sections are then made and mounted in dammar or glycerin. Successful preparations show the nerves alone stained. 365. Eanvibe's Formic Acid Method {Quart. Journ. Mic. Sci. [N.S.], Ixxx, 1880, p. 456). — The tissues are placed in a mixture of chloride of gold and formic acid (4 parts of 1 per cent, gold chloride to 1 part of formic acid) which has been boiled and allowed to cool (Ranvier's Traite, p. 826), They remain in this until thoroughly impregnated (muscle twenty minutes, epidermis two to four hours) ; reduction is affected either by daylight in acidulated water, or in the dark in dilute formic acid (1 part of the acid to 4 parts of water). 366. Ranvier's Lemon-juice Method {Traite, p. 813). — Ranviee finds that of all acids lemon juice is the least hurtful to nerve- CHAPTER XVn. 205 endings. He therefore soaks pieces of tissue in fresh lemon juice until they become transparent (five or ten minutes in the case of muscle). They are then rapidly washed in water, brought for about twenty minutes into 1 per cent, gold chloride solution, washed again in water, and brought into a mixture of 50 c.c. of distilled water and 2 drops of acetic acid. They are exposed to the light for twenty-four to forty-eight hours. The preparations thus obtained are good for immediate study, but are not permanent, the reduction of the gold being incomplete. In order to obtain perfectly reduced, and there- fore permanent, preparations, the reduction should be done in the dark in a few cubic centimetres of dilute formic acid (1 part acid to 4 of water), which takes about twenty-four hours. 367. Viallane's Osmic Acid Method {Hist, et Div. des Insectes, 1883, p. 42). — The tissues are treated with osmic acid (1 per cent, solution) until they begin to turn brown, then with 25 per cent. formic acid for ten minutes ; they are then put into solution of chloride of gold of 1 : 5000 (or even much weaker) for twenty-four hours in the dark, then reduced in the light in 25 per cent, formic acid. I find this an excellent method. Kerschnee {Arch. mik. Anat., Ixxi, 1908, p. 522) puts tiU brown into a mixture of 10 parts 5 per cent, formic acid with 1 part 2 per cent, osmic acid, washes, puts- for two to six hours into 1 per cent. gold chloride in the dark, washes, puts for twelve hours into 25 per cent, formic acid in the dark and then for twenty-four in the light, and mounts in 50 per cent, glycerin with 1 per cent, of formol. 368. Other Methods. — The numerous other methods that have been proposed differ from the foregoing partly in respect of the solutions used for impregnation, but chiefly in respect of details imagined for the purpose oi facilitating the reduction of the gold. Thus Bastiajst employed a solution of gold chloride of a strength of 1 to 2000, acidulated with HCl (l^drop to 75 c.c), and reduced in a mixture of equal parts of formic acid and water kept warm. Henocque {Arch, de I' Anat. et de la Physiol, 1870, p. Ill) impreg- nates in a 0-5 per cent, solution of gold chloride, washes in water for twelve to twenty-four hours, and reduces in a nearly saturated solution of tartaric acid at a temperature of 40° to 50° C. Eeduction is effected very rapidly, sometimes in a quarter of an hour. HoYER {Arch. mik. Anat., ix, 1873, p. 222) says that the double chloride of gold and potassium has many advantages over the simple gold chloride. He impregnates in solutions of 0-5 per cent, strength, and reduces in water containing 1 or 2 drops oi a pyrogallic add 206 METALLIC STAINS {IMPREGNATION METHODS). developing solution, such as is used in photography, or in a warm concentrated solution of tartaric acid, at the temperature of an incubating stove. I have myself used the double chloride of gold and sodium with good results. CiACCio {Joum. de Microgr., vii, 1883, p. 38) prefers the double chloride of gold and cadmium. Flechsig {Die Leitungsbahnen in Gehirn, 1876 ; Arch. Anat. u. Phys., 1884, p. 453) reduces in a 10 per cent, solution of caustic soda. Nesteeopfsky treats impregnated preparations with a drop of ammxmium sulphide, and finishes the reduction in glycerin (quoted from Gierke's Fdrberei z. mik. Zweoken). BoHM reduces in Pritcha/rd's solution — amyl alcohol, 1 ; formic acid, 1 ; water, 98. Manfeedi {Arch, fer le Sci. med., v, No. 15) puts fresh tissues into gold chloride, 1 per cent., for half an hour ; then oxalic acid, 0-5 per cent., in which they are warmed in a water-bath to 36°. Mount in- glycerin. Sunny weather is necessary. BoccARDi {Lavori Instit. Fisiol. Napoli, 1886, i, p. 27 ; Joum. Roy. Mic. Soc, 1888, p. 155) recommends oxalic acid of 0-1 per cent, or of 0-25 to 0-3 per cent., or a mixture of 5 c.c. pure formic acid, 1 c.c. of 1 per cent, oxalic acid, and 25 c.c. of water, reducing in the dark not longer than two to four hours. KoLOSSOW {Zeit. wiss. Mik., v, 1888, p. 52) impregnates for two or three hours in a 1 per cent, solution of gold chloride acidulated with 1 per cent, of HCl, and reduces for two or three days in the dark in a 0-01 per cent, to 0-02 per cent, solution of chromic acid. Geber'g {Intern. Monatsschr., x, 1893, p. 205) states that previous treatment of tissues for twenty-four hours with lime-water (Arn- stein's method) greatly helps the reduction. Bernheim {Arch. Anat. Phys., Phys. Abth., 1892, Supp., p. 29) adds to Lowit's dilute formic^ acid a piece of sulphite of sodium (must be fresh and smell strongly of sulphurous acid). Dr. Lindsay Johnson writes to me that besides the " sunning " of the impregnating solution recommended above (§ 349), the gold should be carefully acidulated with a neutral acetate or formate, or acetic or formic acid, at least twenty-four hours before using ; and then afterwards the tissue must be washed until no reaction occurs to test-paper. ApAthy {Mikrotechnik, p. 173 ; Mitth. Zool. Stat. Neapel, xii, 1897, pp. 718 — 728) lays stress on the necessity of having the objects th&roughly penetrated by light from all sides during the process of CHAPTER XVII. 207 reduction. Objects, therefore, should always be so thin that light can readily stream through them. He impregnates for a few hours in 1 per cent, gold chloride (§ 361) in the dark, then brings the objects without washing out with water, the gold solution being just superficially mopped up with blotting-paper, into 1 per cent, formic acid. They are to be set up in this, in a tube or otherwise, so that the Ught may come through them from all sides, and exposed to diffused daylight in summer, or direct sunlight in winter, for six to eight hours without a break. They must not be moved about more than can be helped in the acid. If the acid becomes brown it may be changed for fresh. The temperature of the acid should not be allowed to rise over 20° C, whence direct sunlight is to be avoided during the summer. He mounts in glycerin or his syrup (§ 343). He finds such preparations absolutely permanent. Post-Impregnation. 369. Gerlach's Method (Strickee's Handb., 1872, p. 678) : Spinal cord is hardened for fifteen to twenty days in a 1 to 2 per cent, solution of bichromate of ammonia. Thin sections are made and thrown into a solution of 1 part of double chloride of gold and potassium to 10,000 parts water, which is very shghtly acidulated with HCl, and after ten to twelve hours are washed in hydrochloric acid of 1 to 2 : 3000 strength, then brought for ten minutes into a mixture of 1 part HCl to 1000 parts of 60 per cent, alcohol, then dehydrated and mounted in balsam. See further, for Nerve .Centres, under " Nervous System." 370. GoLGi (Mem. Accad. Torino [2], xxxii, 1880, p. 382) puts tissues previously hardened in 2 per cent, solution of bichromate of potash for ten to twenty minutes into 1 per cent, solution of arsenic acid, then into ^ per cent, solution of chloride of gold and potassium for half an hour, washes in water, and reduces in sunlight in 1 per .cent, arsenic acid solution, which is changed for fresh as fast as it becomes brown. Mount in glycerin. Sunny weather is necessary. 371. Apiray's Method (Zeit. wiss. Mit, x, 1893, p. 349 ; Mitth. Zool. Stat. Neapel, xii, 1897, p. 729) : The material to be used must have been fixed either in subhmate or in a mixture of equal parts of saturated solution of sublimate in 0-5 per cent, salt solution and 1 per cent, osmic acid (this more particularly for Vertebrates). The material should be imbedded as quickly as possible, either in parafiin or in celloidin. Sections are made and fixed on slides, and after the usual treatment with iodine, etc., are either put into distilled 208 METALLIC STAINS {IMPREGNATION METHODS). water for from two to six hours, or are rinsed in water, treated for one minute with 1 per cent, formic acid, and again well washed with water. They are then put for twenty-four hours, or at least overnight, into the gold-bath, which is preferably 1 per cent, gold chloride (see § 361), but may be weaker, down to 0-1. per cent., after which they are just rinsed with water or superficially dried with blotting- paper. The shdes are then set up on end in a sloping position, the sections looking downwards, so that precipitates may not fall on them, in glass tubes filled with 1 per cent, formic acid. The tubes are then exposed to light until the gold is reduced, as directed in § 368 sub fin. I seem to have found it advantageous to reduce in weak solution oi formaldehyde, either with or without formic acid. SzuTZ (Zeit. wiss. Mik., xxix, 1912, p. 292) reduces as Apathy for one day, then rinses and puts back for the night into the gold, then for the next day again into the formic acid. 372. Impregnation of Marine Animals. — For some reason the tissues of marine animals do not readily impregnate with gold in the fresh state. It is said by Fol that impregnation succeeds better with spirit specimens. 373. Preservation of Impregnated Preparations. — Preparations may be mounted either in balsam or in acidulated glycerin (1 per cent, formic acid). Theoretically they ought to be permanent if the reduction of the metal has been completely effected, but they are very liable to go wrong through after-blackening. Ranvier states that this can be - avoided by putting them for a few days into alcohol, which he says possesses the property of stopping the reduction of the gold. Blackened preparations may be bleached with cyanide or ferri- cyanide of potassium. Redding employs a weak solution of ferri- cyanide, Cybulsky a 0-5 per cent, solution of cyanide. Preparations may be double-stained with the usual stains (safranin being very much to be recommended), but nuclei will only take the second stain in the case of negative impregnation. Other Metallic Stains. 374 Osmic Acid and Pyrogallol.— This method was first published by me in 1887 {La Cellule, iv, p. 110). It consists in putting tissues that have been treated with osmic acid into a weak solution of CHAPTER XVII. 209 pyrogallol, in which they quickly turn greenish black, sometimes much too much so. Hekmaiw {Arch. mik. Anat, xxxvii, 4, 1891, p. 570) put platino- aceto-osmic material hardened in alcohol for twelve to eighteen hours into raw pyroligneous acid. This acid ought {Ergebnisse der Anat., ii, 1893, p. 28) to be as raw as possible, and to be of a dark brown colour and evil-smelhng. (The stain obtained in this way is not due to a mere reduction pf the osmic acid, but also to coloration by the brown pyroligneous acid ; for Hermann has obtained the same stain with sublimate material, or alcohol material {op. cit., i, 1891 [1892], p. 7). I find this gives much better results than the pure osmic acid process, but not the best possible. I now proceed as follows : Hermann or Flemming material is brought in bulk, directly after fixing, into a weak aqueous solution of pyrogallol. The tissues may remain in it for twenty-four hours, but for small objects an hour or less is sufficient. An alcoholic solution of pyrogallol may be taken if desired. Eawitz {Lehrhuch, p. 60) takes 20 per cent, aqueous sol. of tarmin. There is thus obtained a black stain, which is at the same time a plasma stain and a nuclear stain, chromatin being so far stained that it is not necessary to have recourse afterwards to a special chromatin stain. With Invertebrates it sometimes gives very elegant differentia- tions of nervous tissue. It is a very easy method, and if pyrogallol be used a very safe one (with pyrohgneous acid not so safe). If it be desired to add a chromatin stain, I greatly recommend safranin (stain very strongly, twenty-four hours at least, and start the extraction with acid alcohol). This method has been attributed to von Maeheenthal. See also under " Nervous System " modifications of this method by Azoulat and Hellee and Gumpektz ; also one by Kolossow (Zeit. wiss. Mile., ix, 1892, p. 38, and ix, 1893, p. 316). 375. Perchloride of Iron.— This reagent, introduced by Polaillon {Journ. de VArutt, iii, 1866, p. 43), sometimes gives useful results, especially in the study of peripheral nerve-gangUa, in which it stams the nervous tissue alone, the connective tissue remaining colourless. The HOGGANS proceed as follows (Journ. QuekeU Cluh, 1876 ; Journ. Boy. Mio. Soc, ii, 1879, p. 368) :— The tissue (having been first fixed with silver nitrate, which is somewhat reduced by a short exposure to diflused light) is dehydrated in alcohol, and treated for a few minutes with 2 per cent, solution of perchloride of iron in spirit ; tben with a 2 per cent, solution of pyrogaUic acid in spirit, and in a few minutes more, according to the depth of tint required, may be washed in water and moimted in glycerin. 14 M. 210 METALLIC STAINS (IMPREGNATION METHODS). Pol fixes in percMoride (§ 80) and treats for twenty-four hours with alcohol containing a trace of gallic acid. PoLAiLLON {loo. eit.) reduces in tannic acid. The method is not applicable to chromic objects. GrOLODETZ and Unna {Monats. prakt. Derm., xlviii, 1909, p. 153) put sections of skin for five minutes into fresh mixture of 1 per cent, perchloride of iron and 1 per cent. sol. of ferricyanide of potassium. See also Unna and Golodetz, ibid., xlix, 1909, p. 97. Roosevelt {Med. Bee., ii, 1887, p. 84 ; Journ. Boy. Mio. Soc., 1888, p. 157) employs a stain composed of 20 drops of saturated solution of iron sulphate, 30 grms. water, and 15 to 20 drops pyrogalhc acid. 376. Palladium Chloride (see Schulze, § 77). Prussian Blue (see Leber, Arch. OpMhalm., xiv, p. 300 ; Ranvier, Traite, p. 108). Cuprie Sulphate (see Leber, ibid.). Lead Ghromate (see Leber, ibid.). Sulphides (see Landois, Gentralb. med. Wiss., 1885, No. 55 ; and Gierke, in Zeit. wiss. Mik., i, 1884, p. 497). Molybdate of Ammonia (Merkel ; Krause) (see Gierke, ibid., i, 1884, p. 96). Oxychloride of Ruthenium (Nicolle and Oantacuzene) (see Ann. Inst. Pasteur, vii, 1893, p. 331). Ruthenium Red (Ruthenium Sesquichloride) (Eisen, Zeit. wiss. Mik., xiv, 1897, p. 200 ; in my hands totally useless). Oxide of Manganese (Golodetz and Unna, Monats. prakt. Derm., xlviii, 1909, p. 151). CHAPTEE XVIII. OTHER STAINS AND COMBINATIONS. 377. Kemschwarz (Platnee, Zeit. wiss. MiL, iv, 1887, p. 350).— A black liquid on sale by G-riibler & HoUborn. Mayer {Grundziige, Lee & Mayer,. 1st ed., p. 202) finds tbat it contains iron, combined with some gallic acid. I use it as follows : Sections (I bave not tried material in bulk) are fixed on slides and treated with Kemschwarz until the required depth of stain is obtained, which will be from a few minutes to twenty-four hours, according to the material. There is obtained a black or neutral-tint stain, which is either a pure chromatin stain, or at the same time a plasma stain. If overstaining should have occurred, the stain is easily differentiated by means of any weak acid, either in water or alcohol. Platnee took alkalies, preferably carbonate of lithia, for differentiation. It may be well, if a good plasma stain has been obtained, to after- stain for twenty -four hours with safranin, followed by differentiation in either neutral or acid alcohol, and clove oil. The stain is perfectly permanent in balsam, and is stated to be a good one for preparations that it is desired to photograph. I greatly recommend this stain, which is safe and easy. The combination with safranin gives a better chromatin stain than safranin alone. 378. Brazilin, the colouring matter of Brazilian redwood or Pernam- buco wood, has been recommended by Eisen {Zeit. wiss. Mik., xiv, 1897, p. 198) and Hickson (Nature, Ixii, 1900, p. 689, and Quart. Journ. Mio. Sci., 1901, p. 469). Mater (Chrundziige, p. 203) finds that, in alum solution, it gives a stain similar to that of hsematein, but much weaker. Iron-Brazilin (Hickson, Quart. Journ. Micr. Sci., xliv, 1901, p. 470) is better. Sections are mordanted for one to three hours in 1 per cent. sol. of iron alum in alcohol of 70 per cent, (made by dissolving 1 grm. of the salt in 23 c.c. of water, warm, and adding 77 c.c. of 90 per cent, alcohol after Cooling), rinsed with alcohol, and put for three to sixteen hours into 0-5 per cent. sol. of BraziUn in alcohol of 70 per cent. 379. Orchella (Orseille), see Wbdl (Arch. path. Anat, Ixxiv, p. 143) and FoL (Lehrb., p. 192), and early editions of this work. 14—2 212 OTHER STAINS AND COMBINATIONS. 380. Orcein (Israel, Virejiow's Arehiv, ov, 1886, p. 169 ; and Prakti- cum der path. Hist., 2 Auif, Berlin, 1893, p. 72) is a dye obtained from the liclien, Leoanora parella, and is not to be confused with orein, another derivative of the same lichen. It is said to unite in itself the staining properties of the basic and acid stains, and also the combination of two contrast colours. Israel stains sections in a solution containing 2 grms. of orcein, 2 grms. of glacial acetic acid, and 100 o.c. of distilled water, washes in distilled water, and passes rapidly through absolute alcohol to thick cedar oil, in which the preparations remain definitely mounted. Nuclei blue, protoplasm red. See also " Connective Tissues " in Part II, and Laueent, Zeii. wiss. Mile, xiii, 1896, p. 302 ; Ruzicka, ibid., xiv, 1898, p. 455 ; and Wolff, ibid., xix, 1903, p. 488. 381. Purpurin, see Eanviee's Traite technique, p. 280 ; Duval's PrScis de Technique histologique, p. 221 ; and Geenachbr's formula in Arch. mik. Anat., xvi, 1879, p. 470. A very weak stain. 382. Indigo. — Indigo is employed in histology in the form of solutions of sOicaUed indigo carmine, or sulphindigotate of soda or potash. The simple aqueous solution gives a diffuse stain, but is of use when employed in conjunction with carmine. See below. Thiersch's Oxalic Acid Indigo-Carmine (see Arch. mik. Anat., i, 1865, p. 160). 383. Other Vegetal Dyes. — See early editions. Those recommended by Claudius (Zeit. wiss. Mik., xvii, 1900, p. 62) are superfluous. Carmine Combinations. 384. Seiler's Carmine followed by Indigo-Carmine {Am. Quart. Mic. Journ., i, 1879, p. 220). — Stain in borax-carmine, wash out with HCl alcohol, wash out the acid, and after-stain in an extremely dilute alcoholic solution of indigo -carmine (2 drops of saturated aqueous solution added to an ounce of alcohol and filtered). I find this method gives good results with sections, but not if it be attempted to stain in bulk. 385. Merkel's Carmine and Indigo-Carmine in One Stain (Merkel, Unters. anat. Anst. Bostock, 1874 ; Month. Mic. Journ., 1877, pp. 242 and 317). Also NoERis and Shakespeare, Amer. Journ. Med. Sci., January, 1877 ; Merkel, Mon. Mic. Journ., 1877, p. 242 ; Marsh, Section Cutting, p. 85 ; Baterl, Arch. Mik. Anat, xxiii, 1885, pp. 36, 37 ; Macallum, Trans. Ganad. Instit, ii, 1892, p. 222 ; Journ. Boy. Mic. Soc, v, 1892, p. 698. 386. Mayer's Carmalum (or Hsemalum) and Indigo-Carmine in One Stain.— Mayer {Mitth. Zool. Stat. Neapel, xii, 1896, p. 320) obtains very good results by taking a solution of 0-1 grm. of indigo- carmine in 50 c.c. of distilled water, or 5 per cent, alum solution, and combining it witb from 4 to 20 volumes of carmalum or bsemalum. 387. Carmine and Picro-Indigo-Carmine (Eamon y Cajal, Rev. CHAPTER XVIII. 213 de Cienc. med., 1895 ; Calleja, Rev. trim. Microgr., ii, 1897, p. 101 ; Zeit. wiss. Mik., xv, 1899, p. 323). — For use after a carmine stain, Ram6n takes a solution of 0-25 grm. of indigo-carmine in 100 grms. saturated aqueous solution of picric acid. Stain (sections) for five to ten minutes, wash in weak acetic acid, then in water, then remove the excess of picric acid with absolute alcohol, clear and mount. Ram6n also {Elementos de Histologia, 1897 ; quoted from La Cellule, xix, 1901, p. 212) employs the picro-indigo mixture after Magenta ; stain strongly in saturated solution of magenta, rinse in water until no more colour comes away, and pass into the indigo mixture. See also Boreel, Ann. Inst. Pasteur, 1901, p. 57, or Lee et Henneguy, Traite, p. 268. 888. Carmine and Anilin Blue (or Bleu Lumiere, or Bleu de Lyon) (Duval, Precis de Technique Microscopique, 1878, p. 225). — Stain with carmine ; dehydrate, and stain for a few minutes (ten minutes for a section of nerve-centres) in a solution of 10 drops of saturated solution of anilin blue in alcohol to 10 grms. of absolute alcohol. Clear with turpentine, without further treatment with alcohol, and mount in balsam. Other authors recommend, instead of anilin blue, bleu de Lyon, dissolved in 70 per cent, alcohol acidulated with acetic acid (Maueice and Schulgin), or bleu lumiere. The solutions of both these colours should be extremely dilute for sublimate material, but strong for chrom-osmium material. It is possible to use them for staining in bulk. Baumgaeten (Arch. mik. Anat., xl, 1892, p. 512) stains sections (of material previously stained in borax-carmine) for twelve hours in a 0-2 per cent, solution of bleu de Lyon in absolute alcohol, and washes out for about half that time before mounting in balsam. He recommends the process for cartilage and nerve-centres. 389. Carmine and Malachite Green. — ^Maas (Zeit. wiss. Zool., 1, 4, 1890, p. 527) recommends borax-carmine followed by weak alcoholic solution of malachite green, with a final washing out with stronger alcohol. 390. Carmine and Plcro-nigrosin (Pianesb). — See Journ. Boy. Mie. 8oo., 1892, p. 292. 391. Carmine and Picric Acid. — See § 298. Hwmatein or HcBmatoxylin Combinations. 392. Hsematoxylin and Picric Acid. — See § 298. 393. Hsematoxylin and Eosin. — This popular combination gives results that are aesthetically beautiful, but (for most objects) is not 214 OTHER STAINS AND COMBINATIONS. so useful as many others, the eosin lacking in electivity. Objects may be stained with hsematoxylin (either in the mass or as sections) and the sections stained for a few minutes in eosin. I think it is better to take the eosin weak, though it has been recommended (Stohe, see Zeit. wiss. Mil., i, 1884, p. 583) to take it saturated. Either aqueous or alcoholic solutions of eosin may be used. HiCKSON (Quart. Journ. Mic. Sci., 1893, p. 129) stains sections for one hour in a strong solution of eosin in 90 per cent, alcohol, washes with alcohol, and stains for twenty minutes in a weak solution of hsematoxylin. This method is most particularly recommendable for embryo- logical sections, as vitellus takes the eosin stain energetically, and so stands out boldly from the other germinal layers in which the blue of the hsematoxylin dominates. See also List (Zeit. wiss. Mik., ii, 1885, p. 148) ; Busch (Yerh. Bed. Phys. Gses., 1887) ; Gierke (Zeit. wiss. Mik., i, 1884, p. 505). Sections should be very well washed before being passed from eosin into hsematoxylin or the reverse, as eosin very easily precipitates hsematoxylin. For the complicated and superfluous mixtures of Rbnaut and of EvBRARD, Demoor and Massart, see Fol's Lehrbuch, p. 196, Ann. Inst. Pasteur, vii, 1893, p. 166, or early editions. See especially Scott's method, § 676. 393a. Hsematoxylin and Azoeosin or Biebrich Scarlet. — See under " NucleoH," § 676. 394. Hsematoxylin and Congo. — See § 305. 395. Hsematoxylin and Safranln. — Kabl {Morph. Jahrb., x, 1884, p. 215) stained very lightly with very dilute Delafield's hsematoxylin for twenty-four hours, then for some hours in (Ppitznee's) safranin, and washed out with pure alcohol. The plasma stain is here given by the hsematoxylin. Similarly Eegaud, Verh. Anat. Ges., xiv, 1900, p. 112. FoA (Festschr. Virchow, 1891, p. 481) stains in a mixture of 25 c.c. of Bohmer's hsematoxylin, 20 of 1 per cent, solution of safranin, and 100 of water for one to three minutes. 396. Hsematoxylin and Saurefuchsin. — Stain first with iron hsematoxylin or hsemalum, then stain (sections) in 0-5 per cent, aqueous solution of Saurefuchsin, dehydrate and mount. 397. Hsematoxylin and Saurehichsin and Orange. — Proceed as above, using for the second stain the following mixture : Saure- fuchsin, 1 grm. ; orange, 6 grms. ; rectified spirit, 60 c.c. ; water. CHAPTER XVIII. 215 240 c.c. (from Squire's Methods and Formulce, p. 42). Using orange G (not mentioned by Squire), I have had very good results. The method of Cavazzani (Riformd Med., Napoh, 1893, p. 604 ; Zeit. wiss. Mik., xi, 3, 1894, p. 344) is far too complicated. 398. Hsematoxylin and Picro-Saurefuchsin (van Gieson, New York Med. Journ., 1889, p. 57 ; quoted from Moeller, Zeit. wiss. Mik., XV, 2, 1898, p. 172, which see for further details). — Proceed as above, using for the second stain the picro-Saurefuchsin mixture, § 299. The second stain must not be too prolonged. Weigert {Zeit. wiss. Mik., xxi, 1904, p. 1) stains first in his irpn- hsematoxylin mixture (§ 244), rinses in water, and stains for a short time in his picro-Saurefuchsin (§ 299), rinses, dehydrates with 90 per cent, alcohol, and clears with carbolic acid-xylol mixture (§ 167). CHAPTER XIX. EXAMINATION AND PRESERVATION MEDIA. 399. Introductory. — I comprehend under this heading all the media in which an object may be examined to advantage. All preservative media may be used for mounting, though the only media that will afford an absolutely sure preservation of soft tissues are the resinous ones. 400. Refractive Indices of Examination Media. — An examination medium should be of such a refractive index as to afford a due degree of visibility of colourless (unstained) elements. The visibility of these is inversely as their transparency when penetrated by the medium. It is directly proportional to the difference between the refractive indices of the object and of the medium in which it is mounted. The greatest transparency is obtained when the refraction of the medium is the same as that of the tissue elements. Media having a lower index than that of the tissues give diminished trans- parency, but greater visibility. Media having a higher index than that of the tissues give great transparency, but diminished visibility of (unstained) details. Now the index of refraction of most tissue elements, after fixation and dehydration, is occasionally higher than that of Canada balsam : so that media of the greatest clearing power (i.e. giving the greatest transparency) must be looked for amongst reagents having an index superior to that of balsam, whilst for enhanced visibility of detail we must employ less refractive media, such as castor oil, glycerin, or water. The following short list, extracted from Behrens' Tabellen zum Gebrauxih bei mikrosJcopischen Arbeiten, Braunschweig, 1892, p. 42, and other sources, may be useful as a guide to the optical effects of various media. The figures give the approximate indices of refrac- tion. They should be accepted with some caution, on account of the' variability of samples. The figures given for balsam refer evidently to the resin in the solid state and not to the solutions used for mounting, which are certainly much lower, according to the lower index of the solvent. CHAPTER XIK. 217 Air Methyl alcohol Distilled, water Sea water Solution of white of egg Absolute alcohol Acetate of potash, satu rated aqueous sol. Grlycerine with an equal quantity of water . Chloride of calcium, 90 per cent, in water Glycerine, Price's OU of bergamot Parafflnum liquidum Olive oil on of turpentine Glycerine, "concentrated " Gilson's Baume au Camsal GUson's Euparal Terpinol Castor oil . . . 1-000 1-323 1-336 1-343 1-350 1-367 1-370 1-397 1-411 1-460 1-464 1-471 1-473 1-473 1-473 1-478 1-483 1-484 1-490 Xylol . . 1-497 Cedar-wood oil, not thick ened . . 1-510 Crown glass . . . 1-518 Cedar- wood oil, thickened 1-520 Gum damar . 1-520 Xylol balsam . 1-524 Oil of lemons . 1-527 Oil of cloves . 1-633 Canada balsam (solid) 1-535 Creasote 1-538 Golophonium . 1-545 Carbohc acid . 1-549 on of anise seed 1-557 Oil of cinnamon (or cassia 1-567 Anilin oil . . . 1-580 Sulphide of carbon . 1-630 Tolu balsam . 1-640 Monobromide of naphtha- lin . 1-660 Solution of sulphur in sul- phide of carbon . 1-750 It will be seen that cedar oil has nearly the index of crown glass (this is true of the oil in the thick state to which it is brought by exposure to the air — not of the new, thin oil, which is less highly refractive) ; it therefore clears to about the same extent as Canada balsam. Clove oil has a much higher index, and therefore clears more than balsam ; cinnamon oil higher stiU. Turpentine and bergamot oil have much lower indices, and therefore clear less. Watery Media. 401. Isotonic and "Indifferent" Liquids. — The old distinction of " Indifferent " liquids, and those which have some action on tissues, appears to be misleading more than helpful ; for no medium is without action on tissues except the plasma with which they are surrounded during the life of the organism ; and this plasma itself is only " indifferent " whilst all is in situ ; as soon as a portion of tissue is dissected out and transferred to a slide in a portion of plasma the conditions become artificial. Water may be employed for the examination of structures that have been well fixed ; but this is by no means applicable to the examination of fresh tissues. It is very far from being an " ia- different " liquid ; many tissue elements are greatly changed by it (nerverend structures, for instance), and some are totally destroyed by its action if prolonged (for instance, red-blood corpuscles). 218 EXAMINATION AND PRESERVATION MEDIA. In order to render it inoffensive to fresh tissues it must have dissolved in it substances of similar diffusibility to those of the liquids of the tissue, so as to prevent the occurrence of osmosis, to which process the destructive action of pure water is mainly due. Now cell contents are a mixture of colloids and crystalloids ; conse- quently, in order to reduce osmotic processes to a minimum, it is necessary that the examination medium contain a due proportion of both crystalloids and colloids. By adding, for instance, white of egg to salt solution this end may be in some measure attained ; and, as a matter of fact, the Kquids recommended as " indifferent " are generally found to contain both crystalloids and colloids. Liquids thus composed, in which tissue-elements are in osmotic equilibrium — that is, neither swell nor shrink — -are said to be isotonic to the tissues ; whilst those in which they shrink are called hypertonic, and those in which they swell hypotonic. Solutions of common salt, in different concentrations, form the base of the most commonly employed isotonic liquids. For marine Invertebrates, sea-water is generally isotonic. 402. Salt Solution (" normal salt solution," " physiological salt solution "). — 0-75 per cent, sodium chloride in water. Caenoy recommends the addition of a trace of osmic acid. Ringer's solution, much used in physiology, consists of sodium chloride 0-8 parts, calcium chloride 0-02, potassium chloride 0-02, sodium bicarbonate 0-02 and water 100 (with or without 0-1 dex- trose). According to Locke {Boston Med.-Surg. Journ., 1896, p. 514) there should be added to salt solution (which to be isotonic should contain, according to Hamburger, 0-9 to 1 per cent, of salt) 0-01 per cent, chloride of potassium, and 0-02 per cent, chloride of calcium, in order to obtain an " indifferent " liquid. Malassez (C R. Soc. Biol., iii, 1896, pp. 504 and 511) takes for erythrocytes about 1 per cent, sodium chloride. Dekhuyzen (Onderz. Phys. Lab. Leiden., 1900, p. 149) takes for blood of Rana 0-8 per cent. For Selachians, MusKENs'XTijd. Nederb. Dierk. Ver., 1894, p. 314) finds 2 J per cent, right ; and Rodin {Comptes Rend., 1900, p. 1009) 1-5 to 2-6 per cent., according to the species. Engblmann {Deutsch. med. Wochenschr., xxix, 1903, p. 64) finds that 0-9 per cent, is isotonic with human blood-serum, and 0-64 per cent, for red blood corpuscles of the frog. Kronecker's " Artificial Serum " (from Vogt et Yung, Traite ■CHAPTER XIX. 219 d'Anat. Comp. Prat, p. 473) consists of common salt 6 parts, caustic soda 0-06, distilled water 1000. BoHM und Oppel {Taschenbuch, 3 Aufl., p. 19) take carbonate of soda instead of caustic soda. 403. Pictet's Liquid (Mitth. Zool. Stat. Neapel, x, 1891, p. 89).— 5 to 10 per cent, solution of chloride of manganese. These propor- tions are for marine animals, and for terrestrial animals will generally be found much too high. For these from 1 to 3 per cent, will be nearer the mark. I find this liquid excellent. 404. Aqueous Humour, Simple White of Egg.—Require no pre- paration beyond filtering. They may be iodised if desired (see next §), or mixed with salt solution. 405. Iodised Serum. — Max Schultze {Virchmv's Archiv., xxx, 1864, p. 263). I take the following from Ranvier (Traite, p. 76). The only serum that gives reaUy good results is the amniotic liquid of mammals. Flakes of iodine are added to it, and the flask frequently agitated for some days. The flask should have a wide bottom, so that the serum may form only a shallow layer in it. Another method is as follows : Serum is mixed with a large proportion of tincture of iodine ; the precipitate that forms is removed by filtration, and there remains a strong solution of iodine in serum. This should be kept in stock, and a Httle of it added every two or three days to the serum that is intended for use. In general for maceration purposes a serum of a pale brown colour should be employed. 406. Artificial Iodised Serum (Frey, Das Mikroskop, 6 Aufl., 1877, p. 75). — Distilled water 270 grms., white of egg 30, sodium chloride 2-5. Mix, filter, and add tincture of iodine. 407. Migula's Glyeerised Blood-serum (see the paper in Zeit. f. wiss. Mik., vii, 2, 1890, p. 172). 408. Chloride of Calcium (Haeting, Bas Mihroskop, 2 Aufl., p. 297). — ^The aqueous solution, either saturated or diluted with 4 to 8 parts of water, has a low refractive index and does not dry up. 409. Acetate of Potash (Max Schultze, Arch,, mik. Anat, vii, 1872, p. 180). — ^A nearly saturated solution in water. The index of refraction is lower than that of glycerin. 410. Syrup. — A good strength is equal' parts of loaf sugar and water. Dissolve by boihng. To preserve it from mould, chloral hydrate may be dissolved in it (1 to 5 per cent.) — I have used as much as 7 per cent., and found no disadvantage — or carboKc acid (1 per cent.). 220 EXAMINATION AND PRESERVATION MEDIA. It may be used as a mounting medium, but there is always risk of the sugar crystallising out. Fabre-Domergue {Bull. Soc. Philomath, ix, 1899, p. 115) dis- solves 200 parts of sugar in 400 of water, and adds 1 part of formalde- hyde, and camphor to saturation. 411. Chloral Hydrate.— 5 per cent, in water (Ladowsky, Arch. f. mik. Anat., 1876, p. 359). Or, 2-5 per cent, in water (Beady, British Copepods). Or, 1 per cent, in water (Munson, Joum. Roy. Mic. Soc., 1881, p. 847). Mercurial Liquids. (I give these as examination media only, not as permanent mounting media Media containing suhUmate always end by mahing tissues granular.) 412. Gilson's Fluid (Carnoy's Biologic Cellulaire, p. 94). Alcohol of 60 per cent. Water ...... Glycerin ...... Acetic acid (15 parts of the glacial to 85 of water) ...... Bichloride of mercury 413. Gage's Albumen Fluid {Zeit.f. wiss. Mik., 1886, p. 223). White of egg ..... Water ....... Corrosive subhmate .... Salt ■ Mix, agitate, filter, and preserve in a cool place, for the study of red blood-corpuscles and cihated cells. 414. Pacini's Fluids (Joum. de Mik., iv, 1880 ; Joum. Boy. Mie. Soc, [N.S.] ii, 1882, p. 702, and early editions of this work). — Antiquated and superfluous. They consist essentially of corrosive sublimate of from J to ^ per cent, strength, with the addition of a little salt or acetic acid. 415. GrOADBT's Fluids (Micro. Diet, art. " Preservation," or early editions of this work). — Quite unsuited for histological purposes. Other Fluids. 416. Chloride and Acetate of Copper (Ripart et Petit's fluid, see § 90). 417. Tannin (Caknot, Biol. Oellulai/re, p. 95). — ^Water 100 grms., powdered tannin 0-40 grm., as an examination medium only. 418. Wickeesheimeb's Fluid (Zool. Anz., 1879, p. 670). — Worthless for histological purposes. 60 c.c. 30 „ 30 „ 2 „ 0-15 grm. ?86, p. 223). 15 c.c. 200 „ 0-5 grm. 4 grms. Recommended CHAPTER XIX. 221 419. Medium of Farrants (Beale, How to Work, etc., p. 58).— Picked gum arable 4 ozs., water 4, glycerin 2. See also the Micro- graphic Dictionary, and A. F. Stanley Kent, in Joum. Roy. Mic. Soc., 1890, p. 820. 420. Gum and Glycerin Medium (Langebhans, Zool. Anzeig., ii, 1879, p. 575). Gummi arab. . . . . . .5-0 Aquae 5-0 to which after twelve hours are added — . Glyoerini . . . ... . . 5-0 Sol. aquosa acid, oorbal. (5-100) . . .10-0 421. Allen's Gum and Glycerin.— Prof. F. J. Allen (in litt.). Solution of gum* arable of the consistency of glycerin, strained, and \ volume- of glycerin and ^ of formol gradually incorporated. Sets hard. 422. Hotek's Gum with Chloral Hydrate or Acetate of Potash (Biol. Gentralb., ii, 1882, pp. 23, 24). — ^A high 60 c.o. glass with a wide neck is flUed two-thirds full with gum arable (in pieces), and then either a solution of chloral (of several per cent.) containing 5 — 10 per cent, of glycerin is added or officinal solution of acetate of potash or ammonia. Filter after solution. The solution with chloral is for carmine or haematoxyKn objects — that with acetate for aniKn objects. 423. Cole's Gum and Syrup Medium. See § 183. 424. Apathy's Gum and Syrup Medium (see § 343). — This medium sets very hard and may also be used for ringing glycerin mounts. 425. Fabre-Domergue's Glucose Medium (La Nature, No. 823, 9 Mars, 1889, supp.).— Glucose syrup diluted to 25° of the areometer (sp. gr. 1-1968) 1000 parts, methyl alcohol 200, glycerin 100, camphor to saturation. The glucose is to be dissolved in warm water, and the other ingredients added. The mixture, which is always acid, must be neutraUsed by the addition of a little potash or soda. It is said to preserve without change almost all animal pigments, but the mounts do not keep indefinitely. 426. Brun's Glucose Medium (from Fabre-Domergue's Premiers Principes du Microscope, 1889, p. 123).— Distilled water 140 parts, camphorated spirit 10, glucose 40, glycerin 10. Mix the water, glucose, and glycerin, then add the spirit, and filter. Henneguy informs me that this liquid preserves the colour of preparations stained with anihn dyes, methyl green included. 427. Levulose is recommended by Bbhrens, Kossbl u. Schief- PEKDECKER {Das MikrosJcop, etc., 1889). It is uncrystallisable, and preserves well carmine and coal-tar stains (hsematoxylin stains fade 222 EXAMINATION AND PRESERVATION MEDIA. somewhat in it). The index of refraction is somewhat higher than that of glycerin. Objects may be brought into it out of water. 438. Amann's Lactophenol (from Langeeon, C. R. Soc. Biol., Iviii, 1905, p. 750). — Carbolic acid, 20 ; lactic acid, 20 ; glycerin, 40 ; water, 20. For Nematodes, Acarids, etc. Add gradually drop by drop to the water containing the organisms. Not for mounting. Mount in glycerin jelly. Glycerin Media. 429. Glycerin. — Glycerin diluted with water is frequently em- ployed as an examination and mounting medium. Dilution with water is sometimes advisable on account of the increased visibility that it gives to many structures. But for efficacious preservation undiluted glycerin, the strongest that can be procured, should be used (see Bbale, How to tvork, etc.). For closing glycerin mounts, the edges of the cover should first (after having been cleansed as far as possible from superfluous glycerin) be painted with a layer of glycerin jelly ; as soon as this is set a coat of any of the usual cements may be applied. See next chapter. * Glycerin dissolves carbonate of lime, and is therefore to be rejected in the preparation of calcareous structures that it is wished to preserve. 430. Extra-refractive Glycerin. — ^The already high index of refraction of glycerin (Price's glycerin, n = 1-46) may be raised by dissolving suitable substances in it. Thus the refractive index of a solution of chloride of cadmium (CdCla) in glycerin may be 1-504; that of a saturated solution of sulphocarbolate of zinc in glycerin may be 1 -SOI ; that of a saturated solution of Scheeing's chloral hydrate (in crusts) in glycerin is 1-510 ; that of iodate of zinc in glycerin may be brought up to 1 -56. For further details see previous editions, or Journ. Boy. Mio. Soc, ii, 1879, p. 346 ; iii, 1880, p. 1051 ; (N.S.), i, 1881, pp. 943 and 366. 431. Glycerin and Alcohol Mixtures. — These afford one of the best means of bringing delicate objects gradually from weak into strong glycerin. The object is mounted in a drop of the liquid, and left for a few hours or days, the mount not being closed. By the evaporation of the alcohol the liquid gradually increases in density, and after some time the mount may be closed, or the object brought into pure glycerin or glycerin jelly. 1. Calberla's Liquid (Zeit. wiss. Zool, xxx, 1878, p. 442). — Glycerin 1 part, alcohol 2, water 3. 2. I strongly recommend the following for very delicate objects :— Glycerin 1 part, alcohol 1, water 2. CHAPTER XIX. 223 3. Hantsch's Liquid. — Glycerin 1 part, alcohol 3, water 2. 4. Jagee's Liquid (Vogt and Yung's Traite d'Anat. Comp. Prat., p. 16). — Glycerin 1 part, alcohol 1, sea water 10. Glycerin Jellies. 432. Glycerin JeUies have a higher index than pure glycerin, and set hard enough to make luting unnecessary, though it is well to varnish the mount. To use them, you melt a small portion on a slide, introduce the object (previously soaked in water or glycerin), and cover. They seem very plausible, but for delicate work I do not recommend them, and should advise instead either pure glycerin or euparal. 433. Lawrence's Glycerin Jelly (Da vies. Preparation and Mount- ing of Microscopic Objects, p. 84). — Soak some gelatin for two or three hours in cold water, pour off the superfluous water, and heat until melted. To each fluid ounce of the gelatin, whilst it is fluid but cool, he adds a fluid drachm of the white of an egg. Boil until the albumen coagulates and the gelatin is quite clear, and to each ounce of the solution add 6 drachms of a mixture composed of 1 part of glycerin to 2 parts of camphor water. 434. Brandt's Glycerin Jelly {Zeit. wiss. Mik., ii, 1880, p. 69).— Melted gelatin 1 part, glycerin IJ parts. The gelatin to be soaked in water and melted as above. After incorporating the glycerin, filter through spun glass pressed into the lower part of a funnel. He describes a simple arrangement for keeping the fimnel warm during the filtering (see early editions). Some drops of carbolic acid should be added. 435. Kaiser's Glycerin Jelly has been given § 155. 436. Squire's Glycerin Jelly (Squire's Methods and Formulce, etc., p. 84). — Soak 100 grms. of French gelatin in chloroform water, drain when soft, and dissolve with heat in 750 grms. of glycerin. Add 400 grms. of chloroform water with which has been incorporated about 50 grms. of fresh egg-albumen ; mix thoroughly, and heat to boiling-point for about five minutes. Make up the total weight to 1550 grms. with chloroform water. Filter in a warm chamber. 437. Heidenhain (Zeit. wiss. Mik., xx, 1905, p. 328) takes of gelatin 9 parts, glycerin 7, and water 42, and to the filtrate adds drop by drop 14 parts of absolute alcohol. 438. Fischer (ibid., xxix, 1912, p. 65) takes 5 grms, of borax dissolved in 240 c.c. of water and adds 25 c.c. of glycerin. To this 224 EXAMINATION AND PRESERVATION MEDIA. he adds 40 grms. of gelatin, dissolves with heat, and continues to heat gently until the solution has somewhat thickened. This remains fluid at ordinary temperatures. 439. GtIlson's Chloral Hydrate Jelly (communicated by Gilson). — 1 vol. of gelatin, melted secundum artem, and 1 vol. of Price's glycerin. Mix, and add crystals of chloral hydrate until the volume has increased by one-half ; warm till dissolved. This gives a very highly refractive medium. GrEOFFROY, Joum. de Botan., 1893, p. 55 (see Zeit. wiss. Mik., ix, 1893, p. 476), dissolves, by the aid of as little heat as possible, 3 to 4 grms. of gelatin in 100 c.c. of 10 per cent, aqueous solution of chloral hydrate. High Refractive Liquids. 440. Stephenson's Biniodide of Mercury and Iodide of Potassium (Joum. Roy. Mic. Soc. [N.S.], ii, 1882, p. 167). — A solution prepared by adding the two salts to water until each is in excess ; the liquid will then be found to have a refractive index of 1-68. (If [Amann, Zeit. wiss. Mik., xiii, 1896, p. 21] glycerin be taken instead of water, it rises to 1-78 or 1-80. Beheens [Tahellen, 1898, p. 71] takes biniodide 65 parts, iodide 50, and water 25. n = 1-71.) Any lower index can be obtained by suitable dilution with water. This fluid is very dense, its specific gravity being 3-02. It is highly antiseptic. For marine animals a weak- solution is probably well adapted, as about a 1 per cent, solution (5 minims to the ounce) will give the specific gravity of sea water. Covers should be sealed with white wax, and the mounts finished with two or three coatings of gold size and one of shellac. I have experimented both with strong and with weak solutions. They are not adapted, I find, for the purposes of a permanent mount- ing medium, for the preparations are ruined by a precipitate which forms in the fluid. But as a temporary examination medium I have occasionally found this solution valuable. Its optical properties are wonderful ; it allows of the examination of watery tissues, without any dehydration, in a medium of refractive index surpassing that of any known resinous medium. See further details in early editions. ' 441. Monobromlde of Naphthalin. — See Joum. Boy. Mic. Soc, 1880, p. 1043 (Abbe and Van Heueck), and Zool. Anz., 1882, p. 665 (Max Plesch). CHAPTER XIX. 225 Resinous Media. 442. Resins and Balsams. — Eesins and balsams consist of a vitreous or amorphous substance held in solution by an essential oil. By distillation or drying in the air they lose the essential oil and pass into the solid state. It is these soUdified resins that should be employed for microscopical purposes ; for the raw resins always contain a certain proportion of water, which makes it difficult to obtain a clear solution with the usual menstrua, is injurious to the optical properties of the medium and to the preservation of stains. All solutions should therefore be made by heating gently the balsam or resin in a stove until it becomes brittle when cold, and then dissolving in an appropriate menstruum. Solutions made with volatile menstrua, such as xylol and chloro- form, set rapidly, but become rapidly brittle. Solutions made with non-volatile media, such as turpentine, set much less rapidly, and pass much less rapidly into the brittle state. Turpentine media preserve the index of visibility of the prepara- tions much longer than do media made with more volatile menstrua. Preparations made with these often become so transparent in course of time that much fine detail is often lost. (Such mounts may, however, be revivified without removing the cover by putting them for a day or two into a tube of benzol ; the benzol penetrates the balsam, and brings it down to a lower refractive index.) For a permanent mounting medium of somewhat low index I unhesitatingly recommend Ewparal. For cases in which a still lower index is desired, 'Gilson's camsal balsam. Turpentine colo- phonium is a safe and excellent medium, but is injurious to alum- hsematein stains. For these, and in general where a strongly clearing medium is desired, xylol balsam is about the most recom- mendable, though it is not perfectly safe, the mounts sometimes developing granules. Seller's alcohol balsam is a fine medium, and perfectly stable. Oil of cedar is sometimes useful, it keeps perfectly, and with time it thickens sufficiently to hold the cover in place ; or if desired, preparations may be luted with Bell's cement. 443, Canada Balsam. — Prepare with the solid balsam as described last §. The usual menstrua are xylol, benzol,' chloroform, and turpentine. Turpentine has the advantages pointed out last §, but the defect that it does not always give a homogeneous solution with Canada balsam, as it does with colophonium. For most purposes the xylol solution is the best. If time be an object, a benzol solution should be preferred, as it sets much quicker than the xylol solution. 15 226 EXAMINATION AND PRESERVATION MEDIA. The chloroform solutions become very brown with age, and are injurious to stains made with tar dyes. Benzol is good when chemi- cally pure simA. free from water. Sahli {Zeit. wiss. MiL, ii, 1885, p. 5) dissolves in cedar oil. ApAthy {Fauna Flora Golf. Neapel, xxii, 1909, p. 18) takes balsam 2 parts, cedar oil (immersion) 1, and chloroform 1. Samples of balsam that are acid are frequently met with, and are injurious to some stains. 443a. How to make Neutral Balsam. — Take balsam crystals dry. Grind up in mortar and add an equal part of sodium bicarbonate : mix thoroughly. Transfer to a bottle and add enough xylolor benzole to make a quite liquid solution. Allow this to stand for three or four days, shaking occasionally. Filter, and place filtrate on a thermostat to evaporate down to the right consistency. S. G. Scott (Journ. Path., xvi), recommends keeping a piece of marble in the balsam bottle, and either placing the latter in a tin box, or painting it black outside in order to protect the balsam from the Hght. See Colucci {Giorn. Ass. Med. Natural Napoli, vii, 1897, p. 172). 444. Seiler's Alcohol Balsam (Proc. Amer. Soc. Mic, 1881, pp. 60-2 ; Journ. Roy. Mic. Soc. [N.S.], ii, 1882, pp. 126-7).— Dissolve solid balsam in warm absolute alcohol, and filter through absorbent cotton. Objects may be mounted in it direct from absolute alcohol. I find it for most purposes admirable. It is one of the most stable solutions Jcnown to me. Care should be taken not to breathe on it, as this may cause cloudiness. 445. Damar (Gum Damar, or Dammar, or d'Ammar).— Tte menstrua are the same as for balsam. I find xylol the best. For directions for preparing solutions, by various authors, see early editions. After ample experience I am convinced tbat not one of these solutions can he depended on for permanent preservation. Sooner or later, sometimes after a few weeks or days, or it may be only after months or years, granules make their appearance in the mounts. 446. Colophonium.— A solution of pale colophonium in oil of turpentine keeps well and gives very good definitions. The solution should not be too thick, as it thickens with age. This medium dries very slowly (so that ample time is afforded for arranging objects in it). In the winter a slide wiU take about a month before it will be hard enough to be safe with oil-immersim lenses ; whereas an alcohol-balsam mount will be dry enough in a couple of days. It injures alum-hsematein stains ; as it sometimes develops clouds of globules it is not to be depended upon. CHAPTER XIX. 227 Dr. S. G. Scott of Oxford used both damar and colophonium instead of balsam, and very few of his preparations left after his decease are good. A large number have become granular. Eehm (Zeit. wiss. Mik., ix, 1893, p. 387) dissolves 1 part colophonium in 10 of henein. Solutions in chloroform or xylol are also used by some, see NissL in Encycl. mik. Techn., ii, p. 274. 447. Venice Turpentine (Vosselek, Zeit. wiss. Mik., vi, 1889, pp. 292 et seq.). — Commercial Venice turpentine is mixed in a tall cyhnder glass with an equal volume of 96 per cent, alcohol, allowed to stand in a warm place for three or four weeks, and decanted. Preparations may be mounted in this medium direct from absolute alcohol. CeUoidin sections can be mounted direct from 96 per cent. Stains keep well, according to Vosselek, but Mateb finds hsemalum stains fade in it. StrcHANNEK (ibid., vii, 1896, p. 463) prepares it with equal parts of Venice turpentine and neutral absolute alcohol. 448. Thickened Oil of Turpentine has been used as a mounting medium by some workers. To prepare it, pour some oil into a plate, cover it lightly so as to protect it. from dust without excluding the air, and leave it until it has attained a syrupy consistency. 449. GtILSon's Sandarac Media (La Cellule, xxiii, 1906, p. 427 : the formulae have not been published, on account of the extreme difficulty of preparation, but the products are on sale by Griibler & HoUborn, even if not Hsted). There are three of these. They are all of them solutions of gum Sandarac in " Camsal " and other solvents (" Camsal " is a liquid formed by the mutual solution of the two soUds salol and camphor). (1) Camsal balsam (baume au camsal), propylic alcohol formula ; a mixture of sandarac, camsal, and propylic alcohol, n = 0478. (2) Camsal balsam, isobutyUc alcohol formula, n = 1-485. (3) Euparal,* a mixture of camsal, sandarac, eucalyptol, and paraldehyde, n == 1-483. There are two sorts of this, the colourless and the green (" euparal vert "), the latter containing a salt of copper, which intensifies hcematoxylin stains. '• Objects may be prepared for mounting in camsal balsam by a bath of propyUc or isobutylic alcohol ; and for euparal by a bath of the special solvent (suppUed by Griibler & Hollborn imder the name of " essence d' euparal "). But this is not necessary. Objects may always be mounted direct from absolute alcohol, and even at a pinch from alcohol of 70 per cent. I myself generally prefer alcohol of 95 per cent, (absolute is dangerously volatile for sections). In * Now inanufactured by Flatters & Garnett (§11) according to Gilson's original formula. 16—2 228 EXAMINATION AND PRESERVATION MEDIA. diflScult cases you may pass through a mixture of the medium and the solvent. These media work very kindly, and do not dry too rapidly. They are not oxidant, and preserve delicate stains (perfectly, so far as I know). The mounts seem to keep perfectly, without scaUng : all of mine, the oldest being eight years old, have kept without the shghtest deterioration in any respect. The primary intention of these media is to spare delicate objects the usual treatment with absolute alcohol and essential oils. But they have another useful property — their low index of refraction. I find that that of euparal is just right for most delicate cytological researches, giving just the desired increase of visibility to unstained elements. Thus I frequently find that unstained spindles which are totally invisible in balsam become strongly visible in the most minute details in euparal. The camsal balsam, n = 1-478, I have also sometimes found valuable, but its index is a little too low for most things, and I generally prefer euparal, which I find I am now using almost as much as balsam. I consider that all the media which have been recommended on the score of a slightly lower index than balsam, such as damar, colophonium, Venice turpentine, castor-oil, are now superseded by these media. 450. Sandarac (Lavdowskt, form Bef. Handbook Med. 8ci., Supp., p. 438). — Griim sandarac 30 grs., absolute alcohol 50 c.c. Not trust- worthy, the mounts scale badly. 451. Photographic Negative Varnish (for mounting large sections without cover-glasses). — See Weigeet, Zeit. wiss. Mile., iv, 1887, p. 209. 452. Castor Oil. — See Gkenachek, Ahhandl. naturf. Oes. Halle-a.-S., Bd. xvi ; Zeit. wiss. Mik., 1885, p. 244. I have not had good results with it. 453. Terpinol.--)i = 1-484. See § 131. 454. Parolein (a pure form of paraffinum liquidum) is recommended by Coles (Lancet, 1911, p. 878) as being quite neutral and preserving certain coal tar stains. Eing mounts with Apathy's gum syrup, § 343. Its index is 1-471, which I find too low for most things. 455. Cedar Oil.— See § 442, sub fin. 456. Gum Thus, dissolved in xylol, is recommended by Eisen, Zeit. wiss. Mile, xiv, 1897, p. 201. 457. Styrax and Liquidambar. — See Journ. Boy. Mie. Soc, 1883, p. 741 ; ibid., 1884, pp. 318, 475, 655, and 827; and the places there quoted. Also Bull. Sac. Beige de Mie., 1884, p. 178 ; and Pol, Lehrb., p. 141. These are very highly refractive media, therefore seldom useful in histology. CHAPTEE XX. CEMENTS AND VARNISHES. 458. Introduction. — Two, or at most three, of the media given below will certainly be found sufficient for all useful purposes. For many years I have used only one cement (Bell's). I recommend this both as a cement and varnish ; gold size may be found useful for turning cells ; and Miller's caoutchouc cement may be kept for occasions on which the utmost soUdity is required. Marine glue is only necessary for making glass cells. For the operations of mounting in fluids, and of making cells and ringing, see Carpenter's The Microscope. Carpenter lays great stress on the principle that the cements or ■ varnishes used for fluid mounts should always be such as contain no mixture of solid particles, for those that do always become porous after a certain lapse of time. All fluid mounts should have the edges of the cover carefully dried and be ringed with glycerin jelly before applying a cement ; by this means all danger of running in is done away with. See §§ 460 and 461. But no method yet devised will make a glycerine mount absolutely permanent. See also Aubert, The Microscope, xi, 1891, 150, and Journ. Roy. Mic. Soc, 1891, p. 692 ; Beck, The Microscope, xi, 1891, pp. 338, 368, and Journ. Roy. Mic. Soc, 1892, p. 293 ; Behrens' Tabellen zum Gebrauch bei mikroskopischen Arbeiten (Bruhn, Braunschweig, 1892) ; RoussELET, Journ. Quek. Mic. Club, vii, 1898, p. 93 ; and as to the comparative tenacity of divers cements, Behrens, Zeit. wiss. Mik., ii, 1885, p. 54, and Aubert Amer. Mon. Mic. Journ., 1885, p. 227 ; Journ. Roy. Mic. Soc, 1886, p. 173.— Aubert places Miller's caoutchouc cement at the head of the list, Lovett's cement coming haKway down, and zinc white cement at the bottom, with less than one-quarter the tenacity of the caoutchouc cement. 459. Paraffin. — Temporary mounts may be closed with paraffin, or white wax, by applying it with a bent wire, as described § 471, and be made more or less permanent by varnishing. 460. Gelatin Cement (Marsh's Section-cutting, 2nd ed., p. 104). — Take half an ounce of Nelson's opaque gelatin, soak well in water, 230 CEMENTS AND VARNISHES. melt in the usual way, stir ia 3 drops of creasote. It is used warm. When the ring of gelatin has become quite set and dry, it may be painted over with a solution of bichromate of potash made by dissolving 10 grs. of the salt in an ounce of water. This should be done in daylight, in order to render the gelatin insoluble. The cover may then be finished with Bell's cement. This process is particularly adapted for glycerin mounts. 461. The Paper Cell Method. — By means of two punches I cut out rings of paper of about a millimetre in breadth, and of about a millimetre smaller in diameter than the cover-glass. Moisten the paper ring with mounting fluid, and centre it on the slide. Fill the cell thus formed with mounting fluid ; arrange the object in it ; put the cover on ; fill the annular space between the paper and the margin of the cover with glycerin jelly (a turn-table may be useful for this) ; and as soon as the gelatin has set turn a ring of gold-size on it, and when that is quite dry, varnish with Bell's cement. For greater safety, the gelatin may be treated with bichromate, according to Marsh's plan, last §. 462. Eousselet's Method for Aqueous Mounts (op. cit., § 458). — Close the mount with a ring of a mixture of 2 parts of a solution of damar in benzol and 1 part gold-size. When dry, put on three or four thin coats of pure gold-size at intervals of twenty-four hours, and finish with a ring of Ward's brown cement. 463. Miller's Caoutchouc Cement. — Composition unknown. May be obtained from the opticians. A very tenacious and quickly drying cement. It may be diluted by a mixture of equal parts of chloroform and strong alcohol (see Rousselet, Journ. Quek. Club, V, ii, 1895, p. 8). 464. Asphalt Varnish [Bitume de Judee). — Unquestionably one of the best of these media, either as a cement or a varnish, provided it be procured of good quality. It can be procured from the ppticians. 465. Brunswick Black. — See early editions, or Beale, How to Work, etc., p. 49. 466. Gold-Size. — Best obtained from the opticians. It is soluble in on of turpentine. A good cement, when of good quality, and very useful for turning cells. 467. Turpentine, Venice Turpentine (Csokoe, Arch. mik. Anat., xxi, 1882, p. 353 ; Parker, Amer. Mon. Mik. Journ., ii, 1881, CHAPTER XX. 231 pp. 229 — 30). — Venice turpentine, or common resinous turpentine, evaporated by heat until brittle on cooling. It is used for closing glycerin mounts in the following manner : Square covers are used, and superfluous glycerin is cleaned away from the edges in the usual way. The cement is then put on with a piece of wire vent at right angles ; the short arm of the wire should be just the length of the side of the cover-glass. The wire is heated in a spirit lamp, plunged into the cement, some, of which adheres to it, and then brought down flat upon the slide at the margin of the cover. The turpentine distributes itself evenly along the side of the cover, and hardens immediately, so that the slide may be cleaned as soon as the four sides are finished. It is claimed for this cement that it is perfectly secure and never runs in. It sets hard in a few seconds. 468. ApIthy's Cement for Glycerin Mounts {Zeit. wiss. Mik., vi, 1889, p. 171). — Equal parts of hard (60° C. melting-point) paraffin and Canada balsam. Heat together in a porcelain capsule until the mass takes on a golden tint and no longer emits vapours of turpen- tine. Used by warming and applying with a glass rod or brass spatula. One appUcation is enough. Does not run in, and never cracks. 469. Canada Balsam, or Damar. — Cells are sometimes made with these. They are elegant, but in my experience are not reliable for permanent mounts. 470. Tolu Balsam Cement (Carnoy's Biol. Cell, p. 129).— Tolu balsam, 2 parts, Canada balsam 1, saturated solution of shellac in chloroform, 2 parts. Add enough chloroform to bring the mixture to a syrupy consistence. Carnoy finds this cement superior to all others. 471. For the cements of Ward, Bell and Clarke and for Kronig's Colophonium and Wax, Marine Glue, Amber and Copal, and Seahng Wax Varnish, see last edition. PART II. SPECIAL METHODS AND EXAMPLES. CHAPTEK XXI. INJECTION — GELATIN MASSES (wARM). 472. Introduction. — Injection masses are composed of a coloured substance called the colouring mass, and of a substance with which that is combined called the vehicle. For instructions as to the operation of injecting, and the neces- sary apparatus, see the Micrographic Dictionary, Ruthbrpoed's and Schafer's Practical Histology, the treatises of Robin and Ranvier, Beale's How to Work with the Microscope, the Lehrbuch der vergleichenden Mihroscojnschen Anatomic of Fol, and (for apparatus especially) the article in the Encycl. d. mik. Technik. For injections for the study of the angiology of Vertebrates the practice of Robin and Ranvier may safely be followed. For injec- tions of Invertebrates (and indeed, for vertebrates if it is desired to demonstrate the minute structure of environing tissues at the same time as the distribution of vessels) masses not containing gelatin are, I think, generally preferable to gelatin masses ; and I would recommend as particularly convenient the Prussian blue glycerin masses of Be ale. Glycerin masses have the great advantage that they are used cold. All formulse which only give opaque masses, or are only suitable for coarse injections for naked eye study, have been suppressed. In § 592 is a section on injection of embryos. 473. Vaso-dilators. — In order that an injection may run freely it is necessary that the vessels of the subject be in a relaxed state. To this end the older anatomists used to wait until rigor mortis had passed off before injecting. But it is evidently preferable in the interest of the proper preservation of the tissues to inject before rigor mortis has set in. Unfortunately, when this is done, it is found that most injection masses — glycerin masses especially — CHAPTER XXI. 233 stimulate the contraction of the vessels, so that frequently it is very difficult to get the injection in. In these cases it may be advisable to use a vaso-dilator. The animal may be ansesthetised with a mixture of ether and nitrite of amyl, and finally killed with pure nitrite. Or, after killing by nitrite, a little nitrite of amyl in salt solution may be injected before the injection mass is thrown in. In any case it is advisable to add a httle nitrite to the mass just before using. The relaxing power is very gr6at (see Oviatt and Sargent, in St. Louis Med. Journ., 1886, p. 207 ; and Journ. Roy. Mic. Soc, 1887, p. 341). Bayliss (in personal communication) suggests for prevention of coagulation, to wash out in citrate of soda (4 per cent.) instead of •75 NaCl, or to add |- per cent, oxalate of calcium to -75 per cent. NaCl. To relax arterial walls, add sodium nitrite 1 in 500 to the washing out fluid. Or, morphia may be added to the injection mass, or 1 per cent. of lactic acid. Mozejko {Zeit. wiss. Mih., xvi, 1909, p. 545) prefers a saturated solution of neutral Peptonum siccum, which has the advantage of hindering coagulation. For warm-blooded animals the mass should be warmed to body-temperature ; and in all cases masses that tend to dehydrate tissues should be avoided if possible. Eobin's Masses. 474. Robin's Gelatin Vehicle {Traite, p. 30). — One part of gelatin soaked and melted in 7, 8, 9, or even 10 parts of water, on a water- bath. This vehicle, hke all gelatin masses, is liable to be attacked by mould if kept long ; camphor and carbolic acid do not suffice to preserve it. Chloral hydrate 2 per cent, is said to do so. 475. Robin's Glycerin-Gelatin Vehicle {Traite, p. 32). — Dissolve in a water-bath 50 grms. of gelatin in 300 grms. of water, in which has been dissolved some arsenious acid ; add of glycerin 150 grms., and of carbolic acid a few drops. Unlike the pure gelatin vehicles, this mass does keep indefinitely. Fkankl {Zeit f. wiss. Zool., Ixiii, 1897, p. 28) prepares a similar vehicle, and adds to it a little solution of corrosive sublimate and a crystal of thymol. 476. Robin's Carmine Colouring Mass {Traite, p. 33). — Rub up 3 grms. of carmine with a little water and enough ammonia to dissolve it. Add 50 grms. of glycerin and filter. 234 INJECTION— GELATIN MASSES (WARM). Take 50 grms. of glycerin with 5 grms. of acetic acid, and add it by degrees to the carmine-glycerin, until a slightly acid reaction is obtained (as tested by very sensitive blue test-paper, moistened and held over the mixture). One part of this mixture is to be added to 3 or 4 parts of the vehicles given above. 477. Robin's Ferrocyanide of Copper Colouring Mass {ibid., p. 34).— Take — (1) Ferrocyanide of potassium (concentrated solution) . . . . . .20 c.c. Glycerin . . . . . . 60 ,, (2) Sulphate ot copper (concentrated solution). 35 „ Glycerin . . . . . . 50 ,, Mix (1) and (2) slowly, with agitation ; at the moment of injecting combine with 3 volumes of vehicle. 478. Eobin's Prussian Blue Colouring Mass (ibid., p. 35, and 2nd ed., p. 1013). Take- (a) Ferrocyanide of potassium* (sol. sat.) . . 90 c.c. Glycerin 50 „ (b) Liquid perchloride of iron at 30° Baume . . 3 „ Glycerin 50 „ Mix slowly and combine the mixture with 3 parts of vehicle. It is well to add a few drops of HCl. Carmine-gelatin Masses. 479. Ranviee's Carmine-Gelatin Mass (Traite technique, p. 116). — Take 5 grms. Paris gelatin, soak until quite swollen and soft, wash, drain and melt it in the water it has absorbed over a water-bath. When melted add slowly, and with continual agitation, 2J grms. of carmine rubbed up with a little water, and just enough ammonia, added drop by drop, to dissolve the carmine into a transparent solution. The mixture is now neutralised by adding cautiously, drop by drop, with continual agitation, a solution of 1 part of glacial acetic acid in 2 parts of water. (When the mass is near neutrality, dilute the acetic acid stiU further.) The instant of saturation is determined by the smell of the solution, which gradually changes from ammoniacal to sour. As soon as the sour smell is perceived the liquid must be examined u^der the microscope. If it contains a granular precipitate of carmine, too much acid has been added, and it must be thrown away. * Erratum " Sulphocyanide " in 1st ed. of Kobin's TraM. CHAPTER XXI. 235 The mass, having been perfectly neutralised, is strained through new flannel. 480. How to Neutralise a Carmine Mass (Yille, Gaz. hebd. d. Sci. med, de. Montpellier, Fev., 1882 ; may be had separately from Delahaye et Lecrosnier, Paris). — ^Ville points out that when carmine is treated with ammonia a certain proportion of the ammonia combines with the carmine and the rest remains in excess. It is this excess that it is required to neutrahse precisely, not the whole of the ammonia employed. To neutralise the acidity of commercial gelatin, it should be washed for an hour or so in running water. As to the neutralisation of the colouring mass, Ville is of opinion that the sour smell cannot be safely relied on in practice, and prefers to employ dichroic litmus paper (litmus paper sensitised so as to be capable of being used equally for the demonstration of acids and bases). For directions for preparing this see loc. cit. or previous editions. 481. Hoyee's Carmine-Gelatin Mass (Biol. Centralh., 1882, p. 21). — Take a concentrated gelatin solution and add to it the needful quantity of neutral carmine staining solution (loc. cit., p. 17). Digest in a water-bath until the dark violet-red colour begins to pass into a bright red tint. Then add 5 to 10 per cent, by volume of glycerin, and at least 2 per cent, by weight of chloral, in a con- centrated solution, and strain. 482. Fol's Carmine-Gelatin Mass (Lehrb., p. 13). This can be kept in the dry state for an indefinite length of time. Gelatin in sheets is cut into strips which are macerated for two days in carmine solution (prepared by diluting one volume of strong ammonia with three of water and adding carmine to saturation, and filtering after a day or two). The strips are then rinsed and put for a few hours into water acidulated with acetic acid, then washed on a sieve for several hours in running water, dried on parch- ment paper, or on a net, and preserved for future use. To get the mass ready for use, the strips are soaked for an hour in water and melted on a water-bath in 10 to 20 parts of water. For anotlier process, which is said to give somewhat better results, but is more complicated, see toe. cit, or Zeit. wiss. Zool, xxxviii, p. 492, or previous ed/iUons. 483. Keause's Carmine-Gelatin Mass (Zeit. wiss. Mile, xxvi, 1909, p. 1). — 100 grms. gelatin soaked in- water, put for two to three days into a solution of 15 grms. carmine in 2 litres of water with 100 grms. of borax, 236 INJECTION— GELATIN MASSES (WARM). washed, treated for a short time with hydrochloric acid of 2 per cent., washed, melted and preserved with camphor. 484. Other Carmine- Gelatin Masses.— Thiersch's, see Arch. mik. Anat., 1865, p. 148. Geelach's, see Kanviek, TraiU, p. 118, Caktee's, see Beale, p. 113. Davies, see his Prep, (md Mounting of Mic. Objects, p. 138. Blue Gelatin Masses. 485. Eanvier's Prussian Blue Gelatin Mass {TraiU, p. 119). — Make a concentrated solution of sulphate of peroxide of iron in distilled water, and pour it gradually into a concentrated solution of yellow prussiate of potash. There is produced a precipitate of insoluble Prussian blue. Wash this on a felt strainer, underneath which is arranged a paper filter in a glass funnel, for some days, until the liquid begins to run off blue from the second filter. The Prussian blue has now become soluble. The strainer is turned inside out and agitated in distilled water ; the Prussian blue will dissolve if the quantity of water be sufiicient. The solution may now be injected just as it is, or it may be kept in bottles tiU wanted, or evaporated in a stove, and the solid residuum put away in bottle. For injections, if a simple aqueous solution be taken, it should be saturated. Such a mass never transudes though the walls of vessels. Or it may be combined with one-fourth of glycerin, or with one twenty-fifth of gelatin soaked for an hour in water and melted over a water-bath in the water it has absorbed. The gelatin is to be poured gradually into the Prussian blue, on the water-bath, stirring continually until the curdy precipitate that forms at first has disappeared. Filter through new flannel and keep at 40° C. until injected. 486. Bkijcke's Soluble Berlin Blue (Arch. mik. Anat., 1865, p. 87).— Make a solution of ferrocyanide of potassium containing 217 grms. of the salt to 1 litre of water, and one of 1 part commercial chloride of iron in 10 parts water. Take equal volumes of each, and add to each of them twice its volume of a cold saturated solution of sulphate of soda. Pour the chloride soJution into the ferrocyanide solution, stirring con- tinually. Wash the precipitate on a filter until soluble, dry it, press between blotting paper in a press, break the mass in pieces, and dry in the air. The concentrated solution of the colouring matter is to be gelatinised with just so much gelatin that the mass forms a jelly when cold. For another method, see previous editions. 487. Other Blue Gelatin Masses.— Hotee's, Arch. mik. Anat, 1876, p. 649 ; Guignet's, Journ. de Microgr., 1889, p. 94 ; Journ. Boy. Mic. CHAPTER XXI. • 237 Soc, 1889, p. 463 ; Thiersch's, Aroh. mik. Anal, i, 1865, p. 148 ; Pol's, Zeit. wiss. Zool., xxxviii, 1883, p. 494. ; and previous editions. Other Colours. 488. Hoyee's Silver Nitrate Yellow Gelatin Mass (Biol. Centralbl., ii, 1882, pp. 19, 22). — A concentrated solution of gelatin is mixed with an equal volume of a 4 per cent, solution of nitrate of silver and warmed. To this is added a very small quantity of an aqueous solution of pyrogallic acid, which reduces the silver in a few seconds ; chloral and glycerin are added as directed § 489. This mass is yellow in the capillaries and brown in the larger vessels. 489. Other Colours.— Hotbr's Green {Biol. Centralb., ii, 1882, p. 19). Made by mixing a bhie mass and a yellow mass. Thiersch's Green {Arch. mik. Anat., 1865, p. 149). Robin's Scheele's Green (Eobin, TraiU, p. 37). Harting's White (see Frey, Le Microscope, p. 190). Fret's White (ibid.). Teichmann's White {ibid., p. 191). Fol's Brown {Zeit. wiss. Zool, xxxviii, 1883, p. 494). Miller's Purple (see Amer. Mon. Mic. Jowrn., 1888, p. 50 ; Journ. Boy. Mic. 8oc., 1888, p. 518). Fol's Lead Chromate {Lehrb., p. 15). Robin's Cadtaium (his Traits, p. 36). Thiersch's Lead Chromate {Arch. mik. Anat, 1865, p. 149). Hoter's Lead Chromate {ibid., 1867, p. 136) ; or, for any of these, see early editions. 490. Ranvibr's Gelatin Mass for Impregnation {Traite, p. 123).— Concentrated solution of gelatin, 2, 3, or 4 parts ; 1 per cent, nitrate of silver solution, 1 part. Neuville {Ann. 8ci. Nat, xiii, 1901, p. 36) takes a solution of 10 grms. of soaked gelatin in 100 c.c. of 1 per cent, solution of nitrate of silver. 491. Friedbnthal's Hardening Mass {Centralb. Phys., xiii, 1899, p. 267). — ^A 10 per cent, solution of gelatin, combined with a colouring mass, and with 1 volume of 4 per cent, formol, serves for injecting vessels and hardening the tissues at the same time. CHAPTER XXII. INJECTIONS — OTHER MASSES (COLD). 492. Fol's Metagelatin Vehicle (Lehrb., p. 17).— If a slight pro- portion of ammonia be added to a solution of gelatin, and the solution be heated for several hours, the solution passes into the state of metagelatin, that is, a state in which it no longer coagulates on cooling and can be injected without warming. Colouring masses may be added to this vehicle, which may also be thinned by the addition of weak alcohol. After injection the preparations are thrown into strong alcohol or chromic acid, which sets the mass. According to the Encycl. mik. Technik., metagelatin is usually prepared by warming with concentrated acetic or oxalic acid. It may be neutrahsed afterwards with carbonate of lime. 493. Tandler's Gold Gelatin Mass {Zeit. wiss. Mik., xviii, 1901, p. 22). — Five grms. of gelatin are soaked in 100 c.c. of water, warmed and melted, and combined with Berlin blue. Then 5 to 6 grms. of iodide of potassium are slowly incorporated. The mass generally remains liquid enough for injection down to a temperature of 17° C, but if it should coagulate a little more iodide should be added. After injection you may fix with 5 per cent, formol. The specimens will bear decalcification with hydrochloric or sulphurous acid. Peabl {Journ. Afpl. Micr., v, 1902, p. 1736) takes 8 to 10 per cent, of the iodide. Mayer {Grundziige Lee and Mayer, 1910, p. 250) takes simply 10 grms. gelatin, 10 grms. hydrate of chloral and 100 c.c. water. MozEJKO {Zeit. wiss. Mik., xxvii, 1910, p. 374) finds that 10 per cent, (or more) of sodium salicylate will retard the setting of gelatin for hours at normal temperatures. Any of these masses may be made to set in the tissues by means of weak formol. Glycerin Masses. 494. Beale's Carmine Glycerin Mass {How to Work, etc., p. 95). — Five grains of carmine are dissolved in a little water with about 5 drops of ammonia, and added to half an ounce of glycerin. Then add half an ounce of glycerin with 8 or 10 drops of acetic or hydro- chloric acid, gradually, with agitation. Test with blue litmus etc., p. 93). 1 ounce. • 1 „ . 12 grains. 1 drachm 4 ounces. CHAPTER XXII. 239 paper, and if necessary add more acid till the reaction is decidedly acid^ Then add half an ounce of glycerin, two drachms of alcohol, and six drachms of water. I have found this useful, but not so good as the two following. 495. Beale's Prussian Blue {How to Work, Common glycerin Spirits of wine .... Ferrocyanide of potassium . Tincture of perchloride of iron - . Water Dissolve the ferrocyanide in one ounce of the water and glycerin, and add the tincture of iron to another ounce. These solutions should be mixed together vei-y gradually, and well shaken in a bottle, the iron being added to the solution of the ferrocyanide of potassium. Next the spirit and the rest of water are to be added very gradually, the mixture being constantly shaken. Injected specimens should be preserved in acidulated glycerin (e.g., with 1 per cent, acetic acid), otherwise the colour may fade. 496. Beale's Acid Prussian Blue (ibid., p. 296). Price's glycerin ..... 2 fluid ounces. Tinct. of sesquichloride of iron . . 10 drops. Ferrocyanide of potassium ... 3 graius. Strong hydrochloric acid ... 3 drops. Water ...... 1 ounce. Proceed as before, dissolving the ferrocyanide in one half of the glycerin, the iron ia the other, and adding the latter drop by drop to the former. Finally add the water and HCl. Two drachms of alcohol may be added to the whole if desired. I find this excellent. 497. Ranvteb's Prussian Blue Glycerin Mass {Traits, p. 120).— The Prussian blue fluid, § 494, mixed with one fourth of glycerin. 498. Thoma's Indigo-Carmine (Arch. Anat. Phys., Anat. Ahih., 1899, p. 270). — Dissolve 0-15 grm. sulphindigotate of soda in 50 c.c. water, filter, add 40 c.c. glycerin and gradually, with agitation, 10 c.c. of a filtered 10 per cent, solution of sodium chloride in water. If desired, 3 c.c. of a 1 per cent, solution of morphia may be added to dilate arteries. A fine precipitate is formed, which is injected with the mass. 499. Gamboge Glycerin (Harting, Das Mikroskop, 1866, 2, Theil, p. 124).— Gamboge rubbed up with water and added to 240 INJECTIONS— OTHER MASSES (COLD). glycerin ; or a saturated alcoholic solution of gamboge added to a mixture of equal parts of glycerin and water. Any excess of alcohol may be got rid of by allowing the mass to stand for twenty-four hours. 500. Other Colours. — Any of the colouring masses, §§ 485 to 498, or other suitable colouring masses, combined with glycerin, either dilute or pure. Purely Aqueous Masses. (See § 592.) 501. Ranvier's Prussian Blue Aqueous Mass {Traite, p. 120). — The soluble Prussian blue, § 494, injected without any vehicle. It does not extravasate. 502. MiJLLER's Berlin Blue (Arch. mih. Anat, 1865, p. 150).— Precipitate a concentrated solution of Berlin blue by means of I" to 1 volume of 90 per cent, alcohol. The precipitate is very finely divided ; and the fluid may be injected at once. 503. Mayer's Berlin Blue (Mitth. Zool. Stat. Neapel, 1888, p. 307). — A solution of 10 c.c. of tincture of perchloride of iron in 500 c.c. of water is added to a solution of 20 gr. of yellow prussiate of potash in 500 c.c. of water, allowed to stand for twelve hours, decanted, the deposit washed with distilled water on a filter until the washings come through dark blue (one to two days), and the blue dissolved in about a litre of water. It is well to add a little acetic acid and to put up the objects in an acid liquid. 504. Emery's Aqueous Carmine (ibid., 1881, p. 21). — To a 10 per cent. ammoniacal solution of carmine is added acetic acid, with continual stirring, until the colour of the solution changes to blood-red. The supernatant clear solution is injected cold without further preparation. The injected organs are thrown at once into strong alcohol to fix the carmine. For injection of fishes. 505. Taguchi's Indian Ink {Arch. mik. Anat., 1888, p. 565). — Chinese or (better) Japanese ink well rubbed up on a hone until a fluid is obtained that does not run when dropped on thin blotting- paper, nor form a grey ring round the drop. Inject until the preparation appears quite black, and throw it into some hardening liquid (not pure water). Dblla Rosa (Fer. Anat. Ges., 1900, p. 141) recommends the liquid Chinese ink sold in the shops. Partially Aqueous Masses. 506. Joseph's White-of-Egg {Ber. naturw. Sect. Schles. Ges., 1879, pp. 36—40 ; Journ. Roy. Mic. Soc, ii, 1882, p. 274).—" Fil- CHAPTER XXII. 241 tered white-of-egg, diluted -yitli 1 to 5 per cent, of carmine solution. . . . This naass remains liquid when cold, coagulates in dilute nitric acid, chromic or osmic acid, and remains transparent in the vessels." For invertebrates. Grosser {Zeit. wiss. Mik., xvii, 1900, p. 178) rubs up Indian ink with white-of-egg ; Hoffmann {Zeit. Morph. Anthrop., iii, 1901, p. 240) with blood-serum ; so also Hamburger, Zeit. wiss. Mik., XXV, 1908, p. 1 (2 vols, of the ink — " Perltusche " — to 3 of serum). 507. Bjbloussow's Gum Arabic Mass (Arch. Anat. Phys., 1885, p. 379). — Make a syrupy solution of gum arable and a saturated solution of borax in water. Mix the solutions in such proportions as to have in the mixture 1 part of borax to 2 of gum arable. Rub up the transparent, almost insoluble mass with distilled water, added little by little, then force it through a fine-grained cloth. Repeat these operations until there is obtained a mass that is free from clots. It should then coagulate in the presence of alcohol, undergoing at the same time a dilatation to twice its original volume. The vehicle thus prepared may be combined with any colouring mass except cadmium and cobalt. After injection the preparation is thrown into alcohol, and the mass sets inmiediately, ^swelling up as above described, and conse- quently showing vessels largely distended. Cold-blooded animals may be injected whilst alive with this mass. It does not flow out of cut vessels. Injections keep well in alcohol. If it be desired to remove the mass from any part of a preparation, this is easily done with dilute acetic acid. 508. Milk has been recently recommended by Fischer {Centralb. allg. Path., xiii, 1902, p. 277 ; Zeit. wiss. Mik., xx, 1903, p. 224). It runs weU, does not extravasate, and can be used for auto-injection of the living subject. After injection it should be coagulated by putting the organs for at least twenty-four hours into a mixture of 75 parts of formol 15 of acetic acid, and 1,000 of water (pure formol will not do). They are then sectioned, and the sections stained with Sudan HI or Scharlach R, which stain the milk. They cannot be mounted in balsam. Celloidin and other Masses. 509. Schiefferdecker's Celloidin Masses {Areh. Anat. Phys., 1882 [Anat. Ahth.-], p. 201). (For Corrosion preparations.)— See previous editions ■ Hochstetter's Modifleation of Schiefferdecker's Mass (Anat. Am., 1886, p. 51) ; Budge's Asphaltum Mass (ArcTi. mih. Anat., 16 242 INJECTIONS— OTHER MASSES {GOLD). xiv, 1877, p. 70), or early editions ; Hotee's Shellac Mass [Arch. mik. Anat., 1876, p. 645). For this and that of Bellabminow {Anal. Anz., 1888, p. 605), see early editions ; Hoteb's Oil-coIour Masses {Internat. Monatsschr. Anat., 1887, p. 341); Seveeeanu's, Terh. Anat. Ges.,21 vers, 1906, p. 275 ; Pansch's Starch Mass {Arch. Anat. Entw., 1877, p. 480; 1880,pp. 232, 371 ; 1881, p. 76; 1882, p. 60; 1883, p. 265; and a modification of the same by Gage, Arner. Man. Mic. Journ., 1888, p. 195) ; Teichmann's Linseed-Oil Masses {8. B. Math. Kl. Kralcau Akad., vii, pp. 108, 158 ; Journ. Boy. Mic. Soc, 1882, pp. 125 and 716, and 1895, p. 704) ; Flint's Celluloid {Amer. Journ. Anat, i, 1902, p. 270) ; Hubee's {ibid., vi, 1907, p. 393) ; Keassuskaja's Photoxylin {Anat, Heft. 2, xiii, 1904, p. 521). 610. Natural Injections (Eobin, Traite, p. 6). — To preserve these throw the organs into a liquid composed of 10 parts of tincture of perchloride of iron and 100 parts of water. Eetteeer and Zenker use solution of Miiller, see Journ. Anat. Phys., 1894, p. 336, and Arch. Path. Anat, 1894, p. 147. 511. Starch Masses. See " Guides for Vertebrate Dissection," Kingsley, New York, 1907. CHAPTER XXIII. MACERATION, DIGESTION, AND CORROSION. Maceration. 512. Methods of Dissociation. — It is sometimes necessary, in order to obtain a complete knowledge of tlie forms of the elements of a tissue, that the elements be artificially separated from their place in the tissue and separately studied after they have been isolated both from neighbouring elements and from any interstitial cement-substances that may be present in the tissue. Simple teasing with needles is often insufficient, as the cement-substances are frequently tougher than the elements themselves, so that the latter are torn and destroyed in the process. In this case recourse must be had to maceration, by which is meant prolonged soaking (generally for days rather than hours) in media which have the property of dissol-^ing, or at least softening, the cement substances or the elements of the tissue that it is not wished to study, whilst preserving the forms of those it is desired to isolate. When this softening has been effected, the isolation is completed by teasing, or by agitation with liquid in a test-tube, or by the method of tapping, which last gives in many cases (many epithelia, for instance) results which could not be attained in any other way. The mace- rated tissue is placed on a slide and covered with a thin glass cover supported at the corners on four little feet made of pellets of soft wax. By tapping the cover with a needle it is now gradually pressed down, whilst at the same time the cells of the tissue are segregated by the repeated shocks. When the segregation has proceeded far enough, mounting medium may be added and the mount closed. A good material for making wax feet is obtained (Vosseler, Zeit. wiss. Mik., vii, 1891, p. 461) by melting white wax and stirring into it one-half to two-thirds of Venice turpentine. The most desirable macerating media are those which, whilst dissolving intercellular substances, do not attack the cells them- selves. Those which contain colloids have been found to give the best results in this respect. Iodised serum is an example. 613. Iodised Serum (Chap. XIX.). — The manner of employing it for maceration is as follows : A piece of tissue smaller than a pea 16—2 244 MACERATION, DIGESTION, AND CORROSION. must be taken, and placed in 4 or 5 c.c. of weakly iodised serum in a well-closed vessel. After one day's soaking the maceration is generally sufficient, and the preparation may be completed by teasing or pressing out, as indicated last § ; if not, the soaking must be continued, fresh iodine being added as often as the serum becomes pale by the absorption of the iodine by the tissues. By taking this precaution the maceration may be prolonged for several weeks. This method is intended toT^e applied to the preparation oi fresh tissues, the iodine playing the part of a fixing agent with regard to protoplasm, which it slightly hardens. 514. Iodide of Potassium (Abnold, Arch. mile. Anat., lii, 1898, pp. 135 and 763). — 10 c.c. of 10 per cent, aqueous sol. of potassic iodide with 5 to 10 drops of a similar solution, containing also 5 per cent, of iodine. 515. Alcohol. — Eanvier employs one-third alcohol (1 part of 90 per cent, alcohol to 2 parts of water). Epithelia will macerate well in this in twenty-four hours. It macerates more rapidly than iodised serum. Other strengths of alcohol may be used, either stronger (equal parts of alcohol and water) or weaker (J alcohol, for isolation of the nerve-fibres of the retina, for instance — Thin). . 516. Salt Solution. — Ten per cent, solution of sodium chloride is a valuable macerating medium. Weaker strengths, down to 0-6 per cent., are also used. 517. MoLEscHOTT and Piso Borme's Sodium Chloride and Alcohol (Moleschott's Vntersuchungen zur Naturlehre, xi, pp. 99 — 107 ; Ranvier, Traite, p. 242). — ^Ten per cent, solution of sodium chloride, 5 volumes ; absolute alcohol, 1 volume. For vibratile epithelium Ranvier finds the mixture inferior to one-third alcohol. 518. Sodium Chloride and Formaldehyde.— Gage recommends the addition of 2 parts of formalin to 1,000 parts of normal salt solution (quoted from Fish, Proc. Amer. Mic. Soc, xvii, 1895,. p. 328). 519. Caustic Potash, Caustic Soda.— These solutions should be employed strong, 35 to 50 per cent. (Moleschott) ; so employed they do not greatly alter the forms of cells, whilst weak solutions destroy all the elements. ("Weak solutions may, however, be employed for dissociating the cells of epidermis, hairs, and nails.) The strong solutions may be employed by simply treating the tissues with them on the slide. To make permanent preparations, the alkah should be neutralised by adding acetic acid, which forms CHAPTER XXIII. 245 with Caustic potash acetate of potash, which constitutes a mounting medium (see Behrens, Kossel, and Schiefeeedeckee, Das Mikroskop, i, 1889, p. 156). See also Gage, Proc. Amer. Soo. of Microscopists, 1889, p. 35. 520. Baryta-water, Lime-water (Fol, Lehrb., p. 110).— Baryta-water will macerate nerve, muscle, and connective tissue in a few hours, lime- water in a few days. 521. Sulphoeyanldes of Ammonium and Potassium (Stirling, Journ. Anat. cmd Phys., xvii, 1883, p. 208).— 10 per cent, solution of either of these salts, for epithelium. Macerate small pieces for twenty-four to forty-eight hours. SouLrBR {Travaux de VInst. Zool. de Montpellier, Nouv. S6r., 2, 1891, p. 171) has found that Stirling's solution greatly deteriorates cellular elements, but that good results are obtained by combining it with a fixing agent. The best results were obtained with a 2 per cent, solution of sulphocyanide combined with liquid of Ripaet and Petit ; good ones, by combining liquid of Eipabt and Petit with artificial serum of Kroneckbr instead of sulphocyanide, or with pepsin, eau de JaveUe, 10 per cent, sulphate of soda, or 1-5 per cent, solution of caustic soda ; also by combining solutions of chloride of sodium, or solutions of caustic potash 6r soda, with any of the usual fixing agents. 582. Landois's Solution {Arch. mikr. Anat., 1885, p. 445). Saturated sol. of neutral chromate of ammonia ..... 5 parts. Saturated sol. of phosphate of potash . 5 „ Saturated sol. of sulphate of soda . 5 „ Distilled water . . . • . . 100 „ Small pieces of tissue are macerated for one to three, or even four to five days, in the liquid, then brought for twenty-four hours into ammonia carmine diluted with 1 volume of the macerating Hquid. GiEEKE particularly recommends this liquid for all sorts of macerations, but especially for the central nervous system, for which he finds it superior to all other agents. It is also recom- mended for the same piurpose by Nansbn (v. Zeit. wiss. Milk., v, 1888, p. 242). 523. Bichromate of Potash. — 0-2 per cent. EisiG {Fauna u: Flora Golf. Neapel, 16 Monog., 1887, p. 297) macerates CapiteUidse in 0-5 to 1 per cent, solution for months or years, a little thymol being added against mould. Mailer's Solution, diluted to same strength, or combined with saliva, has also been used. Beock (for nervous system of Mollusca, Intern. Monatssch. Anat,, 246 MACMATION, DIGESTION, AND CORROSION. i, 1884, p. 349) takes equal parts of 10 per cent, solution of bichro- mate of potash and visceral fluid of the animal. 524. Permanganate of Potash is recommended, either alone or combined with alum, as the best dissociating agent for the fibres of the cornea (Eollett, Stricker^s Handbuch, p. 1108). I have found it, for some objects, very energetic. 525. Chromic Acid. — Generally employed of a strength of about 0-02 per cent. Specially useful for nerve tissues and smooth muscle. Twenty-four hours' maceration will suffice for nerve tissue. About 10 c.c. of the solution should be taken for a cube of 5 millimetres of the tissue (Ranviee). 526. Osmic and Acetic Acid (the Hertwigs, Das Nervensystem u. die Sinnesorgane der Medusen, Leipzig, 1878, and Jen. Zeitschr., xiii, 1879, p. 457). 0-05 per cent, osmic acid ... 1 part. 0-2 „ acetic acid . . . 1 „ MeduscB are to be treated with this mixture for two or three minutes, according to size, and then washed in repeated changes of 0-1 per cent, acetic acid until all traces of free osmic acid are removed ; they then remain for a day in 0-1 per cent, acetic acid, are washed in water, stained in Bbale's carmine, and preserved in glycerin. For Actiniw the osmic acid is taken weaker, 0'04 per cent. ; both the solutions are made with sea water ; and the washing out is done with 0-2 per cent, acetic acid. If the maceration is complete, stain with picro-carmine ; if not, with Beale's carmine. 527. MoBius's Media {Morph. Jahrh., xii, 1887, p. 174). 1. One part of sea water with 4 to 6 parts of 0-4 per cent, solution of bichromate of potash. 2. 0-25 per cent, chromic acid, 0-1 per cent, osmic acid, 0-1 per cent, acetic acid, dissolved in sea water. For Lamellibranchiata. Macerate for several dayis. 528. Nitric Acid. — Most useful for the maceration of muscle. The strength used is 20 per cent. After twenty-four hours' macera- tion in this, isolated muscle-fibres may generally be obtained by shaking the tissue with water in a test-tube. Preparations may afterwards be washed with water and put up in strong solution of alum, in which they may be preserved for a long time (Hopkins, Proc. Amer. Soc. of Microscopists, 1890, p. 165). Maceration is greatly aided by heat, and at a temperature of 40° to 50° C. may be sufficiently complete in an hour (Gage). CHAPTER XXIII. 247 A mixture of equal parts of nitric acid, glycerin, and water is recommended by Marcacci {Arch. Ital. Biol, iv, 1883, p. 293) for smooth muscle. 529. Nitric Acid and Chlorate of Potash (Kuhne, Ueber die peri- pherischm Endorgane, etc., 1862 ; Eanvier, Traite, p. 79). — Chlorate of potash is mixed, in a watch-glass, with four times its volimie of nitric acid. A piece of muscle is buried in the mixture for half an hour, and then agitated with water in a test-tube, by which means it entirely breaks up into isolated fibres. 530. Nitric and Acetic Acid (Apathy, Zeit. wiss. Mih., x, 1898, p. 49). — 3 volumes glacial acetic acid, 3 of nitric acid, and 20 each of water, glycerin, and absolute alcohol. Macerate leeches for twenty-four hours, and bring them into 70 per cent, alcohol, in which they swell ; then after twenty-four hours, 50 per cent, glycerin, changed till the acid is removed. 531. Hydrochloric Acid. — Konigstein (Sitzb. Ahad. Wien, Ixxi, 1875) takes (for gold-impregnated cornese) equal parts of the concentrated acid, glycerin, and water ; Freud (ibid., Ixxviii, 1879, p. 102, for nerve- impregnations), 10 parts of acid, 7 of water, 3 of glycerin ; and Schuberg and Schroder [Zeit. wiss. Zool., Ixxvi, 1904, p. 516) take (for fresh muscles of Hirudinea) hydrochloric acid of 5 per cent. 532. Bela Haller's Mixture {Morphol. Jahrb., xi, p. 321). — One part glacial acetic acid, 1 part glycerin, 2 parts water. For the central nervous system of MoUusca a maceration of thirty to forty minutes may be sufficient. 533. Sulphuric Acid (Ranvibr, Traite, p. 78). — Macerate for twenty-four hours in 30 grms. of water, to which are added 4 to 5 drops of concentrated sulphuric acid. Agitate. For nasal mucosa, crystalline, retina, etc. Odenius foujid very dilute sulphuric acid to be the best reagent for the study of nerve endings in tactile hairs. He macerated hair- follicles for from eight to fourteen days in a solution of from 3 to 4 grs. of " EngHsh sulphuric acid " to the ounce of water. Hot concentrated sulphuric acid serves to dissociate horny epidermic structures (horn, hair, nails). 534. Oxalic Acid. — Maceration for many days in concentrated solution of oxalic acid has been found useful in the study of nerve- endings. 535. Schiefferdecker's Methyl Mixture (for the retina) {Arch, mik. Anat., xxviii, 1886, p. 305).— Ten parts of glycerin, 1 part of 248 MACERATION, DIGESTION, AND CORROSION. methyl alcohol, and 20 parts of distilled water. Macerate for several days (perfectly fresh tissue). 536. Gage's Picric Alcohol (Proc. Amer. Soc. of Microscopists, 1890, p. 120).— Ninety-five per cent, alcohol, 250 parts ; water, 750 ; picric acid, 1. Kecommended especially for epitheha and muscle. A few hours suffice. 537. Chloral Hydrate. — In not too strong solution, from 2 to 5 per cent, for rastance, chloral hydrate is a mild macerating agent that admirably preserves deUcate elements. Lavdowsky (Arch. mih. Anat., 1876, p. 359) recommends it greatly for saUvary glands, HiCKSON (Quart. Journ. Mic. Sci., 1885, p. 244) for the retina of Arthropods. 538. Lysol (Reinke, Anat. Anz., viii, 1892, p. 582). — Ten per cent, solution in distilled water or in water with alcohol and glycerin. Spermatozoa of the rat or cortical cells of hairs are said to be resolved into fibrils in a few minutes, epithelial cells of salamandra to be dissociated instantaneously. Digestion. 539. Digestion is maceration in organic juices, which by dissolving out some of the constituents of tissues earUer than others serves to isolate those which resist. The chief liquids employed are gastric juice (or pepsin) and pancreatic juice (pancreatin or trypsin). Pepsia is best employed in acidified solution, pancreatin in alkaliae. The most favourable temperature for digestion is about 40° C. Pepsia digests albuminoids, coUagen substance and mucin more or less readily, elastin more slowly. Nucleiu is either not dissolved or very slowly. Keratin, neurokeratin, chitin, fat and carbo- hydrates are not attacked. Pancreatin (trypsin) digests albuminoids, nuclein, mucin, and elastic tissue ; whilst collagen substance, reticular tissue, chitin, horny substances, fat and carbohydrates are not attacked. Tissues for digestion should be fresh, or fixed with alcohol, not with chromic acid or other salts of the heavy metals. 540. Pepsin (Beale's, Archives of Medicine, i, 1858, pp. 296— 316). — The mucus expressed from the stomach glands of the pig is rapidly dried on glass plates, powdered, and kept in stoppered bottles. Bight-tenths of a grain will dissolve 100 grs. of coagulated white of egg. CHAPTER XXIII. 249 To prepare the digestion fluid, the powder is dissolved in distilled water, and the solution filtered. Or the powder may be dissolved in -glycerin. The tissues to be digested may be kept for some hours in the liquid at a temperature of 100° F. (37° C). Brucke's (from Caenoy's Biologic cellulaire, p. 94). Glycerinated extract of pig's stomach . 1 volume. 0-2 per cent, solution of HCl . . 3 volumes. Thymol, a few crystals. BiCKFALVi's iCentrabl. med. Wiss., 1883, p. 838).— One grm. of dried stomachal mucosa is mixed with 20 c.c. of 0-5 per cent, hydrochloric acid, and put into an incubator for three or four hours, then filtered. Macerate for not more than half an hour to an hour. KuSKOw's (Arch. mik. Anat., xxx, p. 32). — ^One part of pepsin dissolved in 200 parts of 3 per cent, solution of oxaUc acid. The solution should be freshly prepared, and the objects (sections of hardened Ligamentum Nuchse) remain in it at the ordinary tem- perature for ten to forty minutes. 541. Fancreatin. — Schieiferdecker's {Zeit. wiss. Mik., iii, 1886, p. 483). — A saturated solution of the " Pankreatinum siccum," prepared by Dr. Witte, Rostock, is made in distilled water, cold, and filtered. Pieces of tissue (epidermis) are macerated in it for three to four houis at about body temperature. KtJHNE's (Unters. a. d. Phys. Inst. Univ. Heidelberg, i, 2, 1877, p. 219). — Very complicated. See also Gedoelst, La Cellule, iii, 1887, p. 117, and v, 1889, p. 126 ; Maas, Festschr. Kupffer, 1899, p. 211, and Hoehl, Arch. Anat. Phys., Anat. Ahfh., 1897, p. 136 (J to % per cent, solution of Mall's or Merck's pancreatin, with 0-3 per cent, of carbonate of soda ; for demonstrating adenoid tissue in paraffin sections). G&rrosion. 542. Corrosion is the operation of destroying the soft parts that surround hard parts that it is desired to study — in short, a means of cleansing hard parts for microscopic study.' It has been applied to the removal of surrounding tissue from injected vessels or cavities. For this, see Altmann's Method [Arch. mik. Anat., 1879, p. 471, or previous editions) ; also Rejsek {Bibliogr. Anat., iv, 1897, p. 229) ; Bbuhl {Anat. Anz., ziv, 1898, p. 418) ; Denker (Anat. Heftc, 1900, p. 300) ; Thoma and Feomherz (Arch. Entwickelungsmech, vii, 1898, p. 678) ; Peabody (Z. Bull, Boston, 1897, p. 164). The following sections relate chiefly to the cleansing of native hard parts. 250 MACERATION, DIGESTION, AND CORROSION. 543. Caustic Potash, Caustic Soda, Nitric Acid.— Boiling, or long soaking in a strong solution of either of these is an efficient means of removing soft parts from skeletal structures (appendages of Arthropods, spicula of sponges, etc.). 544. Eau de Javelle (Hypochlorite of Potash) (Noll, Zool. Anzeig., cxxii, 1882, p. 528). — A piece of sponge, or similar object, is brought on to a slide and treated with a few drops of eau de Javelle, in which it remains until all soft parts are dissolved. (With thin pieces this happens in twenty to thirty minutes.) The preparation is then cautiously treated with acetic acid, which removes all precipitates that may have formed, dehydrated, and mounted in balsam. The process is applicable to calcareous structures. 545. Eau de Labarraque (Hypochlorite of Soda) may be used in the same way as eau de Javelle. Looss {Zool. Anzeig., 1885, p. 333) finds that either of these solutions will completely dissolve chitin in a short time with the aid of heat. For this purpose the commercial solution should be taken concentrated and boiling. If solutions diluted with 4 to 6 volumes of water be taken, and chitinous structures be macerated in them for twenty-four hours or more, according to size, the chitin is not dissolved, but becomes transparent, soft and permeable to staining fluids, aqueous as well as alcoholic. The most dehcate structures, such as nerve-endings, are stated not to be injured by the treatment. The method is applicable to Nematodes and their ova, and also to the removal of the albumen from ova of Amphibia, etc. CHAPTER XXIV. DECALCIFICATION, DESILICIFICATION, AND BLFIACHING. Decalcification. 546. Decalcification. — In order to obtain the best results, it is important to employ only material that has been duly fixed and hardened, and it is well not to put too much confidence in reagents that are said to have the property of hardening and decalcifying fresh material at the same time. It is generally well also to employ fluids that contain substances having a shrinking action on tissues, so as to neutralise the swelling frequently brought about by the decalcifying acids. Large quantities of liquid should be employed. After decalcification the excess of acid should be carefully re- moved_ by washing, not in water, which favours swelling, but in some liquid that has rather a shrinking action, e.g., alum solution. Lastly, the tissues should be neutralised by treatment with car- bonate of lime, or a salt of lithium or sodium or the Uke. Rousseau (Zeit. wiss. Mile., xiv, 1897, p. 207) imbeds fixed material in celloidin, brings it into 85 per cent, alcohol, decalcifies in a very acid mixture (15 to 40 per cent, of nitric acid in alcohol) washes out the acid in alcohol containing precipitated carbonate of Hme, then cuts sections. This for Porifera, corals, Echinoderms, etc. Tissues are said to be well preserved. This process has been applied to the study of the temporal bone of Mammals by Stein (Anat. Anz., xvii, 1900, p. 318). Similarly Bodecker {Zeit. wiss. Mik., xii, p. 190 ; xxv, p., 21 ; xxvi, p. 206 ; and xxviii, p. 158), in a complicated way, adding the acid (6 to 10 per cent.) to the thin celloidin solution taken for imbedding. 547. Decalcification of Bone. — I take the following from Busch : Arch. mik. Anat., xiv, 1877, p. 481 ; see also Hauu, in Zeit. wiss. Mik., viii, 1891, p. 1 ; and Schaffer, ibid., xix, 1903, pp. 308 and 441, and his paper in the Encycl. mik. Technik. The most widely used, though not the best, agent for decalcifica- tion is hydrochloric acid. Its action is rapid, even when very 252 DECALCIFICATION,' DESILICIFICATION, ETC. dilute, but causes serious swelling of the tissues. To remedy this, chromic acid or alcohol may be added to it. Or a 3 per cent, solution of the acid may be taken and have dissolved in it 10 to 15 per cent, of common salt. Or (Waldeyer) to a ^-^^ per cent. solution of chloride of palladium may be added -^ of its volume of HCl. Chromic acid is also much used, but has a very weak decalcifying action and a strong shrinking action on tissues. For this reason it should never be used in solutions of more than 1 per cent, strength, and for delicate structures much lower strengths must be taken. Phosphoric add has been recommended for young bones. Acetic, lactic and pyroligneous acids have considerable decalci- fying power, but cause great swelling. Picric acid has a very slow action, and is only suitable for very small structures. 548. Nitric Acid (Busch, loc. cit.). — To aU other agents Busch prefers nitric acid, which causes no swelling and acts most effica- ciously. One volimie of chemically pure nitric acid of sp. gr. 1-25 is diluted with 10 volumes water. It may be used of this strength for very large and tough bones ; for yoimg bones it may be diluted down to 1 per cent. Presh bones are first laid for three days iu 95 per cent, alcohol ; they, are then placed in the nitric acid, which is changed daily, ioi eight or ten days. They must be removed as soon as the decalcifica- tion is complete, or else they will become stained yellow. When removed they are washed for one or two hours in. running water and placed in 95 per cent, alcqjiol. This is changed after a few days for fresh alcohol. Young and foetal bones may be placed in the first instance in a mixture containing 1 per cent, bichromate of potash and ^ per cent, chromic acid, and decalcified with nitric acid of 1 to 2 per cent., to which may be added a small quantity of chromic acid (■iC P®^ cent.) or bichromate of potash (1 per cent.). By putting them afterwards into alcohol a green stain is obtained. 549. Nitric Acid (Schapeee, Zeit. wiss. Mih., xix, 1903, p. 460).— ScHAFFEK also fiuds uitric acid the best reagent. It should be taken pure ; the addition of formol, alcohol, or the like, slows 'the reaction. The best strength is from 3 to 5 per cent. Objects must not be washed out directly with water, and washing in salt solution, alcohol, phloroglucin, or formol is not sufficient to prevent swelling. Alum in 5 per cent, solution is good, but not necessary. Material should CHAPTER XXIY. 253 be well fixed and imbedded in celloidin (§ 546)^ harden in alcohol ; remove the alcohol with water ; put for twelve to twenty-four hours (large specimens longer) into nitric acid of 3 to 5 per cent., then into a 5 per cent, solution of sulphate of lithium or sodium, to be changed once in the course of twelve to twenty-four hours ; running water, forty-eight hours ; alcohol. 550. Nitric Acid and Alcohol. — Three per cent, of nitric acid in 70 per cent, alcohol. Mayer has long used 5 per cent, acid in 90 per cent, alcohol. Soak specimens for several days or weeks. Pure nitric acid, even if weak, readily exercises a gelatinising action on bone ; whilst the addition of alcohol (or of alum) counteracts this action (Fish, Ref. Handb. Med. Sci., Supp., p. 425). Thoma {Zeit. wiss. Mik., viii, 2, 1891, p. 191) takes 5 volumes of 95 per cent, alcohol and 1 volume pure concentrated nitric acid. Leave bones in this mixture, changing the liquid every two or three days, until th'broughly decalcified, which should happen, even with large bones, in two or three weeks. Wash out until every trace of acid is removed {i.e., for some days after no acid reaction is obtained with Utmus paper) in 95 per cent, alcohol containing an excess of precipitated carbonate of lime. This may take eight to fourteen days, after which the tissues will stain well and may be treated as desired. 551. Nitric Acid and Formol. — Schridde {Hcematol. Techn., Jena, 1910, p. 21) decalcifies material fixed in formol or formol- Miiller in a mixture of 1 part of formol, 1 of nitric acid, and 9 of water. 552. Nitric Acid and Alum (Gage, quoted from Fish, § 550). — A saturated aqueous solution of alum is diluted with an equal volume of water, and to each 100 c.c. of the dilute solution is added 5 c.c. of strong nitric acid. Change every two or three days, until the decalcification is complete. For teeth this is said to be, perhaps, a better decalcifier than the alcohol mixture. 553. Sulphurous Acid (Zieglee, Festschr.f. Kupffer, 1899, p. 51). — A saturated solution in water. Wash out for twenty-four houjs. Acts rapidly and preserves well. Best used after fixation with formol. 554'. Hydrochloric Acid (see §647).— Eanvier says that it may be taken of 50 per cent, strength, and then has a very rapid action. To counteract the swelling action of the acid, sodium chloride may be added (voN Ebner), see HAUG'spaper quoted § 547. He takes either 100 c.o. 254 DECALCIFICATION, DESILlCIFICATION, ETC. cold saturated solutioii of sodium chloride in water, 100 c.c. water, and 4 c.c. hydrochloric acid. Preparations to be placed in this, and 1 to 2 c.c. hydrochloric acid added daily until they are soft. Or, 2-5 parts of hydrochloric acid, 500 of alcohol, 100 of water, and 2-5 of sodium chloride. Haug prefers the proportions of 1-0 to 6-0 of acid, 70 of alcohol, 30 of water, and 0-5 of salt. 555. Hydrochloric Acid and Chromic Acid {Bayeel, Areh. milt. Anat., 1885, p. 35). — Equal parts of 3 per cent, chromic acid and 1 per cent, hydrochloric acid. For ossifying cartilage. Haug recommends equal parts of 1 per cent, hydrochloric acid and 1 per cent, chromic acid (loc. cit.). 556. Hydrochloric Acid and Glycerin. —Glycerin, 95 ; hydrochloric acid, 5 (Squiee's Methods and Formulas, p. 12). 557. Trichloracetic Acid. — Partsch {VerJi. Ges. D. Naturf. Aertze, 1895, 2 Theil, 2 Halfte, p. 26) uses a 5 per cent, aqueous solution, and Neu^erger {Centralb. Phys., xi, 1897, p. 494) a 4 per cent. one. Action energetic, preservation said to fee excellent. 558. Picric Acid should be taken saturated and changed frequently. Its action is weak, but it gives good results with small objects. Picro-nitric or Picro-hydrochhrie Acid. — Action very rapid. 559. Phosphoric Acid.^10 to 15 per cent. (Haug, loc. cit. in §547). Somewhat slow, staining not good. According to Sciiaffee, § 549, it produces swelling. 560. Lactic Acid. — 10 per cent, or more. Fairly rapid, preserves well, and may be recommended (Haug, loc. cit.). 561. Chromic Acid is employed in strengths of from 0-1 per cent, to 2 per cent, (but see § 547), the maceration lasting two or three weeks (in the case of bone). It is better to take the acid weak at first, and increase the strength gradually. Action excessively slow. 562. Chromic and Nitric Acid. — Seilee (For., Lehrb., p. 112) takes 70 volumes of 1 per cent, chromic acid, 3 of nitric acid, and 200 of water. The action is still excessively slow, frequently requiring months to be complete. 563. Chromo-aceto-osmic Acid (Van ver Stricht, Arch. Biol., ix, 1889, p. 29 ; and Schaffer, Zeit. wiss. MiL, x, 1893, p. 179).— Objects to be left in it for months, the liquid being changed at first every two days, afterwards less frequently. Structure well pre- served. 564. Arsenic Acid.— 4 per cent, aqueous solution, used at a tempera- ture of 30° to 40° C. (Squiee's Methods and Formulce, etc., p. 11). 565. Phloroglucin with Acids (Andeee, Centralb. med. Wiss., xii, xxxiii, pp. 193, 579 ; Intern. Monatsschr., i, p. 350 ; Haug, Zeit. wiss. CHAPTER XXIV. 255 Mik., viii, 1891, p. 8 ; Feeeeri, ibid., ix, 1892, p.^236 ; Bull. li. Aead. Med. di Boma, 1892, p. 67).— This is said to be the most rapid method of any. Phloroglucin by itself is not a solvent of lime salts ; its function in the mixture given below is so to protect the organic elements of tissues against the action of the mineral acid that this can be used in a much more concentrated form than would be otherwise advisable. Andeer takes a saturated solution in warm water, and adds to it 5 to 50 per cent, of hydrochloric acid. Wash out in running water. Other acids than hydrochloric may, of course, be taken. See Haug, Zeit. wiss. Mile, viii, 1891, p. 8, and Pekkbei, Bull. Acad. Med. Boma, 1892, p. 67, or (for both) fifth edition. Desilicification . 566. Hydrofluoric Acid (Mayer, Zool. Anz., 1881, p. 593).— The objects are brought in alcohol into a glass vessel coated internally with paraffin. Hydrofluoric acid is then added drop by drop (taking great care to avoid the fumes, which attack mucous mem- branes with great energy). Small pieces of siliceous sponges will be completely desilicified in a few hours, or at most a day. The tissues do not suffer. For sponges I iind that this dangerous method can be avoided. If well imbedded, sections may be made from them without previous removal of the spicula, which appear to break off sharp before the knife. Rousseau imbeds the objects in celloidin, as described § 549, then brings the block, in a covered caoutphouc dish, for a day or two into a mixture of 50 c.c. alcohol and 20 to 30 drops of hydrofluoric acid, and washes out the acid with alcohol containing carbonate of litliia in powder. Bleaching. 567. Mayer's Chlorine Method (Mitth. Zool. Stat. Neapel, ii, 1881, p. 8). — Put into a glass tube a few crystals of chlorate of potash, add 2 or 3 drops of hydrochloric acid, and as soon as the green colour of the evolving chlorine has begun to show itself, add a few cubic centimetres of alcohol of 50 to 70 per cent. Now put the .objects (which must have previously been soaked in alcohol of 70 to 90 per cent.) into the tube. They float at first, but eventually sink. They will be found bleached in from a quarter of an hour to one or two days, without the tissues having suffered. Only in obstinate cases should the liquid be warmed or more acid taken. Sections on slides may be bleached in this way. Instead of hydro- chloric acid nitric acid may be taken, in which case the active agent evolved is oxygen instead of chlorine. This method serves both for removing natural pigments, such as those of the skin or of the eyes of Arthropods, and also for bleaching 256 DEOALQIFIGATION, DESILIOIFIGATION, ETC. material that has been blackened by osmic acid, and, according to renewed experiments of Mayer's, is to be preferred to the peroxide of hydrogen method. , For bleaching chitin of insects, not alcohol but water should be added to the chlorate and acid (Mayek), Arch. Anat. Phys., 1874, p. 321). See also MATEBin Zeit. wiss. Mile, xxiv, 1907, p. 353 (paraffin sections exposed to the vapour of chlorine water). Grtnfeltt and Mestebzat (0. B. 8oo. Biol., Ixi, 1906, p. 87) add 2 c.c. of 20 per cent, solution of chloric acid (HClOs) to 15 c.c. of alcohol and put sections (of retina) into it for several hours at 42° C. 568. Eau de Labarraque. Eau de Javelle (see §§ 544, 545).— These arc bleaching agents. For the manner of preparing a similar solution see ea/rly editions, OTjourn. de Microgr., 1887, p. 164, oiJourn. Boy. Mie. Soc, 1887, p. 518. Of course, the method cannot be used for bleaching soft parts which it is desired to preserve. 569. Peroxide of Hydrogen (Oxygenated Water) (Pouchet's method, M. Duval, PrScis, etc., p. 234). — Macerate in glycerin, to which has been added a Uttle oxygenated water (§ 35), 5 to 6 drops to a watch-glass of glycerin. Solgee {Centralbl. med. Wiss., xxi, 1883, p. 177) takes a 3 per cent, solution of peroxide. Furst {Morph. Arb. Schwalbe, vi, 1896, p. 529) points out that after a time it macerates. The method serves both for removing pigments and for bleaching osmic and chromic material. 570. Peroxide of Sodium (Caeazzi, Zool. Ann., 444, 1894, p. 135).— See previous editions. 571. Peroxide of Magnesium (Mater, GrundzUge, p. 290).— Use as chlorine, § 567. A slow but delicate method. 572. Sulphurous Acid. — Prof. Gilson writes me that he finds alcoholic solution of sulphurous anhydride (SO2) very convenient for the rapid decoloration of bichromate objects. A few drops suffice. MoNCKEBERG and Bethe (Arch. mik. Anat., liv, 1899, p. 135)' obtain the acid by adding to 10 c.c. of a 2 per cent, solution of bisulphate of sodium 2 to 4 drops of concentrated hydrochloric acid. Objects are put into the freshly prepared solution for six to twelve hours. 573. Permanganate of Potash.— Alfieri {Monitore Zool. Ital., viii, 1897, p. 57) bleaches celloidin sections of the choroid, etc., for eight to twenty-four hours in a 1 : 2000 solution of permanganate of potash, then washes them out for a few hours in a solution of oxalic acid of 1 ; 300 strength, or weaker. CHAPTER XXIV. 257 574. Geenacher's Mixture for Eyes of Arthropods and other Animals (Abh. not. Oes. Halle-a.-8., xvi ; Zeit. wiss. Mik., 1885, pi 244). Glycerin 1 part. 80 per cent, aloohol . . . .2 parts. Mix and add 2 to 3 per cent, of iiydrocliloric aoid. Pigments (i.e. thope in question) dissolve in this fluid, and so doing form a stain which suffices in twelve to twenty-four hours for staining the nuclei of the preparation. 575. Nitric Acid. — Parker '{Bull. Mtis. Oomf. Zool., Cambridge, U.S.A., 1889, p. 173) treats sections (of eyes of scorpions) fixed to the slide with Schallibaum's medium, for about a minute with a solution of up to 50 per cent, of nitric acid in alcohol, or, still better, with a 35 per cent, solution of a mixture of equal parts of nitric and hydrochloric acid in alcohol. To make the solution, the acid should be poured slowly into the alcohol (not vice versd), and the mixture kept cool. Jander {Zeit. wiss. Mile, xv, 1898, p. 163) takes for removal of pig- ments Seiler's chromo-nitric aoid (§ 562) ; twelve to forty -eight hours is enough for small objects. See also under " Arthropoda." 576. Caustic Soda. — -Rawitz {Leitfaden, p. 29). dissolves the pigment of the mantle of Lamellibranchia by means of 3 to 9 drops of officinal caustic soda solution added to 15 to 20 c.c. of 96 per cent, alcohol. 17 CHAPTER XXV. 577. New Advances, — In nearly every case the newest advances in ordinary embryological teclinique are constituted by the improve- ments in fixation reported in the new sections on Cytology (§ 681). It would be a great mistake for observers to consider that fixation and staining methods, such as those of Champy-KuU, Kopsch, or Flemming-without-acetic acid, and iron hsematoxylin, are of no concern to embryologists. For instance, amphibian embryos, such as those of Triton (Molge) prepared by Champy-Kull's method are extremely beautiful and instructive for study, for not only does one procure cytological perfection, but also a staining which is poly- chromatic. For the study of invertebrate embryology, the mito- chondrial methods open up a new field for research. The reliability of many of. the new neurological methods (see §§ 865 to 921) has been brought to a state which should now induce embryologists to devote even rare material for preparation, and to use the neurological methods much more widely than at present is the case. In § 768 is a special treatment of the study of fats and lipoids, which can readily be used for embryological studies. In § 646 is a section on " Glycogen," and in § 650 one on " Iron and Copper." In §§ 1035 — 1045 is a Chapter on the " Tissue Culture " methods. In various parts of the book further notes on intravital staining have been inserted. 578. Artificial Fecundation. — This practice, which affords the readiest means of obtaining the early stages of development of many animals, may be very easily carried out in the case of the amphibia * The sections in this chapter treating of Mammalia, Aves, and Pisces, closely follow the TraiU des Methodes Techniques, Lee et Hennegut, and are due almost entirely to Henneguy. The corresponding parts of the Qrimdzilge, Lee and Mayee, are taken from this work, and there- fore also due to Henneguy, which Dr. Lee regrets to observe has not always been understood, though duly pointed out in the Preface to the first edition of the Qrundziige. This foot-note does not apply to any new material introduced into the present edition (J. B. Gr.). CHAPTER XXV. 259 Anura, Teleostea, Cyclostomata, Ecliinodermata, and many Vermes and Coelenterata. In the case of the Amphibia, both the female and the male should be laid open, and the ova should be extracted from the uterus and placed in a watch-glass or dissecting dish, and treated with water in which the testes, or, better, the vasa differentia, of the male have been teased. Females of Teleostea are easily spawned by manipulating the belly with a gentle pressure ; and the milt may be obtained from the males in the same way. (It may occasionally be necessary, as in the case of the Stickleback, to kill the male, and dissect out the testes and tease them.) The spermatozoa of fish, especially those of the Salmonidse, lose their vitality very rapidly in water ; it is, therefore, advisable to add the milt immediately to the spawned ova, then add a little water, and after a few minutes put the whole into a suitable hatching apparatus with running water. Artificial fecundation of Invertebrates is easily performed in a similar way. For methods of artificial Parthenogenesis see Hakvey, Biol. Bull. Wood's Hole, 1910, p. 269. 579. Superficial Examination. — The davelopment of some animals, particularly some invertebrates, may be to a certain extent followed by observations of the living ova under the microscope. This may usefully be done in the case of various Teleosteans, such as the Stickleback, the Perch, Macropodus, and several pelagic forms, and with Chironomus, Asellus aquaticus, Ascidians, Planorbis, many Coelenterata, etc. Some ova of insecta and Arachnida which are completely opaque under normal conditions become transparent if they are placed in a drop of oil ; if care be taken to let their surface be simply impreg- nated with the oil, the normal course of development is not interfered with (Balbiani). 580. Fixation. — Osmic acid, employed either alone or in com- bination with other reagents, is an excellent fixing agent for small embryos, but not at all a good one for large ones. It causes cellular elements to shrink somewhat, and therefore brings out very clearly the slits that separate germinal layers, and any channels or other cavities that may be in course of formation. In virtue of its property of blackening fatty matters, myelin amongst them, it is of service in the study of the development of the nervous system. Chromic acid is indispensable for the study of the external forms 17—2 260 EMBRYOLOGICAL METHODS. of embryos ; it brings out elevations and depressions clearly, and preserves admirably the mutual relations of the parts ; but it does not always preserve the forms of cells faithfully, and is a hindrance to staining in bulk. Picric hquids have an action which is the opposite of that of osmic acid ; they cause cellular elements to swell somewhat, and thus have a tendency to obliterate spaces that may exist in the tissues. But notwithstanding this defect, the picric compounds, and especially Kleinenberg's picro-sulphuric acid, are amongst the best of embryological fixing agents. ScHEiDDE (Zeit. wiss. Mik., xxvii, 1910, p. 362) finds Orth's " Formol-Miiller " in general the best fixative. Fix for not more than twenty-four hours, and pass through graded alcohols (twenty minutes in each) into absolute (one to two hours), cedar oil, xylol, and paraffin. Eabl (Zeit. wiss. Mik., xi, 1894, p. 165) recommends for embryos of Vertebrates, and also for other objects, his platinic subhmate, § 76. This serves for a large number of blastoderms and young embryos (Pisces, Amphibia, Aves, Mammalia). Advanced embryos of Teleostea ought to be fixed in the warmed mixture, in order to avoid rupture of the muscles and shrinkage of the chorda. Some of his best results were obtained by a not too prolonged fixation in a mixture of Platinic chloride, 1 per cent, solution . 1 volume. Picric acid, saturated aqueous . . 2 volumes. Distilled water . . . . .7 Kabl's picro-sublimate mixture has been given § 70. 1 recommended especially for somewhat advanced embryos, such as embryo chicks from the third or fourth day, and other embryos of a similar size. BovEKi (Verli. Phys. Med. Ges. Wiirzburg, xxxix, 1895, p. 4), in order to imbed and out together numbers of ova of Echinoderms, wraps them in pieces of sloughed epidermis of Oryptohrcmchus (of course, other Urodela will do). Sobotta (Arch. mile. Anat., 1, 1897, p. 31) takes pieces of amnios of Mammalia. Sanzo (Zeit. wiss. Mik., xxi, 1904, p. 449) describes an automatic apparatus for fixing material at definite stages. 581. Peter's Double-stam for Yolk and Tissue, see § 224. 582. Removal of Albumen.— The thick layers of albumen that surround many ova are a serious obstacle to the penetration of reagents. Child {Arch. Entwickelungsmech., ix, 1900, p. 587) gives the following as of very general applicability. After fixation CHAPTER XXV. 261 (in any way except with chromic acid) the ova are brought through graduated alcohols up to that of 80 per cent., in which they are hardened. They are then brought down again through successive alcohols into water acidified lightly with any acid (except chromic acid), and the albumen is found to become transparent and dissolve. 583. Reconstruction o£ Embryos from Sections. — To facihtate the study of series of sections, recourse may be had to graphic or plastic reconstruction of the objects. In simple cases it may be sufficient to adopt the plan described by ScHAFFER {Z&it. wiss. Mik., vii, 1890, p. 342). Careful outHnes of the sections to be constructed are drawn on tracing paper with the aid of the camera lucida, superposed, and held up against the light for examination by transparence. Vosmaee [Anat. Am., xvi, 1899, p. 269) draws on plates of celluloid, and sets them up in a rack for examination. Kere [Quart. Journ. Mic. Sd., xlv, 1902, p. 1) draws on plates of ground glass which he afterwards superposes and makes transparent by oil of cloves run in between them. Pensa {Zeit. wiss. Mikr., xxvii, 1910, p. 48) takes sheets of lithographic gelatin. Woodwoeth {Zeit. wiss. Mik., xiv, 1897, p. 15) proceeds as follows : (1) Draw an axial line of the length of the object multiplied by the magnification employed. (2) Measure with a micrometer the greatest diameter of each section. (3) Plot these diameters down transversely on the axial line at distances corresponding to the thickness of the sections multiplied by the magnification. (4) Join the extremities of these diameters ; this wiU'give you an outline of the object. (5) Measure off on each section the nearest and farthest limits (from the margin) of the organs to be filled in, and plot them down on the transverse lines (3), and join the points as before, i.e. from section to section ; this will give you the outlines of the organs. This process is best applicable to reconstruction from transverse sections, but it can be applied to reconstruction from sections in any plane if the object can be provided with a plane of definition at right angles to the plane of section. This may be established by cutting ofE one end of the object, or the like (see also Orientation, §§ 142. 161). To make a simple plastic reconstruction, camera drawings (or photographs) of the sections (all made at the same magnification) are pasted on pieces of cardboard of a thickness equal to that of the sections multiplied by the magnification employed. Then the parts of the drawings representing the cavities of the objects are 262 EMBRYOLOGICAL METHODS. cut out with a knife or fretsaw, cutting through the cardboard ; and the pieces of fretwork thus obtained are pasted together. Many useful modifications of this method have been devised. Cardboard is rather hard to cut, and not conveniently got of the required thickness. Professor Arthur Thompson, of Oxford, uses numbers of sheets of blotting paper to the required thickness, soaked in beeswax ; this makes a very tough substance, and the models, when made, can be handled without chance of injury: other workers use beeswax plates alone, drawing the outline with some sharp instrument and cutting out with a hot knife. Mr. Pittock, of the Zoological Laboratory, University College, London, uses a modification of K. Peter's method {vide infra). Rather thin paper is used for drawing the outline of the object. In this laboratory (Professor J. P. Hill), special rolls of paper are used, so that the diagram of each of hundreds of sections may be safely rolled up in' order till wanted. A large flat stone is used for the manufacture of the wax plates, with two brass gauges of the required thickness placed at a distance which will accommodate in between them the square of paper with the drawing. Instead of treatuig the paper with turpentine, according to Mr. Pittock's method the drawing is rapidly floated over the surface of a dish of water, drawing side down, then laid upon the stone, between the metal gauge and the superfluous moisture smoothed off with a sheet of blotting paper. The melted wax is poured on to the paper, and a heated metal roller passing over the metal gauge leaves just the required amount of wax on the paper. The latter easily peels off the surface of the stone. For more elaborate processes of plastic reconstruction (very compli- , cated and seldom necessary) see Boen, " Die Plattenmodellirmetliode," in Arch. mile. Anat, 1883, p. 591, and Zeit. wiss. Mile, v, 1888, p. 433 ; Steassek, ibid., iii, 1886, p. 179, and iv, pp. 168 and 330 ; k!astsohbnko, iUd., iv, 1887, pp. 235-6 and 353, and v, 1888, p. 173 ; Schapee, ibid., xiii, 1897, p. 446 ; Alexandee, ibid., p. 334, and xv, 1899, p. 446 ; Petee, ibid., xxii, 1906, p. 530 ; Boen and Petee, ibid., xv, 1, p. 31 ; and Verh. Anat. Qes., xiii, 1899, p. 134 ; Johnston, Anat. Ann., xvi, 1899, p. 261 ; Pol, Lehrb., p. 35 or previous editions ; Broman, Anat. Hefte, xi, 1899, p. 557 ; Petee. " Die Methoden d. Rekonstruction " (Fischer, Jena, 1906) ; Schonemann, Anat. Hefte, xviii, 1901, p. 117 ; Gage, Anat. Record, i, 1907, p. 167 ; Neumatee, Festschr. f. Eupffer, 1899, p. 459 ; Maek, Proc. Amer. Acad. Sci., xiii, 1907, p. 629 (electric wax-cutter for cutting out plates). Hill {Bull. Johns Hopkins Hosp., xvii, 1906, p. 114) finds that embryos of mammalia taken from 95 per cent, alcohol and put into caustic potash of 1 per cent, become so transparent that they can be studied without cutting and reconstructing. CHAPTER XXY. 263 Mammalia. * 584. Times for Early Development.— The entry of the sperm into the egg of the mouse takes place from six to ten hours after copula- tion (SoBOTTA, Arch. mikr. Anat. Bd., 45). The pronuclei stage of fertilisation is found from eighteen to twenty-two hours, two-cell stage twenty-six hours, four-cell, fifty hours, eight-cell, sixty hours after copulation : the egg remains in the tube about eighty hours. J. A. Long and E. L. Mark {Contrib. Zool. Lab. Museum, Harvard, Gameg. Inst. Wash., No. 142, 1911) find in the mouse that ovarian eggs within fifteen or sixteen hours after parturition have formed the first maturation spindle. Fertilised eggs are obtained from animals killed between twenty-three and thirty-one hours post partum. The time required for the spermatozoa, after introduction* into the uterus (either artificially or by coitus) to reach the eggs in the first part of the oviduct varies from four to seven hours in mice inseminated about the same number of hours post partum. To obtain free eggs for study, Mark and Long kill mice fourteen to seventeen hours after parturition, the ova being found in a fold of the oviduct. In the rat the eggs are found in the oviduct about 18-7 hours and ovulation occurs in less than eighteen hours post partum. In the rabbit the pronuclei stage of fertilisation occurs about fourteen hours, in the guinea-pig, twenty-two to twenty-four hours after copulation (Sobotta). The rabbit's egg, hke that of the guinea-pig, remains about eighty hours, the dog's egg eight to ten days in the tube (Rothig, Embryol. Technik). Condition of Ovary as Index to Pregnancy. — On opening the body cavity of a mammal, first of all examine the ovary. By so doing one can estimate roughly the time that has elapsed since the dis- charge of the ovum or ova. Prominent stigmata or areas with a blood-shot centre indicate recent ovulation, while a smooth surface of yellowish appearance indicates a corpus luteum, which means that some time has elapsed since ovulation. 585. Isolation of the Eggs and Early Stages. — The tubse and uterus or uteri are dissected out and treated in one of two ways : either the isolated tuba after straightening is washed out from the funnel opening with warm salt solution, or with some fixative like formalin or weak osmic acid, or on the other hand the whole length of the tube is laid open and spread out with a scalpel or sharp scissors and needles, and the eggs are looked for under a dissecting microscope. * Revised by J. B. G. 264 EMBRYOLOGICAL METHODS. If the method of washing out- is adopted, it is best to use a good rubber bulb attached to a glass tube which has been drawn out finely enough to pass into the oviducal opening. KoUiker used Miiller solution or weak osmic acid for injection, collecting the fluid in a series of watch glasses ; J. P. Hill uses solid crystal dishes, which can easily be examined under a stereoscopic binocular microscope. As a fluid for washing out Hill's picronitric osmic (vide infra), weak formalin, or weak osmic acid are probably as good as anything. The success of this injection method depends on the amount of mucous in the tuba and on the condition of the folds in its mucosa ; if the eggs are not found after the injection, the walls of the tube may be opened up with scissors and the lining •Scraped away with a small scalpel ; the mucus thus procured may be diluted with a little indifferent fluid and examined on a slide under the microscope. Both operations of injection or of opening the tuba may succeed with comparatively large animals like the rabbit and dog. It is practically impossible to slit open the tuba of the cat. In cases where the subject is small, as, for instance, the mouse, it is necessary to preserve the whole oviduct and use a fixative sufficiently penetrative to act quickly. Even with the guinea-pig the lumen of the tube is so small that it is difiicult to remove the ova ; we consider that attempts to press out the contents of the tubes are dangerous. In such cases it seems better to cut the tube into lengths with a razor and to fix whole (vide infra). Bischoff in his study on the guinea-pig (Giesson, 1852), and Ballowitz (Arch. Anat. Physiol, 1883) both resorted to the method of squeezing out the contents of the tubes. When found the ova are picked up with the point of a cataract needle or a scalpel, on a piece of black paper cut to a point, or with a pipette, and either examined fresh in the peritoneal fluid or blood serum of the animal, or in Kronecker's or other artificial serum media, or better fixed immediately. In the case o± a large animal such as the rabbit, the same doe may be made to serve for two observations, at some hours' or days' interval. A longitudinal incision of 8 to 10 centimetres' length is made on the median or a lateral line of the abdomen ; an assistant keeps the intestines in their place ; a ligature is placed at the base of one of the uterine cornua, beneath the neck, and a second ligature around the mesometrium and mesovarium. The ovarj, the tuba, and the cornu of that side are then detached with scissors. The abdomen is then closed by means of a few sutures passing through the muscle-layers and the skin. The animals support the operation perfectly well, and the development of the ova of the opposite side is not in the least interfered with. When it CHAPTER XXV. 265 is desired to study these tlie animal may be killed, or may be subjected to a secondary laparotomy if it be desired to preserve it for ulterior observations. This method, however, cannot be carried out in this country owing to the Vivisection Acts. This procedure was also adopted by Hartmann in his study on Didelphys (vide infra). 586. Fixation of the Isolated Ova.— These can be fixed in a chrome- formalin fluid of some kind : Miiller-formol, Helly, Zenker-without- acetic acid and formol are indicated. Eggs may be left in one of these fluids overnight, then washed in distilled water and transferred either to 1 per cent. OSO4, or to some chrome-osmic fluid, this to preserve the fat. The chrome fixation will form insoluble compounds with lipoids, but less so with fats of the type of olein. It seems likely that the fixation technique of Champy-KuU, of Schridde and of Murray (see § 689) wiU be of great value. For a study of the Golgi elements the methods of Cajal and Da Fano and of Mann-Kopsch are worthy of trial, but rather more difficult to work than chrome-osmic or chrome-formol techniques. Where there may be a difficulty of penetration chrome-formol fluids will be found better than chrome-osmium. A perusal of the sections on Mitochondria and Golgi apparatus wiU provide sugges- tions for the treatment of the early stages in manlmalian develop- ment. Van Beneden {Arch, de Biol., 1880, p. 149) brings the living ovum into a drop of 1 per cent. OSO4 on a slide, and thence into a solution of Miiller. After an houi the liquid is changed, and the whole is put into a moist chamber, where it remains for two or three days. It is then treated with glycerine of gradually increasing strength, and at last mounted in pure glycerine acidffied with formic acid. I am inclined to beHeve that the Champy-Kull or Eegaud fixation (the latter with a post-osmication) would be much superior to the above method, that is, for sectiomng. Many authors have used picro-nitric, picro-sulphuric, picro- formol with or without corrosive, chromic-acetic acid, Flenmiing and Hermann, and so on, but one cannot help thinking that the more modern and logical fixation methods will be better. This seems borne out by the late work of Lams {Arch, de Biol., t., xxiii), and Levi {Arch. f. Zellf., xiv). J. P. Hill {Quart. Jour. Micr. Soc, 1910) gives the formula of a " Marsupial mixture " for fixation of ova and blastocysts of Mar- supials. This fluid is made by adding to 96 c.c. of Mayer's picro- nitric, 2 c.c. of 1 per cent. OSO4. Two c.c. of glacial acetic acid may be added, but the picric acid is sufficiently penetrative without the addition of acetic acid. 266 EMBRYOLOGICAL METHODS. J. A. Long {Contrib. Zool. Lab. Museum Compar. Zool. Earvrnd, 1912) describes an ingenious constant temperature bos for working with fresh egg of mammalia. A circulation sMe is also described in detail. So far J. A. Long has succeeded in keeping mice eggs alive and under observa- tion for only twelve hours. J. A. LoxG and E. L. Mark {op. oit.) use a modified Zenker for their study on mouse eggs. They fix for from twenty to sixty minutes. (A) 4 per cent, bichromate of potash. (B) 4 percent, (aq. sol. ) sublimate and 20 per cent, acetic acid. For use, mix equal portions of A and B. Wash out in warm water for twelve to fourteen hours, 70 per cent, alcohol and iodine twelve to fourteen hours, quickly dehydrate, clear in xylol and embed in parafiin. Mark and Long's fixative appears to me (on paper at least) to be far too acid. It may be indicated for chromosome work. 58'?. Subsequent Treatment of Ova. — After fixation the eggs or blastocysts should be brought into 30 per cent, alcohol and slowly- upgraded to 90 per cent, alcohol : at this stage they may be stuck on pieces of liver or brain by Minchest's albumen method ; the egg is placed on the liver and albumen is gently pipetted over it. The alcohol coagulates the albumen, and enables the object to be handled more easily. Another method used by J. P. Hill {Quart. Jour. Micr. Science, 1910) is to bring the ova into alcohol absolute and then into equal parts of alcohol absolute and ether. Then take a hand-cut section of liver or brain (which has been stored in absolute) place 1 drop of 0-5 per cent, solution of photoxyUn (or celloidin) in equal parts of absolute alcohol and ether ; then transfer the egg on a flat camel hair brush to this drop, and harden the object in 15 per cent, chloroform in 90 per cent, alcohol. Transfer to equal parts of absolute alcohol, xylol and chloroform. Then equal parts of chloroform and xylol, and embed in paraffin wax. i The process of sticking the eggs to the hard cut liver or brain section should be carried out under a dissecting microscope. 588. Uterine Eggs. — During the fourth, fifth, and sixth days after copulation the ova of the rabbit are free in the uterine cornua ; they are easily visible to the naked eye, and may be extracted by the same manipulations as those of the tubes. After the sixth day they are at rest in the uterus, but have not yet contracted adhesions with the mucosa, so that they can still be extracted whole. At this stage the parts of the cornua where the ova are lodged are easily distmguishable by their pecuHar aspect, the ova forming eminences of the size of a pea. The cornua should be cut up transversely into as many segments as there are eminences, care being taken to have the ova in the centre of the segments. You then fix each segment CHAPTER XXV. 267 by means of two pins on the bottom of a dissecting dish, with the mesometrial surface downwards and the ovular eminence upwards. The dissecting-dish is then filled up with serum or liquid of Muller, or 0-1 per cent, solution of osmic acid, Bouin's fluid. Hill's fluid, Helly's fluid or 10 per cent, formol. See sections on " Cytology," §§ 673 to 696. With a small scalpel a longitudinal incision is made on the surface of the ovular eminence, not passing deeper than the muscular layer ; the underlying uterine mucosa is then gently dilacerated with two pairs of small forceps, and the ovum set free in the liquid' From the moment the ova have become adherent to the uterine mucosa they can no longer be extracted whole. The embryo being always situated on the mesometrial surface, the ovular eininence is opened by a crucial incision, and th« strip of mucosa to which the embryo remains adherent is fixed with pins on the bottom of the dish. Ed. v. Beneden (see Arch, de Biol., v, fasc. iii, 1885, p. 378) has been able by operating in this way in serum of Kronecker, and keeping the whole at blood temperature, to observe the circulation of the embryo for hours together. (If this be desired to be done, the crucial incision should not be too extended, so as to leave the terminal sinus intact.) Eetterer (C R. Soc. de Biol., 1887, p. 99) advises that for ova of the seventh day the segment of uterus containing them be opened on the mesometrial surface, for at that date no adhesion has yet been contracted with that side. By running in liquid of Kleinenberg by means of a pipette between the ovum and the free surface of the uterus, the ovum may be got away in the shape of a closed vesicle. C. G. Hartmann {Jour. Morph., 1916), in his study of th« develop- ment of the opossum, used Carnoy's, Bouin's, Fleming's and Hill's fluids. He found Hill's " Marsupial mixture " a perfect fixing fluid for marsupial eggs. J. P. Hill now recommends leaving out the acetic acid for delicate objects. 589. Blastoderms and Later Embryos. — The routine methods of embryology apply here in general. Great care must be exercised to avoid rough treatment caused by upgrading the object too quickly. The same remark applies even more particularly to clear- ing, which to get the best result should be done very gradually. In order to bring out the outlines of blastoderm cells the living ovum may be brought into | per cent, solution of nitrate of silver. After remaining there for half a minute to two minutes, according to the age of the vesicle, it is brought into pure water and exposed to the light. The preparations thus obtained are 268 EMBRYOLOGICAL METHODS. instructive, but blacken rapidly, and cannot be permanently preserved. The blastodermic vesicle can be opened with fine needles, and the blastoderm washed, stained, or impregnated with gold, and mounted in glycerin or balsam. For embryonic areas and more advanced embryos, refer to " Cytology," §§ 673 — 696. Kolliker recommends putting the ovum into 0-5 per cent, solution of osmic acid until it has taken on a somewhat dark tint, which happens in about an hour, and then treating it with successive alcohols for several hours. If the ovum be adherent to the uterine mucosa the portion of the membrane to which it is fixed should be left, stretched out with pins, in 0-1 per cent, solution of osmic acid for from four to six hours. The blasto- dermic vesicle can then easily be removed, and further treated as before. For sections Kolliker fixes with osmic acid. v. Beneden treats the ova for twenty-four hours with 1 per cent, solution of chromic acid, then washes well, and brings them through successive alcohols. Chromic acid has the advantage of hardening thoroughly the vesicle, and maintaining at the same time the epiblast cells perfectly adherent to the zona pellucida. v. Beneden also recom- mends the liquid of Kleiaenberg. Hennbguy writes that he fre- quently employs it for embryonic areas and embryos of various ages, always with excellent results. Fol's modification of the liquid of Flemmiag, and Ranvier and Vignal's osmic acid and alcohol mixture (§ 36) also give excellent, results. For staining, Henneguy recommends borax-carmine, or DelafieH's hsematoxylin for small embryos ; for large ones he found that his acetic acid alum-carmine was the only reagent that would give a good stain in the mass. For sections imbed m paraffin, or double imbed. 590. On the Fixation of Whole Tubes. — This may be done in Carnoy, Bouin or Helly. For rapidity of fixation, and faithfulness of preservation of cell aggregates Carnoy's fluid or preferably Sansom's modification of Carnoy are to be recommended. Chrome- formalin mixtures penetrate less readily, but often give fine results. Bouin's fluid I have found capricious. On the whole I think that warm Helly or Mliller-formol as a prehminary fixation are to be recommended for small tubes. Eegaud's or Schridde's methods should give efi&cient fixation (§§ 684 — 689). Many workers have used the picric m ixtures like picro-sulphuric and nitric, and Kleinen- berg's picric acid. Flemming's fluid has also been used. In later stages of development some workers open the uterus CHAPTER XXV. 269 under fixative, or ligature one end of the organ and inject some fixing medium. Corrosive f ormol mixtures have been much used for this purpose. Neutral formalin of from 3 to 10 per cent, strength is often used for preserving later stages, after the uterus has been opened out. The advantage of this procedure from the cytological point of view is that any methods such as those of Eegaud, Bensley- Cowdry, Sjovall, or formol-silver nitrate neurological techniques inay subsequently be used. The chrome-picric or alcoholic acetic formol mixtures are not so suitable if one has cytological study in view. 591. On Clearing Mammalian Material. — This is an important matter, because delicate embryos are easily shrunken up, or even not properly dealcoholised, by injudicious methods. J. P. Hill clears in two stages. Dehydrated embryos are brought into cedar "wood oil in which they are left overnight. The cedar wood oil is subse- quently washed out in benzole for several hours according to size of object. Paraffin parings are then added to the benzole, contained preferably in a tube, and the latter is then left overnight uncovered on the top of the bath, and subsequently put into pure' wax. This method insures a gentle dealcoholisation, and an efficient imbedding. Neither cedar wood oil nor benzole cause the tissue to become brittle as happens often when one uses xylol or chloroform (see §§ 120—135). Imbedding. — For embryological work of a critical character, especially with post-blastoderm stages, double-imbedding in cel- loidin and wax is generally indispensable. It is only necessary to contrast serial sections of chick blastoderms prepared by this method, with those obtained by wax imbedding alone to become convinced of the iaability of the latter method to do complete justice to the details of the structure and relations of the embryonic tissues (Wilson and Hill, Phil. Trans. Roy. Soc, 1907). See also J. P. Hill {Anat. Ans., Bd. xviii, 1900 ; Quart. Journ. Micr. (Soiewce, Ivi, 1910) ; Haetmann (Jow. Jlf orp^., xxvii, 1916). The latter recommends punching a hole in the side of larger blastoderms to facili- tate penetration of dehydrating and clearing fluids. Wetsse, Proe. Amer. Acad. Arts and Sci., 1894, p. 285 (blastodermic vesicle of Sus scrofa) ; Sobotta, Arch. mile. Anat, xlv, 1895, p. 15 (ovum of the Mouse ; fixation in Flemming's weak mixture, sections stained with Benda's iron hsematoxylin), and Anat. Hefte, I Abth., viii, 1897, p. 476 (Rabbit ; fixation with liquid of Flemming or picro-sublimate with 2 per cent. aeetic acid) ; Bonnet, ibid., ix, 1897, p. 426 (Dog ; fixation in sub- limate) ; Selenka, Stud. Entw. d. Thidre, Wiesbaden, 1883, p. 5, and 270 EMBRYOLOGICAL METHODS. 1887, p. 107 (pioro-sulphuric acid for the mouse, and picric acid with ^ per cent, of chromic acidfor Didelphys) ; Keibel, Morph. Arb., ii, 1893, p. 11 (Sus scrofa) ; Neumatbr, Festschr. f. Kupffer, 1899, p. 458 (embryos of the sheep best fixed in Carney's acetic acid, alcohol, and chloroform, § 85) ; Winiwabtee, Arch. Biol, xvii, 1900, p. 39 (mixture of 50 parts saturated sublimate in salt solution, 50 parts alcohol, 20 of 1 per cent, platinum chloride, and 5 of acetic acid) ; Spee, Eneyel. mik. Techn., 1910, p. 353 (cornua of Oai)ia fixed for twelve to twenty -four hours in sublimate, and put into 0-5 per cent, osmic acid till light brown, then into iodine alcohol, in which the osmium is reduced) ; Widakowich, Zeit. wiss. Zool., xciv, 1909, p. 243 {Mus rattus, fixation in Zenker's mixture, or 2 parts of alcohol of 80 per cent, with 1 of formol ; also instructions for dissection). 592. Injection and Clearing of Larger Embryos. — A con- siderable amount of useful work has lately been carried out on embryonic blood and lymph vessels, and on the cerebro-spinal cavities, by micro-injection apparatus. A suitable injection medium is bloASTi or forced into the vessels of an embryo, the latter is fixed and then dehydrated, and cleared by the Spalteholz method (Tiber das Durchsichtigmachen von menschlichen und tierschen Prd- parat^n, und seine theoretischen Bedingungen, Leipzic, S. Herzel, 1911 ; 2 Attfl., 1914). In an early stage in the formation of embryonic vessels and cavities the walls are thin and often ill-marked, and care must be taken not to burst through boundaries by excessive pressure. Very fine metal needles or, better, finely drawn out glass cannulaa are used for injecting the specimens ; the tube leading to the cannula is filled with the injection medium, which, by means of a rubber tube leading to the operator's mouth, is blown carefully into the per- forated vessel or cavity. Or, one may use a rubber bulb either worked by hand, or placed on the floor and compressed by the foot. See E. M. Gregoey, Anat. Record, xi, 1917. The injection media most commonly used are india-ink, a saturated solution of Prussian blue, an aqueous suspension of lamp black, or silver nitrate (5 per cent.). The Prussian blue and india-ink give about equal results, the blue clearing better, the ink being more opaque. The ink flows the better. Silver nitrate preparations are very beautiful and easy to analyse, but its caustic action prevents the finer vessels from filling. Lamp black tends to precipitate in fine flakes (Cunningham, vide infra). Evans (vide infra), for cerebro-spinal spaces of pig embryos, injected potassium ferro- cyanide, 0-5 grms., iron ammonium citrate, 0-5 grm., aq. dest., 100 c.c, and afterwards immersed the embryo for one to ten minutes in a 10 per cent, formaldehyde solution containing 1 per cent. HCl. CHAPTER XXV. 271 The embryo was then fixed in Bouin's fluid, but the Prussian blue faded after about a year. Sabin [vide infra) and Cunningham, after india-ink injection, fix in Carney's fluid, place in 80 per cent, alcohol, dehydrate in graded alcohols, clear thoroughly, first in benzine (or benzol), and then in oil of wintergreen (Spalteholz). Embryos cleared by Spalteholz's method may later be embedded from oil of wintergreen by transferring to half wax, half oil of wintergreen, and then pure wax. Tissues left in oil of wintergreen do not go brittle even after a year or two (Sabin). For areas of osteoblastic activity, see § 780, and cartilaginous skeletons, § 779. See also E. S. Cunningham (Contrib. Carng. Inst. Wash., 1916, No. 12) ; L. H. Weed (ibid., No. 14, 1917) ; F. Sabin (Johns Hopkins Hosp. Report' Monographs, N.S., No. 5, Baltimore, 1913) ; Contrib. to Embryol. Carneg. Inst. Wash., No. 7, 1915) ; P. G. Shipley and C. C. Macklin {Anat. Record, x, 1915-16). Aves. 593. Superficial Examination. — Instructions on this head are given in Foster and Balfour's Elements of Embryology. The following is of more recent publication. If it be desired to observe a living embryo by transmitted light, the egg should be opened under salt solution, as described below. A httle of the white is then removed through the window, the egg is lifted out of the liquid, and a ring of gumined paper is placed on the yolk so as to surround the embryonic area-. As soon as the paper adheres to the vitelline membrane, which will be in a few minutes, a circular incision is made in the blastoderm outside the paper ring. The egg is put back into the salt solution, and the paper ring removed, carrying with it the vitelline membrane and the blastoderm, which may then be brought into' a watch-glass or on to a slide and examined under the microscope (Duval). Gerlach's Window Method {Nature, 1886, p. 497).— Remove with scissors the shell from the small end of the egg ; take out a little white by means of a pipette ; the blastoderm will become placed underneath the window just made, and the white that has been taken out may be replaced on it. Paint the margins of the window with gum mucilage, and build up on the gum a little circular wall of cotton wool ; place on it a small watch-glass (or circular cover-glass), and ring it with gum. When the gum is dry the cover is further fixed in its place by means of collodion and amber varnish, and the egg is put back in its normal 272 EMBRYOLOGICAL METHODS. position in the incubator. The progress of the development may be followed up to the fifth day through the window. A description of further developments of this method, with figures of special apparatus, will be found in Anat. Am., ii, 1887, pp. 583, 609. See also Paton, Jo'um. Exper. Zool, xi, 1911, p. 469 (cultivation of the embryo in vitro). 594. Preparation. — During the first twenty-four hours of incuba- tion, it is extremely difficult to separate the blastoderm from the yolk, and they should be fixed and hardened together.* In later stages, when the embryo is conspicuous, the blastoderm can easily be separated from the yolk, which is very advantageous. To open the egg, lay it on its side and break the shell at the broad end by means of a sharp rap ; then carefuUy remove the shell bit by bit by breaking it away with forceps, working away from the broad end untU the blastoderm is exposed. The egg should be opened in salt solution, then lifted up a little, so as to have the blastoderm above the surface of the liquid ; the blastoderm is then treated with some fixing solution dropped on it from a pipette (1 per cent, solution of osmic acid, or Ranvier and Vignal's osmic acid and alcohol mixture, iodised serxun, solution of Kleinenberg, 10 per cent, nitric acid, etc.). By keeping the upper end of the pipette closed, and the lower end in contact with the Uquid on the blasto- derm, the blastoderm may be kept well immersed for a few minutes, and should then be found to be sufficiently fixed to be excised. (Of course, if you prefer it, you can open the egg in a bath of any fixing liquid [10 per cent, nitric acid being convenient for this purpose] of such a depth as to cover the yolk ; and having exposed the blastoderm, leave it tiU fixed [fifteen to twenty minutes] ; but I think the procedure above described will generally be found more - convenient.) The egg is put back into the salt solution, and a circular iacision made roujid the embryonic area. The blastoderm may then be floated out and got into a watch-glass, in which it may be examined, or may be brought into a hardening liquid. Before putting it into the hardening fluid, the portion of vitelline membrane that covers the blastoderm should be removed with forceps and shaking. * Andrews {Zeit. wiss. Mile, xxi, 1904, p. 177) separates the blasto- derm at this stage by injecting pioro-sulphuric acid (not any rapidly acting fixative) firstly, between the blastoderm and the vitelline mem- brane, so as to separate the two above, and then between the blastoderm and the yolk, so as to free the blastoderm below and float it up. This done, the membrane may be incised and the blastoderm removed. The injection is best done with a pipette having a fine point bent upwards, CHAPTER XXV. 273 Fixation in 10 per cent, nitric acid has the advantage of greatly facilitating the separation of the blastoderm. The acid should be allowed to act for ten minutes, after which it is well to bring the preparation into 2 per cent, solution of alum (c/. Hofmann, Zeit. wiss. Mik., X, 1893, p. 485). Mitrophanow (Anat. Hefte, xii, 1899, p. 200) fixes with nitric acid of 3 per cent. ; Suschkin {Nouv. Mem. Soc. Nat. Moscow, xvi, 1899, p. 34) with sublimate ; Fischel (Morph. Jahrb., xxiv, 1896, p. 371) with Kabl's platino-sublimate, § 76 (embryos of the duck) ; Patterson (Biol. Bull. Wood's Hole, xiii, 1907, p. 252) with picro-sulphuric acid containing 8 per cent, of acetic acid, for an hour (ova of Columba) ; Hoskins (Kansas Univ. Sci. Bull., iv, 1907, p. 176), after removing shell, for five to fifteen minutes in a mixture of 3 parts of 10 per cent, formol with 1 of 10 per cent, nitric acid, and then excises the embryo. In order to cormteract the turning up of the edges of the blasto- derm that generally happens during the process of hardening, it is well to get the blastoderm spread out on the convex surface of a watch-glass, and leave it so during the hardening. For hardening Henneguy prefers the osmic acid and alcohol mixture of Ranvier and Vignal, or Flemming's mixture followed by successive alcohols. Stain and imbed by the usual methods. Up to about the fiftieth hour embryos may be mounted entire in glycerin or balsam. 595. M. Duval's Orientation Method (Ann. Sc. Nat., 1884, p. 3).— In the early stages of the development of the ova of Aves, before the appearance of the primitive streak, it is difiicult to obtain a correct orientation of the hardened cicatricula, so as to be able to make sections in any desired direction. Duval, starting from the fact that during incubation the embryo is almost always found to be lying on the yolk in such a position that the big end of the egg is to the left and the little end to the right of it, marks the position of the blastoderm in the following way. With a strip of paper 5 millimetres wide and 50 millimetres long you construct a sort of triangular bottomless box. You lay this on the yolk, enclosing the cicatricula in such a position that the base of the triangle corresponds to what will be the anterior region of the embryo, and its apex to the posterior region ; that is to say, if the big end of the egg is to your left, the apex of the triangle will point towards you. You now, by means of a pipette, fill the paper triangle with 0-3 per cent, solution of osmic acid. As soon as the preparation begins to darken you put the whole egg into weak M. 1^ 274 mBRYOLOGICAL METHODS. ckromic acid, remove the white, and put the rest into clean chromic acid solution for several days. After hardening you will find on the surface of the yolk a black triangular area, which encloses the cicatricula and marks its position ; you cut out this area with scissors and a scalpel, and complete the hardening with chromic acid and alcohol. See also the method of Hirota, Journ. Roy. Mic. Soc, 1895, p. 118. 596. Kionka's Orientation Method {Anat. Hefte, 1 Abth., iii, 1894, p. 414). — Open the egg under salt solution, free it from the shell and albumen, and mark the poles by sticking into it, at about' a centimetre from the blastoderm, two hedgehog spines, the one at the obtuse end being marked with a red thread. Put the whole for ten minutes into water at 90° C, then bring into 70 per cent, alcohol, and after twenty-four hours cut out the blastoderm and a little yolk round it in the shape of an isosceles triangle, whose base marks the anterior end of the blastoderm. Paraffin sections stained with borax-carmine, washed out with acid alcohol containing 1 drop of concentrated solution of Orange G for each 5 c.c, which stains the yolk. 597. Vialleton's Method (Anat. Anz., vii, 1892, p. 624).— Egg opened in salt solution, blastoderm excised and removed to a glass plate, then treated with 1 per cent, nitrate of silver solution, washed with water, and put into 70 per cent, alcohol for six to twelve hours in the dark. Borax-carmine, alcohol, damar. 598. Chick and Reptile Blastoderms. — Gerhaedt {Anat. Anz., xx) uses : — ■ Chromic acid 1 per cent. .... 150 c.c. Sat. corr. subl. ...... 150 „ Aq. dest 135 „ Acetic acid . . . . . . 15 „ FormaUn 150 „ Leave in twenty-four hours. Wash twenty-four hours in running water, upgrade from 70 per cent, alcohol, 90 per cent, with iodine, pure 90 per cent., etc. Recommended by Prof. J. P. Hill. Rejptilia. 599. General Directions. — The methods described above for birds are applicable to reptiles. During the early stages the blastoderm should be hardened in situ on the yolk ; later the embryo can be isolated, and treated separately. BoHM and Oppel (Taschenbuch, 1900, p. 186) remove the shell CHAPTER XXV. 275 under salt solution, fix in sublimate with 20 per cent, acetic acid, or in Lo BiANCo's«hromo-sublimate (§ 72), then remove the blastoderm and bring it into alcohol. 600. Special Cases. — Mitsukuri {Journ. Coll. Sc. Japan, vi, 1894, p. 229) fixes embryos of tortoises chiefly with picro-sulphuric acid. To study the blastoderm he removes the whole of the shell and as much as possible of the albumen, marks the place where the blasto- derm lies with a hair, brings the whole, with the blastoderm upper- most, into the fixative, and after a few hours cuts out the blasto- derm and further hardens it by itself. Young embryos generally adhere to the shell and can, therefore, be fixed in a piece of it made to serve as a watch-glass, then after half-an-hour can be removed from it and further hardened alone. If the embryonal membranes have been formed, the shell may be scraped away at some spot and there treated with picro-sulphuric acid until a small hole is formed ; then by working away from this spot, by means of scraping and dropping acid on to it, the whole of the shell may be removed. Will (Zool. Jahrb., Ahth. Morph., vi, 1892, p. 8) opens ova of Platydactylus in the fixative (chiefly chromic acid, or chromo- aceto-osmic acid with very little osmic acid) and hardens the embryos on the yolk ; so also for Cistudo and Lacerta (1893 and 1895). Mehnert {Anat. Am., xi, 1895, p. 257) does not approve of these methods ; for his own see Morph. Arb. Schwalbe, i, 1891, p. 370. Gerhardt (Anat. Anz., xx, 1901, p. 244) fixes ova of Tropido- notus for twenty-four hours in Nowak's mixtufe, § 112. Ballowitz (Entmckl. d. Kreuzotter, 1903, p. 19) first fixes seg- ments of the uterus, each containing an ovum, for one or two hours, then tears them open with forceps, isolates the ova, and puts them into fresh fixative, and thence into alcohol of 40 per cent. Nicolas (Arch. Anat. Mic., 1900, p. 457) finds the best fixative for ova of the slow-worm, as for other large ova, is Bouin's picrO' formol (§ 110). See also Perenyi, § 48, and Zool. Anz., 1888, pp. 139 and 196, and other methods in early editions. Amphibia. 601. Preliminary. — In order to prepare ova for section-cutting, it is essential to begin by removing their thick coats of albimien. This may be done by putting them for two or three days into 1 per - cent, solution of chromic acid, and shaking well ; but ova thus treated are very brittle, and do not afford good sections. A better 18—2 276 EMBRYOLOGICAL METHODS. method is that described by Whitman (Amer. Natural, xxii, 1888, p. 857), and by Blochmann (Zool. Anz., 1889, p. 269). Whitman puts the fixed eggs into a 10 per cent, solution of sodium hypo- chlorite diluted with 5 to 6 volumes of water, and leaves them there till they can be shaken free, which happens (for Necturus) in a few minutes. Blochmann takes eau de Javelle (potassium hypo- chlorite), and dilutes it with 3 to 4 volumes of water, and agitates the eggs previously fixed with solution of Flemming, for fifteen to thirty minutes in it. Lebrun (La Cellule, xix, 1902, p. 316) advises fixing ova of Anura for not less than one and a half hours in liquid of Gilson, § 69. The outer envelopes are then hard, and may be easily incised and the ovum extracted by pressing on the pole opposite to the incision. The operation should not be delayed until after hardening in alcohol. Similarly (ibid., xx, 1902, p. 12) for Urodela. GrUYER (Amer. Nat., xli, 1907, p. 400) finds it suffice to roll the ova (either fresh or fixed, but before bringing into alcohol) on blotting paper. 602. Imbedding. — A great difficulty with the ova of Amphibia lies in their becoming extremely brittle on imbedding in paraffin. Caenoy and Lebrun (La Cellule, xii, 1897, p. 212) fix ovaries or ovarian ova for fifteen minutes to three-quarters of an hour (but see last §) in Gilson's mercuro-nitric fluid, § 69, and preserve them in 80 per cent, alcohol. To imbed, they are brought for a quarter of an hour into 95 per cent, alcohol, five minutes in absolute alcohol, then into a mixture of alcohol and chloroform in equal parts, and as soon as they sink in that they are put into pure chloroform. Paraffin is added to the chloroform, enough to about double the volume of the whole, and the whole is put for about three hours into a stove at 35° C. Lastly, the ova are put for not more than five minutes into a bath of pure paraffin at 52° C. Later (ibid., xix, 1902, p. 317) Lebrun explains that it is important not to dehydrate completely with absolute alcohol ; the ova should be left in alcohol of 96 per cent, until chloroform can be added without the mixture becoming turbid, and a second bath of clean paraffin should be added. See also Morgan, Devel. of the Frog's Egg, New York, 1897, p. 171. 603. Siiedon. — The ova are easier to prepare than those of the Anura, because the yolk is separated from the albuminous layer by a wide space filled with a Hquid that is not coagulated by reagents. Put the eggs for a few hours into picro -sulphuric acid, then pierce CHAPTER XXV. 277 the inner chorion with fine scissors or needles, and gently press out the ovum. Harden in alcohol. FiCK {Zeit. wiss. Zool., Ivi, 1893, p. 529) uses a mixture of 250 parts of 1 per cent, chromic acid, 1 of acetic acid, and 750 of water. 604. Triton (Scott and Osborn, Quart. Journ. Mic. Soc, 1879, p. 449). — The albumen is here present in the form of several con- centric coats, which are very delicate. Incise each of them separately with fine scissors, turn out the ovum, and fix it in solution of Kleinenberg. Hertwig (Jen. Zeit. Naturw., 1881 — 2, p. 291) puts the eggs into a mixture of equal parts of 2 per cent, acetic acid and 0-5 per cent, chromic acid. After ten hours he incises the membranes, opening one end of the inner chorion, and turns out the embryos and brings them into successive alcohols. MiCHAELis (Arch. mik. Anat., xlviii, 1896, p. 528) fixes ova, with their envelopes, in a mixture of concentrated sublimate solution and concentrated picric acid, 20 parts each, glacial acetic acid 1, and water 40, but removes the envelopes before bringing into alcohol. 605. Salamandra (Rabl, Morphol. Jahrb., xii, 2, 1886, p. 252).— For his more recent methods see § 580. Gkonkoss (Anat. Anz., xiv, 1898, p. 461) fixes the ova with a mixture of 50 parts each of saturated sublimate and 0-5 per cent, chromic acid with 1 part of acetic acid. 606. Bana. — I have foimd that the following mixture often gives very good results for the eggs of Rana temporaria ; it dissolves away the albumen coat, preserves yolk and mitochondria, and leaves the eggs soft enough to cut in paraffin with a rotary micro- tome : — Bichromate of potash of 2 per cent. . 100 c.c. Chromic acid of 1 per cent. . . . 100 „ Nitric acid . . . . . . 6 „ Use at least 40 c.c. to twenty or thirty eggs for fifteen to twenty- four hours. Slightly shake, and the albumen coats fall off if not already dissolved. Wash out for about one hour in running water and then upgrade from 70 per cent, alcohol (one half-hour), 90 per cent, (one hour), to absolute alcohol, two changes of one hour each. Clear in benzol for fifteen minutes. Add chips of wax and place in thermostat for half an hour. Transfer to pure wax for one-half (to three-quarters) of an hour. Avoid unnecessary heat. 278 EMBRYOLOGICAL METHODS. The eggs thus treated can often be cut 6/x on a rotary microtome provided with a sharp knife. The method is indicated where large numbers of stages of the early development of the frog are required for jimior class purposes. The main objection to the method is that the chromic acid attacks pigment. Fertilisation and segmenta- tion stages and general cytology are often extremely good ; stain in any way (J. B. G.). 0. Heetwig {Jen. Zeit. Naturw., xvi, 1883, p. 249). — The ova are thrown into nearly boiling water (90° to 96° C.) for five or ten minutes. The albuminous envelope of the ovum is then cut open, and the ovum extracted under water. The ova are then brought into 0-5 per cent, chromic acid for not more than twelve hours, or into alcohol of 70, 80, and 90 per cent. Chromic acid makes ova brittle and attacks the pigment, whilst alcohol preserves it, which is frequently important for the study of the germinal layers. Morgan (Amer. Nat., xxv, 1891, p. 759, and Devel. of the Frog's Egg, 1897, p. 171) has the following. During the periods in which it is difficult or impossible to remove the inner jelly-membrane the eggs can be freed as follows : Each egg is cut out with scissors from the general jelly-mass, and put for from one to twelve hours into - saturated solution of picric acid in 70 per cent, alcohol containing 2 per cent, of sulphuric acid. Wash in several changes of alcohol of 70 per cent; About the second day in this the inner membrane begins to swell, and on the third or fourth day may be pierced by a ' needle, and the egg removed and placed in 80 per cent, alcohol. See also Whitman, Meth. of Research, p. 156. ScHULTZE {Arch. mik. Anat., Iv, 1899, p. 174) removes with scissors the outer layers of albumen, and puts the ova for five minutes in 2 per cent, formol warmed to 75° or 80° C. The membrane left on the ova then rises up sufficiently to allow the ova to be got out with needles. See also Born {ibid., xUii, 1894, p. 1). King {Journ. Morph., xvii, 1901, p. 295, and xix, 1908, p. 370) fixes (for a few minutes) the spawn (of Bufo) in sublimate (saturated with 5 per cent, of acetic acid), or in Flemming, Zenker, or Hermann, brings into alcohol, first of 50 and then 80 per cent., and removes the jelly after a few days. Bles {Trans. Roy. Soc. Edinburgh, xli, 1905, p. 792) takes for ova formol of 10 per cent., but for embryos and larvae the mixture given § 109. Bouin takes for larvae of Rana the formol-sublimate mixture § 112. CHAPTER XXV. 279 607. Sulphate of Copper Liquid (Fol, lehrbuch, p. 106, after Remak and GrOETTE) ; for hardening ova of Amphibia : 2 per cent, solution of sulphate of copper. 50 c.c. Alcohol of 25 per cent. . . . . 50 ,, Eectifled wood vinegar . . . .35 drops. Pisces. 608. Teleostea in General. — The ova of many of the .bony fishes can be studied by transmitted light in the living state ; but those of the Salmonidse must be hardened and removed from their envelopes for the study of the external forms of the embryo. To this end they may be put for a few minutes into water con- taining 1 to 2 per cent, of acetic acid, and thence into 1 per cent. chromic acid. After three days the capsule of the ovum may be opened at the side opposite to the embryo, and be removed with fine forceps. The ovum is put for twenty-four hours into distilled water, and then into successive alcohols. Embryos thus pre- pared show no deformation, but the vitellus rapidly becomes excessively hard and brittle, so as greatly to interfere with section- cutting. The following processes give good results as regards section- cutting. Put the ova for a few minutes into 1 per cent, osmic acid ; as soon as they have taken on a light brown colour bring them into Mtiller's solution. Open them therein with fine scissors — ^the vitellus, which immediately coagulates on contact with air, dissolves, on the contrary, in Miiller's solution — and the germ and cortical layer can be extracted from the capsule of the ovum. They should be left in clean Miiller's solution for a few days, then washed with water for twenty-four hours, and brought through successive alcohols. Another method (Hennbguy) is as follows : The ova are fixed in solution of Kleinenberg containing 10 per cent, of acetic acid. After ten minutes they are opened hi water containing 10 per cent, of acetic acid, which dissolves the vitellus. The embryos are put for a few hours into pure solution of Kleinenberg, and are then brought through alcohol of gradually increasing strength. Child (quoted from Sumner, Mem. New York Acad. Sci., ii, 1900, p. 78) fixes for about a minute in sublimate with 10 per cent. of acetic acid, and brings into formalin of 10 per cent., which is said to give a good fixation of the embryo without the yolk becoming hard. 280 EMBRYOLOGICAL METHODS. 609. Kollmann's Fixative (Kollmann, Arch. Anat. Phys., 1885, p. 296). Bichromic of potash . . . .5 per 100. Chromic acid . . . . . 2 ,, Concentrated nitric acid . . . 2 „ For ova of Teleostea. Fix for twelve hours, wash with water for twelve hours, then remove the chorion, and put the ova into 70 per cent. alcohol. 610. Rabl's Method, see § 587 ; for Kowalewsky's see Zeit. wiss. Zool, xliii, 1886, p. 434, or Third Edition. 611. Salmonidse. — Henneguy's methods have been given, § 609. KopscH (Arch. mik. Anat., li, 1897, p. 184), on the suggestion of ViROHOW, fixes embryos for five or ten minutes in a mixture of 1 part of chromic acid to 50 of glacial acetic acid and 450 of water, then removes into chromic acid of 1 : 500, and as soon as may be removes the capsule and yolk under salt solution, and completes the hardening in the chromic acid or the saturated sublimate solution. Similarly, Beheens (Anat. Hefte, x, 1898, p. 233). He opens the ova in the salt solution from the antipolar side, and frees the embryo from the yolk that remains by blowing the latter away with a fine-pointed glass tube. Similarly also Sobotta (ibid., 1902, p. 579). GuDGER {Proc. U.S. Nation. Mus., xxix, 1906, p. 448) fixes blastoderms in fresh liquid of Perenyi, which does not make the yolk too hard ; later stages in Worcester's liquid (9 parts of saturated solution of sublimate in formol of 10 per cent, and 1 part of acetic acid), for half an hour to an hour, and brings gradually into alcohol of 70 per cent. Bourn [C. R. Soc. Biol, Iv, 1903, p. 1691) fixes for thirty-six to forty-eight hours in picro-formol. RABL-EtJCKHAED's Msthod {Arch. Anat. Entw., 1882, p. 118).— Fix in 10 per cent, nitric acid for fifteen minutes. Remove the membranes to avoid deformation of the embryos, and put the ova back into the acid for an hour. Wash out in 1 to 2 per cent, solution of alum for an hour and harden in alcohol. Modification of this method by Goronowitsch (see Morph. Jahrb., x, 1884, p. 381). 612. Selachia.— Beard {Anat. Anz., xviii, 1900, p. 556) has found that the best fixatives for embryos of Raja are Eabl's picro- platinic mixture, § 587, and sublimate. Living embryos can be observed by scraping the shell thin with a knife (Kastschenko, Anat. Anz., iii, 1888, p. 445, and His, Arch. CHAPTER XXV. 281 Anat. Phys., Anat. Abth., 1897, p. 3). See also Beaus, Morph. Jahrb., xxxv, 1906, p. 250. 613. Amphioxus. — Sobotta (Arch. mih. Anat., 1, 1897, p. 20) fixes for twenty-four hours in liquid of Flemming ; Hatschek {Arh. Zool. Inst. Wien., iv, 1881) in picro-sulphuric acid. Impregnation takes place in the evening, and segmentation is completed during the night. Legros (Grrundziige, Lee and Mayer, 1910, p. 288) fixes ova and embryos in equal parts of formol and Flemming. Sublimate is not good ; Rabl's mixtures are better. Larvae aiid young animals ought first to be anaesthetised with cocain in sea-water. After fixation they should remain only for as short a time as possible in alcohol. Cereontaine (Arch. Biol., xxii, 1906, p. 287) fixes with Flemming or Hermann. For study of ova in toto he orients them on a slide in clove-oil-collodion which he sets with chloroform, and adds balsam. For sectioning, he orients in the same way on a layer of paraffin spread on a cover glass and imbeds the whole in parafiin. 614. Pelagic Fish Ova. — ^Whitman {Amer. Natural, xvii, 1883, pp. 1204-5 ; and Methods of Besearch, etc., p. 162). — Fix by treatment first for five to ten minutes with a mixture of equal parts of sea-water and i per cent, osmic acid solution, and then for one or two days with a solution (due to Eisig) of equal parts of 0-25 per cent, platinum chloride and 1 per cent, chromic acid. Prick the membrane before transferring to alcohol. See also Agassiz and Whitman, in Proo. Amer. Acad. Arts and Sciences, xx, 1884 ; and Collinge, Ann. and Mag. Nat. Hist, x, 1892, p. 228. Eaffaele {Mitth. Zool. Stat. Neapel, xii, 1895, p. 169) fixes chiefly with liquid of Hermann (1 to 2 days), or with a mixture of Mingazzini { absolute alcohol 1, acetic acid 1, saturated sublimate solution in water 2). Heinke and Ehrenbaum {Wiss. Meeresunt. Komm. Wiss. Tint. D. Meere, iii, Heligoland, 1900, pp. 205 and 213) prefer formol with 39 volumes of sea-water. Tunicata. 615. Ova. — Davidopf [Mitth. Zool. Stat. Neapel, ix, 1, 1889, p. 118) fixes the ova of Distaplia with a mixture of 3 parts of saturated solution of corrosive sublimate and 1 of glacial acfetic acid for from half an hour to an hour ; or with a mixture of 3 parts of saturated solution of picric acid and 1 of glacial acetic acid for three to four hours ; then 70 per cent, alcohol. Castle (Bull. Mm. Harvard Coll., xxvii, 1896, p. 213) advises for ova of Ciona liquid of Perenyi for twenty minutes, followed by 282 EMBRYOLOGICAL METHODS. 70 per cent, alcohol for twenty-four hours, and for the larvae picro- nitric acid. 616. Test-Cells of Ascldians (Morgan, Joum. of Morphol, iv. 1890, p. 195). — Tease fresh ovaries in very weak osmic acid, wash in distilled water, treat for half an hour with. 1 per cent, silver nitrate, wash for half an hour in 2 per cent, acetic acid and reduce in sunlight. Imbed in paraffin. By this process the limits of the follicle cells are demon- strated. 617. Buds. — PizoN {Ann. Sc. Nat., xix, 1893, p. 5) studies the gemmation of the. composite Ascidians either on entire corms, which he first bleaches with peroxide of hydrogen and then stains, or by making sections, after anaesthetising the colonies with cocain of 1 : 1000, fixing in glacial acetic acid or picro-sulphuric or liquid of Flemming, and staining in toto with borax carmine or alum carmine, or with a strong solution of methylen blue in alcohol of 90 or 100 per cent, (after Bernard, ibid., ix, 1890, p. 97). RiTTER (Joum. of Morph., xii, 1896, p. 150) recommends for fixing Perophora and Goodsiria picro-sulphuric acid. Bryozoa. 618. Statoblasts. — Braem {Bihl. Zool., Chun and Leuchart, 6 Heft, 1890, p. 95) fixes statoblasts of Cristatella with hot con- centrated solution of sublimate for ten minutes, brings them into water and there incises them with a razor, and after half an hour passes them gradually into alcohol. He stains with picro-carmine. Mollusca. 619. Cephalopoda (Ussow, Arch, de Biol., ii, 1881, p. 582). — Segmenting ova are placed in 2 per cent, solution of chromic acid for two minutes, and then in distilled water, to which a little acetic acid (1 drop to a watch-glassful) has been added, for two minutes. If an incision be now made into the egg-membrane, the yolk flows away and the blastoderm remains ; if any yolk stUl clings to it, it may be removed by pouring away the water and adding more. WATAsi; (Joum. of Morphol., iv, 1891, p. 249) kills the ova in the macerating mixture of the Hertwigs (§ 534), and as soon as the blastoderm turns white and opaque removes it imder dilute glycerin. Treatment with liquid of Perenyi is recommended for surface views. Vialleton (Ann. Sc. Nat., vi, 1887, p. 168) brings ovarian ova of Sepia into a freshly prepared mixture of picro-sulphuric acid and 2 per cent, solution of bichromate of potash in equal parts, CHAPTER XXV. 283 and after one or two minutes incises them in the equator, fixes for an hour and a half in picro--sulphuric acid the halves that contain the formative vitellus, separates this from the nutritive vitellus with a spatula, spreads it out, and hardens it in alcohol of 70 to 90 per cent. He fixes entire ova in liquid of Flemming or osmic acid. KoRSCHELT {Festchrift Leuckart, Leipzig, 1892, p. 348) fixes advanced embryos of Loligo in liquid of Flemming, sublimate, picro-sulphuric acid, or 0-2 per cent, chromic acid. This last is specially good for young embryos if it is washed out with many changes of picric acid. Faussek (Mitth. Zool. Stat. Neapel, xiv, 1900, p. 83) recommends picro-nitric acid. Fix in this, harden in alcohol, bring the ova, still in their albumen, into hsemalum, stain for twenty-four hours, wash in 1 per cent, alum solution for twenty-four hours, when the albumen will be found softened so that the ova can easily be extracted. 620. Gastropoda (Henneguy). — Ova of Helix may be fixed for from four to six hours in Mayer's picro-nitric acid. The carbonate of lime that encrusts the external membrane is thus dissolved, and the albuminous coat of the egg is coagulated. The egg is opened with needles, the albumen comes away in bits, and the embryo can be removed. Henchman (Bull. Mus. Comp. Zool., Harvard, xx, 1890, p. 171) fixes ova of Limax with 0-33 per cent, chromic acid, or with liquid of Perenyi. It is best to remove only the outer envelope before putting into the chromic acid, the inner membrane being removed after two or three minutes therein. Where Perenyi is used the membranes must be removed first, as the albumen will else coagulate in such a way as to prevent the removal of the embryos. Meisenheimeb (Zeit. wiss. Zool., Ixii, 1896,' p. 417) dissects out the embryos of Limax and fixes them with picro-sidphuric acid or concentrated sublimate. Advanced embryos are first got into extension by means of 2 per cent, cocaine, or are rapidly killed with hot sublimate. Schmidt (Entw. Pulmonaten, Dorpat, 1891, p. 4) fixes the ova in toto with concentrated sublimate, and dissects them out afterwards. Similarly Kopoid {Bull. Mus. Harvard Coll., xxvii, 1895, p. 35). Or, preferably, the ova are put into salt solution, the shell removed, the albumen removed with a pipette full of salt solution, which dissolves it ; the ova are then fixed for one minute in Fol's modification of liquid of Flemming, and brought direct into Orth's picro-Hthutn- carmine. See also Linville, iMd., 1900, p. 215, who adopts this 284 EMBRYOLOGTCAL METHODS. method of shelling, but prefers fixing in acetic-acid sublimate, or liquid of Perenyi. Heyder (Zeit. wiss. Zool., xciii, 1909, p. 92), before imbedding embryos of Arion that have been fixed with sublimate, treats them for an hour or two with carbonate of soda of -^ to ^V P^^" cent., which makes the stomach and intestine less brittle. Gatenby {Quart. Journ. Micr. Science, 1919), for Limnwa stagnalis ova, used Flemming's strong fluid without acetic acid, Champy's fluid for two days to a week, and Kopsch's method. Holmes (Journ. of Morph., 1900, p. 371) teases the egg-capsules of Plcmorbis in nitrate of silver of | per cent., exposes to sunlight until the oell-Umits come out, rinses with 0-2 per cent, hyposulphite of soda, puts for a few minutes into picric acid, and then through alcohol into balsam. See also Washburn, Amer. Anat, xxviii, 1894, p. 528 (liquid of Flem- ming, or 0-3 per cent, chromic acid, or 1 per cent, osmic acid, followed by liquid of Merkel). CoNKLiN (Journ. of MorpJi., xiii, 1897, p. 7) fixes ova of Grepidula for fifteen to thirty minutes in picro-sulphuric acid, and stains with dilute acidified h»matoxyIin of Delafield. KosTANECKi and Wierzejski {Arch. mik. Anat., xlvii, 1896, p. 313) fix the spawn of Phy sa fontinalis -eithei in 1-|- to 2 per cent, nitric acid, or in " sublimate and 3 per cent, nitric acid in the pro- portion of 2:1," and bring through successive alcohols. They imbed entire ova in paraffin, but isolated embryos in celloidin. 621. Chiton, see Metcalf, Stud. Biol. Lab. Johns Hopkins Univ., v, 1893, p. 251. (Ova with young embryos put for twenty to forty-five seconds into eau de Labarraque, then into water, in which the chorion swells and can easily be removed.) 623. Lamellibranchiata. — Stauffacher {Jena Zeit., xxviii, 1893, p. 196) fixes erhbryos of Cyclas in sublimate, stains with hsemalum, and cuts in paraffin. LiLLiE {Journ. of Morph., x, 1895, p. 7) fixes ova of Unio for ten to twenty minutes in liquid of Perenyi, and preserves them in 70 per cent, alcohol, or advanc'ed embryos with liquid of Merkel or sublimate, larvae with 0-05 to 0-1 per cent, osmic acid, preserving them in glycerin. Glochidia may be cut with the shell in paraffin of 58° melting-point ; they may be anaesthetised with chloral hydrate before fixing. Arthropoda. 623. Fixation of Ova. — In many cases the ova of Arthropods are best fixed by heat (§ 13). This may be followed either by alcohol or some watery hardening agent. If it be desired to avoid heating, picro-nitric acid may be tried. CHAPTER XXV. 285 624. Removal of Membranes.— It may often be advisable not to attempt to remove them, but to soften them with eau de Javelle or eau de Labarraque. See § 545. Morgan {Amer. Natural, xxii, 1888, p. 357) recommends (for the ova of Periplaneta) eau de Labarraque diluted with 5 to 8 volumes of water, and slightly warmed. This will soften the chitin mem- branes sufficiently in thirty to sixty minutes, if employed before fixing. Fixed ova take longer. The fluid must, of course, not be allowed to penetrate into the interior of the ovum. 625. Henking's Methods (Zeit. wiss. Mik, viii, 1891, p. 156).— Henking generally kills ova by plunging them into hot water, or by pouring hot water on to them in a watch-glass, and then removing into 70 per cent, alcohol. He thinks that eau de Javelle for softening membranes is best avoided. They should either be dissected away or left in situ, and cut with the rest of the egg, according to the nature of the case. To avoid brittleness of the yolk proceed as follows : After fixing and treating with alcohol, prick the chorion and stain with borax- carmine. Put the stained ova for twelve hours into a mixture containing 20 c.c. of 70 per cent, alcohol, 1 drop of concentrated hydrochloric acid, and a knife pointful of pepsin (it is not necessary that all the pepsin should be dissolved). The ova may then be treated with alcohol, oil of bergamot, and paraffin, and (with some exceptions, amongst which is Bombyx mori) will be found to cut without crumbling. 626. Diptera (Henking, Zeit. wiss. Zool, xlvi, 1888, p. 289).— Ova still contained within the fly may be fixed by plunging the insect for some time into boiling water, then dissecting out and bringing them into 70 per cent, alcohol. Laid eggs may have boiling water poured over them, or be put into solution of Flemming in a test-tube which is plunged into boiling water until the eggs begin to darken (about a minute). Cold solution of Flemming easily causes a certain vacuolisation of the contents of the ova. Open the ova at the larger end, stain with borax-carmine for fifteen to thirty hours, and cut in parafiin. Beuel {Zool. Jahrb., Abth. Morph., x, 1897, p. 569) fixes larvae and pupae in absolute alcohol heated to 70° to 75° C, and con- taining a " little " sublimate. See also Van Eees, ibid., iii, 1888, p. 10. Bengtsson (Handl. Fysiogr. Scellsk Lund., viii, 1897) finds hot alcoholic solution of sublimate (Frenzel's, § 69) the best fixative for 286 EMBRYOLOGICAL METHODS. larvae of Phalacrocera. . He could not succeed in softening the chitin with eau de Javelle. Perez (Arch. Zool. exper., (4), v, 1910, p. 11) fixes pupae in Bouin's picro-formol, or Marchoux's mixture, for twenty-four hours. 627. Lepidoptera (BoBRETZKy, Zeit. wiss. Zool., 1879, p. 198). — Ova are slightly warmed in water and put for sixteen to twenty hours in 0'5 per cent, chromic acid. The membranes can then be removed. 628. Hymenoptera. — CARRifeRE and Burger [Nova Acta Acad. Leop. Car., Ixix, 1897, p. 273) kill ova of Chalicodoma by warming in water to 60° C, and fix in aqueous picric acid, or alcohol of 70 per cent. Petrunkewitsch (Zool. Jahrb., Abth. Morph., xiv, 1901, p. 576) fixes for twenty-four hours in his sublimate mixture, and passes into alcohol of 70 per cent, with iodine. 629. Orthoptera (Patten, Quart. Journ. Mic. Sci., 1884, p. 549). — The ova or larvae (of Blattida) are placed in cold water, which is gradually raised to 80° C. You leave off heating as soon as the ova have become hard and white. Pass very gradually through succes- sive alcohols, beginning with 20 per cent. Wheeler (Journ. of Morph., iii, 1889, p. 292) dissects out ovarian ova in salt solution and fixes in liquid of Per6nyi (fifteen minutes), then treats with alcohol, and staias with borax-carmine. Laid eggs may be killed by Patten's method. After heating, the two lips of the crista of the capsule may be separated with fine forceps and pieces of the walls torn away, and the eggs pushed out of the compartments formed by their choria and hardened as desired. Good results are also obtained by heating to 80° C. for ten minutes in liquid of Kleinenberg, and preserving in 70 per cent, alcohol. This causes the envelopes to dilate and stand off from the surface of the egg, so that they can easily be dissected away. Heymons (Zeit. wiss. Zool., liii, 1892, p. 434), for young embryos, incises the cocoon at the end by which it adheres in the body of the mother, brings it for two minutes into water heated to 90° C, and opens in Flemming, in which the embryo is dissected out. Morgan (Amer. Natural., xxii, 1888, p. 357) puts ova of Peri- planeta for thirty minutes or an hour into eau de Javelle diluted with 4 to 8 volumes of water and slightly warmed, which softens the capsules. 630. Coleoptera. — Hirschlbr (Zeit. wiss. Zool., xcii, 1909, p. 628) fixes ova of Donacia (after incising the chorion) for two to three CHAPTER XXV. 287 hours in equal parts of sublimate of 6 per cent, and nitric acid of 3 per cent. G-ATBNBY {Quart. 'Journ. Mic. Sci., 1917) for Donacia uses Petrunkewitsch or picro-nitric. In the latter case the chorion must be incised. Baling (Dissert. Marburg, 1906, p. 10) fixes ova of Tenebrio for about two minutes in a hot mixture of 40 parts of alcohol of 96 per cent., 4 of nitric acid, and 50 of saturated aqueous subhmate ; or for three minutes in a hot mixture of 1 part of formol with 3 of water. Karawaiew {Biol. Centralb., xix, 1899, p. 124) kiUs larvae of Anobium in hot water, freezes them with ether spray, cuts away a lateral strip, lets them thaw, and puts for twenty-four hours into picro-sulphuric acid. 631. Phalangida. — The ova of Phalangium opilio possess a chorion covered with yellow corpuscles that render them opaque. Balbiani puts them into water with a few drops of caustic potash, and raises to boiling point. The ova are then laid on filter paper, and the chorion removed by rubbing with a camel's hair brush, the viteUine membrane remaining intact, so that the embryo can be studied through it. Henking's method {Zeit. wiss. Zool., xlv, 1886, p. 86). — Fix with boihng water or Flemming. Preserve the ova in 90 per cent, alcohol. To open the chorion, bring them back into 70 per cent, alcohol, which causes them to swell up so that the chorion can easily be pierced with needles, and the ovum turned out. 632. Araneida. — Kishinoxjye {Journ. Coll. Sci. Imp. Univ. Japan, iv, 1891, p. 55 ; Zeit. wiss. Mik., ix, 1892, p. 215) fixes in water warmed to 70° or 80° C, puts into 70 per cent, alcohol, and after twenty-four hours therein pierces the membranes and passes through stronger alcohol. See also Logy, Bull. Mus. Camp. Zool. Harvard, xii, 3, 1886. Fix by hot water. The hquid of Per6nyi may also be used ; it has the advantage of not making the yolk so granular. Montgomery {Journ. Morph., xx, 1909, p. 628) fixes ova of Theridium for one or two hours in Carnoy & Lebrun's mixture. Lambert {ibid., p. 420) fixes ova of Epeira in picro-sulphuric acid warmed to 70° or 80° C. PuRCELL {Quart. Journ. Micr. Sd., liv, 1909, p. 7) fixes ova of Atta in boiling saturated sol. of sublimate in alcohol of 70 per cent. Hamburger {Zeit. wiss. Zool., xcvi, 1910, p. 3) fixes ova of Argyroneta in Gilson's mixture. 288 EMBRYOLOGICAL METHODS. 633. Limulus.— KiNGSLEY (Joum. Morph., vii, 1892, p. 38) kills ova by heating in sea-water to 70° or 75° C. and brings into alcohol of 30 to 70 per cent. Similarly Kishinouye, Joum. Coll. Sci. Japan, v, 1893, p. 56. 634. Decapoda. — Reichenbach {Abh. Senckenberg Ges. Frank- furt, xiv, 1886, p. 2) fixes ova of Astacus in water gradually warmed to 60° or 70° C. (if the chorion should burst, that is no evil), hardens for twenty-four hours in 1 to 2 per cent, bichromate of potash or 0-5 per cent, chromic acid, washes out for the same time in running water, and brings into alcohol. Remove the chorion, and remove the embryo from the yolk with a sharp knife. Herrick (Bull. U.S. Fish, Confm., xv, 1896, p. 226) kills the ova in hot water, shells and fixes in picro-sulphuric acid. For Homarus, see Waite, Bull. Mus. Comp. Zool, xxxv, 1899, p. 155. 635. Amphipoda. — Bella Valle (Fauna u. Flora Golf. Neapel XX, Monog., 1893, p. 170) puts ova of Orchestia by means of a pipette into boihng, cold-saturated sublimate solution, removes them instantly into sea-water, and thence into weak alcohol. If the chorion does not burst of itself it must be pricked with a needle. 636. Cladocera. — Haeker (Zellen. u. Befruchtungslehre, 1899, p. 60) fixes females of Sida with winter eggs in a hot mixture of 100 c.c. alcohol of 70 per cent, with 1 to 2 c.c. saturated sol. of subUmate. See also Samter, Zeit. wiss. Zool., Ixviii, 1900, p. 176. 637. Copepoda. — Krueger (Arch. Zellforsch., vi, 1911, p. 173) fixes ovaries of Harpactida in Zenker's mixture with 10 per cent, of formol added. No other liquids give good results. Vermes. 638. Rotatoria. — Jennings (Bull. Mus. Harvard Coll., xxx, 1896, p. 101) finds the best fixative for pregnant females is the strong liquid of Flemming, but the ova must then be bleached with chlorate of potash (§ 575). Lenssen (La Cellule, xiv, 1898, p. 428) fixes ova of Hydatina with sublimate for twenty seconds. 639. Turbellaria. — Gardiner (Joum. of Morph., id, 1895, p. 158) finds the best fixative for ova of Polychoerus is a mixture of equal parts of absolute alcohol and glacial acetic acid. Bresslau (Zeit. wiss. Zool., Ixxvi, 1904, p. 219) fixes Mesosto- CHAPTER XXV. 289 midse with summer-eggs in Tellyesniczky's mixture (either cold or warmed, to 60° or 70° C.) for ten to twelve hours, and washes out for the same time. He incises winter-ova at one pole, fixes and brings into alcohol of 95 per cent., then makes an incision at the other pole, and imbeds in paraffin through cedar oil. In the paraffin, shces of the shell may be removed with a scalpel, and the ova re- imbedded when sufficiently shelled. Van dek Stricht [Arch. Biol., xv, 1898, p. 370) finds that ova of Thysanozoon wiU only cut weU when they have been not more than two minutes in absolute alcohol followed by chloroform and paraffin as used by Carnoy and Lebrun, § 602. See also, for Polyclads, Francotte, Arch. Zool. Exper., vi, 1898, p. 196 ; and, for fresh- water Planaria, Iijima, Zeit. wiss. Zool., xl, 1884, p. 359. 640. Cestoda (v. Bbnedbn, Arch. Biol., ii, 1881, p. 187).— Ova of Tcenia in which a chitinous membrane has formed around the embryo are impervious to reagents. They may be put on a shde with a drop of some hquid and covered. Then, by withdrawing the liquid by means of blotting-paper, the cover may be made to gradually press on them so as to burst the membranes, and the embryo may then be treated with the usual reagents. Haswell {Qvnrt. Journ. Micr. Sci., hv, 1909, p. 417) fixes ova of . Tenmocephala in " subhmate alcohol," brings them into 90 per cent. alcohol with iodine added, and thence gradually back into water, softens the shells in weak sodium hypochlorite, washes and imbeds. 641. Trematoda. — Coe {Zool. Jahrb., Ahth. Morph., ix, 1896, pp. 563, 566), for the special study of the excretory system of the Miracidia of Distomum, kills with osmic acid, rinses with distilled water, and puts for a couple of days into J per cent, solution of silver nitrate. Egg-capsules may be softened with 5 per cent, caustic potash and then burst open (Heckert, Bibl. Zool., iv, 1889). 642. Nematoda. — The ova of Ascaris megalocephala, a classical object of study, are one of the most impervious things in the animal kingdom. Years ago Fol related to me that he had had ova seg- menting right through absolute alcohol into balsam. Bataillon {Arch. Entwichelungsmech., 1901, p. 149) has had ova showing living embryos after having been for six months in liquid of Flemming, and found them to remain alive for months after drying for twenty- four hours at 35° C, and mounting in balsam, and for weeks in acids or alkalies. 19 290 EMBRYOLOGICAL METHODS. Doubtless the best fixative yet made known for ova furnished with their capsules will be found to be that of Carnoy and Lebrun, § 86 {La Cellule, xiii, 1897, p. 68). After fixation the ova are carefully brought into 80 per cent, alcohol, in which they are preserved. Imbedding should be carefully done as recommended for the ova of Amphibia (§ 602), but they ought not to remain in the pure paraffin for more than a minute to a minute and a half. But these authors prefer the ceUoidin method. At least six weeks' soaking in the different strengths of celloidin will be necessary to ensure penetration. They stain with iron hsematoxylin. ZuR Strapsen (Arch. Entwichelungsmech., iii, 1896, p. 29) fixes for twenty-four hours in a mixture of 4 parts 96 per cent, alcohol and 1 part acetic acid, brings into pure alcohol, stains with hydro- chloric acid carmine, and brings gradually into glycerin. Similarly ZoJA {Arch. mik. Anat., xlvii, 1896, p. 218) and Brlanger {ibid., xlix, 1897, p. 309). Zoja stained with Bismarck brown and examined in dilute glycerin ; Erlanger made paraffin sections and stained with iron hsematoxylin. KosTANECKi and Siedlecki {ibid., xlvui, 1896, p. 184) employed concentrated sublimate solution, or 3 per cent, nitric acid or mixtures ■ of these two, for ovarian ova. Van Beneden and Neyt {Bull. Acad. Belg., 1887, p. 214) took equal parts of alcohol and acetic acid. Boveri {Jena Zeit., xxi, 1887, p. 423) fixed in his picro-acetic acid, § 95 — a. clearly inadequate method. Gulick {Arch. Zellforsch., vi, 1911) has " fixed " ova of Heterakis for twenty-two Kovirs in one-third saturated picric acid with 3 per cent, of glacial acetic acid, and had them develop in alcohol of 70 per cent, to stages representing a normal development of several weeks. Boring {Arch. Zellforsch., iv, 1909, p. 121) spreads ova of Ascaris on a layer of Mayer's albumen on a slide, sets the albumen with a drop of formol, fixes with 4 parts of alcohol to 1 of acetic acid, stains in alcoholic hydrochloric acid carmine, and mounts in glycerin. Artom {Zeit. wiss. Mik., xxv, 1908, p. 5) freezes segments of the uteri of Ascaris in salt water, and cuts them with the freezing microtome into disks 30 (j. thick, and fixes these with divers liquids. Cerpontaine {ibid., xxix, 1912, p. 305) brings fixed ova from alcohol into absolute alcohol with 1 per cent, of clove oil, evaporates this down to one-tenth, puts into absolute alcohol with 5 per cent, of clove oil, evaporates again down to one-tenth, then into the same CHAPTER XXY. 291 with 5 per cent, of collodion added, evaporates almost entirely away, and passes through cedar oil into paraffin. For methods for the Mitochondria and Golgi apparatus' it is necessary to treat uteri as does Artom (above explained), and then fix in the proper fluid. Echinodermata, Coelenterata, and Porifera. See the chapter on " Zoological Methods." 19--2 CHAPTEE XXVI. CYTOLOGICAL METHODS. 643. Study of Living Cells. — In the young larvae of Amphibia, both Ariiira and Urodela, the gills and caudal " fin," and sometimes other regions, may be studied in the living state. The larvse may be fixed in a suitable cell, or wrapped in moist blotting-paper, or may be curarised ; or the tail may be excised. (It is preferable to cut through the larva, close in front of the hind limbs.) In the living animal the epithelial cells and nuclei (in the state of repose) are so transparent as to be ha,rdly visible in the natural state. They may, however, be brought out by curarising the larva ; or, still better, by placing the curarised larva for half an hour in 1 per cent, chloride of sodium solution. Normal larvae may be used for the study of the active state of the nucleus, but much time is saved by using curare. Curare. — ^Dissolve 1 part of curare in 100 parts water, and add 100 parts of glycerin. Of this mixture add from 5 to 10 drops (according to the size of the larva), or even more for large larvae, to a watch-glassful of water. From half to one hour of immersion is necessary for curarisation. The larvae need not be left in the solu- tion until they become quite motionless ; as soon as their move- ments have become slow they may be taken out and placed on a slide, wrapped in blotting-paper. If they be replaced in water they return to the normal state in eight or ten hours, and may be re-curarised several times. Other Narcotics. — Three per cent, alcohol or 3 per cent, ether, or infusion of tobacco, may be used in a similar way. These reagents cause no obstruction to the processes of cell-division. Indifferent Media. — One per cent, salt solution, iodised serum, syrup, cold water (-|- 1° C), and warm water (35°— 40°C.). The taU may be excised from the living animal and studied for a long time in these media (Peremeschko, Arch. mik. Anat., xvi, 1879, p. 437). For the processes of staining living cells see § 208. CHAPTER XXVI. 293 644. Study of Fresh and Lightly Fixed Cells.— So-called " in- different " liquids must not be believed to be without action on nuclei. Iodised serum, salt solution, serum, aqueous humour, lymph, better deserve the name of weak hardening agents. Between these and such energetic hardening agents as Flemming's mixture come such Kght fixing agents as picric acid or very dilute acetic acid. These it is whose employment is indicated for the study of fresh isolated cells. A typical example of this kind of work is as follows : Tease out a piece of living tissue in a drop of acidulated solution of methyl green (0-75 per cent, of acetic acid). This is a delicate fixing agent, killing cells instantly without change of form. Complete the fixation by exposing the preparation for a quarter of an hour to vapour of osmium, and add a drop of solution of Eipart and Petit and a cover. Or you may fix the preparation, after teasing, with vapour of osmium for half a minute to two minutes, then add a drop of methyl green, and after five minutes wash out with 1 per cent, acetic acid, and add solution of Eipart and Petit and cover. Or you may kill and fix the cells by teasing in solution of Eipart and Petit (to which you may add a trace of osmic acid if you like), and afterwards stain with methyl green. I have found Pictet's chloride of manganese (§ 403) useful as an examination medium. A little solution of dahlia may be added to it. Henking {Zeit. wiss. Mik, viii, 1891, p. 156) recommends a liquid composed of — Water 80 c.c. Glycerin Formic acid Osmic acid of 1 per cent. Dahlia 16 „ 3. „ 1 „ 0-Oi grm. Other fixing agents, such as picric acid or weak sublimate solution, may of course be used. Other stains, too, such as Bismarck brown, and of course other examination media than solution of Eipart may be employed. But, for general purposes, the methyl-green-osmium- and-Eipart's-medium method gives such good results, and is so very convenient, that it may be called a classical method for the study of fresh cells. 645. Some Microchemical Reactions.— ilfe%Z green is a test for chromatin, in so far as {with, fresh cells) it colours nothing but the chromatin in the nucleus, see § 276. It is, however, not a perfect 294 CYTOLOGICAL METHODS. test, for the intensity of the coloration it produces varies greatly in different nuclei, and may in certain nuclei be extremely weak, or (apparently) even altogether wanting. In these cases other tests must be applied in order to establish with certainty the presence or absence of that element. Chromatin is distinguished from albuminoids by not being soluble, as these are, in water and in weak mineral acids, such as 0-1 per cent, hydrochloric acid. It is easily 'soluble in concentrated mineral acids, in alkalies, even when very dilute, and in some alkaline salts, such as carbonate of potash and biphosphate of soda. In the presence of 10 per cent, solution of sodium chloride it swells up into a gelatinous mass, or even, as frequently happens, dissolves entirely (Carnoy, Biol. Cell., pp. 208 — 9). It is only partially digestible (when in situ in the nucleus) in the usual laboratory digestion fluids. The solvents of chromatin that are the most useful in practice are 1 per cent, caustic potash, fuming hydrochloric acid, or cyanide of potassium, or carbonate of potash. These last generally give better results than dilute alkalies. They may be employed in solutions of 40 to 50 per cent, strength. If it be desired to remove all the chromatin from a nucleus the reaction must be prolonged, sometimes to as much as two or three days, especially if the opera- tion be conducted on a slide and under a cover-glass, which is the safer plan. These operations must be performed on fresh cells, for hardening agents render chromatin almost insoluble in ammonia, potash, or sodic phosphate, etc. Hydrochloric acid, however, still swells and dissolves it, though with difficulty. Chromatin resists the action of digestive fluids much longer than the albumins do ; so that a moderate digestion serves to free the chromosomes from any caryoplasmic granulations that may obscure them, whilst at the same time it clears up the cytoplasm. Unna {Monatschr. prakt. Derm., xxxiii, 1901, p. 342) digests tissues in solutions of sodium chloride, to remove the granoplasm. See also §§ 652, 664 and 668. Glycogen. 646, Glycogen is a carbohydrate which occurs in many cells, both glandular and genital : it is found in both inter- and intra- cellular positions, in the form of small areas of flocculent appearance. For its study in a tissue or organ two methods should be used : — (1) An iodine technique ; and (2) that of Best's carmine. The specificity of the latter method has been questioned, and both techniques must be used for comparison (see p. 338). CHAPTER XXVI. 295 647. Iodine Method. — Fix tissue in Carney, or alcohol absolute, 4 parts, acetic add glacial, 1 part ; or in absolute alcohol ; or in alcoholic fixatives not containing alcohol lower than a" strength of 90 per cent. It is better if the tissue is cut small. Fix for one hour, then transfer for twenty-four hours or longer, in two changes of absolute alcohol ; then xylol and paraffin wax. Fix sections to sUde with a mixture of 50 per cent, alcohol with a few drops of glycerin and albumen, using the alcohol as you would water ; drain the shdes dry. Eeraove wax in xylol, bring to 70 per cent, alcohol. Stain sections in Bhrlich's hsematoxyhn for five or ten minutes. Blue in tap water substitute, § 669. Pass to a 2 per cent, solution of potassium iodide saturated in iodine (a Lugol solution) ; leave five minutes ; pour away, wipe around slide, and dehydrate in absolute alcohol saturated in iodine. Clear in oleum origanum cretici for about ten minutes. Mount in origanum balsam. Such preparations should keep for years without fading much. I have some shdes of human placenta which after six years still show the glycogen. 648. Best's Carmine Stain. — Material is fixed as for the iodine method and may be imbedded in celloidin. If paraffin sections are used the slide must be placed in 1 per cent. ceUoidin overnight, drained and allowed to dry partly, and then plunged into chloroform and absolute alcohol (equal parts), then treated as for celloidin sections. Transfer through alcohols 90 per cent, and 70 per cent, to water. Stain in Ehrlich's or iron hsematoxylin as usual, but differentiate in acid alcohol. Then proceed to Best's carmine stain {Zeit. f. mikros., Bd. xxiii). Make up this stock carmine solution : — Carmine 2 grms. Potass, carbonate . . . . .1 grm. Potass, chloride 5 grms. Aq. dest 60 c.c. Boil gently for a few minutes ; cool. Add strong liq. ammon. 20 c.c. Keep this solution in a well- stoppered bottle in a cupboard. It may go bad in a month during summer. Wash sectioms in distilled water after staining in hsematoxylin. Stain in following solution : — Stock carmine solution .... 2 parts. Liq. ammon. fort. . . . . . 3 „ Methyl alcohol (pure) . . . . 3 „ for five minutes. 296 CYTOLOGICAL METHODS. DifEerentiate in — Absolute alcohol . . . . , 80 parts. Methyl alcohol 40 „ Aq. dest 100 „ till no more red comes out (three to five minutes). Waah in 80 per cent, alcohol, absolute and clove oil, xylol and xylol balsam ; nuclei and cytoplasm, blue, glycogen red. It is a good plan when working on glycogen to prepare triplicate slides, one for iodine stain, one for Best ; the other slide is brought down to water and spat upon and set aside : the glycogen is dissolved by the diastase of the saliva, the latter is washed off in water and the slide stained as usual for Best's carmine. Comparison between the first slide and this one wiU assist in properly identifying glycogen ; (procedure of Dr. B. R. G. Russell, Imperial Cancer Besearch Bureau). One generally succeeds at first trial with such material as the liver of a rabbit, but with invertebrate materials, especially from paralHn sections, even though soaked in 1 per cent, celloidin, the results are often disappointing. This can be overcome by practice and by slight modi- fication in the time used for differentiation. For delicate material it seems best to work with celloidin sections. 649. Zieglwallner's Alcoholic Flemming for Glycogen and Pat.— Neither the iodine nor Best's carmine method preserves fat as well as glycogen. ZieglwaUner has worked out the following method for preserving both fat and glycogen. Fix small pieces of tissue in this mixture for twenty-four to forty-eight hours : — 1 per cent, chromic acid in 80 per cent, alcohol 15-0 2 per cent. O8O4 in water .... 4-0 Acetic acid . . . . . . .1-0 In 100 c.c. of this mixture there would be 50 per cent, alcohol. If a corrosive sublimate fixation is necessary use this mixture in the same way : — Concentrated aq. sol. corrosive sublimate . 20-0 2 per cent. OSO4 in water . . . .20-0 Acetic acid . . . . . . 10-0 Alcohol absolute . . . . . .60-0 . In washing out, a little iodine wiU be necessary. Transfer the pieces of tissue to 70 per cent., then upgrade and imbed in celloidin. In order to preserve the brownish black colour of the osmic stain of fat, which soon disappears when the sections are brought to balsam, one may convert the reduced osmic into its sulphide by adding a small quantity of Na^S to the 70 per cent, alcohol which replac.es the fixative. Imbed in celloidin or wax : stain as by the iodine, or better in Best's carmine method, from celloidin. Dr. J. A. Murray informs me that it is generally necessary to stain sections first in warm iron alum, then warm haematoxyUn, and then to differentiate in the cold with acid alcohol. Afterwards proceed to Best's carmine. Paul Buchner {Prdlctikum der Zellenlehre I., Berlin, 1916) fixes CHAPTER XXVI. 297 overnight in a freshly-made mixture of equal parts of absolute alcohol and strong Flemming. Wash out for two days in 50 per cent, alcohol, imbed in celloidin, stain in Best's carmine. See also Ceeighton, The JE'ormMive Property of Glycogen, London, 1896 ; Gage, Trans. Amer. Micr. Soo., xxviii, 1908, p. 203 ; Kato, Arch. Oes. Phys., cxxvii, 1909, p. 125 ; Btjsch, Areh. Intern. Phys., iii, 1905, p. 51 ; Maybe, Zeit. wiss. MiJcr., xxvi, 1909, p. 513 ; Arnold, Sitzb. Heidelberg. Acad. Wiss., 1909, p. 1, 1910, p. 3, and 1911, 14 Abh. ; Arch. path. Anat., cxciii, 1908, p. 175 ; Arch. mih. Anat., Ixxiii, 1909, p. 265; Ixxvii, 1911, p. 346 ; . Beitr. path. Anat., li, 1911, p. 439; Fraenkbl, Virchow's Arch., 1911, p. 197 ; Neubeet, Beitr. path. Anat., xlv, 1909, p. 38 ; Eehaed, Areh. Zellforsch., viii, 1912, pp. 447 and 507 ; Ehelich and Lazaeus, Bie Anaemic, 1898, p. 30 ; Pekelhaeing, Petrtis Oamper, Deel I-, 1901, -p. 231 ; Deiessen, Zeit. wiss. Mik. , xxii, 1905, p. 422 ; FiscHER, Anat. Ann., xxvi, 1905, p. 399 ; Fiessinger, G. B. Spc. Biol., Ixvi, 1909, p. 183 ; Neukiech, Arch. path. Anat, co, 1910, p. 82. 650. Some Microchemical Tests. — Iron. — Organic compounds of iron, which are not ionisable into ferric and ferrous ions, and in which the iron cannot be detected by the ordinary reagents, are much more frequently present in animal and vegetable tissues than was previously believed to be the case. In addition to the albuminate compounds, there exist iron compounds giving ferric and ferrous ions, detectable with the ordinary reagents, and which, for con- venience, may be designated Inorganic Iron Compounds. The nature of many of the compounds of iron found in placentas, blood-organs, the liver, etc., is obscure ; many of them appear to be formed as degeneration or excretion products, from the breaking down of haemoglobin. See below. Most of our knowledge of the methods for the detection of iron ia tissues and cells is due to A. B. Macallum {Quart. Journ. Micr. Science, xxxviii, 1895 ; Journ. Physiology, 1897 ; Ergehn. d. Physiol. Wiesbaden, 1908). Macallum has shown that, to detect organic iron, one must convert it into inorganic. This can be done by allowing sulphuxic or nitric acid alcohol to act upon sections, or a piece of tissue, for from one to twenty-four hours at 35° C, accord- ing to the strength of acid and the size of the object. When masked iron is liberated in the tissues by acid alcohol, most of it is ia the form of ferric salts, particularly when the oxidising nitric acid is used, and a small part occasionally of ferrous compounds. Inorganic iron compounds in tissues are usually ferric, more rarely ferrous The commonest tests for iron in tissues are the Prussian blue reaction, and Macallum's hsematoxylia. The latter test should never 298 CYTOLOGICAL METHODS. be used alone, because its complete specificity is somewliat doubtful. It is hardly necessary to point out that proper precautions should be taken to avoid contamination of the tissue by vessels or chemicals which may contain iron compounds. Glass needles should be used instead of steel, and the water used should have been distilled from a clean glass retort. The tissues should either be fixed in redistilled formalin (10 per cent.), chemically pure ethyl alcohol or pure 90 to 95 per cent. alcohol, or in redistilled methylated spirit. Bouin's fluid, Flemming, and such mixtures should not be used, as such a practice is almost certain to introduce error. Material should be fixed or hardened for several days in strong alcohol. Sections are made either freehand with a bright rust-free razor wetted with absolute alcohol, or 'by the paraffin method with a dry rust-free knife. Macallum's HiBMAToxYLiN Method. — As an indicator Macallum uses a 0-5 per cent, solution of absolutely " pure haematoxylin " made up in perfectly pure aqua dest. The solution should look brownish yellow, but when alkalies or alkaline earths are added, the colour becomes violet or red. When such a pure haematoxylin is brought into contact with a salt of iron, the yellow colour becomes blue-black, or bluish-black ; with organic iron compounds the hematoxylin is unaffected. Such compounds must be unmasked by sulphuric or nitric acid alcohol as above mentioned. When the compounds of iron to be investigated are found in tissues, the latter are well hardened in alcohol (purified, vide supra), sections prepared and washed in aq. dest., or the tissue simply teased out, and then the hsematoxylin solution is added. Those parts which go blue-black or blue-violet contain inorganic iron ; the remainder of the preparation may go quite dark yellowish brown, especially nuclei, and the presence of iron may thus be obscured. To remove this excess the preparation is treated in a mixture of equal parts of absolute alcohol and ether, but not for longer than one hour. The unaffected haematoxylin is extracted, the blue-black compound remains. Clear in oil of cloves, mount in balsam. Such preparations are permanent. This reaction of inorganic compounds of iron with hsematoxyhn seems to be one of oxidation (Maybe, Mitth. Zool. Stat. Neapel., x, p. 170). Extraordinarily small traces of inorganic iron are thus demon- strated. The method is more sensitive than that of Prussian blue or ammonium sulphide. CHAPTER XXVI. 2&9 Organic Iron Compounds. — These will not give the iron reactions unless the complex iron compound has been broken up, that is, the iron " unmasked " by some reagent : acid alcohol is used for this. Sulphuric acid alcohol (4 per cent, in 95 per cent, alcohol) and nitric acid alcohol (3 per cent, in 95 per cent, alcohol) are better than the hydrochloric acid alcohol (Bunge's fluid). Sulphuric acid alcohol acts very slowly especially on bulk tissues, and even Protozoa take twenty-four hours at 35° C. before their masked iron is revealed. Nitric acid alcohol acts more quickly and extracts very little of the iron it liberates (which is a danger with Bunge's fluid) ; the process is completed in about thirty-six hours. Sections are treated with acid alcohol, 90 per cent, alcohol, and aq. dest., and then Macallum's hsematoxylin is added ; the sections are washed in aq. dest., stained in safranin, as described in next section, dehydrated and mounted in balsam. Prussian Blue Reactions on Organic Compounds. — Sections after being treated in the acid alcohol (nitric or sulphuric) are washed in pure 90 per cent, alcohol and then in aq. dest. They are placed not longer than five minutes in the following solution : aq. potassic ferrocyanide 1-5 per cent., and hydrochloric acid 0-5 per cent, in aq. solution equal parts, freshly made. Again washed carefully in aq. dest. stained in eosin or safranin, dehy- drated in alcohol, cleared in oil of cedar and mounted in benzole balsam. The safranin or eosin are used in 1 per cent, strength in 30 per cent, alcohol, for three minutes for eosin, and for one half- hour for safranin, and differentiated in 90 per cent, alcohol. Ferric and Ferrous Salts both occur in inorganic iron com- pounds. Ferrous salts may be distinguished from ferric by the fact that only the latter give an immediate reaction with ferro- cyanide of potassium, while the former react with ferricyanide of potassium. Fix material in alcohol of about 90 per cent, for several days. Reaction for Ferric Salts. — Wash sections in aq. dest., transfer to 2 per cent. , aq. sol. ferrocyanide of potassium for from three to fifteen minutes. Bring to acid alcohol (1 c.c. in 70 per cent, alcohol) for about ten minutes. The Prussian blue reaction takes place. Wash in pure 70 per cent, alcohol, dehydrate clear and mount in benzole balsam. Counter-stain if desired in eosin or safranin (op. cit.). Ferrmts Salts. — As above, substituting ferricyanide of potassium instead of ferrocyanide. 300 CYTOLOGICAL METHODS. Simultaneous detection of both categories of salts may be made by using a solution of equal parts of ferricyanide and ferrocyanide. Blood, and Iron Salts. — In § 789 is given Okajima's method for elective staining of haematids (bsemoglobin). Degeneration products of hcemoglobin are hcemosiderin and melanin (of malaria). Hsemosiderin is found in the liver in pernicious ansemia, and also in large extravasations of blood. It is said that haemosiderin will, but melanin will not, give the Prussian blue and other iron reactions ; both pigments survive dehydration and a clearing oil. Another pigment derived from blood is hcematoidin or bilirubin, which contains no iron and does not give the iron reactions. Hcemoglobin itself is not unmasked by acid alcohol and will not give the iron reactions, but stains bright red with eosin from Mann's methyl-blue eosin mixture, and orange in Okajima's ahzarin stain, § 789. Most pigments are destroyed by concentrated sulphuric acid, which will not afEect any carbon granules which may have been fed to the cells experimentally. See alsoTiEMANN, Qoerbersdorfer Veroeffentl.,ii, 1898, p. Ill ; Schnei- der, Mitth. Zool. Stat. Neapel, xii, 1895, p. 208 ; Caenot and Lebbun, La Cellule, xii, 1897, p. 275 ; Sumita, Aroh. path. Anat., cc, 1910, p. 230 ; Zaleski, Zeit. Phys. Ghemie, xiv, 1890 ; Wasseemann, Anat. Eefte, xlii, 1910, p. 283, Jones, Biochem. Jour. 1920. Copper. — R. Boyce and W. A. Heedman, in their paper on the Green Leucocytosis in Oysters (Proc. Roy. Soc, Ixii, 1897 — 98), have given directions for the application of the well-known potassic ferrocyanide test of chemists, to sections of tissues in which copper is to be detected. These authors fix with proper precautions (vide supra, under " Iron ") in absolute alcohol, imbed in pure paraffin and cut sections. Care must be taken to avoid acid solutions, such as commercial turpentine or old xylol. Sections are brought from absolute alcohol to distilled water, placed in a 1-5 per cent, solution of freshly prepared potassic ferrocyanide or, preferably, in equal parts of the same ferrocyanide solution, and a 0-5 per cent. HCl solution, and parts where copper is present go a reddish colour. Sections are then washed in aq. dest., dehydrated in absolute alcohol, cleared in cedar-wood oil and mounted in Canada balsam. See also Maoallum, Journ. Phys. Cambridge, xxii, 1897, p. 92 ; Maefoki, Arch. Ital. Biol., xxx, 1898, p. 186. Tor Zinc see Mendel and Bradley, Amer. Journ. Phyg., xiv, 1905, p. 320. For Lime salts see Geandis and Mainini, Aroh. Ital. Biol., xxxiv, 1900, p. 76 ; Schaffer, Zeit. wiss. Zool., Ixxxix, 1908, p. 13 ; Leutert, Encycl. milcr. Technilc, ii, p. 588 ; Stoeltznee, Arch. path. Anat., CHAPTER XXVI. 301 cLsxx, 1906, p. 363 ; Macallum, Ergeb. Phys. Wiesbaden, vii, 1908, p. 612. For Potassium see Macallum, Journ. Phys. Cambridge, xxxii, 1906, p. 95 ; Ergeb. Phys. Wiesbaden, vii, 1908, p. 600. Tor Guanin see Giacomo, Zeit. wiss. Mik., xxvii, 1910, p. 267. Concerning the mioroch.emistry of the cell in general, see tuithe.i fourth edition ; also Cabnot and Lebedn, La Cellule, xii, 2, 1897, p. 194 ; ZiMMEEMANN, Die Morphologie u. Physiologie des Pflamzlichen ZeWcernes, Jena, 1896 (treats also of the animal cell) ; Haeckee, Praxis u. Theorie der Zellenund Befruohtungslehre, Jena ; Peenant, Journ. Anat. Phys., xlvi, 1910, p. 343. 651. Cjrtological Fixing Agents. — A fixing agent that is good for one element of a cell is not necessarily good for all others. As regards the nucleus, all fixatives should be acid ; for if not they will not satisfactorily preserve either chromatin or nucleoli. For instance, bichromate of potash, if not rendered acid, fixes, chromo- somes and nucleoli in a distended state so that clear images of them are not obtained. Acids contract them somewhat, and so give them sharper outlines. The fixatives mostly employed for nuclei are liquid of Flemmino and liquid of Hermann. There is a slight difference between them. Liquid of Hermann, owing to the platinum chloride, causes chromatin to shrink more than liquid of Flemming does, and for this reason is supposed to give clearer images of chromosomes, especially of their splitting. I find that it generally makes them shrink too much, and that it is not at all good for spindles. For many, if not most objects, I prefer to these two reagents Bouin's picro-formol, which gives a highly faithful preservation and a more penetrating and equable fixation. For spindles I recommend Flemming (picro-formol does not give quite such bold images). Some of the finest chromosomes I have seen have been fixed with Lindsay Johnson's mixture (§ 44), and liquid of Tellyesniczky has given me others nearly if not quite as good. As regards the cytoplasm. — Cytoplasm is made up of two elements : a fibrillar element — ^the spongioplasm or mitome ; and a more or less granular liquid that bathes it — the hyaloplasm or enchylema. It does not follow that a reagent that will fix one of these will also fix the other. Nor is it always desirable that both should be equally fixed. If you fix both, you will have a full fixation ; but in that case the granules of the hyaloplasm (be they vital, or be they only " pre- cipitation forms," see § 29), and the secretions or other enclosures that may be present in it, may so mask the fibrils of the spongio- 302 CYTOLOGICAL METHODS. plasm as to interfere with the observation of it. So that if the latter is the principal object of study, a thin fixation, one in which the spongioplasm is entirely preserved, but the hyaloplasm only partly, may be the better. The spongioplasm is the easier to fix of the two, and the majority of acid fixatives will preserve it more or less. The best images I have obtained are those given by liquid of Flemming or Hermann in cells in which the action of the reagent has been moderate, i.e. insufficient to thoroughly fix the hyaloplasm at the same time. Nearly, if not quite, as good, is Bouin's picro-formol, which has the great advantage of being very favourable for plasma-staining. I have also had very good results with vom Eath's picro-osmic and picro-platinosmic mixtures, and with acid sublimate. Hyaloplasm is not nearly so easy to fix, and there are only two reagents in common use that readily give a really full fixation of it ; these are osmic acid and bichromate of potash. Osmic acid acts as a fixative of hyaloplasm in liquid of Flemming or Hermann, but only gives a full fixation in the outer layers of the material ; and in these it easily happens that many or most of the cells are ruined by over-fixation. See § 35. This defect may be to a certain degree corrected by taking the osmic acid weaker than is usual. Thus by successively reducing the proportion of this ingredient in liquid of Hermann, I have found that it can be brought down to one-eighth of the prescribed amount without loss of the distinctive characters of the fixation. The defect of want of penetration seems to be incurable. See §§ 35 and 42. Substitution of more highly penetrating reagents such as picric acid, for the chromic acid or platinum chloride does not help in the least ; you only get the osmic fixation outside, no whit deeper than before, and a picro-acetic fixation, instead of a chromo- or platino-acetic one, in the deeper layers, that is all. In view of these defects' of osmic mixtures, it may often be advisable, where hyaloplasm, or its enclosures, is the chief object of study, to have recourse to bichromate of potash. The formula that has given me the finest fixations is that of Lindsay Johnson, but it has the drawback that there is risk of osmication in the outer layers. In this respect liquid of Tellyesniczky, § 52, is to be preferred. Corrosive sublimate gives a fairly full fixation ; but I believe it frequently produces serious artifacts, Heidenhain's " Lanthanin " being one of them. Heidenhain's solution, § 64, containing as it does some 11 per cent, of sublimate, without the addition of any acid to neutralise its shrinking action, seems to me to be an inad- CHAPTER XXVI. 303 missibly coarse reagent. I have, however, obtained with liquid of Carnoy-Lebrun, § 86, some most excellent fixations of cj^oplasm. The aqueous solutions of sublimate are frequently used in pre- ference to liquid of Flamming on account of the facilities they afford for the emplojninent of certain stains ; but to that end- 1 prefer Bouin's picro-formol. 652. Chromosomes ; Chromatin Stains.* — For fresh tissues, see §645. With hardly an exception modern work on chromosomes in the germ-cell cycle is carried out by use of such fixations as strong Flemming (§ 41), Bouin's picro-formol-acetic (§ 110), Carnoy (§ 85), or an alcoholic nitric corrosive acetic of the Gilson or Petrunkewitsch type. Stains now used much are iron hsematoxylin of Heidenhain or Benda, thionin, safranin, well ripened Delafield, and Mayer's acid hsemalum. Gentian violet is used by many. For the study of the chromosomes, the desideratum seems to be some fixative which will penetrate evenly and rapidly, which will strip out of the cell, fats and lipoids, and which wiU allow the subsequent use of some dense, preferably black or dark blue stain. Some English workers have found Flemming's strong formula, without acetic acid to give beautiful results for chromosomes (e.(/. L. Hogben, Proc. Roy. Soc, B., xci, 1920). In nearly aU my slides fixed for the cytoplasmic inclusions (§ 673) good chromosome plates are found, but I consider that fixatives wbicb contain lipoid solvents are indicated for chromosome work: the preparations are thereby "stripped" and cleaner, and difiicult nuclei are better interpreted when superfluous materials are removed. Sister Monica Taylor (Quart. Jour. Micr. Sci., 1915) spiins first in thionin, mounts and studies the sections ; then, if they prove to be worth it, removes the coverslip by soaking in xylol, and restains in iron alum hsematoxylin. Red stains are not indicated because of the eye-strain they cause. * Iron alum hcematoxyUn is especially recommended. Dr. Lee has sent me the following note on the use of this stain : — •" Some cyto- logists have given up iron hsematoxylin because they have found it to clog the chromosomes ; but this will not occur if the following precautions are observed. Mordant sections (7 to 8 ju) for not more than two and a half minutes in iron alum of 2 to 3 per cent. ; wash for at least a quarter of an hour ; stain in a 0-5 per cent, solution of ripened haematoxylin until the sections appear dark grey, but not black (about twenty-five minutes if the solution is fresh, and not more than four if it has already had several slides passed through * By J. B. G. 304 GYTOLOGIGAL METHODS. it) ', differentiate in the iron alum solution for at least two minutes after the chromosomes, examined in water, appear to have been sufficiently extracted, for chromosomes always appear paler in water than after they have been got into balsam." Personally I have never found iron hsematoxylin to clog chromosomes, and can only assume that something was wrong with the fixation, or a bad specimen of stain was used. Hot or Cold Fixation? — Some workers advocate the use of hot fixatives, others believe that the best results are obtained by keeping the capsule or vial of fixative on ice while the material is being fixed. Possibly cold, not freezing, fixatives are indicated for invertebrates and cold-blooded animals, and fixatives at body heat for warm- blooded. This, however, must be left to the discretion of the worker, who may find that either very cold or hot fixatives may irnprove his preparations in an unexpected manner. Ezra Allb n recommends using his modified chromic Bouin and urea at 38° C, while he believes that Flemmiag should be used in an ice-box {Anat. Record X, 1915 — 16). CowDEY [Contrih. Carnegie Inst. Wash., viii, 1918, recommends the use of Eegaud's formol-bichromate on ice. See also § 31. 653. Dissection of Animals for Chromosome Work. — Some ob- servers have claimed that dissecting out gonads of invertebrates, in a dish of " tap-water," gives clearer chromosome figures than when one uses Ringer or such salt solutions. If possible, avoid dissecting out ia any fluid. Fleas and lice, and such small insects, are to be treated as follows : cut off the end of the abdomen, hold the insect down on a glass slide by its head with the aid of a mounted needle, and with another needle press the viscera out . with a stripping motion from the head backwards. Immediately transfer the viscera to a fixative (Doncaster, Quart. Jour. Micr. Sci., 1920). For bigger insects one may open the body cavity after having out off their heads, and pipette fixative over the viscera before separating away the gonads. Then transfer to a capsule of the fixative. Read also directions in §§ 12 and 676. 654. Fusion of Chromosomes caused by Fixation. —It should be noted that unsuitable or inferior fixation ma/y cause such artifacts as fusion of chromosomes which, intra vitam, may have merely been closely paired. With Diptera it has been found that bodies of mosquitoes, etc., should not be thrown whole into a fixer, but either finely teased or the gonads should be carefully dissected out. This obtains a more rapid and there- fore a more efficient fixation (Hance, Jour. Morph-, 1917 ; Metz, Jour. Bxp. Zool., 1916). CHAPTER XXVI. 305 In the same way more fluid chromosomes such as those of mammals may be caused to run together and so introduce error. This probably explains the discordance in the accounts given by different workers for such material as that of man. 655. Note on Fixatives for Chiomosome Work. — For work on vertebrate tissues there is little doubt that Bouin's picro-formol- acetic, or one of its modifications, is the best mixture to use. For invertebrates in general such Bouin fixatives are also very satis- factory, but Flemming's strong formula should always be tried. Corrosive acetic acid I hold to be a rough and unreliable fixative, and if a corrosive fixation is desired, it is better to use Gilson or Petrunkewitsch — ^these penetrate more rapidly and give a more delicate fixation. I have seen some excellent chromosome plates in mammals got by using Sansom's Carnoy modification (§ 86). It should be noted carefully that many workers use chromosome fixatives for a short time only ; for instance, Flemming may be used on insect gonads for half to one hour only, followed by a washiag out under tap, and then upgrading from 30 per cent, alcohol (from Professor Leonard Doncastbr, in Uteris). 656. Urea and Chromosome Fixation. — It has been claimed by some American workers (McClung, Ezra Allen, R. T. Hance, etc.) that the addition of from 0"5 to 2 per cent, urea crystals to fixatives of the Flemming or Bouin type assists in penetration and gives sharper pictures of the chromosomes. The idea of using urea is, I understand, due to Professor McClung. It is out of the province of this book to discuss whether a solution of urea in such complicated fluids as Bouin or Flemming, has the same peculiar penetrative properties as in water, or even to question whether, after the addition of the crystals to the Bouin or Flemming, disintegra- tion of the urea does not take place. We are prepared to accept the statements of McClung, AUen and Hance, and to recommend a trial of the method. Waeo Nakahaka (Jour. Morph., 1919), working on Perlaj did not find that the addition of urea crystals to his fixative made any appreciable difference. . 657. Fixation of Mammalian Chromosomes. — The material must be absolutely fresh ; even half an hour's delay is fatal ; prepare your fixatives and capsules, knives, etc., before you kill the animal. Apparently chromosomes of mammals will clump together about ten minutes after death. Hance (Anat. Record, xii, 1917) gives the following method : — (1) Obtain fresh specimens of tissue from as many different animals as possible, so as to be sure of obtaining one or more in a " cycle of cell division." (2) Place small or finely M. ' 20 306 CYTOLOGICAL METHODS. teased pieces of fresh tissue immediately into cold Flemming's solution (on ice) plus about 0-5 per cent, urea crystals. Flemming's solution kept on ice registers about 4° to 5° C. Leave in cold solution for twenty-four hours or longer. (3) If this fixation fails^ try the following : Allow small pieces of fresh tissue to remain in the air for from ten to twenty minutes after removal from the animal before placing them in the cold Flemming.* Then fix as before. (4) Wash in water about twenty-four hours. (5) Dehydrate by very gradual steps. (6) Clear from 95 per cent, alcohol in cedar oil followed by xylol. Embed in parafiin. 658. Ezra Allen's Chromic Bouin and Urea. — Used for work on the spermatogenesis of rat, etc., and generally indicated for mammals. Picric acid, sat. sol. . . . . .75 c.c. Formol (pure) . . . . . . 25 „ Glacial acetic . . . . . . 5 ,, To freshly-made mixture raised to temperature of 38° C. add and dissolve, first, 1-5 grms. of chromic acid crystals, and then 2 grms. of urea crystals. Kill animal by decapitation, remove testis imme- diately, snip into small pieces, fix at 38° or 40° C for from one to two hours. Fixative replaced by " drop method " with 70 per cent, alcohol, picric acid washed out by addition to the alcohol of a sat. sol. of lithium carbonate, a few drops at a time ; the alcohol is replaced by anilin oil (freshly distilled), this by sjmthetic oil of wintergreen, and this by paraffin of 52° melting point. The paraffin is slowly added till the tissue is in a bath of high paraffin concen- tration. It is then passed through several changes of pure paraffin to remove oil (Ezra Allen, Jour. Morph., 1918 ; Anat. Record, X, 1916). 659. Amphibian Chromosomes.^CHARLEs Parmenter {Jour. Morph., 1919) uses Ezra Allen's chromic' Bouin with success for amblystoma. Ordinary Bouin with urea crystals, Hermann, and Flemming were also gqod. 660. Modified Bouin and Urea for Insects.— Miss E. Eleanor Carothers for orthopterous chromosomes uses the following fluid which has been developed in the Zoology Laboratory, Pennsylvania. It is a modification of Bouin'e formula : — Picric acid, sat. sol. aq 75 c.c. Formalin (strong) 15 ^ * This, of course, is directly contrary to all the rules of fixing tech- nique, but it might work satisfactorily. CHAPTER XXVI. 307 Glacial acetic acid lOc.c. Urea crystals ^ grm. Use for twenty-four hours. Stain in iron hsematoxylin or Plemming's 'tricolour mixture {Jour. Morph., xxviii., 1916 — 17). 661. Precautions in Dehydrating and Clearing. — It is well known that too rapid dehydration or clearing will cause shrinkage and distortion. Material to be used for chromosome study should be dehydrated gradually, either by a syphon or some drop method (§ 3). For clearing xylol is not good, as it causes much shrinkage. The least shrinkage occurs with some vegetable oil, hke bergamot, origammi, cedar wood, or cassia (cinnamic aldehyde), which, if it will not mix with paraiSn, should be washed out subsequently in some parafEn solvent, like benzole or xylol. Ezra Allen (Anat. Record, x, 1915—16), following Suchannek (§ 134), uses distilled aniline oil as a substitute for the higher alcohols. One gradually brings brain and genital or such tissue to 75 per cent, alcohol, and then adds distilled anilin oil by the drop method, shaking frequently, ox usiug some system for agitatiag the fluids, or by some diffusion apparatus (§ 3). When nearly pure anilin oil has replaced the alcohol, one transfers to pure oil until the tissue is cleared. From aniUn oil one embeds as follows : warm the oil and tissue slightly, adding every ten minutes a few drops of melted paraffin, mixing thoroughly with a pipette ; continue till the mixture has 85 to 90 per cent, paraffin. Transfer to melted paraffin. If bergamot oil has been used for largish objects, at least four changes of pure parafiin must be made, one haK-hour to each, and the fifth bath for at least one hour. In most cases gradual clearing in cedar-wood oil will give satisfactory results. See also § 5. 662. Mounting Sections between Coverslips.— Agar (Quart. Jour. Micr. Sci., 1911) has devised a method for mounting prepara- tions between coverslips in order that they can be observed on both sides. Use one larger covershp as if it were a slide. Carriers may be made by stamping out a square in stifE cardboard, or thin metal. See also, C. CiipiiDE, G. R. Soc. Biol, civ, 1913. 663. Smear Preparations of Gonads. — In some cases smear pre- parations of testes especially may provide useful evidence ia a research on chromosomes. One may be fortunate enough to find nuclei at the prophase or metaphase of mitosis, with all the chromo- somes spread out so as to be counted with ease. In many cases, to study early stages in synapsis for which very rapid penetration is essential, smear preparations are a sine qua nan. 20—2 308 CYTOLOGICAL METHODS. Eemove the testes ; if it is large take a fragment by a pair of forceps and quickly smear along the length of a dry slide several times, so as to cover as much of the middle part of the slide as possible. If the cells are likely to stick, fix immediately by pouring on some Flemming, Bouin, or Petrunkewitsch. Set aside for a few miautes, wash off in water, upgrade from 30 per cent, alcohol, and leave overnight in 90 per cent, alcohol. Bring back to water, stain in iron hsematoxyliQ, thionin, or gentian violet, etc. See also Goodrich's iodine-Bouin method (under "Protozoa"). Note that smears may be fixed in steam, acetic, osmic, formalin vapour, or " Lucidol " (§§ 107, 783), or stained and fixed simultaneously ia Leishmann, acetic Bismarck brown (§ 277), aceto-carmine (§ 221), or such mixtures. Smears of very liquid testes, like those of Lepidoptera, are liable to be washed away if fixative is added too soon. It is probably best to kill the cells in some toxic vapour, then allow them to dry a little, and then fix in a liquid. Foot and Strobell {Arch. f. Zellf., Bd. xii, 1914) recommend the following : — Place testes in drop of acidulated (acetic) " water " on end of slide, and with fine needle (No. IX.) cut from it the area at stage required (previously ascertained by examination of sections). Push this area by point of needle to middle of slide and break up the tissue by gently tapping with the needle (never roughly spread as recommended by some workers). This should be done under dissecting microscope. See also Kernschwarz, and ' ' Iron Carmine," § 222. For Bataillon and Koehlee's borax -methylen-blue see Oomptes Bendus, cxvii, 1893, p. 521. 664. The So-called Microchemical "Tests" for Chromatin.*— Among these the " digestion tests " have met with some favour. It is well-known that examination of the partly digested tissue from the gut of animals which eat fresh cells (e.g. parasitic hymenoptera) shows that the nucleus of the cell resists digestion for the longest time. On the other hand, there are a whole series of enzymes which are specially concerned in the hydrolysis of nucleic acid, each acting on some particular substrate ; these enzymes are grouped together as " nucleases." The results of tests carried out with proteolytic enzymes on the nature of unidentified cell granules must not be depended upon too much. Extracts of such organs as the spleen and pancreas are known to contain more than one kind of enzyme, '*■ * By " chromatin " I mean that material which forms the substance of the chromosomes. The question of chromatin, nucleoli, and basophil granules is one of the most difficult in Cytology. (See Gatenbt, Quart. Jour. Micr. Sci., vol. 64, 1920, Science Progress, January, 1921, K. LuDFORD, Jour. Boy. Micr. Sac, 1921, and recent numbers of the Quarterly Journal of Microscopical Science.) CHAPTER XXVI. 309 and the zoologist especially is advised to get the help of an expert on enzymes before attempting to interpret any experiments he may have made with such intra-cellular enzymes. Because a certain protease will not dissolve away a given cell granule, while a " nuclease " may do so, does not by itself provide good evidence for the conclusion that the granule in question is " chromatin," or derived from the chromosomes. Van Hekwerden (Aroh. f. Zellf., x, 1913) for instance, using the extract of spleen containing " nuclease," finds that the granules identi- fied as " chromatin " emission by Schaxel are dissolved away. In all probability, however, such granules are phospholipin and not chroma- tinic, and were dissolved away by some lipolytic enzyme in the solution used by Van Herwerden. Successful experimentation on this side of cytology calls for two desiderata : Firstly, an intimate knowledge of the nature of the enzyme solution to be used, and of what effects the latter has on various definitely identified categories of cell substances ; second, a thorough study of the origin and microchemical reactions of the body to be treated by the enzyme solution. See Vernon, Intra- cellular Enzymes, London, John Murray, 1908 ; Bayliss, The Nature of Enzyme Action, Longmans, Green & Co., 1920 ; Euler, General Chemistry of the Enzymes, Pope's Translation ; Bayliss, General Principles of Physiology, 1919. 665. Method of Using Enzyme Solutions.— These should be used on fresh cells. It is best to begin by identifying cell or nuclear bodies or granules in properly fixed and stained preparations ; after this one should familiarise oneself with the appearance of such granules or bodies in the fresh cells, with and without intra-vital staining. The enzyme or digesting fluids may be added to fresh cells, a coverslip provided, and the preparation observed at intervals under a high power. Another method is to immerse pieces of fresh tissue in the digestive fluid for suitable times, and then fix the tissue in some mixture by previous trials indicated for the purpose ; controls should be made by soaking other pieces of tissue for a similar time in some of the same solution inactivated by heat, or without the added enzyme. As indicated above, such tests are of doubtful value as regards the identification of " chromatin " ; the solutions used should be tried carefully on cells whose cytology is thoroughly known by the observer. The, use of proprietary "pepsins " and such-like solutions whose origin and method of manufacture are unknown to the worker, is not likely to lead to the best results. See also C. Beckwith, Jour. Morfh., xxv, 1914, and Jokgensen, Arch. f. Zellf., x, 1913, and.§ 645. 666. Chromophility. — Some workers indiscriminately call all basophil chromatic material chromatinic, which is unjustifiable : 310 CYTOLOGICAL METHODS. the chromatic " blushes " which are sometimes found sixrrounding the nuclei of eggs and other cells are sometimes believed to be chromatinic, but the evidence for this is very slender. Chromatin, moreover, may sometimes stain oxyphil, and it is now a well-known fact that the nature of the fixation and the method of staining are important factors in deciding whether the bodies in question will stain in the basic or the acid dye. Under " Nucleoli " are given a number of staining and fixing methods which may be tried. See also Chapter XI, § 211. 667. "Vital" Staining of the Nucleus. — A. M. Pezbsmycky {C. R. Soc. Biol., Lxxviii, 1915) uses neutral red. The living nucleus is said to have a greater affinity for neutral red than protoplasm, as it stains more strongly and decolourises more slowly. This is not my experience with weak neutral red. Chambers {Science, 1912) uses janus green, which shows both chromosomes and spindle fibres. Certain physiologists doubt whether the living resting nucleus can be stained ; some observations on this will be found in Chapter XI., § 207. Reference may be made to the paper of P. G. Shipley (Amer. Journ. Physiol., xlix, 1919). It has been asserted by some observers that the nucleus may also be stained during the life of the cell by means of Bismarck brown, Congo red, methylen blue, Nile blue, and safranin. But Bolles Lee believes that it is by no means clear from the statements of these writers that the coloration observed by them is localised in the chromatin of the nucleus. It would rather appear to be a diffuse coloration brought about by mechanical and momentary retention of the dye in the nucleus — ^which is a very different thing from a true nuclear stain. And in some of the cases reported it is by no means certain that the coloured nuclei were really in the living state. In any case there is no dye known which is a specific intra- vital test for chromatin. It seems probable that the most " specific " test for chromatin known to cytology is acidulated methyl green, for which, see §§ 278 and 645. 668. Nucleoli. — The word is nowadays generally used to mean any large rounded stainable body in the nucleus. The plasmosome is " acidophilous " in so far as, in fixed material, it selects the acid dye or dyes from mixtures such as the Ehrlich-Biondi, which stains it reddish or orange. So far as known true plasmosomes of unfixed cells do not stain green with the acid methyl green, but while this may hold for the plastin nucleoli of somatic cells in general, there are to be found in eggs complicated nucleoli which have not properly CHAPTER XXVI. 311 been investigated. Even if part of these nucleoli did stain green in acidulated methyl green, it would be unsafe to interpret such material as chromatin, especially in view of the fact that methyl green will stain silk and mucin, and the secretions of many gland cells ; plasmosomes often seem to be related in some way to gland secretion. Most of the work on plasmosomes or true nucleoli has been carried out on fixed material, and, it cannot he too strongly emphasised that by such means it is not possible properly to discriminate between what is true chromatin, and what is not true chromatin. There are no perfectly specific chromatin dyes known, the nucleoli in general may be made to stain differently according as to whether they may have been fixed in acid or non-acid fixatives (this applies especially to acetic acid), or in osmicated or non-osmicated mixtures, while true chromatin may itself stain " acidophile," as, for instance, in the head of the sperm during spermateleosis, and in the egg during oogenesis. These remarks refer also to the use of so-called specific dyes for discriminating between and identifying the various cytoplasmic inclusions. See especially §§ 707 et seq. The case of the so-called karyosome or chromatin nucleolus is even less satisfactory ; one often meets with " solid " intra-nuclear bodies which stain basophil with many so-called nuclear dyes, but which are only doubtfully to be regarded as chromatin. In this connection, see Bayliss (§ 204) on specificity of dyes. Safranin and light green, and iron hsematoxylin, following strong Flemming's fluid, are classic stains for nucleoli, but neither of these methods is to be regarded as providing any useful evidence as to the micro-chemical nature of the bodies they tinge. They simply stain most deeply, solid bodies. The relationship between nucleoli and the chromatin network is quite unknown in general, but Cajal and Cablbton (for references see Quart. Jour. Micr. Sci, Ixiv, 1920) have shown by formalin and silver nitrate methods that both basophil and oxyphil nucleoli may contain an argentophile core. Carleton has followed the core through mitosis, and finds that it keeps its individuality ; the definitive nucleolus of the " resting " nucleus is possibly derived after every mitosis from the core or nuoleolinus. I have found nucleolini in the nucleoli of the gut cells of Saccocirrus, and in foUicle-ceUs of the ovary of insects ; nucleolini are known in many kinds of vertebrate cells. For the study of nucleoli the following methods are advised : — (1) Fixation by a corrosive sublimate, both acidified by acetic acid, and alone. (2) Fixation by hot water or steam. (3) Fixation by a 312 CYTOLOGICAL METHODS. variety of mixtures such as Carnoy, Bouin and Zenker with or without acetic acid, etc. Staining^ in Ehrlich-Biondi, Ehrlich's hsematoxylin and azoeosin or Biebrich scarlet (by Scott's method, described below), by Pappenheim's pyronin and methyl green, by Auerbach's fuchsin and methyl green, Zimmermann's fuchsin iodine green, and by Mann's methyl-blue eosin. The double and triple simultaneous stains are valuable. Auerbach's stain consists of equal parts of 1 per cent, methyl green, and 1 per cent, acid fuchsin ; Pappenheim's stain (§ 292) consists of methyl green and pyronin, a red basic stain instead of the acid fuchsin of the Auerbach. Another staia which is very valuable is the triple stain of Ehrlich, but it is less easy to work than Auerbach or Pappenheim. In all work oh nuclei and nucleoli, Mann's methyl-blue eosin will be found especially helpful, because the eosin-staining from this mixture is generally more restricted and intense than when one stains in some basic dye followed by eosin, or vice versa. Very beautiful results are occasionally procured by using Unna's polychrome methylene blue (§ 337). Eecourse should be made to the methods for the mitochondria, particularly those such as Champy-Kull and Bensley-Cowdry (compare with Auerbach preparations), for many nucleoli are compound bodies almost certainly containing lipoids or fats. The formalin silver nitrate techniques of Cajal or Da Fano should be tried, and wherever possible tests on fresh cells should be carried out (e.g. digestion, methyl green, etc., etc.). It seems indicated that further observations carried out on nucleoli of live cells in tissue cultures will provide new facts, especially with regard to the part played by these bodies during mitosis. See sections on " Tissue Culture." 669. S. Gr. Scott's Standard Hsematoxylin and Biebrich Scarlet for Chromophility (Jour. Path, and Bact., xvi, 1912). — Fix tissue in sublimate formalin, Zenker without acetic, HeUy's Zenker-formalin, Miiller, or formalin. All strongly acid fixatives must be avoided, for the Ehrlich's hsematoxylin will not then stain anything but nuclei, and these only faintly. Paraffin sections are made and fixed to slide. After removal of paraffin with xylol, and this with alcohol, sections of material fixed in sublitnate solutions are treated with iodine for three or four minutes (0-2 per cent, iodine in 80 per cent, alcohol). Einse off excess iodine with a little alcohol and remove all iodine from tissue with a 0-25 per cent, solution of Na2S203 in 50 per cent, alcohol, not in water as recommended by Heidenhain [Arch.f. d. ges. Phys., Bonn, 1902, Bd. xc, § 115). This is a most CHAPTER XXVI. 313 important part of the method, as sublimate and iodine both act detri- mentally to staining. After washing in Na2S203, wash off in a stream of aq. dest. ; roll the water off the slide with 1 or 2 drops of alcohol (90 per cent.), wipe the slide dry around the sections, and pour on 3 to 4 drops of Ehrlich's acid hsematoxylin to every section. Leave exposed on bench ten minutes. Remove the hsematoxylin by rolling it off with drops of alcohol (90 per cent.) from a drop-bottle ; do not wash off the stain with water as this lowers the viscosity of the solution and allows a diffuse staining. Wash away alcohol with a stream of aq. dest. till every trace of stain is removed from the slide. Blue the hsematoxylin lake and remove acid from combiaation with the proteins of the section by dropping on the latter 8 to 10 drops (or more for a number of sections) of the following tap water substitute : — KHCO3 2grms. MgSOi 7H2O 20 grms. Aq. dest 1000 c.C; Sat. with camphor, or thymol, After three to five minutes wash off the alkaline solution thoroughly in aq. dest. Wipe around sections to remove superfluous water, and add a 1 in 2000 solution of azoeosin (not eosin) or Biebrich scarlet dissolved in this medium : — Glycerol 2 per cent. Methyl or 96 per cent, ethyl alcohol . 8 „ Dist. water . . . . . 90 „ Use azoeosin (Bayer) for bichromated material and Biebrich scarlet for non-bichromated material. Leave in the acid dye for ten to thirty minutes, rinse off in aq. dest., rapidly dehydrate in 90 per cent, and absolute alcohol ; xylol, and xylol balsam. In my hands weak watery solutions of good Eosin have not given such satisfactory results. Scott believes that sulphonated monazo colours, such as orange G and Bordeaux K, are useless. Eosin is much less precise than Biebrich scarlet and azoeosin. The special points about this method are : — ( 1 ) Getting rid of all corrosive and iodine. (2) Stain- ing by placing hsematoxylin on slide where the solution can evaporate, and thus arrive at a state of viscosity which automatically prevents overstaining. (3) Washing off stain in alcohol, not water, thus pre- venting diffuseness. (4) A definitely alkaline tap-water substitute. (6) A precise counter-staui. Histologists. and cytologists carrying out work on chromophility are recommended to use this method as a standard for either basophility or oxyphility. Note, however, that granules which stain basophil by this 314 CYTOLOGICAL METHODS. method axe not necessarily chromatinio. Occasionally this method seems to overstain genital cells, but this gives the correct degree of chromophiUty of such cells in comparison with the cells of other tissues. After having used the method critically, I have come to the conclusion that it is more precise than the usual procedure which introduces differentiation in an acid solution. Scott worked out this method mainly with sections of mammalian red bone marrow. 669a. Obst {Zeit. wiss. Zool,, Ixri, 1899) fixes in sublimate, stains in borax carmine, and then stains the sections for three hours in very dilute aqueous methyl green or solid gTeen. Nucleoli blue, chromatin red. BuCHNEE {Arch. Zellforsch., iii, 1909, p. 337) has found this iiseful for distinguishing the accessory chromosome in testis cells of Orthoptera — normal chromosomes red, accessory and chromatin nucleoli blue-violet. ZiMMERMANN {Zeit. wiss. Mik., 1896, p. 463) stains for ten minutes in a fresh mixture of 9 parts 0-1 per cent, aqueous iodine green with 1 part concentrated aqueous solution of fuchsin, and differentiates in absolute alcohol with 1 per cent, of acetic acid and 0-1 per cent, of iodine. Nucleoli red, chromatin blue. Fischer {Fixirung, etc., p. 140) adds 30 drops of hot 0-1 per cent, fuchsin solution to 100 c.c. of 0-3 to 0-5 per cent, solution of methyl green. MONTGOMEET {Joum. Morph., XV, 1899) stains for an hour in Ehrlich's haematoxylin, and then for five minutes in concentrated aqueous eosin, or first with concentrated aqueous methylen blue, and then with con- centrated alcoholic solution of Brazilin. For a series of papers on staining nucleoli, and illustrated by profuse coloured plates, see Max Jorgensen's memoirs in the Arch, f. Zellf., X, 1913. Another paper worth consulting is Champy's magnificent work on the male germ-ceUs of amphibia in the Arch, de Zool. Exper., 1913. See also Rbddingius, Virchow's Arch., clxii, 1900, p. 206. For nucleoli of ova, List, Mitth. Zool. Stat. Neapel, xii, 1896, p. 480 ; of nerve-cells, Euzicka, Zeit. wiss. Mile, xiv, 1898, p. 453, and Levi, Biv. Pat. Nerv. Ment. Firenze, iii, 1898, p. 289. 670. Plasma Stains. — ^Dr. BoUes Lee states that he has been unable to discover a single thoroughly satisfactory one. Almost all of them colour too readily the enchylema or hyaloplasm at the same time as the spongioplasm. And, on the other hand, there are many important elements of cells which cannot be got to stain sufficiently. We consider Saurefuchsin and Biebrich scarlet the most generally recoimnendable, especially after iron hsematoxylin. See also Bordeaux E. Flemming's orange method has been much used. It is very capricious and unreliable, Bhrlich-Biondi mixture is a celebrated plasma stain, CHAPTER XXVI. 315 The Iron-Hcematein Lakes of Benda and M. Heidenhain give good plasma stains, according to the degree of extraction, and would be inferior to none were it iiot that they stain in the same tone as the chromatin. See also Ehrlich's tri-acid, and his addophilous mixture, also gold chloride, Apathy's process, § 371, and Kernschwarz. Imperfectly stained plasma structures can often be well brought out by mounting in Euparal instead of balsam. 671. Centrosomes. — These can be stained by some " acid " anilin dyes, better by a " neutral " dye {e.g. Flemming's orange method, or the Ehrlich-Biondi-Heidenhain stain). But by far the best stain is iron-hcematoxylin. It is said by Heidenhain that the stain is obtained in a sharper form by combining the hsematoxylin stain with a foregoing stain with Bor- deaux B. He directs (Arch. mik. Anat., xlii, 1894, p. 666) that the sections (sublimate sections were used by him) are to be stained for twenty-four hours or more in " a weak " solution of Bordeaux, until they have attained such an intensity of colour as that " they would just be fit lor microscopic examination with high powers " (I. c, p. 440, note), and that they be then brought into the ferric alum. After mordanting and staining, the haematoxylin is to be extracted in the iron alum until the chromatin has become entirely or almost entirely colourless. Instead of Bordeaux, " anilin blue " may be used in the same way. The images of these objects given by iron-hsematoxyhn require to- be interpreted with special care. Globular or even elongated objects, such as chromosomes, do not always yield up their stain simultaneously and equally throughout their whole depth, but lose it suddenly and entirely in their outer layers, whilst retaining it in its full strength in their deeper layers. It seems that certain erroneous observations that have been published have been due to this deception. 672. Cell Granules. — For the study of the conspicuous " granules," undoubtedly metabolic products, occurring in certain gland-cells and blood- and lymph-corpuscles, and in certain elements belonging to the group of connective tissues, see the sections on " Connective Tissues," " Mitochondria " and " Fat." The most generally employed stains are the mixtures of Bhrlich. Intra-vitam staining is useful here. See § 208. See also Arnold, Anat. Am., xxi, 1902, p. 417. Benda {Verh. phys. Ges. Berlin, 1899—1900, Nr. 1—4, and Verh. Anat. Ges., xv, 1901, p. 172) gives the following method for demon- strating secretion-granules and distinguishing them from other granules : Harden for twenty-four hours in 10 per cent, formalin, 316 CYTOLOGICAL METHODS. then for one day in 0-25 per cent, chromic acid, one in 0-33 per cent, and two to three in 0-5 per cent., wash one day in water, dehydrate and make paraifin sections. Then stain with one of Ehrlich's mixtures, according as the granulations are basophilous, acido- philous, or neutrophilous. The methylen-blue and eosin process of Michaelis is recommended. See also Mallory, §§ 271 and 272 ; Mann, § 328. 673. Mitochondria,* Golgi Apparatus,t Yolk, Fat, and other Cytoplasmic Inclusions, t — The mitochondria and Golgi apparatus never clearly appear in stained sections prepared by such methods as fixation in corrosive acid, Gilson, Bouin, Carnoy or Flemming- with-acetic acid, and staining in Ehrlich's hsematoxylin and eosin, toluidin-blue and eosia, paracarmine and borax carmine. Though the mitochondria and Golgi apparatus are properly fixed by formalin, Miiller, Flemming-without-acetic acid, Champy, Altmann, etc., they will rarely appear visible in stained sections which have been pre- pared in EhrHch's or Delafield's hsematoxylin or carmine stains, or in fact in any of the current laboratory stains used for general zoological purposes. The mitochondria and Golgi apparatus may appear visible in sections fixed in formalin, Miiller, etc., and stained in Altmann's acid, fuchsin-picric acid, iron-heematoxylin, Benda's alizarin and crystal-violet, etc. The Golgi apparatus rarely becomes ■visible after any of the above methods, and to study it one must use more specialised methods ; to study the Golgi apparatus and the mitochondria by routine zoological laboratory technique is not possible, simply because these methods will not demonstrate the bodies in question. Nearly all of the older fixing mixtures contain either alcohol, chloroform, or acetic acid, but the last few years of cytological research have shown that the picture given by a fixing mixture containing them is incorrect and inadequate, and one cannot fail to be surprised at the improvement produced when these reagents are omitted. Nearly all the modern research on the cyto- plasm has to be carried out by observers using chrome- or platinum- osmium fixatives, followed by iron-alum hsematoxylin, Benda's crystal violet, or Altmann's acid fuchsin ; or by the important Kopsch and Mann-Kopsch, and Sjovall osmium tetroxide methods ; or by the useful methods of Cajal, Golgi or Da Fano's modification * Chondriosomes, cliondriokonts, plastoohondria, " ohromidia," bio- blasts, chondriome, chondriomites, etc., etc. t Nebenkern batonettes, idiozome rods, " Golgi-Kopsch apparat," apparato interno reticolare, dictyosomes, Binnennetz, etc. J By J. B. G. CHAPTER XXYI. 317 of Cajal, which consist of silver nitrate impregnation following formalin fixation. Intra-vitam methods, such as janus green, neutral red, or dahlia violet are also used extensively. The mito- chondria are extremely fuchsinophile, and after chrome-osmium fixation stain strongly in iron-alum hsematoxylin. The Golgi apparatus of somatic cells and of ovarian cells rarely stains by these methods (Altmann or Heidenhain) unmodified, although the Golgi apparatus of the male germ cells nearly always stains in fuchsin or hsematoxylin after chrome-osmium or formalin fixation. Besides observing the Golgi apparatus and mitochondria, certain workers have turned their attention to the study of fats and yolk (vitellus) in cells. In § 768 is a special article on fats and lipoids, and on methods for their study ; on the following pages are set forth various techniques for the investigation of defnite cell organs known to be partly lipoid in nature. The application of all these methods to embryological study opens the way to a valuable field for research. In § 768 will be found definitions of the terms " fat," " lipoid," and " lipin." Fats or lipoids form a special part of almost all cell-organs, as seems to be indicated by fixing tests, and so far as we know such substances are always intimately associated with protoplasm. Many of the lipoids appear to be able to form with certain metallic salts or oxides such as CrOa, KaCrgOv, PtCU, OSO4, etc., compounds insoluble or only slowly soluble in alcohol or such clearing oils as xy^pl, benzole, or chloroform ; this is one of the several reactions which take place when a cell is fixed in such a fluid as that of Flemming (without acetic acid), Champy, or Altmann, and subse- quently dehydrated and cleared. See §§ 29, 30 and 31. 674. Choice of Method.* — We have given below a number of methods for lipoid granules, mitochondria, and other cell inclusions, and not all are suitable for every piece of work. It is very rare to find that one single method will produce the same good result in both vertebrate and invertebrate tissues. In the same way, methods which act satisfactorily with amphibia will often give disappointing results with mammaUa. Osmic-chrome fixation will nearly always be found excellent for all classes of invertebrata ; Flemming- without- acetic acid and Champy-Kull can be highly recommended. For amphibia the addition of some K2Cr207 to the Flemming is necessary before a correct fixation of the mitochondria is obtained ; thus * The beginner is recommended to master such techniques as those of Bouin's fluid and borax carmine, or Zenker and Ehrlich's hsematoxylin before trying these methods. 318 CYTOLOGICAL METHODS. Champy's fluid was invented for amphibia and gives very satis- factory results (§ 43). For mammalian tissues a preliminary fixation in osmic acid fixatives is not generally indicated ; the tissues of mammals are far more " fatty " than those of in vertebra ta or amphibia, and one finds that the OsOi becomes reduced very rapidly and penetration is very poor. For mammalian tissues formalin-chrome (Eegaud, Bensley-Cowdry), formalin-corrosive or formalin alone are indicated as a preliminary treatment at the least. Formalin does not destroy lipoids, and by subsequently placing small pieces of chrome-formalin fixed tissues in osmic acid (post-osmica- tion), a fixation of lipoids and fats is obtained (Schridde) ; the same result may be got by fixing tissues in chrome salts and then trans- ferring to osmic acid. It should be noted, however, that previous fixation in a chrome salt prevents the blackening of the Golgi apparatus ; the formalin-osmic acid method (Sjovall) is indicated especially for mammalian tissues, when an impregnation of the Golgi apparatus is required by means of an osmic method, but the formalin silver nitrate methods of Golgi, Cajal and Da Fano are always clearly indicated for work on the Golgi apparatus of mammalian tissues. So far as possible intra vitam and fresh smear preparations should be used, as these nearly always give valuable results. 675. Specificity of Techniques for Cytoplasmic Inclusions, Fats, and Lipoids. — As, a rule the lipoid granules, vacuoles, and cell organs containing fats or Upoids are formed not of one puje substance, but of a mixture of several. Consequently it is necessary to proceed with caution in claiming a specificity for the techniques for various lipoid substances : properly used, however, microchemical methods may give valuable evidence as to the precise chemical nature of any special body : microchemical methods, which depend for their application on the use of complicated fixing and staining methods are to be used cautiously. For example, Benda (§ 683) and Altmann- Bensley methods (§§ 680 and 686), will stain granules other than mitochondria, while the Cajal formalin uranium and silver nitrate technique impregnates bodies apart from the Golgi apparatus. In all these cases, however, the number of such exceptions to a specificity is small, and suitable differentiation between two doubtful bodies can be made by some other method. See Bayliss on " Specificity " of staining methods, § 211. 676. On Killing Animab for Cytological Purposes.— So far as possible avoid narcotics of any sort. Either cut off the heads of invertebrates, or, if delicate like some worms, drop them whole in CHAPTER XXVI. 319 the fixative ; kill vertebrates by a blow on the head, or by pithing. If for a study of brain, bleed, or anaesthetise in coal gas, less pre- ferably chloroform or ether. Insects can be killed with cyanide or xylol. Hints on removing Tissues and Cutting. — ^Avoid pinching the material with forceps, as this will introduce artifacts ; it is preferable to remove tissue without recourse to dissection under tap-water or salt solution ; . for Kopsch techniques, quickly remove blood or lymph, etc., from surface of material with aq. dest. before placing in fixer ; for cutting tissue the best instrument is a new safety razor blade stuck in a special holder made for the purpose, or in a split penholder, or held by artery forceps. When working on arthropods, it is best to dissect the organ from the animal, instead of preserving the whole body ; surrounding fat, etc., should be removed. See also §§ 12 and 653. 677. Chrome-osmium Techniques. — Potassium bichromate or chromium trioxide, used in watery solutions will not oxidise true fats (see § 768) in such a way that full vacuoles of the latter will appear in fiinished sections prepared by routine methods ; but combinations of such salts with osmium tetroxide provide fixatives which will preserve almost all cell elements in finished sections. The basis of all chrome-osmium techniques consists in a preliminary fixation of small pieces of tissue, small embryos or eggs, in such a fluid as Benda, Champy, Flemming-without-acetic acid, or Altmaim, for from at least twelve hours to a week. The osmic reaction is then, in some methods (KuU, Benda), " set " or strengthened by the reducing effect of pyroligneous acid ; following this treatment is a further " chroming " in 3 per cent, bichromate of potash, and, finally, a thorough wash out under the tap. Material treated in this way is generally perfectly preserved, and fit for selective staining. Arranged below are chrome-osmium techniques of progressive intensity and difficulty. 678. Modified " Flemmings " for Cell Inclusions. — Benda ; 15 c.c. chromic acid 1 per cent., 4 c.c. osmic acid 2 per cent., 3 to 6 drops of acetic acid. Meves : 15 c.c. of chromic of 0-5 to 1 per cent., containing 1 per cent, sodium chloride, with 3 to 4 c.c. of 2 per cent, osmic acid, and 3 to 4 drops of acetic {Encycl. mik. Techn., 1910). GrATENBY : Strong Plemming without acetic acid ; and same solution diluted by one-haK or one-third {Qumt. Journ. Micr. Sci.,_ 1919). The presence of a small quantity of acetic acid is always liable to introduce distortion, but less so among vertebrates 320 CYTOLOGICAL METHODS. than among invertebrates. See also " Champy's Fluid " (§ 43) and " Altmann's Fluid " (§ 42), and § 41. 679. Flemming's Strong Fluid without Acetic Acid, and Iron Hsematoxylin. — Small organs freshly dissected out in normal saline, or parts of organs cut with safety razor blade, not more than 5 milli- metres in diameter, are placed in about 15 c.c. of one of the above- mentioned fixing fluids, preferably Gatenby's modification. A glass-covered capsule or vial is the best vessel to use, and the material is left for at least twenty hours, and not longer than one week. We find about twenty-four hours gives a satisfactory fixation of most tissues. After fixation the liquid is poured away, and the material is washed for at least two hours, and not necessarily longer than five, in running tap-water. It is then passed through up-graded alcohols, beginning at 30 per cent., giving the material at least three hours in the strengths 30, 50 and 70 per cent., and overnight in 90 per cent. The pieces of tissue are dehydrated two or three hours in two changes of absolute alcohol, and then transferred to a mixture of half absolute alcohol and half xylol for a quarter of an hour ; then pure xylol, and embedded in wax. Sections are cut from 4 to 8 ju, but I generally find 6 /a to be convenient. Leave eight to ten hours in iron alum, twelve to twenty hours in hsema- toxylin. This method gives a delicate and precise stain of the mitochondria (and Golgi apparatus or nebenkern batonettes of male germ-cells only), fat is black, while yolk is generally greenish brown. Especially recommended for germ-cells, and histology of Inverte- brata, but with vertebrate tissues, and especially mammalian material (not embryos), it often gives atrocious reisults ; for such material, Helly, Zenker, or Eegaud's methods are indicated (Gatenby, Quart. Journ. Micr. Sci., 1919). Note that Meming-without-acetic acid is not a suitable fixative for after-staining in Altmann's acid fuchsin. For this the material must be washed in distilled water for a short time after fixation, and then transferred to 3 per cent, bichromate of potash for three days ; or the more elaborate mordantage as for Champy-KuU may be used (§ 681). 680. Altmann's Acid Fuchsia and Picric Acid {Die Elementaror- ganismen, Leipzig, 1890). — Fix twenty-four hours in mixture of equal parts of 5 per cent, bichromate of potash and 2 per cent, osmic acid. Embed in parafiin, stain sections on slide for one minute over flame, with a solution of 20 grms. (sic) of acid fuchsin in 100 c.c. of aniline oil-water (§ 286). Cool, and wash out in a saturated alcoholic solution of picric acid diluted with 2 volumes of water, CHAPTER XXVI. 321 heat being used as before to aid differentiation ; blot, dip into 90 per cent, or absolute alcohol, xylol, balsam. This method only stains granules which can be seen intra vitam ; properly used it never produces artifacts, and Fischer's critique is quite wrong {Wiximng Faerbung u. Ban des Protoplasmas). Altmann's original method has been superseded more or less by the following method of Champy-Kull. (Both Dr. J. A. Murray and I find that the 20 grms. of acid fuchsin will not dissolve in 100 c.c. of aniline oil- water ; only about 5 to 7 grms. will dissolve, and this quantity will make a perfectly efficient solution.) 681. Champy-Kull's Acid Fuchsin, Toluidin Blue and Aurantia (KuLL, Anat. Anz., Bd. xlv, 1913).— The following method, while being generally useful, will be found very convenient for work on Invertebrata. It gives results intermediate between those of Benda and Altmann, but is shorter and undoubtedly better than the method of Benda. It will be found very useful for embryological research, and probably also for protozoology. Fix in Champy (§ 43) (we find Flemming-without-acetic acid will do, too) for twenty- four hours. Pieces to be fixed must be small. After fixation wash half an hour in aq. dest., and then transfer to a mixture of 1 part acid acet. pyrolignosum rect., and 2 parts 1 per cent, chromic acid, for twenty hours. Wash half an hour in aq. dest., and transfer to a 3 per cent, solution of potassium bichromate for three days. Wash imder tap for twenty-four hours ; pass through up-graded alcohols to xylol ; embed in paraffin wax (or celloidin method, if desired). Section 4 or 5 /^. Proceed as follows : — (1) Stain in Altmann's acid fuchsin aniline oil mixture (5 to 10 grms. of acid fuchsin in 100 c.c. of aniline oil-water), and heat till steaming. (2) Set slide aside to cool for six minutes (this is important), pour off, and wash quickly in aq. dest. (3) Counter-stain in either a 0-5 per cent, solution of toluidin blue or a saturated solution of thionin in aq. dest. for one to two minutes. Wash in aq. dest. In some cases the time in the blue stain must be shortened. Transfer to a 0-5 per cent, solution of aurantia in 70 per cent, alcohol for from twenty to forty seconds, watching extraction of fuchsin stain under microscope. Differentiate the blue stain in 96 per cent, alcohol, then absolute, xylol, and balsam. The chromatin is generally blue, mitochondria (and occasionally Golgi apparatus) are red, and the ground cytoplasm is golden-yellowish to green. This modification of Altmann's method is a most brilliant three-colour stain which is highly recommended. We have found that it is useful for histo- logical as well as cytological purposes ; sections of Annelids, or of M. 21 322 CYTOLOGICAL METHODS. flat-worms, for instance, prepared by Champy-Kull show beautiful colour graduations in their different tissues. The preparations begin to fade after a year. After Champy-Kull fixation you can : (a) stain in iron hsema- toxylin (long method, § 242), (6) stain as for Benda (§ 683), (c) mount unstained for examination of osmicated granulations, (d) stain in safranin and light green (§ 286). For a chart illustrating Champy- KuJl technique, see below. Maximow (0. B. Soo. Biol., Paris, Ixxix, p. 462) fixes in Champy, washes slightly in water, transfers to mixture of 1 per cent, chromic acid 1 part, glacial acetic 2 parts, for twenty-four hours. Wash again for half-an-hour, place for three days in 3 per cent. £201^07. Wash in running water. Stain sections as above. 683. Champy-Kull Fixation. Subsequent method. Mito- chondria, Momit unstained Yellowish. Champy- EuU stain . Bed or pink. Iron lumiatoxylin . Black. Benda stain (alizarin and crystal violet) Violet. ' Fat. Black (extract- able in tur- pentine). Black. Black, pro- vided it has not been ex- tracted in turpentine. Same as above. Yolk. Yellow to black. Yellow to black. Same as above. Same as above. Golgi apparatus. Yellowish or does not show. Generally will not show in somatic cells or ovaries, red in male germ cells. Same as above, but black in male germ cells. Same as above, but violet in male germ cells. Nucleus. Yellowish. Chromatin blue to greenish (nucleolus red). Chromatin grey to black (nucleolus black). Chromatin brown or yellowish (nucleolus violet). 683. Benda's Alizarin Method {Ergebnisse der Anat., xii, 1902 (1903), p. 752, and other places) is as follows : — Harden for eight days in strong liquid of Flemming, the acetic acid therein being reduced to 3 drops (or as for Champy-Kull or Eegaud). Wash for an hour in water and put for twenty-four hours into a mixture of equal parts of pjoroligneous acid and 1 per cent, chromic acid, then for twenty-four hours into bichromate of potash of 2 per cent., wash for twenty-four hours and embed in parafiin. Sections on the slide are mordanted for twenty-four hours with 4 per cent, solution of ferric alum or diluted liq.ferri sulfur, oxydat., then rinsed with water and put for twenty-four hours into an amber-yellow aqueous solution of Kahlbaum's sulfalizarinate of soda, prepared by dropping 1 c.c. of saturated alcohoUo solution thereof into 80 to 100 c.c. of water. Einse in water, flood the slides with the solution of crystal CHAPTER XXVI. 323 violet (§ 330) diluted with an equal volume of water, and warm till vapour is given of£. Rinse, differentiate one or two minutes in 30 per cent, acetic acid (till the nuclei come out reddish), wash in running water for five to ten minutes, dry with blotting-paper, dip into absolute alcohol, pass through bergamot oil into xylol and balsam. Mitochondria violet, chromatin and " archoplasm " brown- red, certain secretion granules pale violet, centrosomes red violet. Instead of the staining solution prescribed above (which may be kept in stock) you may take {Encycl., ii, p. 198) a, freshly prepared mixture of equal parts of anilin water and saturated alcoholic solution of crystal violet — and this is to be preferred. Some workers prefer to harden, as Benda, but to stain with iron haematoxylin instead of by the alizarin process ; the special hardening rendering the hsematoxyhn stain sufficiently specific. Arnold {Arch. Zellf., viii, 1912, p. 256) stains first with iron hsema- toxylin, differentiates, stains for twenty to thirty minutes with saturated aqueous solution of thionin, passes up to absolute alcohol, stains for two minutes with Orange G dissolved in clove oil, and passes through xylol into balsam. Chromatin blue, chondriosomes black. 684. Formalin-Chrome Techniques.* — The methods of Regaud, Bensley-Cowdry, Schridde, Murray, etc., are of importance on account of their suitability for vertebrate, and especially mammalian tissues. The tissues are fixed either in neutral formalin or in formalin-chrome mixtures, washed, and then mordanted in 3 per cent. K2Cr207. As with Champy-KuU, it is possible to stain after such fixation by a variety of methods : — iron hsematoxylin, acid fuchsin, alizarin and crystal violet, safranin, etc. The Regaud and Bensley-Cowdry methods do not preserve ^ijeutral fat in the finished sections, but by post-osmication, as for Schridde (§ 687), or Murray (§ 689), this can be done. 685. Rbgaud's Formol- Bichromate and Iron Hsematoxylin {Arch. d'Anat. micr., t. xi, 1910). — Fix in a mixture of 3 per cent, potassium bichromate 80 volumes, commercial formalin 20 volumes, for four days, changing every day. Mordant in potassium bichromate for seven days, changing every second day. Wash in running water twenty-four hours, dehydrate (twenty-four houi's each strength), * Note that formalin-chrome mixtures consist of a reducer and an oxidiser, and will not keep. Such solutions should always be made up just before use. Zenker's fluid, too, keeps better without the acetic acid, which, if being used, should be added just before the material is put in the fixative. 21—2 324 CYTOLOGIC AL METHODS. clear and embed in parafi&n. Pass sections on slide down to water ; 5 per cent, iron alum at 35° C. for twenty-four hours ; rinse in aq. dest., not tap-water. Stain twenty-four hours in this solution : — 1 grm. of pure crystals of hsematoxylin in 10 c.c. of absolute alcohol, added to 10 c.c. of glycerine and 80 c.c. of aq. dest. Differentiate in 5 per cent, iron alum, watching process under microscope. The main point is to avoid washing out the mordant too much when the slides are being transferred from the iron alum to the haematoxylin. Permanent stain, very good for vertebrate tissues . See also Co wdry, ihner. Journ. Anat., xix, p. 441. I find ordinary iron hsematoxylin is quite good after Regaud fixation. 686. Bensley-Cowdry Acid Fuchsin and Methyl Green Stain (CowDEY, Contrib. Carnegie Inst. Wash., viii, 1918). — Fix as for Regaud, either by immersion or injection ; formalin should be neutrahsed in magnesium carbonate, and, if possible, the fixation should be done in an ice-box, but this- is not necessary. Pass sections down to aq. dest. through toluol (or xylol), absolute alcohol, etc., thirty seconds in each ; transfer to 1 per cent, potassium per- manganate for thirty seconds, but time must be determined experi- mentally ; then 5 per cent, oxalic acid for thirty seconds. (Note. — The permanganate and oxalic acid may generally be omitted.) Then rinse in several changes of aq. dest. for about one minute (incomplete washing prevents staining in acid fuchsin). Stain in Altmann's fuchsin (§ 680) as follows : dry around sections with duster, add stain, warm over spirit lamp until fumes come ofi ; cool for six minutes ; wipe around sections with duster, rinse off in aq. dest., so that the only remaining stain is in the sections (or a precipitate forms with the methyl green) ; pipette a little 1 per cent, methyl green over the sections for about five seconds at first, modify time as experimentally found convenient ; drain off excess, plunge into 95 per cent, alcohol for a second or two. Rinse in absolute alcohol, clear, in toluol, mount in balsam. Difficulties are that the methyl green may remove the fuchsin (due to incomplete chrome mordanting during fixation), or the fuchsin may have over- stained (due to too much mordanting). Sometimes, if the methyl green is too weak, it is better to omit the 95 per cent, alcohol, dehydrating in absolute. The difficulties of this modification of Altmann's stain are easily overcome ; I have used it for a senior histology class, and with success. Like the Champy-Kull method, this stain is not so permanent as Iron Hsematoxylin. See also Bbnsley, Amer. Journ. Anat., xii, p. 308 ; Duesberg, ibid., xxi, p. 469. CHAPTER XXVI. 325 687. Schridde'm Method for Mitochondria, modified (Ergeb. Anat. u. E. Merk. Bonnet, xx, 1911). — Fix in this mixture : formol (1 part), Miiller (9 parts), for two days ; then place in Miiller, two to four days ; then 2 per cent. OSO4, for two days. Wash overnight, dehydrate, clear in xylol, cut parafiin sections 5 jji. Stain as follows ; iron alum hot for a quarter of an hour, then hsematoxylin hot, a quarter of an hour. Differentiate in alum in the cold. This has the advantage over pure formol-chrome techniques in that the introduction of the OsOi preserves- fat ; recommended by Duesberg. With this mordanting it should be possible to stain either as for Altmann, Bensley-Cowdry, Champy-KuU, or Benda. Levi, G. {Arch. f. Zellf., Bd. xi), ovary of mammals. 10 c.e. . 2-5 per cent. KjCraO,. 10 „ . 5 per cent, sublimate containing 2 c.c. of formol. 2 ,, . 2 per cent. OsOi. Leave for three or four days. Wash out well in running water. Stain in Kegaud, Benda. etc. 688. A. H. Drew's Formol-Chrome-Hsematoxylin Method {Journ. R. Micr. Soc, 1920). — This method is used for demonstrating rod- like bodies in the cytoplasm of plant cells. These rods are supposed to be the homologue of the Golgi apparatus of animal cells. The method will undoubtedly be useful for studying animal tissues. Fix plant root tips, etc., for twenty-four hours in a mixture of formol, 20 c.c. ; cobalt nitrate, 2 grms. ; sodium chloride, 0-8 grm. ; water to 100 c.c. (preferably at temperature of 37° C). Soak fixed tissues in gum-syrup for at least an hour, and cut sections on freezing microtome. Wash in water, and fix on gelatin-coated slides with formalin. See §§ 172 and 182. Rinse in water to remove excess formalin, mordant at 50° to 55° C. in chromic acid 4 per cent., osmic acid 2 per cent., equal parts, on slide for varying periods — fifteen minutes to one hour, or longer. Einse in water and stain with iron alum 3 per cent, for fifteen minutes, followed by ^ per cent, hsematoxylin for fifteen minutes, at 50° C. Differentiate in the cold in iron alum till the nuclei show pale brown. Transfer to 2 per cent, pyridin for two minutes, dehydrate^ and mount in xylol- balsam. In speciriiens chromed for short periods the mitochondria alone are visible, while in those chromed for a longer time the mitochondria stain less well, while gradually the long Golgi elements appear in the best chromed cells. In animal cells, too. Drew finds short chroming shows the mitochondria, while it requires longer treatment in the chrome to demonstrate the Golgi apparatus. This is my own experience with the Golgi elements or " nebenkern batonettes " of MoUusoa. 326 CYTOLOGICAL METHODS. 689. J. A. Murray's Chrome-Osmic Method for Mitochondria and Bacteria of MammaUan Tissue. — Fix tissue in formol-salt or f ormol- Miiller overnight. Thin shoes are then placed in Miiller's fluid for from two to seven days, and then transferred to 2 per cent. OsO^ for two days more. Wash overnight in running water, dehydrate, embed in paraffin. Sections to be not more than 5 /j. thick, fixed on slide, and stained in 3^ per cent, iron alum at 50° C. for fifteen minutes, followed by |- per cent, aqueous hsematoxylin in same way and for same time. Sections should now be jet black. If such sections be decolourised in the ordinary way in iron alum, both mitochondria and bacteria (if present) will retain the stain, and nuclei are decolourised. If such sections are decolourised in 0-5 per cent. HCI in 70 per cent, alcohol, the mitochondria give up the lake and the bacteria remain deeply stained. At the same time the details of the nuclei are sharply stained. Wash sections for twenty minutes in tap-water, counterstain in Van Gieson, mount in balsam (Report Imp. Cancer Research Bureau, 1919). 690. Double-Staining in Haematoxylin and Acid Fuchsin.— It is well known that diflerent cell elements have varying powers of resisting deoolourisation or diSerentiation after iron alum or such, hematoxylin, stains. Thus in a hermaphrodite gonad or during fertilisation it is sometimes noticed that the mitochondria of the egg hold the hsematin lake much faster than those of the spsrm or spermatogenesis stages. It is possible in certain cases to make use of this fact for studying differen- tially cell granules, etc. Fix tissue by some prolonged mordanting method, such as that of Champy-KuU, or Eegaud. Wash out well in running water and prepare thin paraffin sections. Stain by some intense haematoxylin method, such as that of Benda or Heidenhain ; differentiate the cell element which you wish to be stained subsequently a red colour, till it looks pale greyish under the microscope : wash well in water, and counter- stain in Altmann's acid fuchsin. Extract the fuchsin to the right stage in 95 p?r cent, alcohol, quickly dehydrate and clear in xylol ; mount in balsam. If necessary, after staining in acid fuchsin, you may apply the picric acid of Altmann's method (§ 680), but this necessitates under- diflerentiation in the iron alum. I have found that after staining in the acid fuchsin you may differen- tiate partly in aurantia as for the KuU method (§ 681). A method to be tried only by experienced cytologists. The difficulty is to differentiate the hsematoxylin just to the right stage, and to avoid washing away the acid fuchsin (GtAtenbt, Journ. Boy. Mier. Soc, 1919 ; Hans Held, Arch. f. mikr. Anat., Bd. Ixxxix). 691. On Post-Chroming and Post-Osmication in General.— By soakiiig tissues in KgCrgO,, with or without CrOj, one produces CHAPTER XXVI. 327 stainable compounds of cell proteins and lipoids wMch are not easily dissolved out by alcoliol and a clearing oil. One may fix in almost any mixture not containing alcohol, chloroform and acetic acid. Wash the tissue in aq. des^. for a short time (say half an hour or less), cut into small pieces, and transfer to 3 per cent, potassium bichromate for several days, and then to 1 or 2 per cent. OsO^ for a day. Wash under tap overnight and stain in Heidenhain or an Altmann. Thus tissues or embryos fixed in special formol-chrome, corrosive formol, chrome-corrosive, and other mixtures which one has found most suitable for one's purpose, may be post-chromed, or post-osmicated as well. Schridde's and Murray's methods (above) include both post-chroming and post-osmication. 692. Kopsch's Osmium Tetroxide Method {Sitzungberg. d. h. preuss. Alcad. d. Wiss. zu Berlin). — Osmium tetroxide solution will fix both fats and lipoids, and proteid substances. As has been mentioned above, the various cell inclusions, such as mitochondria, Golgi apparatus, yolk and fat, are nearly always mixtures of different quantities of several definite substances, and consequently will reduce the osmic solution in varying degrees of intensity. Kopsch methods are somewhat capricious, but one gets results unequalled by other methods ; for chrome-osmium, or chrome-formol, followed by iron hsematoxylin, or Altmann, generally will not demonstrate the Golgi apparatus (except in male germ-cells), while the Kopsch methods preserve and demonstrate Golgi apparatus, mitochondria, yolk, fat and chromatin structures, and occasionally neurofibrils of embryos. For this method dissect out organs, and cut tissue into small pieces ; dip these quickly into aq. dest. to remove blood or cell detritus from surface, and then transfer to a small glass-stoppered or glass-covered capsule of 2 per cent. OSO4. Leave in a darkened cupboard for tviro weeks (fourteen days) at room tempera- ture. Wash in running water for several hours, dehydrate, embed in hard wax ; section about 3 //. Mount unstained, or stain chromatin in safranin or methyl-blue eosin. Unsaturated fats black, others yellowish, Golgi apparatus, and sometimes mitochondria, black. This method succeeds for mollusc and many invertebrate and vertebrate tissues, but the following Mann-Kopsch method is generally superior. Note that a trace of chromic add, potassium bichromate, or platinum chloride, in the OsO^ solution will inhibit the blackening of the Oolgi apparatus, but not of fat. See also §776. 328 CYTOLOGIGAL METHODS. 693. The Mann-Kopsch Method (Wbigl, Bull. Acad. Scien. Cracovie, 1912 ; Hieschler, Arch. mikr. Anat. 89 ; G-atenby, Joum. R. Micr. Soc, 1919). For a study of cell structure, and in general cytology, the Mann-Kopsch method gives invaluable results. It is an alternative to the formalin-silver nitrate tech- niques of Grolgi, Cajal or Da Fano, but in addition preserves fatty substances. The Mann-Kopsch technique in itself is easy to work, but the subsequent steps in staining are often extremely difficult. The ordinary Kopsch technique may cause extreme shrinkage, and is not generally so specific. .First fix in Mann's osmo-sublimate fluid (§ 71) for from one quatter-hour to two or three hours or more. Pieces to be fixed must be small (not exceeding a centimetre in diameter) and should only be left in the osmo-sublimate long enough to complete the penetration of the fluid. For an insect ovary, or small invertebrate, one half -hour is sufiicient ; for solid tissues like nerve, longer is necessary. These times must be ascer- tained experimentally. After fixation the pieces are washed in two changes of distilled water for half an hour or less, according to the size of the tissue and its accessibility to the water. The pieces are transferred to a glass-stoppered bottle containing just enough 2 per cent. OsO^ in aq. dest. to cover them. Then they are left in a cupboard at room temperature, for at least ten days, and preferably two weeks. Every few- days the bottle should be examined to see whether the OsO^ is evaporating, or whether it has completely disintegrated. Should either have happened the pieces should be washed quickly in aq. dest., and new OSO4 solution added. It should be noted, however, that the osmic solution nearly always becomes slightly dark, but not until it has gone black or no longer smells of OSO4 should new liquid be added. When the right period has elapsed the objects are taken out of the osmic, and preferably washed for several hours in running water before transference to 50 per cent, alcohol. They are upgraded and embedded in hard paraffin. Sections to be cut from 3 to 6 jn. They are stuck on the shde with albumen and water in the usual way and dried over- night. One of the slides is taken, the wax removed in xylol, and it is moxmted in xyol balsam. Examination of this slide will enable one to ascertain to what extent the process has acted successfully. In completely successful preparations the Golgi apparatus, yolk and fat alone are blackened, while nuclear organs, mitochondria and cytoplasm are stained in shades of yellow and greenish brown. Having studied this untreated slide, and noted the extent of the CHAPTER XXVI. 329 blackening effect of the OSO4, one may then proceed to make experiments., Several alternative methods may be tried : — (a) The blackening may be extracted step by step in turpentine, and the appearance of tte cell granules studied at intervals. (b) If the mitochondria are not stained black by the OsOj, one may proceed directly to Altmann's method (but preferably after cautious treatment in -125 per cent, permanganate of potash). (0) The nuclear structures may be stained in safranin, crystal violet, or acid fuchsin. The sections are brought down to distilled water and transferred to watery solutions of the dye. A few minutes generally suffice to stain the nuclei. We find that in successful Mann-Kopsch preparations especially of Invertebrata, the mitochondria do not generally become black, but are either unstained or go yellowish. In many, but not aU, cases it wiU be found that where the mitochondria do become black after OsOj, the colour is more readily extracted from them than from the Golgi elements, so that a distinction can nearly always be made by the Mann-Kopsch method itself, without recourse to other methods which wiU generally stain mitochondria and not Golgi apparatus (Regaud, Flemming, as described in § 679). Among the most useful differentiation or extraction methods after Mann-Kopsch, turpentine is probably the best. The wax is removed from the sections on the slide by means of xylol, and the slide is transferred to a jar of turpentine. After about half a minute the section is examined under a |th.inch objective, and the effect of the tur- pentine is noted; one sometimes finds that. the black colour in fat globules and yolk spheres is extracted before a quarter of an hour has elapsed, while the Golgi apparatus retains its black condition. In most cases it is therefore possible to distinguish between yolk and fat on the one hand, and the Golgi apparatus on the other. 694. Mann-Kopsch-Altmann Combination (Gatenby, Journ. Roy. Micr. Soc, 1921). — If examination of the first Mann-Kopsch section showed that the Golgi apparatus was blackened, and the mito- chondria were either not stained or only straw or light-brown coloured, one may proceed directly to the Altmann stain. Should the examination show that the mitochondria as well as the Golgi apparatus have become blackened, the sections must be extracted in turpentine in an endeavour to remove the blackening from the mitochondria. If the latter treatment does not succeed properly the only course is to make new Mann-Kopsch preparations, allowing less time in the OSO4, say seven or eight days instead of the two weeks. When one has succeeded in procuring sections in which the Golgi apparatus alone is blackened, it is possible to stain in Altmann's aniline acid fuchsin and picric acid method, so that the mitochon- dria (and nucleoli) become red, the Golgi apparatus is black and the ground cytoplasm yellowish. The Mann-Kopsch sections are 330 CYTOLOGICAL METHODS. brought down to distilled water, and cautiously treated in a '5 to -125 per cent, solution of potassium permanganate, in order to recover the staining properties of the tissue. A very short time suffices. Wash in water ; then stain (§ 680). An important part of the technique is to ascertain the optimum length of time to leave the tissue in the Mann's fluid. Examples are as follows : — (1) Saccocirrus (entire) overnight in Mann. (2) Cavia testis about five hours. (3) Chick embryos about one quarter-hour. The Mann-Kopsch technique can be used in combination with the Kull staining method (§ 681). We find that the cells are rather liable to overstain in the toluidin blue, which must be left on for a very short time. Explanation. — ^Mann's osmo-sublimate fixes the cells successfully, because the HgCU aids penetration and the OsO^ is not so strong as to cause shrinkage. Thus, before the tissue is transferred to the Kopsch iluid (OSO4 of 2 per cent.), a complete fixation has taken place and the distorting effect of the strong OsO^ is avoided. Left for two weeks, the lipoid materials which partly form the substance of the Golgi apparatus, the unsaturated fats, and the special lipoids of the mitochondria, are all able to reduce the OsO^ in varying degrees. The subsequent treatment of the sections by turpentine (oxidiser) introduces a further differentia- tion, and so the various inclusions can be distinguished. The acid fuchsin stains presum ably the lipoid content of the mitochondria. See also § 768 . 695. Osmic Vapour Method (W. Ceamer, Imp. Cancer Resemch Fund Report, 1919). — Choose a small glass-stoppered bottle, and place a piece of wide glass tube open at both eiids, in the bottom. Arrange a piece of gauze over the top of the inner tube. Add some 2 per cent. OsO^ to the outer vessel. Objects to be fixed by the osmic vapour are placed on the gauze. All the surrounding (fatty) tissue should be removed from the organ or material to be treated ; if too dry the outside of the material should be slightly wetted. The bottle, with object suspended in the OSO4 vapour, is kept at temperature of 40° C. for one and a half hours. Removed from bottle, the tissue is placed in 50 per cent, alcohol and upgraded and embedded in paraffin. Cut sections, mount in balsam without staining. Such wax sections may be treated in 10 per cent, hydrogen peroxide in 80 per cent, alcohol for two hours, after which they may be stained in ordinary methods {e.g. iron haematoxylin). GrATENBT (Qworf. Joum. Micr. ScL, 1920) suggests two modifications. [a) Fix as above for one and a half hours, and then transfer to 2 per CHAPTER XXYI. 331 cent. OSO4 ill water at 37° C. for several days as for Kopscli. Then wash in water for several hours, dehydrate, embed and section. Mount unstained, or cautiously treat in permanganate of potash or hydrogen peroxide and then stain in acid fuchsin (Altmann) or iron hsematoxylin. (6) Tissues may also be fixed as above, and then transferred to Alt- mann's or Champy's fluid, and subsequently stained in Altmann's fuchsin and picric acid. Cramer fixes wet films for about three minutes. We think that a subsequent treatment of films as in above two paragraphs should be useful. The main point to note is that substances in a tissue which might be dissolved out or altered by the water added to the OsOj crystals are fixed in situ, and without the danger of alteration. This method should be of value to histologists and oytologists. 696. Sjovall's Formol Osmic Acid Method {Anat. Hefte., Bd. xxx, 1906). Material fixed in formalin, but without chrome salts or platinum chloride, may ba used for Sjovall's technique {Anat. Hefte., Bd. xxx). Fix pieces of tissue or small embryos in neutral formalin (5 to 20 per cent, neutralised with magnesium carbonate) for two days. Cut into smaller pieces and wash in several changes of aq. dest. Transfer to 2 per cent. O8O4 solution for from two to fourteen days at room temperature, as for Kopsch. Wash well in water, dehydrate clear and embed. Cut sections 3 jx, it necessary decolourise in peroxide (§ 695) and mount unstained in balsam. This technique is capricious, much more so than Kopsch, and depends firstly on a suitable fixation in formalin, and secondly on the right time in OsOj : it is convenient to cut the tissue into several pieces, which are removed from the OsOj at different intervals. At times Sjovall demon- strates mitochondria and not Golgi elements, and in aU probability it is not of such value as Mann-Kopsch. We consider that the method is of use to the skilled cyiologist, although it may not be applicable for general purposes. Note. — Corrosive-formalin (§ 112) and osmicated picric (§ 100) may also be used as preliminary fixers before a Kopsch or Sjovall osmica- tion. We cannot recommend the latter, as the picric acid probably introduces maceration ; the former is good. 697. Champy'.'? Iodide of Osmium Method [Journ. de I'Anat. et Phys., xlix, 1913). — Champy finds that osmium iodide prepared as below has the power of blackening fat and certain other cytoplasmic inclusions whose identity seems doubtful, but which he calls for the time being catalyosomes or lyosomes. Just before use prepare the following solution : — Osmic acid 2 per cent. ... .1 part. Iodide of sodium 3 per cent. . . . 1 ,, It produces a golden yellow colour. Place relatively large pieces of tissue (5 to 6 millimetres) in the solution. If one takes very small pieces as for the Kopsch techniques, one gets nothing but the osmic reaction while the iodine reaction, which takes place deeper in, is masked. Leave tissue in for at least twenty-four hours, and use a good deal of the liquid 332 CYTOLOGIOAL METHODS. for a few pieces of tissue. Upgrade in alcohol, pass through toluol, embed in wax. The bodies reducing the osmium iodide go black, on a pale grey background. One may subsequently stain in Altmann (mito- chondria red, lyosomes black) or in iron hsematoxylin. It is possible to distinguish between lyosomes and fat, by fixing some of the same tissue in Flemming and comparing the two preparations ; or by comparing the central part of the osmium iodide preparation with the periphery where the fixation is due to the OsO^ exclusively. I have tried this method, at present with disappointing results. 698. Marine or Fresh Water Organisms and Techniques for the Cell Inclusions. — If you are going to use a formalin-chrome technique, kill the animals by adding neutral formalin to the water ; if an osmic technique is to follow, kill with OsO^ ; wash slightly in aq. dest. in both cases and then transfer to the special fixative. See also under " Protozoa " and " Plankton." 699. The Centrifuge and Folariscope Microscope In Oogenesis Studies, etc.- — In examining large differentiated cells, such as ovarian or nerve, most valuable help can be obtained by use of a powerful centrifuge. Tissues or small gonads or whole invertebrates are placed in a tube and centrifuged at high speeds {circa 3,000 revolutions) for from a quarter of an hour to one hour. The centrifuged tissues or animals are imme- diately divided out among several capsules and fixed by several methods which have previously been found to show the various cytoplasmic inclusions : the specifically stained layers can then be examined. We are of the opinion that no study of the inclusions during cell differentia- tion is complete without recourse to this method. See also Gatenbt, Quart. Journ. Micr. Sci., 1920 ; Lillie, Biol. Bull, 1908—9. FADEi-FKEMiET (0. B. 8oe. Biol., Paris, Ixxxiii) attaches a small platform to the centrifuge, so that preparations on the slide, under covershp, can bo centrifuged and examined from time to time. The pola/riscope microscope has proved very useful, not only for studying the musculature of small animals, but also for discriminating between various fatty and lipoid materials. In working on accumu- lations of masses of metaplastic substances in embryos, or in eggs and other differentiated cells, polarised light is often most helpful. The .subject is dealt with in § 768 by Cramer. 700. Vital Staining of the Mitochondria. — There is probably no specific intra vitam stain for the mitochondria alone, most of the so-called specific stains will tinge other bodies. E. V. Cowdry has summarised the various methods used for this purpose [Contrib. to Embryol. Carney. Inst., Washington, viii, 1918). Four stains (Janus green B^ Janus blue, Janus black I, and diethylsafranin) will give an inten"sely positive reaction on the mitochondria of freshly drawn human lymphocytes. All these are Hoechst proprietary preparations ; I find the Janus green of CHAPTER XXVI. 333 Griibler is much less satisfactory. Nile blue B, Janus green G, methylen blue med., pyronin, Bismarck brown, and methyl violet 5 B, will all tinge the mitochondria, but faintly. Cowdry claims that in dilution 1 : 500,000 Janus green is specific, and will not stain graQules other than mitochondria : it seems certain at any rate that exceptions to its specificity for mitochondria alone are rare. Cowdry {loc. cit.), in his admirable discussion on the Jamis colours (pp. 86 — 93), states that the specificity of these dyes is due to a diethylsafranin group. Janus black consists partly of Janus green and another substance ; so also Janus blue and grey. See also " Bayliss," § 207. For a treatment of the Benzidine dyes, see § 790. 701. Mitochondria of Blood and Cell Smears (Cowdry, Internal. Monat. f. Anat. u. Phys., Bd. xxxi). — Janus green B, 1 : 10,000 in 0-85 per cent. NaCl. Place a drop on a series of six or more slides : add a small amount of freshly drawn blood (etc.), and then apply coverslip. Do not attempt to mix the cells and the Janus green. The mitochondria stain rapidly, and the preparation, after ringing with vaseline, will last hours. 702. Injection of Janus Green B (Bensley, Amer. Journ. Anat., xiii, 1911). — Kill the animal (we believe coal gas seems preferable for this), inject 1 : 10,000 Janus green in salt solution, through left ventricle or aorta by gravity pressure ; in order to obtain a good penetration clamp up the return flow momentarily. After ten minutes' perfusion small pieces of the gland (pancreas in this case) may be removed and examined microscopically for mitochon- dria. When the desired depth of staining has been reached the entire gland is placed in salt solution pending further study. 703. Neutral Red. — Use as above for Janus green (§ 700) in 1 : 2,000 to 1 : 10,000 solution (see §§ 308, 790, and 804). 704. Toxic and other Examining Media for Mitochondria of Blood, Protozoa, and Fresh Cells. — The mitochondria can be examined successfully in toxic solutions such as weak OSO4, with or without an added dye. Meves' Victoria Green. — Add a "small" quantity of Victoria green (malachite green) to a 4 per cent, iodic acid solution. Place a drop on a slide with the cells to be examined (Anat. Anz., Bd. xxvi). Osmicated solution of Ripart and Petit, cf . § 90, recommended to leave out the acetic acid of the original formula. Dahlia saturated solution in 0-75 per cent. NaCl. — Stains mito- 334 CYTOLOGICAL METHODS. chondria and Golgi apparatus (nebenkern) of male cells distinctly violet. Both methyl violet 5 B (Griibler) and methyl blue in aqueous solutions will stain the mitochondria. Bismarck brown gives a less clear picture. See under these dyes in Part I. 705. Cajal, Golgi, and Da Fano Silver Nitrate Methods for the Golgi Apparatus. — For vertebrate tissue Cajal or Golgi, and for invertebrate tissue Cajal and Da Fano methods are a great aid to research on the cell. Every cytologist is recommended to master either Cajal's or Da Fano's method. These methods will be found in the neurology section (§§ 844, 849). 706. Differentiation between Cell Inclusions. — It is frequently somewhat difficult to distinguish between the various categories of cell inclusions. In this section we have provided a series of tables intended to act as a tentative guide to the interpretation of the various images got by representative cytological techniques. These tables are based on work carried out on animals of most orders, but it would he injudicious for the researcher to depend upon them implicitly, because many exceptions are met with, and the personal factor is to be taken into consideration. The use' of such tables, if made with several methods and in conjunction with a careful study of the origin and morphology of any doubtful cell body, will, however, provide reliable evidence for identification. Another warning must be given — ^never try to ascertain the nature of granules in developing eggs without first studying the oogenesis of the animal in question. Eggs after spawning or laying are difficult objects to study by these methods, and even the most experienced worker is unable to give a valuable interpretation until he has worked at the oogenesis. It should also be remembered that there are periods in the development of the cell during which the mito- chondria are often able to resist becoming dissolved in lipoid solvents, these periods are in the early spermatogonium in some animals, and during the last stages of spermatogenesis (spermateleosis) in all animals, and sometimes in large oocytes. See also the work of Eegaud, Arch. d'Anat. micr., xi. Nota Bene. — With regard to the oil used for clearing and im- bedding, it should be pointed out that all these tables are based on preparations cleared and imbedded in xylol, which occasionally tends to extract lightly osmicated fat. Vegetable oils like cedar wood oil seem to be less active in this way. I have not found that chrome-osmicated fat, or " Kopsched " fat, is extracted either by xylol or xylol-balsam. See also § 768. CHAPTER XXYI. 335 707. Differentiation between Mitochondria and Fat (Olein, Stearin and Palmitin Mixtures especially). Tresh tissue stained Fixed in Hem- Kopsch or Mann- Regaud or for- Fresh tissue in Herxheiraer's scarlet ming with Kojisch. malin fixation, stained in Mellwi J red or such alcoholic acetic acid iron hsema-' Janus green employed. fluids. examined in unstained sec- toxylin. 1 : 10,000. tions on slide. Mitochon- Will not stain bright- Do not show, Yellow or black : if Black. Green. dria. ly, generally dis- generally dis- black, colour often solved away. solved away, except in cer- tain cases where the mitochondria are more re- sistant to acetic acid. difficult to extract in turpentine ; if yellow, can be stained in acid f uch- sin of Altmann. Fat (see § Stains brightly. (It Black (see § Black ; colour easily Wot stained, as Does not 768). should be noted here 768). extracted after a it has been stain. that while Herx- few hours in turpen- dissolved out heimer's scarlet red tine. by the clear- will not stain mito- ing reagent chondria it may pos- (xylol or sibly stain lipoids chloroform. other than true fat.) not vegetable oils). 708. Differentiation between Golgi Apparatus and Mitochondria."' 1 2. 3. 4. 6. Tormol- Kopsch, Flemming-without- Janus _ Method silver Mann- acetic, Regaud, green smployed. nitrate Kopsoh Champy, formalin. 1 : 10,000. methods of (osmium etc., followed by iron Golgi, tetroxide alum hsematoxylin. Oajal, Da methods). Fano. Oolqi appara- Black Black. Does not show except Rarely In addition to these ius (dictyo- (even when in odgonia or very stains methods a study of the aomes, ne- untoned). young oocytes, and (e X cept morphology and origin of b enk e rn in male germ cells ; in male bodies found in differen- batonettes, rarely in other cells. germ tiated cells will aid in idiozome When stained gene- cells). settling the nature of rods, etc.). rally less intense than the mitochon- dria. granules or rods in ques- tion. See CowDRT (Jn- tcrnat. Monat., Bd. xxix); Gatenby (Journ. Roy. Mitochondria. Golden Not stained, Stain black, or dark Green. Micr. Soc, 1919, p. 93) ; (untoned) y e llowish. grey. and HiKSOHLEK lArch. greyish or more f. mikr. Anat., Ixxxix). (toned). rarely more black. rarely black. 709. Mitochondria, Golgi Apparatus, Chromatin, True Chromidia, and Nucleoli. — In § 710 is a table giving the main fixing and staining reactions of these bodies. It will be found to hold for a discrimina- tion between chromatin of the nucleus and mitochondria and Golgi apparatus, especially in somatic cells ; but the chief diffi- * See addendum, § 713. 336 CYTOLOGICAL METHODS. culties arise in the study of oogenesis and gland secretion, where one meets with profound changes in the chromophility of the nucleus, and with complicated nucleoU, whose real nature is as yet undetermined ; moreover, at certain stages in the differentiation of a cell, true chromatin may stain oxyphil. Such exceptions must be taken into consideration ; always ascertain carefully the origin and behaviour of a doubtful body and remember that the safest tests for chromatin lie not in staining, but in the behaviour of the doubtful material during mitosis and in the sexual or reproductive phenomena of the cell. See also Gatenby, Journ. R. Mic. Soc, 1919 and 1920. 710. Chromatin and the Inclusions. 1 1. 2. 3. 4. 5. 6. Fixation in al- Method of A i t m a u n- Chrora e-os- Cajal's for- Mann-Kopsch, cohol acetic, Ch a ra p y - B e n s 1 e y, mium flxa- mol uranium i.e., fixation Carnoy (a t KuU, i.e., flx- chrome - os- t i on and nitrate and in corrosive- least one ati on in mium fixa- staining in silver method osmic, after- TechniQue hour) ; Pet- chrome - os- tion, stain- Ehrlich's for Golgi ap- treatment in employed. runkewitsch mium, stain- ing in acid hsematoxylin paratus coun- OsOd of 2 % or G i 1 s n ing in acid fuchsin and or 1 1 uidin terstained in for 14 days, (overnight) fuchsin au- methyl blue and safranin (or then stain in a and staining rantia and green. eosin. in methyl- basic dye like in hsematoxy- toluidinblue. blue eosin or safranin. lin of Ehr- methyl- lich, or tolui- green). din-blue and \ eosin. Chromatin Blue or red, Blue to green- Green. Bluish. Blue, red or lied in safra- (nucleus) etc. See ish, rarely green, a c - nm, etc. and chfo- §§ 211 and red. cording to midia. 666, colour of basic stain used. Mitochon- Will not show, Mitochondria Mitochondria Do not show. M i tochondria Yellowish fo) dria and because they red, Golgi red, Golgi or faintly golden 1 black, will Golgi ap- have been a p p a r a tus a p p a ratus oxyphil, gra- dark brown. not stain in paratus. nearly o r rarely shows, also as for 2. nules as such G olg i ap- safranin. quite dis- but when it not identifi- p a r a t u s solved away, does so it is able. black. and morpho- red. logically altered b y the fat sol- vents of the preparing media. Nucleolus. Eed generally. Red. Ueddish. Bluish. Ueddish or colour of plasma stain. Ueddish. See also remarks on nucleoli in §§ 664 and 668. 711. Cytoplasmic Inclusions in Gametogenesis.— In the table below is a summary of the fixing and staining reactions of the inclusions during oogenesis and spermatogenesis. In the male germ cells the Golgi apparatus (nebenkern batonettes) show through- CHAPTER XXVI. 337 out ; those of the egg can generally only be demonstrated by methods 1 and 2. The fat-methods of Daddi, Herxheimer Martinotti, and Lorrain Smith should be tried as well, and reference made to tables given below. If yolk granules contain olein or such unsaturated fat they will stain in OSO4 like fat,. but by slow de- colourisation as in paragraph 2 (with turpentine), their proteid basis will be noted, and they will generally be demonstrated in methods 1 and 4, while fat vacuoles disappear completely. See also the special sections on " Fat," §§ 768 to 772. 712. Inclusions in Gametogenesis. 1 1. 2. 3. 4. 5. 6. Method. Cajal or Da Fano Kopsch series. Chrome-osmium and iron hsema- toxylln (or Alt- mann). Bouin and corrosive acetic and Ehr- lich-s hsem. Champy- Kull. Benda. Mitochon- dria. Oolgi ap- paratus. Yolh gra- nules. Fat vacu- oles. Chromatin granules. Nucleoli . Either do not show or greyish or golden brow-n, accord- i n ET as to whether sec- tions have been toned. Black. Either will not show, greyish or golden brown, accord- ing as to tonmg. Do not show. Do not show, but may subse- quently be stained in a basic colour, like methyl green or safra- nin. Yellowish may take s u b 3 e- quent stain. Often will not show, or faintly yellowish, more rarely black or brown, but can often be decolour- ised rapidly in turps. Black, and resists decolourisation in turps longer than mitochondria, fat or yolk. Yellowish, or black easily decolour- ised in turps. Black, easily de- colourised in turps. Yellowish, will sub- • sequently stain in hsematoxylln or safranin. Yellowish. Black (or red). Barely shows, when it does, black (or red), or not stained. May or may not go black (or unstained), very rarely red. Black in un- stained pre- paration. Black to grey (or reddish purple). Black or dark grey. Do not show. Does not show. Not stained or yellowish. Not stained, washed away. Bluish to purple. Heddish, or reddish purple. Red. Barely shows, if so, red. Yellowish or black. Black. Blue. Eed. Violet. Barely shows, if so, violet. Yellowish or black. Black. Browni* yellow. Violet or brown. 713. Plan lor Cytological Research.— It is best to work at some animal which is procurable in sufficient numbers, as frequently your first pre- parations are disappointing. If a vertebrate, begin with Eegaud, and stain in iron alum hsematoxylln ; if an invertebrate try Flamming without acetic acid, and iron hsematoxylin ; the former method will M. 22 338 OYTOLOGICAL METHODS. give mitochondria and yolk ; zymogen but not fat ; the latter will give all these. Champy-KuU and Bensley-Cowdry or Altmann methods are next worth trying. Mann-Kopsch and Sjovall methods may present difficulties, but again one might succeed at the first trial : if invertebrate tissue, the Mann-Kopsch method wiU be best, if vertebrate, the Cajal or Da Fano formalin-silver nitrate methods are most indicated for a study of the Golgi apparatus. If your material is limited to a small amount, the Champy-KuU (or Bensley-Cowdry) and the Mann-Kopsch methods are recommended : successful preparations by both these methods wiU enable you to make an almost complete study (sometimes of the chromosomes and) of the cytoplasmic inclusions of every kind — excepting glycogen.* Recom- mended in the third place is a formaUn-silver nitrate method— Cajal or Da Fano. See also G-atenbt, Quart. Journ. Micr. Science, Ixiv, 1920, p. 296, and E. V. Cowdrt, Oontrib. JEmhryology, Carneg. Inst, 'VVashing- ton, viii, p. 69. Addendum: Saguchi {Amer. Jour. Anal., Nov. 15th, 1920) finds that the Golgi apparatus of acinus cells of the frog pancreas is brought into evidence by Cajal's uranic silver nitrate method, and also by the Weigl, Kopsch, and Sjovall methods, but that in islet cells, the former method exhibits some other sort of apparatus. See also Saguchi, ibid., vol. 26, 1920. * Shun Ichi Ono (Anat, Anthrop. Ass. of China, 1920) finds that osmicated mitochondrial fixatives preserve glycogen, which can be stained in Best's carmine and iron heematoxylin, the mitochondria (grey black) and the glycogen (reddish) showing side by side. CHAPTER XXVII. TEGUMENTAEY ORGANS. 714. Epithelium. — Both for surface views and for sections good results are obtained by the nitrate of silver method, the methylen blue method, the perchloride of iron and pyrogallol method of the Hoggans, § 375, the osmic add and pyrogallol process, § 374, and by iron-hcematoxylin . For the purpose of separating the epidermis from the corium, LoEWY {Arch. mik. Anat., xxxvii, 1891, p. 159) recommends mace- rating for twenty-four to forty-eight hours, at a temperature of about 40° C, in 6 per cent. pyroUgneous acid. Acetic acid of I per cent. (Philippson) is also good. Minot {Amer. Nat., xx, 1886, p. 575) macerates embryos for several days in 0-6 per cent, salt solution, Mitrophanow {Zeit. wiss. Mik., v, 1888, p. 573) for a quarter of an hour in 3 per cent, nitric acid, then one hour in one-third alcohol, and, if need be, twenty-four in stronger alcohol. Mayer {Lotos, 2, xii, 1892) exposes the cornea or membrana nictitans of Rana, Bufo, and Mus for half a minute to the vapour of acetic acid, and then puts it into 0-5 per cent, salt solution. For ciliated epithelium see the methods of Bngelmann under « Mollusca." 715. Intercellular Bridges (and Canals), Prickle Cells. — See Ide, in La Cellule, iv, 1888, p. 409, and v, 1889, p. 321 ; also KoLOSsow, Arch. mik. Anat., lii, 1898, p. 1. Kolossow used an osmic-acid- tarmin stain, § 374. See also Flbmming, Anat. Hefte, lAbth, vi, 1895, p. 1. Besides maceration, impregnation may be useful ; Mitrophanow {Arch. Anat. Phys., Phys. Ahth., 1884, p. 191) has used gold chloride. Unna {Monatsschr. prakt. Derm., xxxvii, 1903, p. 1) has described a highly complicated process with Wasserblau and orcein, see Zeit. wiss. Mik, xxi, 1904, p. 68. 716. Plasma-fibrils of Epithelium. — Kromayer's process {Arch, mik. Anoit., xxxix, 1892, p. 141) is as follows : Sections are stained for five minutes in a mixture of equal volumes of anilin water 22—2 340 TEGUMENTARY ORGANS. (§ 286) and concentrated aqueous solution of methyl violet 6 b. They are well washed in water and treated with solution of iodine in iodide of potassium until they become blue-black (one to thirty seconds). They are again washed with water, dried with blotting- paper, and treated with a mixture of 1 volume of anilin to 2 volumes of xylol until sufficiently differentiated, when they are brought into pure xylol. Very thin sections will require more xylol in pro- portion to the anilin, viz. 1 : 3 or 1 : 4 ; thicker ones may require more anilin, viz., 3 : 5 or 3 : 3. Gentian or Krystallviolett will do instead of methyl violet, but not quite so well. See also Ehrmann and Jadassohn, Arch. Dermatol, u. Syphilis, 1892, 1, p. 303 ; Zeit. wiss. Mik., ix, 1893, p. 356 ; Herxheimer, Arch. mik. Anat., liii, 1899, p. 510 ; and Eosenstadt, ibid., Ixxv, 1910, p. 659 (takes the differentiating mixture much weaker in anilin). Unna {Monatsschr. prakt. Derm., xix, 1894, p. 1 and pp. 277 et seq. ; Zeit. wiss. Mik., xii, 1, 1895, pp. 61, 63) has given a whole series of methods, from which the following are some extracts. (1) Wassbrblau-Orcein. — Stain sections for ten minutes in a neutral aqueous 1 per cent, solution of Wasserblau, rinse and stain for five or ten minutes in a neutral alcoholic 1 per cent, solution of Griibler's orcein. Dehydrate, clear, and mount in balsam. This may be varied as follows : (a) Ten minutes in the Wasserblau and thirty minutes or more in the orcein. (6) Take for the second stain an acid solution of orcein. (c) Stain for only one minute in the Wasserblau, but for thirty or more in the neutral orcein. (2) Stain for half an hour or more in a strong solution of haemalum, rinse, stain for half a minute in a saturated aqueous solution of picric acid, and dehydrate for thirty seconds in alcohol containing 0-5 per cent, of picric acid. (3) Heemalum for two hours, neutral orcein as above for ten to twenty minutes. More recently Unna advocates the process mentioned last section. See also Ranvier, Arch. Anat. Mihr., iii, 1899, p. 1. 111. Keratohyalin.— The keratohyaUn granules of the cells of the stratum granulosum are soluble in mineral acids, and can be digested in pepsin. They can be stained with picro-carmine, alum hsematoxyUn, van Gieson's mixture, or Unna's Wasserblau-orcein, last §. FiCK (Centralb. allg. Path., xiii, 1902, p. 987 ; Zeit. wiss. Mik., XX, 1903, p. 222) stains sections of alcohol material for three CHAPTER XXV 11. 341 to four minutes in concentrated aqueous solution of KresylecMviolett, differentiates in alcohol, clears in xylol, and mounts in balsam. See also Unna, Monatsschr. praM. Derm., xx, 1895, p. 69 ; the article " Haut " in the Encyd. mile. TecJmik. ; and Unna and Golodetz, Monatsschr. prakt. Derm., xlix, 1909, p. 95 ; Laffont, Bibl. Anat., 1909, p. 209. For Trioholiyalin, see Gavazzeni, Monatsschr. praht. Derm., xlvii, 1908, p. 229. 718. Eleidin. — To demonstrate the stratum granulosum and the eleidin granules Ranvier (Arch. Anat. Micr., iii, 1899, p. 1) hardens with alcohol, stains with picro-carmine, and treats with lime-water. The cells swell and show up the granules, which do not change. See loc. cit., other methods for the study of skin. Buzzi (see Encycl. mile. Technik., article " Haut ") stains sections for a few minutes in a watch-glassful of water with 2 to 3 drops of 1 per cent. Congo red. Similarly Weidenrbich, Arch. mih. Anat., Ivii, 1901, p. 583. Other authors recommend nigrosin, or Wasser- blau, or orcein. See also Joseph, " Dermatohist. Technik," Berlin, 1905, and Dreuw, Med. Klinik, Berlin, 1907, Nos. 27 and 28. For Cholesterin see Golodetz and Unna, Monatsschr. prakt. Derm., xlvii, 1908, p. 1. 719. Horn, Hair, and Nails. — The elements of hairs and nails may be isolated by prolonged maceration in 40 per cent, potash solution, or by heating with concentrated sulphuric acid. See also von Nathusius, Zool. Anz., xv, 1892, p. 395. Horny tissues stain well in safranin or gentian violet (Rbinke, Arch. f. mik. Anat., xxx, 1887, p. 183 ; Ernst, ibid., xlvii, 1896, p. 669 ; Rabl, ibid., xlviii, 1896, p. 489). Unna [of. cit. last section, p. 598) stains the tyrosin-bearing keratin in sections of skin for a few seconds or minutes in a mixture of 5 parts of Millon's reagent, 5 of water, and 1 of glycerin, treats shortly with nitric acid of 25 per cent., and mounts in balsam. 720. Skin-nerves and Nerve-endings. — Impregnate with gold chloride. See Chapter XVII., especially § 365. 721. Tactile Corpuscles.— See §§ 364— 366.— Gold methods are indicated. See also Ranvier, Traite, p. 919 ; Langerhans, Arch, mik. Anat., 1873, p. 730 ; Kultschizky, ibid., 1884, p. 358 ; and Smirnow, Intern. Monatsschr. f. Anat., etc., x, 1893, p. 241, who recommends, besides the gold method of Lowit, the rapid bichromate of silver method of Golgi. 342 TEGUMENTARY ORGANS. 722. Corpuscles of Herbst and Corpuscles of Grandry. — Dogibl (Arch. Anat. u. Entwickel., 1891, p. 182) has used the methylen blue method. Four per cent, solution of methylen blue, warmed to 40° C, is injected into blood-vessels of the heads of ducks or geese ; pieces of skin are removed from the beaks, sectioned in pith, and the sections brought on to slides and moistened with aqueous or vitreous humour from the animal and left for ten to thirty minutes exposed to the air, then brought into picrate of ammonia, and treated as described § 343. Geberg {Intern. Monatsschr. Anat., x, 1893, p. 205) made use of a method of Arnstein, according to which pieces of skin are put for twenty-four hours into lime-water, the horny layer removed, the pieces treated for five minutes with 0-25 per cent, gold chloride, reduced in water, and the precipitate that forms on them removed by putting into 0-25 per cent, cyanide of potassium and brushing. NowAK {Armt. Anz., xxxvi, 1910, p. 217) takes Unna's Orcein- wasserblau mixture (Wasserblau O.D., 1 part, orcein 1, acetic acid 5, glycerin 20, alcohol 50, water 100) and adds to it 1 part more of orcein. To 10 c.c. of this he adds at the moment of using 10 c.c. of 1 per cent, solution of eosin in alcohol of 80 per cent, and 3 c.c. of 1 per cent, solution of hydroquinon. Stain for five to ten minutes, rinse, stain for ten minutes in 1 per cent, aqueous solution of safranin, wash, treat for thirty minutes with 0-5 per cent, solution of bichromate of potash, dehydrate and mount. Similarly DoGiEL, Folia Neurobiol., iv, 1910, p. 218 (also employing Bielschowsky's neurofibril method). 72B. Corpuscles of Meissner and of Krause (Cornea and Conjunc- tiva). — DoGiEL {Arch. f. mile. Anat., xxxvii, 1891, p. 602, and xUv, 1894, p. 15) employs the methylen blue method ; for details see jprevious editions. See also Longworth's methods, Arch. mik. Anat., 1875, p. 655. 724. Similar Objects. —Papillae Foliatse of the Rabbit, Hermann, see Zeit. wiss. Mile, v, 1888, p. 524.^ Aenstein, ibid., xiii, 1897, p. 240. Olfactlve Organs of Vertebrates, Dogiel, Arch. mile. Anat., 1887, p. 74. Organs of a " Sixth Sense " in Amphibia, Miteophanow, Zeit. wiss. Mile, V, 1888, p. 513 (details as to staining with " Wasserblau," for which see also Biol. Oentralb., vii, 1887, p. 175). Nerve-endings in Tongue of Frog, Fajerstain, Arch, de Zool. expir. et gin., vii, 1889, p. 705. Tongue of Rabbit, von Lenhossek, Zeit. wiss. Mile, xi, 1894, p. 377 {Ram6n y Cajal's double Golgi method). 725. -Cornea.— There are three chief methods— the methylen blue, the silver, and the gold method. CHAPTER XXVII. 343 For the methylen blue method see particularly § 345. Negative images of the corneal cells are easily obtained by the dry silver method (Klein). The conjunctival epithehum should be removed by brushing from a living cornea, and the corneal surface well rubbed with a piece of lunar caustic. After half an hour the cornea may be detached and examined in distilled water. In order to obtain positive images of the fixed cells the simplest plan (Ranviee) is to macerate a cornea that has been prepared as above for two or three days in distilled water. There takes place a secondary impregnation . The same result may be obtained by cauterising the cornea of a living animal as above, but allowing it to remain on the hving animal for two or three days before dissecting it out, or by treating a negatively impregnated cornea with weak salt solution or weak solution of hydrochloric acid (His). But the best positive images are those furnished by gold chloride. Ranviee prefers his lemon-juice method. It is important that the cornea should not remain too long in the gold solution, or the nerves alone will be well impregnated. Zawaesin {Arch. mik. Anat., Ixxiv, 1909, p. 116) removes the membrane of Descemet for study in the following manner. A cornea, fixed in sublimate, is dissected out and put for some hours into a mixture of alcohol and ether. Then collodion of 4 per cent, is poured on to the inner surface, and after some time a layer of collodion with the membrane attached can be peeled off, and the collodion removed from the tissue by a mixture of alcohol and ether. See also Rollett, in Strieker's Handb., pp. 1102, 1115, oi previous editions; Taetupeei, Anat. Anz., v, 1890, p. 524, or previous editions ; Ciaccio, Arch. ital. Biol., iii, p. 75 ; and Renault, G, R. Acad. Sc, 1880, p. 137. 726. Crystalline. — Geehaedt {Zeit. wiss. Mik, xiii, 1896, p. 306) hardens the lens for one or two days in 4 to 10 per cent, formalin ; it is then easily dissociated with needles into its fibres. Rabl {Zeit. wiss. Zool., Ixv, 1898, p. 272) fixes the enucleated eye for half an hour in his platinum chloride or picro-sublimate, §§ 75 and 70, divides it at the equator, and puts the anterior half back for twenty-four hours into the fixative. For Maceration you may use sulphuric acid, § 533. See also Robinski, Zur Kenntniss d. Augenlinse, Berhn, 1883. CHAPTER XXVIII. MUSCLE AND TENDON (NEEVE-ENDINGS). Striated Muscle. 121. Muscle-cells. — For these and allied subjects see, inter alia, Behrens, Kossel, und Schieffeedecker, Das Mikroskop, etc., vol. ii, pp. 154 — 161 ; and Schaper, Proc. Roy. Soc, xlix, 1891, p. 280. Iron hsematoxyUn gives very fine images of striped muscle, and so does Mallory's phospho-tungstic. For dissociation methods see §§ 512 — 545. To isolate the sarcolemma Solgee {Zeit. wiss. Mik., vi, 1889, p. 189) teases fresh muscle in saturated solution of ammonium carbonate. 728. Nerve-endings — the Methylen Blufe Method. — For Biedee- mann's procedure for the muscles of Astacus see § 342 (see also Zeit. wiss. Mik., vi, 1889, p. 65). After impregnating as there directed the carapace should be opened, and the muscles exposed to the air in a roomy moist chamber for from two to six hours. For Hydrophilus piceus, Biedeemann proceeded by injecting 0-5 c.c. of methylen blue solution between the ultimate and pen- ultimate abdominal rings, in the ventral furrow, and keeping the animals alive in water for three to four hours, then opened the thorax by two lateral incisions, and removed the muscles of the first pair of legs and exposed them to the air for three or four hours in a moist chamber, and finally examined in salt solution. Gerlach (Sitzb. Akad. Wiss. MUnchen, 1889, ii, p. 125) injected frogs, either through the abdominal vein or through the aorta, with 4 to 5 c.c. of a 1 : 400 solution in 1 per cent, salt solution, and examined pieces of muscle in serum of the animal, afterwards fixing with picrate of ammonia and mounting in glycerin jelly. The procedure of Dogiel has been given, § 342. 729. Nerve-endings— the Gold Method.— Fischer (Arch. mik. Anat., 1876, p. 365) used the method of Lowit. Biedeemann (last section) recommends for Astacus a similar CHAPTER XXVIII. 345 procedure, the preliminary treatment with formic acid being omitted, and the muscles being put for a couple of days into glycerin after reduction in the acid. Eanvibr (Traits, p. 813) finds that for the study of the motor terminations of Vertebrates the best method is his lemon- juice process (§ 366). See also the methods of Apathy, §§ 368, 371. 730. Nerve-endings — the Silver Method.— Eanvier employs it as follows (ibid., p. 810) ; Portions of muscle (gastro-cnemius of frog) having been very carefully teased out in fresh serum, are treated for ten or twenty seconds with nitrate of silver solution of 2 to 3 per 1000, and exposed to bright light (direct sunlight is best) in distilled water. As soon as they have become black or brown they are brought into 1 per cent, acetic acid, where they remain until they have swelled up to their normal dimensions. They are then examined in a mixture of equal parts of glycerin and water. This process gives negative images, the muscular substance being stained brown, and the nervous arborescence unstained. The gold process gives positive images, the nervous structures being stained dark violet. 731. Nerve-endings — the Bichromate of Silver Method. — The rapid method of Golgi has been used by E.am6n y Cajal for the terminations of nerves and tracheae in the muscles of insects. See Zeit. wiss. Mik., vii, 1890, p. 332, on fourth edition. A modification is used by Wthstderee, Arch. mik. Anat., Ixxi, 1908, p. 523. 733. Muscle-spindles. — See Cilimbaeis, Arch. mik. Anat., bcxv, 1910, p. 692. Principally intra vitam methylen blue, by injection through the internal carotid. For elastic fibres, Weigert's resorcin- fuchsin, followed by 1 per cent, orcein acidified with HCl. Electric Organs. 733. Electric Organs. — Ranvier (Traite, Chap, xviii), finds that osmic acid is the only reagent that will fix properly the terminal arborisations on the lamellae. He injects a little 2 per cent, solution under the surface of the organ, removes a small portion of it after a few minutes, and puts it into a quantity of the same solution for twenty-four hours. The electric plates may then be teased out and examined in water, and will show the stag's horn ramifications ; and the dissepiments between the columns will show the bouquets 346 MUSCLE AND TENDON (NERVE-ENDINGS). of Wagner. The terminal arborescence may be impregnated with silver. A portion of the surface of the organ is rubbed with lunar caustic until it appears opaque, then removed and the plates teased out in water. This gives negative images. Or, electric plates, isolated by teasing after twenty-four hours in osmic acid as above, and kept for some days in one-third alcohol, are washed and placed on a sUde with their ventral surface upper- most. They are then treated with a few drops of 0-5 per cent, solution of chloride of gold and potassium, and those which become violeb are washed and mounted in glycerin. This gives positive images. These may also be obtained by putting material fixed by osmic acid into 2 per cent, solution of bichromate of ammonia for a few weeks, then teasing, staining with alum haematoxylin, and mounting in damar. Torpedo. — Ballowitz {Arch. mik. Anat., xhi, 1893, p. 460) gets the best results by the rapid Golgi impregnation. An electric column, with about ^ to 1 centimetre of tissue round it, is dissected out, and put for three to four days into the osmium bichromate mixture ; then for one to three days into f per cent, silver, cut without imbedding and mounted in xylol balsam. Im- pregnates all the important elements. See further, on the whole subject, Ballowitz, Encycl. mik. Techn., 1910, p. 298. Cavalie {Bihl. Anat., xiii, 1904, p. 214) takes material fixed with osmic acid of 2 per cent, and impregnates it with gold by the method of Nabias, and mounts in glycerin. Raja. — IwANZOFP {Bull. Soc. Nat. Moscou, ix, 1895, p. 74) fixes the organ in the tail of Raja with liquid of Flemming, stains with hsemacalcium and eosin, and makes paraffin sections. Ballowitz {Anat. Hefle, 1 Abth., vii, 1897, p. 285) finds the method of Golgi excellent for this organ. He also makes sections after fixing in saturated solution of sublimate (in sea-water), or in liquid of Flemming, and examines them in water. Methylen blue may be used, intra vitam. Gold is little good. Gymnotus. — Ballowitz {Encycl. mik. Technik., p. 303) fixes with Flemming, and makes sections. He also commends impregnation with gold chloride, but not the Golgi method. Malapterarus.— Ballowitz {ibid., p. 202) fixes with picro-subli- mate, with Flemming, or with various mixtures of bichromate, sublimate, and formol, and uses gold chloride and Golgi impregna- tions. He macerates in liquid of Mliller or saturated aqueous solution of picric acid. CHAPTER XXV III. 347 Tendon. 734. Tendons.— Eetteree (C. R. Soc. Biol, x, 1898, p. 580) fixes in equal parts of saturated solutions of sublimate and picric acid, puts for one to three days into saturated picric acid with 2 to 3 per cent, of sodium chloride, to remove the mucin, and imbeds in paraffin. 735. Union of Muscle and Tendon. — For this see Ketterer and Lelievre, C. R. Soc. Biol., 1911, No. 12 (orcein for twenty-four hours, followed by iron hsematoxyUn) ; and Schultze (Verh. phys. med. Ges. Wurzburg, 1911, p. 33) (treats for a day or two with a mixture of equal parts of 2 per cent, bichromate of potash and alcohol, in the dark, then for two days with 0-5 per cent, solution of hsematoxyUn in alcohol of 70 per cent., then with Van Gieson's picro.-saurefuchsin) . 736. Corpuscles of Golgi (Ranvier, Traite, p. 929).— Take the tendon of the anterior and superior insertion of the gemini muscles of the rabbit. Treat it by the formic acid and gold method (§ 365), and after reduction scrape with a scalpel, in order to remove the muscle-fibres that mask the musculo-tendinous organs. Marchi's methods for the tendons of the motores bulbi oculi (Archivio per le Scienze Mediche, v, No. 15). — The enucleated eyes, together with their muscles, were put for not less than three days into 2 per cent, bichromate of potash. The muscles and tendons were then carefully dissected out, stained with gold chloride and osmic acid (Golgi's method), and by the method of Manpredi, § 368. Mount in glycerin. The methods only succeed completely during fine, sunny weather. RuFPiNi {Atti R. Ace. Lincei Roma Rend. [5], i, 1892, p. 442) recommends the method of Fischer. CiACOio {Mem. R. Ace. Sci. Bologna [4], t. x, 1890, p. 301) puts tendons of Amphibia into 0-1 per cent, hydrochloric acid or 0-2 per cent, acetic acid until transparent ; then for five minutes into a mixture of O-l per cent, gold chloride and 0-1 per cent, potassium chloride ; then back intothe acetic acid, for a day in the dark, and for two or three hours more in the sunlight. When they have become somewhat violet they are put for a day into 0-1 per cent, osmic acid, and finally mounted in glycerin acidulated with 0-5 per cent, of acetic or formic acid. DoGiEL (Arch. milt. Anat., Ixvii, 1906, p. 638) stretches tendons of eye-muscles on cardboard with hedgehog spines, puts for four 348 MUSCLE AND TENDON (NERVE-ENDINGS). or five days into nitrate of silver of 1 to 2 per cent., reduces for a day in pyrogallic acid with formol, and imbeds in celloidin. Smooth Muscle. 737. Tests for Smooth Muscle. — Picro-saurefuchsin, § 299, stains muscle yellow, connective tissue red. Picro-nigrosin, § 325, stains muscle yellowish, connective tissue blue. Unna (Encycl. mih. Technik., article " Kollagen ") stains for twenty-four hours in orcein 1 part, Wasserblau 0-25, alcohol 60, glycerin 10, water 30, which gives muscle in a mixed tone, collagen blue, elastin reddish. See also a complicated process with methylen blue in Monctssch. prakt. Dermatol., xix, 1894, p. 533, and another with orcein, haematein, saurefuchsin and picric acid. Eetteree {C. R. Soc. Biol., 1887, p. 645) fixes in 10 volumes of alcohol with one of formic acid, washes well and stains in alum carmine. Muscle red, connecbive tissue unstained. 738. General Structure. — Weenee (Hist. d. glatten Musculatur, Dorpat, 1894, p. 22) fixes stretched intestine or bladder in Flemming, washes well and stains in Heidenhain's chrome heematoxylin, § 265. For demonstrating intercellular spaces, fresh intestine is put for twenty-four hours into oil, at 37° C, then for twelve hours into Flemming, and for four to six into chromo-acetic acid. 739. Isolation of Fibres.— Gage's methods, see §§ 518, 528, and 536. MoBius, muscle of Cardium, see § 527. Ballowitz, muscle of Cephalopoda, see Arch. mik. Anat., xxxix, 1892, p. 291. ScHULTZ {Arch. Anat. Phys., Phys. Ahth., 1895-6, p. 521) puts muscle of Vertebrates for twenty-four hours into 10 per cent, nitric acid, rinses with water, and brings pieces for six to eight days (in the dark at first) into a mixture of equal parts of ^ per cent, osmic acid and J per cent, acetic acid, teases and mounts in glycerin. For smooth muscle of Vermes, see Apathy, Zeit.f. wiss. Mik., x, 1893, pp. 36, 319, and § 530, ante. 740. Iris. — DoGiEL {Arch. mik. Anat., 1886, p. 403) puts the anterior half of an enucleated eye for some days into a mixture of two parts one-third alcohol and one part 0-5 per cent, acetic acid. The iris can then be isolated, and split from the edge into an interior and posterior plate, and these stained according to the usual methods. CHAPTER XXVIII. 349 See also Koganei, Arch. miJc. Anat., 1885, p. 1 ; Canfield, ibid., 1886, p. 121 ; and Dostoiewsky, ibid., p. 91. 741. Bladder o£ Frog, Innervation of (Wolff, Arch.f. mik. Anat., 1881, p. 362). — ^A frog is killed and a solution of gold chloride of 1 : 20,000 injected into the bladder through the anus. (If the injection flows out on removal of the syringe, tie the frog's thighs together.) Now open the frog, ligature the intestine above the bladder, and cut away the abdomen so as to have in one piece bladder, rectum and hind legs. Put this into gold solution of 1 : 2000 for four hours ; the bladder is then excised, slit open, and pinned (with hedgehog spines) on to a cork (outside downwards). Place it rmder running water until all the epithelium is washed away. Use a camel' s-hair brush if necessary. Put for twenty-four hours into gold solution of 1 : 6000. Wash in pure water, and put away in the dark " for some time " in acidulated water, and finally reduce in fresh water in daylight. Ranvibr (Traite, p. 854) recommends his two gold processes, the liquids being injected as above. Geunstein {Arch. mik. Anat., 1899, p. 1) injects 1 per cent, methylen blue in normal salt solution through the vena abdominalis, and after twenty to thirty minutes excises the bladder and exposes to the air. Fix the stain with picrate of ammonia and mount in glycerin with the same (§ 343). CHAPTER XXIX. CONNECTIVE TISSUES. Connective Tissue. 742. General Stains for Connective Tissue. — Connective tissue, elastic tissue, and smooth muscle are all normally acidopliilous. Collagen, the distinctive element of connective tissue, absolutely requires " acid " dyes for the production of a permanent stain, whilst elastic tissue and muscle will also fix " basic " dyes. Collagen has a special affinity for Saurefuchsin and Wasserblau. Elastin has a strong affinity for acid orcein, whilst muscle has no special affinity for either, but staius energetically with picric acid. Picro-sdurefuchsin is much used and very convenient as a general difEerentiating staiu, but not to be recommended for cytological detail. See Schafeer, Zeit. wiss. Zool., Ixxx, 1905, p. 176. E. and T. Savini recommend Benda's Picro-Saurefuchsin, § 299. Ehelich-Biondi mixture gives connective tissue red, but smooth muscle redder stiU. Unna's Wasserbluu-orcein for distinguishing connective tissue and muscle has been given, § 676. It works after all . fixatives. Staiu long, and dehydrate preferably with acid alcohol. Fbeeb^en {Amer. Man. Mic. Journ., 1888, p. 231) recommends (for sections) picro-nigrosin, made by mixing 5 c.c. of 1 per cent, aqueous solution of nigrosin, with 45 c.c. of aqueous solution of picric acid. Stain for three to five minutes, wash with water, and mount in balsam. Connective tissue blue, nuclei blackish, the rest yellowish. Ram6n y Cajal's jncro-imdigo-carmine gives connective-tissue fibres dark blue^ with red nuclei. S. Mayer [Sitzh. k. Akad. Wiss., Ixxxv, 1882, p. 69) recommends for staining /resA tissue Violet B, § 330. Elastic fibres and smooth muscle also stain, but of different tints. DuBEEUiL (C. R. Ass. Anat., vi Sess., 1904, p. 62) uses a mixture of 23 volumes 1 per cent, picric acid and 2 volumes 1 per cent, methyl blue — with a foregoing stain with carmalum or safranin. For Ranviee's method of artificial oedemata for the study of areolar tissue, see his Traiti, p. 329. CHAPTER XXIX. 351 743. Unna's Orcein Method. — {Encyd. mik. Technik, 1910, p. 250). Sections are stained for ten minutes in Griibler's polychrome methylen blue. They are then washed with water, mopped up, and brought for fifteen minutes into a neutral 1 per cent, solution of orcein in absolute alcohol, rinsed in pure alcohol, cleared in bergamot oil, and mounted. Collagenous ground-substance dark red, muscle bluish, elastic fibres sometimes dark red. Material may be fixed in almost any way except with nitric or picric acid, formol, or liquids of Miiller and Hermann. 744. Unna's Methylen-blue + Sauiefuchsin (Unna, in Encyol. mik. Technik, 1910, p. 247). Stain for two to five minutes in poly- chrome methylen blue solution (Griibler). Wash and stain for ten to fifteen minutes in " (0'5 per cent.) Saurefuchsin -f (33 per cent.) tannin-mixture (Griibler)." Water, alcohol, essence, balsam. Collagen, protoplasm, and muscle red, nuclei and keratin blue. On Flemming material, elastin blue. Liquids of Hermann and Erlicki, formol and copper fixatives incompatible. 745. Unna's Sa£ranin -f Wasserblau (ibid.). Ten minutes in 1 per cent, safranin. Wash. Ten to fifteen minutes in " (1 per cent.) Wasserblau + (33 per cent.) tannin mixture." Wash. Stains in opposite colours to the last. Formol and liquid of Hermann contra-indicated for fixing.' 746. Flemming's Orange Method is said to give a very sharp differen- tiation of developing fibrils. liil. Malloet (Zeit. wiss. Mik., xviii, 1901, p. 176) stains sections of sublimate or Zenker material for a few minutes in Saurefuchsin of 0-1 per cent, mordants for a few minutes in 1 per cent, phosphomolybdic acid and stains for two to twenty minutes in anihn blue 0-5 grms., Orange Gr. 2, oxalic acid 2, and water 100. His phosphotungstic hsematoxyUn stains connective tissue sharply, but does not differentiate it sufficiently from elastic tissue and muscle. 748. For the complicated procedure of Hobnowski see ibid., xxvi, 1909, p. 138. 749. For Delamaee's mixture or orcein, hsematoxyUn, Saurefuchsin and picric acid see Verh. Anat. Oes., xix, 1905, p. 227. 750. Masson (0. B. 8oc. Biol, Ixx, 1911, p. 673), stains first in hsemalum, then in eosin, and then for a. few minutes in 1 per cent, solu- tion of satfron in tap water (made by boiling). Connective tissue, bone, and cartilage, yellow. 751. Benecke's stain for fibrils (Verh. Anat. Oes., vii, 1893, p. 165) is essentially that of Keomayeb, § 716. 352 CONNECTIVE TISSUES. 753. Bielschowsky's Silver Method {post, under " Neurofibrils ") has been used for connective-tissue fibrils. Snessakew (Anat. Anz., xxxvi, 1910, p. 401) employs it as follows : Tissue is hardened in neutral formol and sectioned with a freezing microtome. The sections are put for at least four days into iron alum of 2-5 to 10 per cent., changed daily. They are then silvered for thirty-six to forty-eight hours in nitrate of silver of 10 per cent., then treated with the oxide bath and reduced in formol of 20 per cent. Collagen fibres grey, but fine connective networks black, nerve fibres un- stained or only weakly stained. See also Maresch, Zeit. wiss. Mile., xxiii, 1906, p. 356 ; Stud- NiCKA, ibid., p. 416 ; Zimmermann, ibid., xxv, 1908, p. 10 ; Levi, Monit. zool. Ital., 1908, p. 290 ; Heinrich, Arch. mik. Anat., Ixxiv, 1909, p. 786 (dentine) : Insabato, Arch. Ital. Anat. Emb., viii, 1909, p. 375 (silvers Flemming material) ; Athanasiu and Deagoiu, C. R. Acad. Sci., cli, 1910, p. 551 (Ramon y Cajal's silver process, with alcohol fixation). Elastic Tissue. 753. Elastic Tissue, Generalities. — Elastic fibres have a great affinity for osmium, staining with much more rapidity than most other tissue elements. They are not changed by caustic soda or potash. They are normally acidophilous, but are easily rendered artificially basophilous by means of chromic acid or other mordants, and then stain with great energy with basic dyes. Hence a group of stains of which those of Lustgarten and Martinotiii are types. They have a natural affinity for orcein, whence stains of the Taenzer- Unna type. For a review of the older methods of Balzer, Unna, Lustgarten, and Hbrxheimer, see the paper by G-. Martinotti, in Zeit. unss. Mik, iv, 1887, p. 31 ; also Encycl. mik. Technik, art. " Blastin." 754. Victoria Blue (Lustgarten). See § 289. 755. Safranin (G. Martinotti, he. cit., § 753).— Fix in a chromic liquid, wash, stain for forty-eight hours in strong (5 per cent. Pfitzner's) solution of safranin, wash, dehydrate, clear, and mount in balsam. Elastic fibres black. The staining will be performed quicker if it be done at the temperature of an incubating stove (Geiesbacii, ibid., iv, 1887, p. 442). See also Pebeia {ibid., r, 1888, p. 342). See also Mibelli, Mon. Zool. Italia/no, 1, p. 17, or Zeit. wiss. Mile., vii, 1890, p. 225 (the report in Journ. Boy. Mic. Soc, 1890, p. 803, is vitiated by a misprint). Other basic dyes have been recommended. CHAPTER XXIX. 353 756. Kresofuchsin (Rothig, see § 289). 757. Orcein. — This method is due to Taenzer, and as modified by UmsTA is known as the Taenzer-Unna method, see third edition, or Monatssch. prakt. Dermatol., xii, 1891, p. 394. Unna's Modified Orcein Method {Monatssch. prakt. Dermatol, xix, 1894, p. 397 ; Zeit. wiss. Mik, xii, 1895, p. 240).— Griibler's orcein 1 part, hydrochloric acid 1 part, absolute alcohol 100 parts. Stain sections for thirty to sixty minutes, or for ten to fifteen at 30° C, rinse in alcohol, clear, and mount. Elastin dark brown, collagen light brown. See also Merle. 8iU. Ahad. Wiss. Wien., oviii, 1899, p. 335 ; Prantbe, ibid., xix, 1903, p. 361 (he takes 2 per cent, of nitric acid instead of the hydrochloric, and stains six to twenty-four hours) ; Wolff, ibid., p. 488 ; the article " Elastin " in Encyel. mik. Technik. ; and E. and T. Savini, Zeit. wiss. Mile, xxvi, 1909, p. 34. 758. Weigert's Besorcin-Fuchsin Method {Centralb. allg. Path., ix, 1898, p. 290). — One per cent, of basic fuchsin and 2 per cent, of resorcin (or of carbolic acid) are dissolved in water. Two hundred cubic centimetres of the solution are raised to boiling-point in a capside, and 25 c.c. of Liquor ferri sesquichlorati P. G. are added, and the whole is boiled, with stirring, for two to five minutes more. A precipitate is formed. After cooUng the liquid is filtered, and the precipitate which remains on the filter is brought back into the capsule, and there boiled with 200 c.c. of 94 per cent, alcohol. Allow to cool, filter, make up the filtrate to 200 c.c. with alcohol, and add 4 c.c. of hydrochloric acid. WoLPEUM {Zeit. wiss. Mik., xxv, 1908, p. 219) adds 10 to 15 per cent, of acetone to the mixture. Stain sections (of material fixed in any way) for twenty minutes to an hour, wash with alcohol, clear with xylol (not with an essence). Elastic fibres dark blue on a light ground. MiNEBViNi(^eif. wiss. Mik., xviii, 1901, p. 161) gives a variant with safranin instead of fuchsin. See also Pranter, ibid., xix, 1903, p. 361 ; B. Fischer, VircJww's Arch., clxx, 1902, p. 285, or Zeit. wiss. Mik., xx, 1903, p. 40 (chemistry of the dyes obtained by these processes, which he calls " Fuchselin," " Safranelin," etc.) ; Hart, Gentralb. allg. Path., xix, 1908, p. 1 ; and CiLiMBARiS, Arch. mik. Anal., Ixxv, 1910, p. 708. 759. Hsematoxylin Methods.— Harris [Zeit. wiss. Mik., xviii, 1902, p. 290) makes an " Elasthaematein " as follows : Hsematoxylin 0-2 grms., aluminium chloride 0-1 grms., alcohol of 50 per cent. 100 c.c, boil and add mercuric oxide 0-6 grms., filter and- add 1 drop of HCI. Keep for 23 354 CONNECTIVE TISSUES. some weeks. Stain lor five or ten minutes, put into alcohol with 1 per cent, of nitric acid for one minute, then pure alcohol. See also De Witt, Anat Bee, i, 1897, p. 74 ; Dubrck, Arch. Path. Anat, clxxxix, 1907, p. 62 ; Vebhoeff, Journ. Amer. Med. Assoc., 1908, No. 11. Mallort's phosphotungstio hsematoxylin is good, but not speciflc. For a haem atoxylin and eosin stain for connective tissues see Ketjger (Zeit.f. w. Mikr., xxxi, or. Journ. B. Micr. Soc, 1914). 760. Other Methods for Elastic Tissue.— For the elastic tissue of the skin see Passarge and Keosing, Derm. Stud., xviii, 1894. See also for staining and dissociation Agababow, Arch. mik. Anat., 1, 1897, p. 566 et seq. For C. Martinotti's silver impregnation see Zeit. wiss. Mik., v, 1888, p. 521, or Arch. Ital. Biol, xi, 1889, p. 257. Schumacher {Arch. mik. Anat., Iv, 1899, p. 151) has had good results (for the spleen) with pioro-nigrosin, § 681. See also § 733. 761. Drew-Mubray van Gieson-Nile Blue Method for Connective Tissues (and Bacteria). — Fix in formol-salt solution. Prepare paraffin (or frozen) sections. Stain one to three minutes in van Gieson's picric acid-acid fuchsin solution. Wash in aq. dest. ; treat in 2 per cent. Nile blue sulphate solution in aq. dest. for from two to twenty-four hours. Wash in changes of aq. dest. till the latter is tinted pale blue. Stain again in van Gieson one to five minutes. Wash in aq. dest. till wash-water is pale yellow. Dehy- drate rapidly with absolute alcohol from drop bottle. Clear quickly in xylol (not more than a minute). Differentiate in clove oil from five minutes to several hours (the longer period is for frozen sections). Wash in xylol — Canada balsam. In successful preparations nuclear chromatin a saturated trans- parent blue, mast cell granules nearly black, collagen red, keratin and erythrocytes orange yellow (if bacteria are present they stain blue). {Report of Imper. Cancer Research Fund, 1919.) Plasma Cells. 762. Plasma Cells and " Mastzellen " ; Generalities. — Plasma cells, of which " Mastzellen " are a sub-species, are cells found in or along with connective tissue, and distinguished by their hyper- trophied and very granular cytoplasm and poorly staining nucleus. The granules are highly basophilous, much more so than the nuclei ; they stain with special energy with basic anilins, and mostly meta- chromatically. They do not, however, stain with pure methyl green. The nuclei either do not stain at all or not in the normal way, except with pure methyl green. CHAPTER XXIX. 355 According to Unna in Encycl. mik. Technik., 1910, ii, p. 411, material should be fixed in chemically pure absolute alcohol and sectioned in celloidin. Care should be taken to avoid contamination of the liquids by tannin ; corks, and supports for imbedding, should be soaked for some hours before use in 2 per cent, carbonate of 763. Mastzellen (Nordmann, Beitr. z. Kenntniss d. Inaugural diss., Helmstedt, 1884). — A concentrated solution of vesuvin containing 4 to 6 per cent, of hydrochloric acid . Stain for a few minutes , and dehydrate with absolute alcohol. 764. Plasma Cells, Unna's Later Methods (Unna, in Encycl. mik. Technik., 1910, ii, p. 411). A. — For Large Plasma Cells. (1) Ten minutes in Griibler's polychrome methylen blue solution, wash and drain. Fifteen minutes in 1 per cent, orcein solution (Griibler), without acid ; absolute alcohol, so long as methylen blue comes away abimdantly ; bergamot oil, balsam. (2) Methylen- blue as above, two minutes. Wash well. Then two minutes in glycerin-ether mixture* (Griibler) diluted with 4 volumes of water. Wash thoroughly (two to five minutes) ; absolute alcohol, bergamot oil, balsam. (3) Modification of a method of Pappenheim (Virchow's Arch., clxiv, 1901, p. 111). Ten minutes in the warm, 20° to 40° C, in Griibler's carbol-pyronin-methyl-green mixture. Cool rapidly, by plrmging the recipient containing the tissues into cold water. Remove the tissues with a platinum wire and rinse. Absolute alcohol, bergamot oil, balsam. Steopeni {Zeit. wiss. Mik., xxix, 1913, p. 302) takes acridin red instead of the pyronin. This will work after various fixatives. B. — For Small Plasmu Cells. (4) As No. 2, supra, but only half a minute in the glycerin-ether. (5) After removal of the celloidin from the sections with alcohol and ether, five minutes in polychrome methylen blue, wash, dry with blotting-paper, dehydrate (about a minute) in a mixture of 2 parts alcoliol to 3 of xylol, then one minute in xylol ; then five to ten minutes in alum-anilin (prepared by allowing anilin to stand * Glycerin ether CgHioOs, is a glycerin anhydride. It is a differen- tiating agent for basic dyes. The glycerin -ether mixture in question contains alcohol and glycerin, and can be obtained from Griibler. 23—2 356 CONNECTIVE TISSUES. over a layer of powdered alum a couple of fingers deep) ; xylol, balsam. (6) As No. 3, supra, after a foregoing stain of two minutes in polychrome methylen blue. See also Ehelich in Virchow's Arch., clxxv, 1904, p. 198. 765. Ehrlich's Original Method for Mastzellen (Arch. mik. Anat., xii, 1876, p. 263). — Stain for at least twelve hours in — Absolute alcohol . . . . 50 c.c. Water 100 „ Acid. acet. glacial ..... 12|^^ ,, — to which has been added enough dahlia to give an almost satu- rated solution. Wash out with alcohol, and mount in resinified turpentine. Sse also Soi-iieffeedeckee and Kossel's Oewehelehre, p. 329. 766. Mastzellen, Unna's Latest Methods (Encycl. mik. Techn., 1910, ii, p. 72). — (1) Stain three hours to overnight in polychrome methylen blue with a knife-pointful of alum to a watch-glass of the stain, rinse ; alcohol, oil, balsam. (2) Stain in polychrome methylen blue quarter of an hour, rinse, then ten minutes in glycerin-ether, § 702, wash thoroughly, alcohol, oil, balsam. These methods give a specific metachromatic stain of Mastzellen on a light ground. See also loc. cit., two other methods demon- strating plasma cells at the same time. 767. Other Methods for Plasma Cells and Mastzellen.— See, inter alios (in 'previous editions) Pappenheim, Virchow's Arch., clxvi, 1901, p. 427 ; Bergonzini, Anat. Anz., 1891, p. 596 ; Scheidde, Anat. Hefte, xxviii, 1905, p. 698 ; Maximow, Arch. mile. Anat, Ixvii, 1906, p. 686 ; Sohae- FEE, Centralb. Phys., xxi, 1907, p. 261 (fixation in absolute alcohol or 2 parts alcohol to 1 of formol, staining ±or half an hour in methylen blue, thionin or toluidin blue, in 70 per cent, alcohol with 1 per cent, of HCl) ; Eanviee, G. B. Acad. 8ci., 1890, p. 165 (his " Clasmatocytes " : fix with osmium, stain with aqueous methyl violet 5B). Fatty Substances.* 768. Fatty Substances. — The general term "falty substances " is used here to denote the true fats and the lipoids. True Fats are esters of the alcohol, glycerol, with the higher fatty acids, chiefly palmitic acid, stearic acid and oleic acid. The fatty acids may be " saturated " as, for instance, palmitic and stearic acid, or they may be " unsaturated " as, for instance, oleic acid. * By Dr. W. Cramer, Imper. Cancer Research Bureau, and partly by J. B. G. CHAPTER XXIX. 357 Lipoids. This term was used originally to denote substances having solubilities similar to those of the true fats, i.e., substances which may be present, together with the true fats in alcohol, ether and chloroform extracts of tissues. This definition is, however, too crude, and the term as now used is restricted to certain chemi- cally well-defined groups of substances, which in fact constitute the bulk of such extracts, namely, cholesterol and its esters, phospha- tides, cerebrosides and phosphorised cerebrosides. The phos- phorised cerebrosides are " compound lipoids." Their molecule is very large and consists of a chemical combination of several groups of simple lipoids, e.g., cerebrosides and a phosphatide in protagon. The existence of such compound lipoids is denied by some authors, who look upon them merely as mixtures of cerebrosides and phos- phatides. These groups of substances, although differing in their chemical constitution from each other and from true fats, frequently occur together in the tissues, and may, therefore, be presumed to have a similar physiological significance. They also resemble each other and true fats in their general staining reactions, but some of them exhibit characteristic differences in this respect. Some authors use the term " lipoid " as including the true fats. Others, morphologists in particular, use the term " fat " so as to include lipoids. It seems advisable, however, to separate the true fats from lipoids, as is done here, since the two groups of substances fulfil different physiological functions. The term " Lipin " has been proposed by some authors to denote certain groups of lipoids. The advantage of this nomenclature is not obvious, and since almost every author who has used this term has given it a new definition it will not be used here. For a detailed account of the chemistry of these substances the reader is referred to the larger text-books and monographs, such as Hammarstbn's Text-hook of Physiological Chemistry, Abdee- HALDEN's'jBtocAemwcfes Handlexicon, Vol. iii, Maclean's monograph on Lecithin and the Allied Substances. The table on p. 358 gives only a few elementary data concerning the chemical constitution of these substances and their solubilities, which are of importance in connection with their staining reactions. In the following, the term " true fats " will always be applied to mixtures of unsaturated and saturated fats, since in the tissues these substances always occur together. The table shows that all fatty substances occurring in the tissues contain somewhere in their molecule a double linkage (marked thus ii in the table overpage), and are therefore unsaturated compounds. This fact is of importance, 358 CONNECTIVE TISSUES. Table of Chemical Constitution of Fatty Substances. Group. Examples. Characteristic central groups. Additional groups. Solubilities. True fats . Palmitin. Glycerine and iatty acids. None. Ueadily soluble in ace- Stearin. li tone, ether, chloro- Olein. form and benzene. Simple (A.) Cholesterin (A.) Cholesterin free. None. (A.) Same as true fats. lipoids. (Cholesterol). ii (B.) Not readily soluble Sterinea. (B.) Cholesteri- (B.) Cholesterin and Iatty in alcohol and ace- nesters. ii acids, ii ton, soluble in ether, chloroform and ben- zene. Phospha- Lecithin. Glycerine, phosplioric acid, A nitrogenous base, Not soluble in acetone, tides Kephalin. and fatty acids. e.g., cholin in the readily soluble in (phos- il case of lecithin. alcohol, ether, chloro- phorised form and benzene. lats). • Kephalin, when pure, is insoluble in alcohol. Cerebro- Cerebron Gala ctose and a fatty acid . The nitrogenous base Insoluble in fatty sol- sides. (Cerebrin, sphingosin. vents in the cold ex- Phrenosin). ii cept pyridine. Soluble Homooerebrin in hot alcohol, ben- (Kerasin). zene, chloroform. Compound Protagon. ,4 chemical combination Sphingomyelin, which Same as cerebrosides. lipoids. of the two cerebrosides, contains phosphoric Phospho- cerebrin and homocere- acid, a fatty acid. rised brin (vide supra), and a sphingosin and cholin cerebro- substance sphingomye- ii ii sides. lin, allied to the phos- phatides (see next column). as, on it depends most of the characteristic staining reactions for fatty substances. The characteristic staining and other reactions for fatty sub- stances fall into five groups : — (1) Staining with Sudan III., or Scharlach E. (2) Blackening with osmic acid, either with the acid itself or mixed with bichromate solution. (3) Staining with hsematoxylin after mordanting with bichromate. (4) Their behaviour in polarised light. (5) Staining with Nile blue. The rationale of these methods will now be considered. (1) Scharlach E, Sudan III. — Mixtures of the true fats, as they occur in the tissues, are readily stained by these dyes. Most lipoids, when pure, do not take this stain at all, or only slightly, except cholesterinesters, which take the stains, although not as readily as the true fats. The staining is probably a purely physical process and depends on the solution of the stain in the fatty material. Such solution occurs only when the fatty material is fluid, and this condition is fulfilled in the tissues where mixtures of the true fats CHAPTER XXIX. 359 are always present together, ^s, for instance, in the cells of adipose tissue, or in cells showing fatty degeneration. Mixtures of the true fats and lipoids which constitute, for instance, many of the fat globules of the adrenal cortex also take these stains. It is to be noted, however, that Scharlach E. and Sudan III. are applied in alcoholic solution, and in the process of staining with these dyes some of the fatty substances tend to be dissolved out. The true fats are apparently not so readily dissolved as some substances, which show a double refraction, presumably cholesterin — fatty acid mixtures. The method of Herxheimer, in which Scharlach R is used in strongly alkaline solution, has been recommended on the ground that it is a more " energetic " fat stain for fatty substances in the sense that it stains many cell inclusions which are not stained by Scharlach or Sudan alone. The chemistry of the reaction has not been worked out. It depends probably on the saponifying action of the alkali, which liberates some fatty acids and then produces mixtures of fatty acids and lipoids, which are more readily stained by Scharlach. The range of staining by this method is, therefore, probably as wide as that of osmic acid alone, and will be found to comprise most fatty substances, but for the purpose of differentiating between the different groups of fatty substances has lost the advantage of restricted staining, which the ordinary staining with Sudan III. and Scharlach R alone possesses. (2) Osmic Acid Methods. — The true fats and the lipoids are all blackened bjy osmic acid. This blackening indicates a reduction of osmic acid to a lower oxide. It is due to the fact that all these fatty substances have a double linkage (marked ii in table on p. 358) in their molecule and are, therefore, more or less easily oxidised. But the various groups of substances differ in the readiness with which they are oxidised, and amsequently in the rapidity with which they are blackened by osmic acid and the depth of the blackening produced. The true fats are blackened most rapidly and most deeply, the phospha- tides, lecithin and kephalin, come next in order, while cholesterin, the cerebrosides and phospho-cerebrosides are least susceptible to the oxidising action of osmic acid. In fact, these substances when solid are not blackened at all, but are so when dissolved in an appro- priate solvent, such as chloroform. In the myelin sheath of peri- pheral nerves they are present in the form of a colloidal solution, since it can be shown that they contribute to the blackening of the sheath by osmic acid. This difference in the reducing power of the various fatty sub- stances can be accentuated by using osmic acid together with bichro- 360 CONNECTIVE TISSUES. mate solutions, as in the Marchi method. The bichromate, which is itself an oxidising agent, acts in the various double linkages, and prevents the osmic acid from being reduced except by the substances having the strongest reducing power ; these are the true fats and mix- tures of cholesterin and unsaturated fatty acids. Since the latter show double refraction in polarised light (see below), while the true fats do not, it is possible to differentiate these two groups of substances. The behaviour of mixtures of cholesterin and fatty acids is para- doxical, since cholesterin alone and fatty acids alone do not blacken with osmic acid after bichromate. The same paradoxical behaviour is exhibited by these cholesterin fatty acid mixtures in their staining reaction with hsematoxylin after mordanting with bichromate (see next paragraph). One must assume that cholesterin is present in these mixtures in a special physical state, in which it exhibits a greater reducing power. The myelin sheath of normal nerves does not contain true fats and gives, accordingly, no blackening with bichromate-osmic acid treatment. In the early stages of degeneration globules of true fat are formed, which stain black with bichromate-osmic acid and then give the positive Marchi reaction. With other tissues rich in lipoids, such as the adrenal cortex, no such clear distinction can be obtained because the lipoids are present therein, not only together with true fats, but also mixed with them, so that a globule of fatty material frequently contains both groups of substances. (3) Staining with Hematoxylin aptee Moedanting with Bichromate. — This method, which was introduced originally by Weigert for the staining of £he medullary sheath of nerves, is applicable to all fatty substances. Like the preceding method, it also depends on the presence of a double linkage in the molecule, as pointed out by Thorpe. Substances having a double linkage are oxidised by the bichromate solution and, in this process of oxidation, a chromium compound is formed which is insoluble in fat solvents and which at one stage of the oxidation has the property of forming a dark blue lake with hsematoxylin. If oxidation be continued, however, hsematoxylin will again cease to stain. The rate of oxidation with bichromate varies, as might be expected, with the concentration of the solution used and with the temperature at which it is carried out. Working under similar conditions, it is found again that the different fatty substances vary in the readiness with which they are oxidised by bichromate and, consequently, reach the stage of staining with hsematoxylin after different periods CHAPTER XXIX. 361 of mordanting with bichromate. The different lipoids, when pure, differ greatly in the ease with which the stainable chromium com- pounds is formed ; thus the cerebrosides and protagon stain after a short mordanting ; the unsaturated true fats require a slightly longer time, while lecithin, and especially cholesterin, are very resistant and require prolonged mordanting. But mixtures of cholesterin and the various fatty substances show quite a. different behaviour and reach the stainable stage very rapidly. This appears to be due to the fact that such mixtures form colloidal solutions in a peculiar physical condition (fluid crystals). It will be clear that a histochemical identification of the various lipoids by means of this method is not possible. Its value lies in the possibility of demonstrating, first, the presence of fatty substances by a niethod which gives good histological details, and secondly, by comparison with normal tissues, the occurrence of chemical changes in these substances under pathological conditions. As introduced by Weigert, the method stains the lipoids of the normal myelin sheath. By prolonging the bichromating the degeneratiug nerve fibres may be made to staiu in the early stage of degeneration, or the fat droplets in fatty organs may be stained. The same principle underlies Altmann's method for the staining of mitochondria, which are by some believed to consist of a central core of protein covered by an envelope of fatty material. The original method consists in mordanting with a bichromate solution and staining with acid fuchsia, just as Weigert originally used acid fuchsin for the stainiug of the myelin sheath. In the staining of mitochondria, the acid fuchsin can again be replaced by hsematoxylin (Heidenhain's hsematoxylin). (4) Behaviour in Polarised Light. — The true fats and the fatty acids are isotropic, i.e., show no double refraction in polarised light, so that with crossed nicols the field appears dark. The lipoids are anisotropic. In fresh teased preparations they can be seen with crossed nicols as luminous droplets with a varying degree of brilliancy. The double refraction disappears on gentle heating to about 60° and reappears on cooling. In formol fixed frozen sections the aniso- tropic lipoids appear chiefly in the form of needles and as droplets. Heating and cooling produces the effect mentioned above. The behaviour in polarised light is, therefore, an easy and im- portant means of differentiating the isotropic true fats from lipoids. (For a detailed description of the technique of the polarisation microscope, see Ambronn, Anleitung zur Benutzung des Polarisa- tions Mikfoskopes bei Histologiscken Untersuchungen ; Adami, 362 CONNECTIVE TISSUES. The Myelin and Potential Fluid Crystalline Bodies of the Organism, 1906 ; Kaiserling and Ogler, " Uber das Auftreten von Myelin in Zellen, etc.," Virchow's Archiv., clxvii.). (5) Nile Blue. — When a solution of Nile blue is boiled with sulphuric acid the solution contains, in addition to the original basic oxaziae dye, which unites with fatty acids to form a blue compound, a red oxazone dye, not basic in. character, but soluble in liquid fat and giving it a red colour. The stain, therefore, enables us to distinguish neutral fat and fatty acid. If a given globule contains neutral fat and no fatty acid, it will be coloured red ; if it contains fatty acid only, it will be coloured blue ; but if it contains both neutral fat and fatty acid, it will be coloured a tint between blue and red, depending on the proportion of neutral fat and fatty acid. The principal histochemical group reactions are summarised in the following table : — Table of some important Histochemical Group Reactions. Sudan. Double refrac- tion. Osmic acid. Osmic after bi- chromate. Solubility in cold Acetone. Alcohol. Chloro- form. True fats + + + - + + + -f +1+1111 + + + Cholesterin Cholesterinesters Phosphatides . Cerebrosides . Phospho-cerebrosides + + + + + + + + + + + + - + + + + + + + Cholesterin - fatty ) acid mixtures. f + + + + + + + Although different groups of fatty substances differ in their behaviour towards different staining methods, it is, nevertheless, necessary to proceed with caution in interpreting the results obtained when these methods are applied to tissues. In the tissues the various groups of fatty substances are mixed, so that one globule may contain two or three different substances. The various substances may then form a special kind of colloidal solution, which modifies their physical state and alters their staining reaction completely. The behaviour of mixtures of cholesterin and fatty acids is an example and has been referred to above. Or one substance encloses another substance belonging to a different group. In such a case the staining reactions of the globule would be those of the material which constitutes the envelope. In the adrenal cortex, for instance, CHAPTER XXIX. 363 many globules contain a core of lipoids surrounded by true fats. In sucli a case examination in polarised light is helpful. Differentia- tion by means of different solvents is also possible within limits. Again, the size of the globules is, for obvious reasons, an important factor in the methods which involve mordanting with bichromate. But even if due consideration is given to these reservations, the application of these staining reactions has yielded important results and has profoundly modified our conceptions of the part taken by the fatty substances as constituents of protoplasm. In the first place, it is important to realise that no one single method is a specific staining •method, either for all fatty substances- or for any one group of them. Thus, Sudan III. or Scharlach R. do not stain all fatty substances, as a glance at the table of the group reactions shows. They stain intensely the true fats, and less intensely cholesterinesters and cholesterin-fatty acid mixtures. But many lipoids are not stained by these dyes, and an examination in polarised light is necessary to detect their presence. Osmic acid alone has the widest range as a reagent for fatty substances, and stains all the different groups. With some substances, however, such as chole- sterin or cerebrosides, it may give negative results if they are not present in a state of colloidal solution in the cytoplasm. But since osmic acid stains also substances which are not fatty in nature, e.g., adrenalin in the cells of the adrenal medulla, it cannot be looked upon as a specific stain. In the case mentioned, a differentiation can still be effected by immersing the section in turpentine, which dissolves the fatty substances, even after osmication. The method giving the highest degree of specificity is the use of osmic acid after bichromate. This will stain only the true fats or cholesterin-fatty acid mixtures. Or, expressed in terms of everyday histological technique, the presence of black cell globules in material fixed either in bichromate-osmic mixtures, such as Altmann's, Champy's or Flemming's fixatives, or first in bichromate fixatives, such as Muller's or Zenker's fiuids, and post-osmicated (§ 691) indicates the presence either of true fats or of cholesterin-fatty acid mixtures. These two can then be differentiated by examination in polarised light. It is perhaps equally important to be able to draw the opposite con- clusion, when other facts have indicated the presence of fatty substances. The absence of blackening of the globules under the conditions just mentioned definitely excludes the presence of true fats. Thus, true fats can be proved to be absent from the myelin sheath of normal nerves. The significance of the staining of fatty substances after mordanting with bichromate has already been 364 CONNECTIVE TISSUES. referred to as revealing the essential similarity of Weigert's method for the myelin sheath of nerves and Altmann's method for mito- chondria. The staining reactions of the Golgi apparatus — blacken- ing after prolonged action of osmic acid, staining with hsematoxylin after prolonged mordanting with bichromate — suggest that it is made up of fatty substances, and, further, that these substances are not readily oxidised. They thus correspond in their behaviour to lipoids such as cholesterin. From what has been said, it is clear that for the study of fatty substances several methods must be applied to the tissues. In so far as the selection of a fixative is concerned, it follows that fixatives containing alcohol or chloroform must be avoided. A fixative such as Carnoy, or such mixtures containing strong alcohol or chloroform, are themselves fat solvents, as well as lipoid solvents, and they dissolve away all fat-vacuoles and shrink up cell organs which may be formed partly of other " fatty " sub- stances, e.g., mitochondria. Other fixatives, such as picric acid or corrosive sublimate, while in themselves not fat or lipoid solvents, are unable to prevent the fat from being dissolved away in any subsequent dehydration and cleariag of the tissue. Very few reagents are known which are able to form with fat substances insoluble or scarcely soluble in alcohol and a clearing medium, hke xylol ; osmium tetroxide and, to a less extent, potassium bichromate, are thus valuable reagents to the histologist. Various oils act differently towards these fat OsO^ compounds ; xylol and chloro- form do not easily disintegrate them, but an oxidising oil like tur- pentine will quickly do so. The " compounds " of OSO4 with fats and lipoids are ill understood, but Partington and Huntingford have recently shown (see § 772) that the reduced black substance is a hydrated form of OsOg. Martinotti (see § 772) has recently introduced a new method which may prove of great importance. According to this observer the orange yellow dye chrysoidin (phenyl-azo-m-phenylene-diamide), when applied to fatty tissue and then treated with an oxidising agent, such as bichromate of potash, has the power of preventing the fat globules from being dissolved away in alcohol, benzol and xylol. In preparations the fat looks a brown orange colour. A complete histochemical investigation of fatty cell inclusions comprises, thus, the following methods : — (1) In fresh preparations : (a) Examination in polarised light. (b) Staining with Sudan III. or Scharlach R. CHAPTER XXIX. 365 (c) Staining with osmic acid in solution or as vapour. (2) In frozen sections, material fixed in formol : {a) Examination in polarised light. (6) Staining with Sudan III. or Scharlach R. (c) Staining with Nile blue. _ {d) Repeat (6) and (c) after immersing sections in cold acetone or cold alcohol for a few minutes. (3) In paraffin sections : (a) Fixation in osmic acid. (6) Fixation in bichromate osmic acid mixtures or fixation in bichromate solutions and post-osmication. (c) Prolonged fixation in bichromate and staining with Sudan III. (Ciaccio, Bell). {d) Staining .with heematoxylin (Weigert, Lorrain, Smith, Dietrich). (e) Treatment with chrysoidin and subsequent fixation in bichromate (Martinotti). Such a plan of investigation refers especially to vertebrate materials, but with certain small modifications is applicable to the study of fatty substances in developing eggs, embryos, and to the tissues of invertebrates. Paragraphs 1 and 3 are both applicable to invertebrate tissues and embryos, while in the case of para- graph 2 the tests can be used after the whole embryos or eggs have been fixed in neutralised and suitably diluted formalin. It should be remembered that in all animal cells (and possibly plant cells, too) there exist two categories of cytoplasmic inclusions, Golgi apparatus and mitochondria, which are partly formed of lipoid materials, and which we now know may produce fats, or may metamorphose into fats. In nearly all eggs where fat granules are present, exami- nation has revealed the fact that such " yolk " is derived either from Golgi elements or mitochondria (Gatenby and Woodger, Journ. Roy. Micr. Soc, 1920), and in certain cells of vertebrate tissues we know that mitochondria may change into fat (Murray, Scientific Report Cancer Research Fund, 1919). Reference should be made to the sections on "Mitochondria," "Golgi Apparatus," "Fat," and "Yolk" (§§ 673—713), and especially to the tables in §§ 702, 708, 710 and 712, where some attempt has been made to illustrate the behaviour of the various inclusions after the application of certain well-knoAvn techniques. It is always necessary to ascertain exactly the condition and behaviour of the mitochondria and Golgi apparatus in tissues or cells being investigated for fatty and lipoid substances, particularly 366 CONNECTIVE TISSUES. in view of any inter-relationship which may exist between the former and the latter. See also Bell (Journ. Med. Research, xxiv, 1911, p. 539 ; Journ. of Pathol, and Bad. xix, 1914, p. 105. CiACCio {Centralblatt f. allg. Pathol, and Path. Anatomie, xx, 1909, p. 771 ; Arch. f. Zellforschung, v, 1910, p. 235. Ceamee, Feiss and Bullock (Proceed. Phys. Soc, 1913 ; Journ. of Physiology, xlvi, p. 51. International Congress of Medicine, London, 1913. Section of Pathology). Dieteich {Erg. d. Allg. Pathologic und Patholog. Anat., xiii, 1909, pt. 2, p. 283 ; Deutsche Patholog. Gesellsch., xiv, 1910, p. 263). Kawamura (Die Cholesterinester verfettung. Jena. Gustav Fischer. 1911). Smith and Mair (Jowrw. Pathol, and Bact., xiii, 1909, p. 14 ; Skand. Arch. f. Physiol., xxv, 1911, p. 247). 769. Fixing and Staining. — The choice Ol the fixative depends on the question whether the material is to be examined in frozen sections or in paraffin sections. In any case all fixatives containing acetone, alcohol, chloroform or other fat solvents are excluded. For paraffin sections the material may be fixed in osmic acid alone (1 per cent, in solution, or 2 per cent, if fixed in vapour), or in osmic acid mixed with bichromate solution (see fixatives of Flemming, Altmann, Champy). Or it may be fixed in formol bichromate and treated subsequently with a bichromate-osmic mixture (see methods of Schridde and Marchi). As stated in the general part (see p. 363) the different methods give different results with the various groups of fatty substances. For all these methods very small pieces of tissue must be used. For the effects of alcohol on the blackening of certain fatty substances by osmic acid, see Hand- WERCK, Zeit. wiss. Mik., xv, 1898, p. 177 ; Mulon, ibid., xxii, 1905, p .138 ; Golodetz, ibid., xxviii, 1911, p. 213 ; and Chem. Rev. Fett u. Harzindustrie, xvii, 1910, p. 70 ; Loisel, C. R. Soc. Biol., 1903, p. 826. Another method consists in fixing and mordanting with strong bichromate solution and subsequent staining with Sudan (see below. Bell's method, also Ciaccio) or with hsematoxylin (methods of Weigert for nervous system ; also Lorrain Smith, Dietrich). For examination in frozen section the tissue may be fixed in formol saline or formol-bichromate, or the stain may be applied directly to the fresh tissue after teasing. For fine cytological work, the formol should be neutralised by shaking with solid calcium carbonate. Hays Bullaed (Atner. Journ. Anat., xix, 1916) recom- mends neutralisation and distillation method of Gustav Mann (Physiological Histology, Oxford, 1902) : neutralise commercial CHAPTER XXIX. 367 solution with sodium or lithium carbonate, and freshly distil. A 20 per cent, solution is then prepared and rendered isotonic : •75 gms. of NaCl to 100 c.c. of fluid. With short fixation (fchirty-five minutes to five hours), the quantity of fat usually does not differ from that seen in fresh tissue (Bullard). Cut on freezing microtome, stain by one of the methods given below, preferably Herxheimer's alkaline scarlet red. As control use also fresh tissue. For quinolein blue, see § 322. Daddi {Arch. Ital. Biol., xxvi, 1896, p. 413) stains fat in tissues by treating for five to ten minutes with concentrated alcoholic solution of Sudan III. washing for the same time with alcohol, mopping up with blotting paper, and mounting in glycerin. Similarly Rieder, see Zeit. wiss. Mik., xv, 1898, p. 211. The alcohol for making the stain should be of 70 per cent., according to most authors, though Sata (Beitr. path. Anat., xxviii, 1900, p. 461 ; Zeit. wiss. Mik., xviii, 1901, p. 67) employs 96 per cent. Rosenthal (ibid., xix, p. 469 ; Verh. path. Ges., September, 1899, p. 440) insists that the washing-out be done with alcohol of exactly 50 per cent. MiCHAELis {Yirchow's Arch., clxiv, 1901, p. 263) recommends Scharlach R (syn. " Fettponceau "). Stain for fifteen to thirty minutes in a saturated solution in 70 per cent, alcohol, and mount in glycerin or levulose. Other authors also commend this stain. Heexheimer {Deutsche med. Wocherischr., xxvii, 1901, p. 607 ; Zeit. wiss. Mik., xix, 1902, p. 66) makes a solution of 70 parts of absolute alcohol, 10 of water. 20 of 10 per cent, caustic soda, and Scharlach R to saturation. This makes a stronger solution, and stains in a couple of minutes. Wash out with alcohol of 70 per cent. With either solution the staining must be done in a covered vessel or the stain will precipitate. Similarly Bell, Amer. Journ. Anat., ix, 1909, p. 401, and Anat. Rec., iv, 1910, p. 199. Herxheimbr also (Centralb. allg. Path., xiv, 1903, p. 841 ; Zeit. wiss. Mik., xxi, 1904, p. B7) recommends a saturated solution of the dye in a mixture of equal parts of acetone and 70 per cent, alcohol. He also [Deutsche med. Wochenschr., xxvii, 1909, p. 607 ; Zeit. wiss. Mik., xix, 1902, p. 67) has had very fine 'results by staining for 20 minutes in a saturated solution of Indophenol in 70 % alcohol. MoLLisoN (Ztit. wiss. Zool, Ixxvii, 1904, p. 529) has had good results by staining gelatin sections for a few minutes in strong 368 CONNECTIVE TISSUES. extract of Alkanna in 96 per cent, alcohol, and mounting in glycerin or syrup. LoEEAiN Smith (Journ. Path. Bad., xii, 1907, p. 1) finds that Nile blue stains fatty acids blue and neutral fats reddish. Similarly Eisenbeeg (Virchow's Arch., cxcix, 1910, p. 502), who recommends aqueous solution of Nilblau BB. Benda (ibid., clxi, 1900, p. 194) finds that free fatty acids can be detected by Weigert's neuroglia mordant. See also Beenee, ibid., clxxxvii, 1907, p. 360, and Fischler, Zeit. wiss. Mik, xxii, 1905, p. 263. Okajima (ibid., xxix, 1912, p. 67) extracts red capsicum berries for some days with alcohol, and evaporates down to one fifth. This stains only fatty bodies : amongst them, myelin. See also Kingsbury, Anat. Rec, v, 1911, p. 313. 770. Removal of Fatty Substances. — If not treated with osmic acid or mordanted with strong bichromate, alcohol, ether, chloroform, pyridine, xylol, will readily dissolve fatty substances. Osmicated fats and lipoids are more resistant, especially if osmic acid and bichromate have been combined. It can then be removed in a few hours by alcoholic hydrogen peroxide (10 per cent. HaOj in 80*per cent, alcohol) or in twenty-four hours by oil of turpentine. Ether, creosote, xylol, clove oil and chloroform will also remove osmicated fats and lipoids if allowed to act sufficiently long. See also Flemming in Zeit. wiss. Mikr., 1889, pp. 39, 178. 771. Differentiation between Fats and various Lipoids. — Fix in for- mol and prepare frozen sections. Stain some with Sudan or Soharlach (see above § 769), others with osmic acid. Leave some unstained. Globules which stain with Sudan or Soharlach and osmic acid, and which in unstained sections show no double refraction, can be identified with certainty as true fats. This may be confirmed in paraffin sections by fixing in bichromate and subsequent treatment with osmic acid as in the methods of Schridde and Marchi. These globules should then reduce osmic acid. But the presence of double refraction must not be taken as excluding the presence of true fats since the globules may be a mixture of true fats and double refracting lipoids. The deduction that true fats are absent can be made when tissue containing fatty material as indicated by blackening with osmic acid fails to give this blackening after previous treatment with bichromate, as for instance in normal peripheral nerve. The histochemical differentiation between true fats and lipoids is much more difficult when these substances are mixed in one and the same globule than when one cell contains several globules of which some are composed entirely of true fats while others contain lipoids. In the latter case methods may be applied which depend on differences in the solubility in various solvents. Dbflandre (Journ. Anat. Fht/s., 1904-, p. 80) fixes in formol of 4 per CHAPTER XXIX. 369 cent, and brings into acetone, in wMch fat is dissolved, but not lecithin, which can then be stained by osmium. See also Ciaccio, Arch. Zelljorsch, v, 1910, p. 235 ; and Fischlee, Zeit. wiss. Mik., xxii, 1905, p. 262 ; Loisel, 0. B. Soo. Biol, Iv, 1903, p. 703. Bell with a modification of Dietrich's and Ciaccio' s methods (Journ. Path, and Bact, xix) claimed to be able to distinguish between fat drops mainly of triolein and those that principally contain " lipoids." The former appear in annular shape, the latter are quite solid. In the former case the centre of the droplet is not chromated, and therefore dissolves out in xylol used for imbedding. Fix at 45° C. to 50° C. in 10 per cent, aqueous KaCraO,, 100 c.c, acidified by 5 c.c. of acetic. Wash, dehydrate and imbed in paraffin. Fasten 3 to 5 ;u. sections to slide with albumen water. Treat sections in xylol, absolute alcohol, and transfer to freshly prepared solution of Sudan III in 80 per cent, alcohol for ten minutes. Einse off excess stain in 50 per cent, alcohol ; transfer to water to stop action of alcohol. Counterstain in Delafleld's haematoxylin, wash in water, differentiate in acid alcohol, wash and mount in glycerin gum-arabic. 772. Mounting Fat. — ^After treatment with osmic acid sections can generally be mounted in balsam without special precautions. Many lipoids, however, fade even after osmication, if kept for a long time in balsam. In some cases it may suffi.ce to avoid absolute alcohol and essences as much as possible, and mount direct in alcohol balsam or euparal, or clear with cedar oil, which has little solvent action. For very delicate fats it may be necessary to avoid alcohol of more than 70 per cent., or avoid it altogether, and mount in glycerin or levulose. Chrysoidin (L. Maetinotti, Zeit. Physiol. Chem., xci, 1914) fixes tissue in 10 per cent, formol, sections on a freezing microtome, washes in aq. dest., and immerses for five to ten minutes up to several hours in a 1 per cent, aqueous solution of chrysoidin. Wash not longer than one minute in aq. dest., and treat in 10 per cent. K2Cr207 or CrOa for one minute, wash, dehydrate in benzol and xylol, and mount in neutral balsam. Bone.* t '?'?S. Bone, Non-decalcified. — Ranvier {Traite, p. 297) has the following : Bones should be plunged into water, without being allowed to dry, as soon as the surroimding soft parts have been removed, and should be divided into lengths with a saw whilst wet. The * For a detailed review of the whole subject, see the paper of Schaf- PBB in Zeit. wiss. Mik., x, 1893, p. 167, or the article " Knochen und Zahne " in Encycl. mik. Technik. ■j- This section has been revised by J. Thornton Carter, Esq., F E.M.S., of the Zoological Department, University College, London, M. 24 370 CONNECTIVE TISSUES. medulla should then be driven out from the central canal by means of a jet of water ; spongy bones should be treated as follows : An epiphysis having been removed, together with a small portion of the diaphysis, a piece of caoutchouc tubing is fixed by a ligature on to the cut end of the diaphysis, and the free end of the piece of tubing adapted to a tap through which water flows under pressure ; they are then put to macerate for several months, the liquid being changed from time to time. As soon as all the soft parts are per- fectly destroyed, the bones may be left to dry. Thin sections may then be cut with a saw and prepared by rubbing down with pumice-stone. Compact pumice-stone should be taken and cut in the direction of its fibres. The surface should be mois- tened with water and the section of bone rubbed down on it with the fingers. When both sides of the sections have been rubbed smooth in this way, another pumice-stone may be taken, the section placed between the two, and the rubbing continued. As soon as the section is thin enough to be almost transparent it is polished by rubbing with water (with the fingers) on a Turkey hone or litho- graphic stone. Spongy bone should be soaked in gum and dried before rubbing down (but see Von Koch's copal process and Eheenbaum's colophonium process). ' ScHAFPER (Zeit. wiss. Mile, x, 1893, p. 171) grinds and polishes on stones of graduated fineness. For the process of Weil for bones and tseth see § 180. Rose {Anat. Anz., vii, 1892, pp. 512-519) follows Koch's process. He penetrates first with a mixture of cedar oil and xylol, then with pure xylol, and imbeds in solution of Damar in chloroform or xylol. The method can be combined with Golgi's impregnation. Panz [Anat. Becord, xiv, 1918, p. 493) employs sand or carborundum paper of diflerent grades of coarseness for grinding, using the back or smooth side, of a piece of sandpaper for polishing the section. He recommends shellac in preference to balsam for attaching the section to the glass slip. White {Journ. Roy. Mic. Soc, 1891, p. 307) recommends the following : Sections of osseous or dental tissue should be cut or ground down moderately thin, and soaked in ether for twenty- four hours or more. They should then be put for two or three days into a thin solution of fuchsin in collodion, then into spirit to harden the collodion. After this they are ground down to the requisite thinness between two plates of old ground glass, with water and pumice powder, and mounted, surface dry, in stiff balsam or styrax, care being taken to use as little heat as possible. Lacrmse, canaliculi, and dentinal tubuli are found infiltrated by the coloured collodion. CHAPTER XXIX. 371 Hanazawa (Dental Cosmos, lix, 1917, pp. 125 et seq.) gives a number of methodfs for staining giound and decalcified sections of dentine to demonstrate its minute structure. Matschinsky [Arch. miJc. Anat., xxxix, 1892, p. 151, and xlvi, 1895, p. 290), after grinding, impregnates with nitrate of silver. For similar method of Rupeecht, see Zeit. wiss. Mile, xiii, 1896, p. 21, wherein see also quoted (p. 23) a method of Zimmeemann. CsoKOE {Verh. anat. Oes., 1892, p. 270) describes a saw which will cut fresh bone to 120 ,i ; and Aendt [Zeit. wiss. Mile, xviii, 1901, p. 146) a double saw which wiU also give very thin sections. 774. Mounting. — To show lacunae and canaliculi injected with air, take a section, or piece of very thin flat bone, quite dry. Place on a slide a small lump of solid balsam, and apply just enough heat to melt it. Do the same with a cover glass, place the bone in the balsam, cover, and cool rapidly. When thin ground sections of enamel are mounted in Canada balsam it is found often that they appear almost structureless. To demonstrate the enamel pattern of such sections they may be etched by immersion in -6 per cent, of hydrochloric acid in 70 per cent, alcohol, or in a weak aqueous solution of picric acid, and mounted in Camsal balsam or Euparal, media which, on account of their low index of refraction, will be found to disclose the structure of the enamel more easily. 775. Sections of Bones or Teeth showing the Soft Parts. — A developing tooth with its epithehal enamel-organ, its mesodermal dentinal papilla, and its layers of partially calcified enamel and den- tine, is made up of very delicate structures of different consistency and so peculiarly liable to unequal shrinkage, with consequent distortion during the period of fixation and in the subsequent processes passed through in the preparation of sections. Further, post-mortem changes in the ameloblasts occur within a very few minutes after death leading to a less precise behaviour to stains than is found in the case of cells which are fixed immediately after death. For the examination of developing teeth in situ, jaws may be fixed in corrosive-formalin-acetic mixture, in Bouin's picro-formol, in Zenker's mixture or Helly's modification thereof, or in Sansom's modification of Carnoy's mixture (§ 86). For the study of the micro-anatomy of the enamel-organ and the dentinal papilla, a young pup or a kitten, two or three days old, is killed, preferably by a blow on the head. The jaws are removed and the bone of the under-surface of the mandible pared away by a sharp scalpel until the bases of the tooth-germs are almost exposed. 372 CONNECTIVE TISSUES. The muco-periosteum is grasped with, a pair of forceps and stripped from the bone, when the tooth-germs will come away attached thereto. Sansom's modification of Carnoy's mixture, employed at blood- heat, is particularly effective when the tooth-germs have been exposed in the manner outlined above, fixation therein being com- plete in from five to ten minutes. They are then passed through successive baths of alcohol of 30 per cent, and 50 per cent., each for fifteen minutes ; 70 per cent., to which is added tincture of iodine, for four hours ; 90 per cent, for thirty minutes ; and into two changes of absolute alcohol, each for fifteen minutes or longer. The tooth-germs are then transferred to a mixture of equal parts of absolute alcohol and carbon disulphide for one hour, two changes of pure carbon disulphide, each of fifteen minutes, then for thirty minutes into carbon disulphide saturated with paraffin at 30° C, transferred to carbon disulphide saturated with paraffin at 42° C. for a like period, and finally into two baths of paraffin, in each half an hour. Imbed for cutting in pure paraffin. By the employment of this method the amount of shrinkage in the tissues is extremely slight and the dentine does not become hardened, so that the tooth-germs of the incisors may be cut without decalcification. In the case of the canine and molar tooth-germs a short period of decalcification may be necessary, and for this purpose a rapid and delicate method lies in the employment of Zeigler's method (Festschr. f. Kupffer, 1899, p. 51), in which, by the use of a 5 per cent, solution of sidphujous acid, the insoluble tricaldum phosphate is changed into the readily soluble mono- calcium phosphate. To demonstrate cytological detail no stain equals iron-hsema- toxylin followed by a counter-stain of picric-lichtgrun or of Rubin S in picrate of ammonia. It cannot be too strongly emphasised that the precision of staining methods depends on the rapidity with which fixation of the tissues is effected after death. Refer to §31. For large jaws imbedding in celloidin, or, when serial sections are required, double imbedding in celloidin, parlodion or photoxylin and paraffin is recommended (§ 171). Mummery {Phil. Trans. B., ccviii, 1917, p. 258) deprecates the employment of paraffin for imbedding the tooth-germs of fishes, considering the heat employed to be very injurious to the delicate fenamel organs, and advocates the use of the freezing method in obtaining sections. See carbon disulphide method above. Nealbt (Amer. Mon. Mie. Journ., 1884, p. 142 ; Journ. Boy. Mic. CHAPTER XXIX. 373 8oG., 1885, p. 348) says that perfectly fresTi portions of bone or teeth may be ground with emery on a dentist's lathe, and good sections, with the soft parts in situ, obtained in half an hour. Hopewull-Smiti-i {Journ. Brit. Dent. Ass., xi, 1890, p. 310 ; Journ. Boy. Mic. Soc, 1890, p. 529) says that for preparing sections of tefeth showing odontoblasts in situ the best plan is to take embryonic tissues. A lower jaw of an embryonic kitten or pup may be taken, and hardened in solution of MiiUer followed by alcohol, then cut with a freezing microtome. Weil (he. eit, § 180) fixes pieces of fresh teeth in sublimate, stains with borax-carmine, brings them through alcohol into chloroform and chloroform-balsam, and after hardening this by heat proceeds to grind as usual (§ 177). See also Rose, § 773. 775a. — For the study of the vessels in teeth, Lepkowsky {Anat. Hefte, viii, 1897, p. 568) injects with Berlin blue, hardens the teeth with a piece of the jaw for one or two days in 50 per cent, formol, decalcifies in 10 per cent, nitric acid (eight to fourteen days, change frequently) and makes ceUoidin sections. For decalcification of teeth, see also § 546 (Kousseau, Bodeckek and Fleischmann). Bodecker finds Rousseau's process not appli- cable to human teeth : the acid must be added to the fluid celloidiu. For the study of the lymphatics in the dental pulp, Dewey and NoYES (Dental Cosmos, lix, 1917, pp. 436 — 44) first' iuject the blood vessels with carmin-gelatin. Then 2 grms. of Prussian blue (oil colour in tubes) is stirred with 3 grms. of turpentine oil in a glass mortar for five minutes ; 15 grms. of sulphuric ether is added, and this fluid filtered through flannel or chamois skiu. After the injection of this fluid the head is placed for twenty-four hours or longer in 20 per cent, formalin, and- then the injected teeth are carefully removed and the pulps examined. Later it was found that more constant results were obtained when the injection of the blood vessels followed that of the Prussian blue.- Prussian blue injected directly into the pulps and trypan blue or lithium carmine injected intravenously or intraperitoneally were also employed. See §§ 780 and 790. Wellings (Proc. Sixth Internat. Dent. Cong., pp. 47 et seq.) demon- strated intra-vitam staining of dental and adjacent tissues by means of trypan blue (§ 780). MuMMEKY (Phil. Trans. B., ccii, 1912), for the fixation of the nerve-tissue of the dental pulp, finds formaliu to be preferable to all other fixing agents, employing 10 parts of the 40 per cent, com- mercial formalin to 90 parts of water. Decalcification is effected by means of 33-3 per cent, formic acid. After thorough washing he leaves for twenty-four hours in a strong 374 CONNECTIVE TISSUES. solution of dextrin (which he finds preferable to gum arable), and sections are cut on the freezing microtome, by the employment of which he is able usually to obtain thinner sections than when paraffin is used for imbedding. The sections are stained either by means of iron and tannin, iron-hsematoxylin (Benda), Congo red, Ranvier's modification of Lowet's gold chloride process, or by Cajal's method, where : — (1) Small pieces of the decalcified tooth, not more than 4 milli- metres thick, are placed in 50 c.c. of rectified spirit, to which 3 or 4 drops of ammonia may be added, and kept in this solution for from four to six hours. (2) Transfer to absolute alcohol for twenty-four hours. (3) Rinse with distilled water. (4) Place in a large quantity of 1-5 per cent, solution of silver nitrate, and keep in .warm incubator at about 35° C. for five or six days. (5) Rinse in distilled water for a few seconds. (6) Place in the following solution for twenty-four hours : — Hydrokinone . . . . . 1 to 1-5 grm. Distilled water ..... 100 c.c. Formol . . . . . . 5 to 10 c.c. Rectified spirit . . . . . 10 to 15 c.c. (7) Wash in water for some minutes. (8) Cut sections, and mount. The presence of nerve-end cells in the dental pulp was demon- strated by Mummery {Phil. Trans. B., ccix, 1920), by means of a modification of the gold method of Beckwith. Teeth, immediately after extraction, are placed in a solution of formol and water or of formol and normal salt solution, preferably 4 per cent, of formol. This is, after a few days, changed to a 10 per cent, solution, and the teeth kept in this for at least a fortnight. Decalcification is effected by means of a 33-3 per cent, solution of formic acid in distilled water, to which 5 per cent, of formol may be added. (Mummery states that neither he nor Dependorf has ever procured good nerve preparations of teeth which have been decalcified in the mineral acids.) Wash in running water for twenty-four hours, then for a few minutes in distilled water. The pieces are taken from the distilled water and suspended by threads in a large quantity of a weak solution of gold chloride (1 in 5,000). Bach piece should be suspended in at least 100 c.c. CHAPTER XXIX. 375 of the solution, in which it is left in the dark for from four days to one week, according to its size. On removal from the gold solution it is washed for a few minutes only in distilled water. Eeduction is effected by placing the pieces in a 20 per cent, solution of caustic soda for four minutes, then rinsing in water and placing in a 10 per cent, solution of potassium carbonate for from half an hour to an hour. This is then drained off, and the pieces are placed in a 10 per cent, solution of potassium iodide for a short time — usually five to ten minutes. As soon as seen to darken, the pieces are removed from this solution to water, placed in gum for twelve hours, and sections cut on the freezing microtome. After dehydration the sections are mounted in camsal (propylic) balsam. 776. ViVANTE {Intern. Monasschr. Am,t. u. Phys., is, 1892, p. 398) impregnates portions of frontal bone of four to six months calves, which, ara not more than 3 to 4 millimetres thick, by Golgi's rapid bichromate and silver process. After impregnation the specimens should be decalcified in von Ebner's mixture (§ 562), well washed with water, and brought into solution of carbonats of soda, and finally imbedded in paraffin. For his quinolein blue method see fourth edition. For Underwood's gold process for teeth, and for that of Lepkowski, see third edition, or Anat. Anz., 1892, p. 294. Law (Proe. Boy. Soc. Med., i, 1908, p. 45) studies nerve^endings in teeth of mammals by treating paraffin sections of decalcified tissue with Bethe's molybdenum toluidin blue (details in Journ. Boy. Mier. Soc., 1908, p. 518). Van der Strioht {Carnegie Instit. Embryol. Contrib., No. 21) fixes the isolated cochlea in a 5 per cent, aqueous solution of tri- chloracetic acid, or in Bouin's or Zenker's fiuid, and stains, before imbedding, in borax-carmine. The sections are afterwards stained in iron-hsematoxylin, Congo red and light green. He obtained the best results with the membrana tectoria by making one or two openings in the bony wall of the fresh cochlea and exposing the piece for fifteen minutes to the vapours from an aqueous solution of osmic acid or by submerging it in a 1 per cent, solution of the same for one hour. Afterwards fixation was completed by immersion in trichloracetic acid, Bouin's fluid or Zenker's fluid, and the series of sections therefrom stained as above. By this method some of the turns of the cochlea give very good preparations of the structure of the membrana tectoria. The mitochondria are also visible within osteoblasts, osteoclasts, connective-tissue cells, all epithelial cells, and the sensorial elements. Mitochondria in odontoblasts and osteoblasts may be demon- strated by fixation in Flemming-without-acetic followed by staining 376 CONNECTIVE TISSUES, in iron-hsematoxylin (§ 679), and the Golgi apparatus in these cells is well shown by the employment of Grolgi's method, Cajal's method, or of Da Fano's modification thereof (§§ 844, 849), though a negative image of this cell-element is clearly shown when the tissues are fixed in Sansom's modification of Carney's mixture. 77'?. Bone, Decalcified (Flemming, Zeit. wiss. Mik., 1886, p. 47).— Sections of decalcified bone are soaked in water, dehydrated with alcohol under pressure, dried under pressure and mounted in hard balsam melted on the slide. They show the lacunar system injected with air as in non-decalcified sections. 778. Stains for Cartilage and Decalcified Bone. — See hereon ScHAFFER in Zeit. mss. Mik., v, 1888, p. 1 ; and Encycl. mik. Technik., art. " Knochen." KoLLiKER (Zeit. wiss. Zool., xliv, 1886, p. 662) treats sections of decalcified bone with concentrated acetic acid until they become transparent, and then puts for one quarter to one minute into a concentrated solution of indigo-carmine, washes and mounts in glycerin or balsam. The fibres of Sharpey appear red, the remaining bone substance blue. ScHAFFEE (Zeit. wiss. Mik., v, 1888, p. 17) employed at one time a safranin method modified from Bouma (Centralb. med. Wiss., 1883, p. 866), for which see previous editions. He now (Encycl. mik. Tech., 1910, i, p. 762) stains sections for twenty-four hours in a bath of 20 c.c. of water with 1 drop of 1 per cent, solution of safranin (or thionin) and (apparently) mounts in balsam. The safranin stain will keep if the material is cartilage which has been fixed in picro- sublimate ; otherwise it must be fixed with ammonium molybdate of 5 per cent, before dehydrating. SoHMOEL (Centralb. allg. Path., x, 1899, p. 745) stains in a mixture of 2 c.c. concentrated solution of thionin in alcohol of 50 per cent, and 10 c.c. of water for ten minutes, rinses and puts into saturated aqueous picric acid for thirty to sixty seconds. Einse and pass through graded alcohols into origanum oil or carbol-xylol and balsam. Matrix yellow, cells red, fat-cells violet. He also describes a more compUcated method with thionin and phosphotungstic or phosphomolybdic acid. Moll (Centralb. Physiol, xiii, 1899, p. 225) stains embryonic cartilage for six to twenty-four hours in orcein 0-5 gr., alcohol 40, water 20, hydrochloric acid 20 drops, and mounts in balsam. Matrix blue, nuclei red. Kallius (Anat. Hefte, xxx, 1905, p. 9) stains first with borax carmine or alum-carmine, then (sections) for ten minutes in satu- CHAPTER XXIX. 377 rated solution of thionin, and washes out with alcohol of 70 per cent. - Said to be specific for embryonic cartilage. Vastaeini-Ceesi (Att. Accad. med.-chir. NapoU, 1907, p. 4) stains sections of embryonic cartilage with borax carmine, then with muchsematein (alcoholic solution without acid), and then with Orange G. in alcohol. Bayeel's method for ossifying cartilage {Arch. mik. Anat., 1885, p. 35) : — Portions of ossified cartilage are decalcified as directed, § 555, cut in paraffin, stained in Merkel's carmine and indigo- carmine mixture, and mounted in balsam. Mayee (Grundziige, Lee and Mayee, 1910, p. 393) prefers to all these resorcin fuchsin, § 758, the precipitate being freed from iron chloride by washing before dissolving in the alcohol. Aqueous solution of henzoaeurin has been commended as a stain for ossifying cartilage by Zschokke, see Zeit. wiss. Mile., x, 1893, p. 381. A process of Baumgakten's has been given, § 388. MoERNEE (Sktmdinavisches Arch. Physiol., i, 1889, p. 216 ; Zeit. wiss. Mik., vi, 1889, p. 508) gives several stains for tracheal cartilage, chiefly as microchemical tests, for which see third edition. See also a critique of these methods by Wolteks in Arch. mik. Anat., xxxvii, 1891, p. 492 ; and on the whole subject of cartilage see Schief- feedeckek's Gewebelehre, p. 331. PusAEi (Arch. Ital. Biol., xxv, 1896, p. 200) makes sections of fresh cartilage, puts them for twenty-four hours into 1 per cent, nitrate of silver, washes, dehydrates, and exposgs to the light in balsam. See also Disse, Anat. Anz., xxxv, 1909, p. 318, a stain for dentine (haemalum followed by a mixture of Saurerubin and Orange G) ; and Eetteeee and Lelievee, O. B. Soe. Biol., Ixx, 1911, p. 630. Skeletons of Embryos. 779. Cartilaginous Skeletons of embryos (Van Wijhb, Proc. K. Akad. Wetensch. Amsterdam, 1902, p. 47) may be studied by staining embryos for a week in a solution of 0-25 grm. methyl en blue in 100 c.c. of 70 per cent, alcohol with 1 per cent, of hydrochloric acid. Wash out in alcohol with 1 per cent, of hydrochloric acid until no more colour comes away (about a week) and moimt in balsam. The cartilage remains blue, all the other tissues being colourless. Similarly, Lundvall {Anat. Anz., xxv, 1904, p. 219, and xl, 1912, p. 639), using toluidin blue. Thionin blue also may be used. Similarly also Bakat {Verh. Anat. Oes., 1902, p. 248), with Bismarck brown (the embryos having been previously treated with nitric acid of 3 per cent.). For flsh embryos. Professor E. S. Goodrich, of Oxford, informs me that thionin is excellent. For the Spalteholz method of clearing such preparations see § 592. 378 CONNECTIVE TISSUES. 780. Demonstration of Centres of Osteoblastic Activity by Trypan Blue (P. G. Shipley and C. C. Macklin, Anat. Record, x, 1915—16). — If an azo dye like trypan blue be administered to a very young animal, the bones are stained quickly and very intensely with vital colour. The dye is injected in a 1 per cent, solution into the peri- toneal cavity (less preferably subcutaneously). The animal is killed forty-eight hours after staining, and the tissues are fixed by 10 per cent, neutral formalin injected through blood vessels, followed by immersion in 10 per cent, formalin for twenty -four to forty-eight hours. Bones are washed thoroughly, hardened in ascending grades of alcohol, after which the soft parts are dissected away. Clear in benzol and then in oil of wintergreen. Study with dissecting microscope. Eefer also to J. Thornton Carter, § 775a. 780a. Potash Method for Osteoblastic Centres (Schultze, Grundriss d. Entwickl. d. Menschens, 1897, and F. P. Mall, Amer. Journ. Anat., v. No. 4, 1905-6). Embryos of mammals after fixation in alcohol may be cleared, for the study of the ossification centres, by means of weak potash. For alcohol specimens Mall considers that Schultze's solution is too strong, and uses instead a 1 per cent. KOH solution for a few hours. With weak solutions the tissues of the smaller embryos remain firm, and, in the end, the specimen is transparent, with the bones held in place. After treatment with the potash, the embryo is placed in the following solution for days, or even months : — Water 79 Glycerin 20 Potash ........ 1 From time to time the embryo may be returned to a 3 per cent, solution of potash for a number of hours to hasten the clearing process ; then returned to the glycerin solution, which helps to hold the parts together. When properly cleared, upgrade gradually to pure glycerin, in which they may remain. Mall (op. cit.) c\ea,is formalin embryos in 10 per cent, potash for about a month or longer. Formalin renders the connective tissues very tough, and this strong KOH solution is necessary. Refer also to § 592. CHAPTEE XXX. BLOOD AND GLANDS. Blood. 781. Fixing and Preserving Methods. — The school of Ehrlich used to fix by heat. A film of blood was spread on a cover-glass and allowed to dry in the air, and then fixed by passing the cover a few times, three to ten or twenty, through a flame, or by laying it face downwards on a hot plate kept for several minutes or as much as two hours at a temperature at which water not only boils, but assumes the spheroidal state (110° to 150° C). For details see GuLLAND, Scottish Med. Journ., April, 1899, p. 312 ; Kubinstein, Zeit. wiss. Mik., xiv, 1898, p. 456 ; Ziblina, ibid., p. 463. But I believe they have now well-nigh abandoned this barbarous practice. In wet methods either the blood is mixed at once, on being drawn, with some fixing and preserving medium, and studied as a fluid mount, or films are prepared and put into a fixing liquid before they have had time to dry, or after drying in the air without heat for a few seconds (at most ten to thirty). To make a film, place a very small drop of blood on a perfectly clean sUde. Bring down on to the slide the edge of another slide held over it at a slope ; move this along till it touches the edge of the drop and the blood runs along the angle between the two slides. Then move the second slide away from the drop, and the blood will follow it and be drawn out into a film without being crushed. Simi- larly with two cover-glasses, to make a cover-glass film, which can be floated face down on to fixing or staining liquids in a watch-glass. Some persons make films by flattening blood between two cover- glasses which are afterwards separated by sliding the one over the other ; but that produces an injurious kneading of the cellular elements. Most of the usual fixing agents are applicable to blood. But it is often necessary to employ only such as are favourable to certain stains. Those most recommended in this respect are alcohol, formol, sublimate (should not be too strong), osmic acid in very light fixation, or absolute methyl alcohol, which is an energetic fixative of dried films. 380 BLOOD AND GLANDS. Air-dried films ought to be fixed before putting iato aqueous or glycerin stains, else they will wash off ; but this is not necessary for alcoholic stains. 782. Fijong and Preserving in Bulk. — Most morphologists are agreed that by far the most faithful fixing agent for blood-corpuscles is osmic acid. A drop or two of blood (Biondi recommends two drops exactly) is mixed with 5 c.c. of osmic acid solution, and allowed to remain in it for from one to twenty-four hours. As a rule the osmic acid should be strong — 1 to 2 per cent. Fixed speci- mens may be preserved for use in acetate of potash solution (Max Flesch, Zeit. wiss. Mik., v, 1888, p. 83). Griesbach also {ibid., 1890, p. 328) combines the osmic acid with certain stains. He mentions methyl green, methyl violet, crystal violet, safranin, eosin, Saurefuchsin, rhodamin, and iodine in potassic iodide. Rossi {ibid., vi, 1889, p. 475) advises a mixture of equal parts of 1 per cent, osmic acid, water, and strong solution of methyl green, permanent mounts being made by means of glycerin cau- tiously added. EwALD {Zeit. Biol., xxxiv, 1897, p. 257) mixes 3 to 4 drops of blood of amphibia or reptiles with 10 c.c. of a solution of 0-5 per cent, osmic acid in 0-5 per cent, salt solution (for mammals 0-6 to 0-7 per cent, salt), siphons off the supernatant liquid after twenty- four hours with his capillary siphon (§ 3, p. 4), and substitutes water, alum-carmine, etc., and lastly, 50 per cent, alcohol. Weideneeich {Arch. mik. Anat., Ixxii, 1908, p. 213) lays a cover with a drop of blood on ib on a layer of agar-agar (1 per cent, in salt solution of 0-8 per cent.), and after five minutes runs in osmic acid of 1 per cent., and after five minutes more removes the cover. Dekhuyzen {Anat. Anz., xix, 1901, p. 536) recommends a mixture of either 3 or 9 volumes of 2 per cent, osmic acid with 1 of 6 per cent, acetic acid, containing \ per cent, of methylen blue, which he calls " Osmacet." The mercurial liquids of Pacini (§ 414) used to be considered good. Hayem (" Du Sang," etc., Paris, 1889 ; see also Zeit. wiss. Mik., vi, 1889, p. 335) has the following formula : sublimate 0*5, salt 1, sulphate of soda 5, and water 200. This should be mixed with blood in the proportion of about 1 : 100. Eosin may be added to it. Lowit's formula {Sitzb. h. Akad. Wiss. Wien, xcv, 1887, p. 144) consists of 5 c.c. cold saturated sublimate solution, 5 grms. sulphate of soda, 2 grms. salt, and 300 c.c. water. Mosso finds that both of these are too weak in sublimate. CHAPTER XXX. 381 DuBOSCQ [Arch. Zool. Exper., vi, 1899, p. 481) uses (for blood of Chilopoda) a solution of acetic acid, copper acetate, copper chloride, osmic acid, thionia, 1 grm. each, water 400, which, mixed with the blood, fixes and stains in about two minutes. Formol has lately been used. Marcano {Arch, de Med. Exper., xi, 1899, p. 434) mixes fresh, blood with a mixture of 100 parts of sodium sulphate of sp. gr. 1-020 and 1 of formol ; or with water 85 to 100 parts, sodium chloride 1, and formol 1. KiZER (Journ. Roy. Mic. Soc, 1900, p. 128) simply mixes 1 drop of blood with 3 of 2 per cent, formalin, and allows to stand for an hour. ScHRiDDE {Hcemat. Techn. Jena, 1910, p. 17) lets blood drop into a mixture of 1 part of formol, 9 of liquid of Mxiller, and 10 of water, fixes therein for two to four hours at 40° C, filters, washes and brings through alcohol and chloroform into paraffin for sectioning. 783. Fixing and Preserving in Films. — Muir {Journ. of Anat. and Phys., xxvi, 1892) makes cover-glass films and drops them into saturated sublimate solution, and after half an hour washes, dehy- drates, and passes through xylol into balsam. GuLLAND {Brit. Med. Journ., March 13th, 1897 ; Scottish Med. Journ., April, 1899) makes cover-glass films, and after a few seconds ' drops them face downwards into a solution of — Absolute alcohol saturated with eosin . . 25 c.c. Pure ether . . . . . . 25 „ Sublimate in absolute alcohol (2 grms. to 10 c.c.) 5 drops. After three or four minutes they are washed, stained, and mounted in balsam. For Jenner's fixing and staining and staining method, see next section. Many recent authors fix wet films with formol. Benario {Deut. med. Wochenschr., 1895, p. 572) mixes 1 part of 10 per cent, formol with 9 of alcohol (the mixture must be freshly prepared), and plunges films into it for a minute. Similarly Gulland, with 1 part of formol to 9 of alcohol. Similarly Wermel (see Zeit. wiss. Mik., xvi, 1899, p. 50), who combines various stains (methylen blue, eosin, gentian, etc.) with the formol. Edington {Brit. Med. Journ., 1900, p. 19) exposes films for fifteen to thirty minutes to vapour of formol under a bell-jar. Scott {Journ. of Path, and Bacter., vii, 1900, p. 131) exposes 382 BLOOD AND GLANDS. films to the vapour for about five seconds and drops into absolute alcohol, and after fifteen minutes stains and mounts. A short exposure (thirty seconds) to vapour of osmium has also been recommended. SzECSi {Deutsch. med. Wochschr., 1913, p. 1584) has recommended Lucidol for blood smears, and smears of faeces containing protozoa and cysts. The formulae for an acetone and a pyridin solution will be found on p. 59, § 107, and also of an acetone-xylol solution for subsequent washing of the smears. It is best to keep a sufiicient quantity of the fixing solutions in staining jars. Make a smear, allow it to dry, and place it in the acetone peroxide of benzol solution for fifteen minutes ; transfer to the acetone xylol solution for ten minutes in order to remove the lucidol ; wash off in pure methyl alcohol ; the slide is now ready for staining. It will be found that most of the current stains used for such smears will act successfully after the lucidol fixation. Pappen- heim's panoptic method (§ 784) is recommended. For smears of faeces a fixation of twenty minutes in the pyridin- benzol peroxide solution is used ; wash as above, in acetone-xylol, or pyridin-xylol, and then in methyl-alcohol. Possibly the substitution of pure acetone for the methyl alcohol bath might prove advantageous in some ways. 784. Stains for Blood. — Fresh (unfixed) blood can be stained on the slide. See also § 1008, et seq. ToisoN (Journ. Sci. med. de Lille, fev., 1885 ; Zeit. wiss. Mik.,- 1885, p. 398) recommends that it be mixed with the following fluid: Distilled water Glycerin (neutral, 30° Baum6) Pure sulphate of sodium . Pure chloride of sodium . Methyl violet 5 B . (The methyl violet is to be dissolved in the glycerin with one half of the water added to it ; the two salts are to be dissolved in the other half of the water, and the two solutions are to be mixed and filtered.) This mixture stains leucocytes sharply, which facilitates enumeration. BizzozERO and Toree (Arch. Sci. Mediche, 1880, p. 390) dilute a drop with normal salt solution containing a little methyl violet, which stains nuclei intensely, cytoplasm less intensely. Similarly Giglio-Tos {Zeit. wiss. Mik., 1898, p. 166), diluting with saturated solution of neutral red in salt solution, which stains 160 c.c. 30 „ 8 grammes 1 gramme. 0-5 „ CHAPTER XXX. 383 hsemoglobigenous granules in five to ten minutes. This is also recommended by Ehelich and Lazaeus. See § 309. Similarly also Eoss (Trans. Path. 8oc., 1907, p. 117), using polychrome methylen blue. Levaditi (Journ, PJiys. 'path. Gen., Paris, 1901, p. 425) allows solution of Brillantkresylblau in alcohol to dry on a slide, puts a drop of blood on the dried layer, and covers. Similarly Cesaeis- Demel {Arch. path. Anat., 1909, p. 92), with a mixture of this dye and Sudan III ; and Nakanishi {Centralb. Bakt., 1901, p. 98), with methylen blue BB. Fixed films may be treated with the usual tissue stains, eosin being an important one, as it stains rose-red all parts of blood-cells that contain hsemoglobia. Ehrlich's acid hsematoxylin, with 0*5 gr. of eosin dissolved in it, is a good general stain. Or, stain with hsemalum, and then with eosin (0-5 per cent, in alcohol or water). Ehelich's triacid (§ 296) gives good general views, and demon- strates neutrophilous granules. His mixture for eosinophilous cells has been given (§ 311). Pappenheim's panoptic triacid (on sale by Griibler) is Ehrlich's triacid with methylen blue in place of the methyl green. Chenzinski's mixture, which is good, has been given (§ 313). Stain for six to twenty-four hours in a. stove. This gives rise to precipitates. To avoid them (Willebrand, Deutsch. med. Wochenschr., 1901, p. 57) you may make a mixture of equal parts of 0-5 per cent, solution of eosin in 70 per cent, alcohol and saturated solution of methylen blue in water, and add acetic acid of 1 per cent, drop by drop till the mixture begins to turn red, and filter before use. Or (Michaelis, ibid., 1899, No. 30) make (a) a mixture of 20 parts 1 per cent, aqueous methylen blue with 20 of absolute alcohol, and (b) a mixture of 12 parts 1 per cent, aqueous eosiu with 28 of acetone, and for staining mix equal parts of these and stain for half a minute to ten minutes. Jennee {Lancet, 1899, No. 6, p. 370) mixes equal parts of 1-2 to 1-25 per cent, water-soluble eosin (Griibler's) and 1 per cent, methylen blue, filters after twenty-four hours, washes the precipitate on the filter, dries it, and dissolves it ia 200 parts of absolute methyl alcohol (the solution can be had ready made from Griibler and HoUborn). (Or, simply mix 125 c.c. of 0-5 per cent, solution of the eosiu in methyl alcohol with 100 c.c. of 0-5 per cent, solution of methylen blue.) Cover-glass films are floated on to this, in which they are fixed and stained in three minutes. Wash off the stain with a little 384 BLOOD AND GLANDS. water (not under the tap), dry, and mount in balsam. Erythrocytes red, all nuclei blue, parasites blue, but with unstained nuclei. The methods of May and Grunwald are closely similar to this. AssMANN (Miinch. med. Wochenschr., 1906, No. 28 ; " Das eosinsaure Methylenblau," Leipzig, 1908, p. 35). treats fresh films for half a minute to three minutes in a Petri dish with a few drops of Jenner's solution (from Griibler and Hollborn), then pours on 20 c.c. of distilled water with 5 drops of -^^ per cent, solution of lithium carbonate, leaves for five minutes, rinses in distilled water, dries with blotting paper, and mounts in neutral balsam. The foregoing mixtures give a stain — seemingly due to the forma- tion of an eosinate of methylen blue — iu which the nuclei of blood- cells are blue and their plasma red to violet. It was made out by EoMANOwsKY {St. Petersburger med. Wochenschr., 1891) that under certain conditions mixtures of these two dyes give a stain which is in some respects the inverse of this, blood-cells being stained in divers hues, according to their kinds, and any protozoan parasites that may be present showing red nuclei and blue plasma, which greatly facilitates their detection and diagnosis. This reaction appears to be due to the formation of an eosinate — not of methylen blue, but — of Methylenazur (§ 377). The method, only vaguely indicated by Eomanowsky, has undergone, at the hands of Ziemann, Zettnow, Nocht, Reutbe, Michaelis, Euge, Maueee, Lbishman, Giemsa and others, numerous modifications which have culminated in the establishment of a process worked out by Giemsa as perhaps the most trustworthy and efficient of " Eomanowsky " stains. This is as follows :. Giemsa's Azur-eosin process. You start with a mixture of eosin with methylenazur (instead of methylen blue). This mixture is very troublesome to prepare, and is best obtained ready made from Griibler and Hollborn (their " Giemsa'sche Loesung fur Eomanowsky- faerbung "*). Air-dried films (Deutsch. med. Wochenschr., 1907, No. 17) are fixed in alcohol or in methyl-alcohol (two to three minutes), and dried with blotting paper. They are treated for ten to fifteen minutes with a dilution of 1 drop of the stock mixture to 1 c.c. of water, washed under a tap, dried with blotting paper, and again dried in the air and mounted in balsam, or (preferably) pre- served unmounted. All reagents, especially the balsam, must be strictly /ree/rom acid. * To make this up from Griibler's powders, dissolve 3 grms. of Azur Il-eosin and 8 decigrammes of Azur II in 125 grms. of glycerin and 375 of methyl-alcohol. CHAPTER XXX. 385 Wet films (ibid., 1909, p. 1751) are treated as follows : Fix them for twelve to twenty-four hours in a mixture of 2 parts saturated aqueous solution of sublimate with 1 of absolute alcohol. Wash and treat for five to ten minutes with a mixture of 2 parts of iodide of potassium, 100 of water, and 3 of Lugol's solution. Wash, and treat for ten minutes with 0-5 per cent, solution of sodium thio- sulphate. Wash, and stain as above (changing the stain for fresh after half an hour), for one to twelve hours. Then pass through mixtures of acetone with first 5, then 30, then 50 parts per cent, of xylol into pure xylol, and mount in cedar oil. This process is applicable to sections. Or (ibid., 1910, p. 2476) a slide is placed in a Petri dish and covered with a mixture of equal parts of methyl-alcohol and stock mixture. After half a minute this is poured off and enough distilled water poured in to cover the slide, and the whole is rocked to mix the two. After three to five minutes, wash in running water, dry, and mount in cedar oil. By any of these processes nuclei (red) are demonstrated not only in hsematozoa, but in many bacteria, spirochsetse, coccidia, sar- cosporidia, etc. See also, for paraffin sections, Schubeeg, in Deutsch. med. Wochenschr., xxv, 1909, No. 48, or Zeit. wiss. Mik., xxvii, 1910, p. 161, who passes through acetone and xylol into balsam. The older Romanowsky stains published by the authors men- tioned above, as also Laveran's " Bleu Borrel " seem to be super- seded by Giemsa's. Leishman's Romanowsky Stain (Brit. Med. Journ., March 16th and September 21st, 1901) is as follows : To a 1 per cent, solution of Griibler's medicinal methylen blue in water add 0-5 per cent, of sodium carbonate, heat to 65° C. for twelve hours and let stand for ten days. Then add an equal volume of 0-1 per cent, solution of Griibler's Eosin extra B, let stand for six to twelve hours, collect the resulting precipitate on a filter, wash it until the wash water comes off colourless, dry and powder. For staining, dissolve0-15grm. in 100 c.c. of pure methyl alcohol. Stain cover-glass films (air- dried) for five to ten minutes ; flood the film with water for one minute, and examine, or dry (without heat) and moxmt in xylol balsam. Nuclei in shades of red, cytoplasm bluish, parasites blue with ruby red chromatin. Raadt (Munch, med. Wochenschr., 1911, No. 27 ; Zeit. wiss. Mik.,. ■ 1912", p. 236) obtains a Romanowsky stain of blood and parasites with Jenner's solution. Films fixed with alcohol and ether are 25 386 BLOOB AND GLANDS. first stained for five to ten minutes in solution of 1 part methy- lenblau med. puriss. Hoechst, 0-5 part of lithium carbonate and 100 of water, kept for at least three weeks and diluted with 10 volumes of water. Eirise with water, dry with blotting paper, flood with Jennee's solution diluted with 2 or 3 volumes of water, and stain for five to ten minutes. Wash, dry with blotting paper, and mount. See also Scott, Folia Haem., xii, 1911. 785. Pappenheim (Anat. Ann., xlii, 1912, p. 525) recommends the following for sections of hcemopoietic tissues, and also of kidney, liver, hypophysis, suprarenals, lung, intestinal epithelium and central nervous system. Fix in Orth's rormol-Miiller, stain sections for twenty minutes in a stove in " aqueous diluted alcoholic " solution of Mat-Geunwald or Jennee diluted with 8 volumes of water ; after-stain for forty minutes in the stove in " aqueous Giemsa solution (15 c.c. of water with 10 drops of glacial acetic acid) " ; differentiate in 100 c.c. of water with 5 to 6 drops of acetic acid ; wash, dry between blotting-paper ; dehydrate in mixture of equal parts of acetone and absolute alcohol, and mount in neutral balsam. The result is not a Komanowsky stain, but a pale methylen-blue-eosin stain. See also Weight, Pub. Massachusetts Gen. Hasp., iii, 1910, p. I, or Journ. B. Micr. Soc, 1910, p. 783. For the special technique of eosinophilous cells see Maetinotti in Zeit. wiss. Mikr., xxvi, 1909, p. 4 (alphabetical bibliography of eight pages). 786. Demonstration of Blood-plates of Bizzozero (Kemp, Stiidies from the Biol. Lab. Johns Hopkins Univ., May, 1886, iii, No. 6 ; Nature, 1886, p. 132). — A somewhat large drop of blood is placed on a sHde, and quickly washed with a small stream of normal salt solution. The blood-plates are not washed away, because they have the property of adhering to glass. They may be stained with solution of 0-02 per cent, of methyl violet or 1 : 3,000 of gentian violet, in salt solution. To make permanent preparations of them, they should first be fixed, by putting a drop of osmic acid solution on the finger before pricking it. They may also be stained m films, especially by the Romanowsky method. According to Pappenheim {Farbchemie, p. 107) Wasser- blau is almost specific for them. Wright {Journ. Morph., xxi, 1910, p. 274) studies them in tissues, after fixation with formol or sublimate (not Zenker) by staining with a modified Giemsa stain, and bringing through acetone and oil of turpentine into turpentine colophonium. Details loc. cit. or Journ. Roy. Mic. Soc, 1910, p. 783. See also Dekhuyzen, Anat. Anz., xix, 1901, p. 533 ; Kopsch, CHAPTER XXX. 387 Intern. Monatschr. Anat. Phys., xxi, 1904, p. 344, and xxiii, 1906, p. 359 ; Deetjbn, Zeit. phys. Chem., Ixiii, 1909, p. 1. 787. Demonstration of a New Body in Red Blood Corpuscles (Golgi, Boll. Soc. Med. Chir. Pavia, 1919, xxxi ; Hcematologica (Napoli), 1920). This original communication of Golgi gives two methods of interest to workers on blood : (1) Blood-films are fixed twenty-four to forty-eight hours in equal parts of saturated solutions of mercury chloride and potassium bichromate. They are then transferred into equal parts of 2 per cent, mercury chloride and potassium bichro- mate, to which 5 to 10 c.c. of 1 per cent, gold chloride and 5 to 10 drops of acetic acid are added. The films are observed in glycerol, starting from the second or third day after the last treatment until the fifteenth or twentieth day ; (2) drops of blood are fixed in a watch-glass by means of a fluid composed of 2 per cent, mercury chloride 60 c.c, saturated solution of picric acid 20 c.c, 1 per cent, osmic acid 10 c.c, acetic acid 5 drops, with the addition either immediately or after eight to twenty-four hours of 10 c.c. of 1 per cent, gold chloride for every 50 c.c. of fixative. Preparations are made from the sediment with some glycerol, from the second until about the tenth day after fixation. Both methods show within the red blood corpuscles a peculiar body with a diamete;: of about one-third that of adult corpuscles and of about half that of foetal ones. The body occupies the central part of the erytrocytes, and particularly by means of the second process it appears to have a fine, sometimes more fibrillar, sometimes more reticular structure. Its contours, though clearly defined, are irregular, and there is no indication whatever of a limiting membrane. This " reticulo-fibrUlar apparatus " is not a nucleus, as the latter remains colourless by the new methods, not only in the white, but , also in the nucleated red corpuscles, in which the apparatus appears concentrically arranged round the unstained nucleus. According to Golgi this apparatus does not correspond to any of the structures already described within the red corpuscles, but it reminds one a little of the endoglobular body recently demonstrated Jjy Petrone, by means of a lead impregnation method, and not to be confused with the well-known Petrone's bodies. Golgi is convinced that the images obtainable by his two new methods are the expression of a real structuTe situated within the red blood corpuscles, but he does not feel able at present to give any opinion about their significance. The new methods stain also the centrosome in the white corpuscles of the blood. 25—2 388 BLOOD AND GLANDS. 788. Weigert's Fibrin Stain {Fortschr. d. Med., v, 1887, No. 8, p. 228). — Sections (alcohol material) are stained in a saturated solution of gentian or methyl violet in anihn water (§ 286). They are brought on to a slide and' mopped up with blotting-pa,per, and a little Lugol's solution is poured on to them. After this has been allowed to act for a sufficient time they are mopped up with blotting- paper, and a drop of anilin is poured on to them. The anilin soon becomes dark, and is then changed for fresh once or twice. The anihn is then thoroughly removed by means of xylol, and a drop of balsam and a cover are added. This stain may be applied to celloidin sections without previous removal of the ceUoidin. See also the modifications of this method by Kkomater (§ 656) ; Benecke (§ 690) ; Unna (Monatssoh. praM'. Dermat, xx, 1895, p. 140) ; Wolff {Zeit. wise. Mile, xv, 1899, p. 310) ; and one of another sort by KocKEL {Gentralb. allg. Path., x, 1899). 789. Elective Staining of Erythrocsrtes (K. Okajima, Anat. Record, xi, 1917). — This stain is based on the fact that the phosphomolybdic acid lake of ahzarin stains, shows a special affinity for haemoglobin. Fix material in formol, sublimate, chrome, etc. Transfer sections on slide to aq. dest. ; mordant in 10 per cent, phosphomolybdic acid solution for thirty seconds to two minutes ; wash in water ; stain in this mixture for twenty minutes to twenty hours : — sodium sulfaKzarinate, saturated aqueous solution, 100 c.c. ; and 10 per cent, phosphomolybdic acid, aq. solution, 30 c.c. (10 to 50 c.c.) ; wash in water ; alcohols, xylol, balsam. Erythrocytes go bright yellow orange. Counter-staining may be done in Ehrlich's hsema- toxylin. The completeness of the " specificity " of this method is open to doubt, but it gives interesting results. 790. Intravital Staining with Benzidine Dyes. — Bouffard {Ann. de I' Inst. Pasteur, xx), then Goldmann (Beit. z. Klin. Chir., Ixiv, have shown that animal tissues may be " stained " intra vitam by the injection of several benzidine dyes. There are some categories of cells in the body which seem to show a special affinity for phagocy- tosing or, at least, ingesting in some way granules of certain of these colloid dyes. These various cells are often called pyrrhol cells (Goldmann), macrophages (Evans), histiocytes (AschofE-Kiyono), or resting wandering cells (Tschaschin). According to Hal Downey {Anat. Record, xii, 1917) the process of " staining " is one of storage or ingestion, and not of true staining, and attempts to classify cells according to their reactions to these colloidal CHAPTER XXX. 389 dyes are a failure. This author believes that bfood cells behave towards these dyes just as they do to any foreign matter. Other authors do not agree. " To dismiss these cells (pyrrhol cells) as scavengers is to do them an injustice, for, however important this fimction may be, their service to the body is a far greater one " (P. G. Shipley, Amer. Journ. Physiol., sirs, 1919, p. 300). Evans and Schttlemann {Science, N.S., 1914), believe that vital staining with azo dyes is the result of " phagocytosis " of ultra-micro- scopic dye particles, existing in a state of fine dispersion as an hydrosol. In using the term " phagocytosis " Evans does not quite mean an engulfing by pseudopodia as with amceba. P. Gr. Shipley {Amer. Journ. Physiol., 1919, p. 285) points out that some cells which are most active in phagocytosing bacteria and other coarse particles, take no part, imder ordinary conditions, in the segregation of vital dyes in the body of the living animal. It has been stated that the benzidine dyes are not characterised by a propensity for staining the mitochondria, as are Janus green or dahlia. The granules in cells which store ultramicroscopic particles of the benzidine dyes seem to be something apart from the mitochondria. Only occasionally the mitochondria, as such, take up a benzidine dye like trypan blue. In tissue cultures from forty to sixty hours old many of the cells are seen to contain large greyish granules which were either not present in the early stages or were not very noticeable. Such gi'anules (" segregation granules " of Shipley, and possibly degeneration or " neutral red " granules of the Lewises, Amer. Journ. Anat, 1915), stain red in neutral red and deep purple in cresyl blue, and by using a combination of trypan red and Janus green, it can be shown, according to Shipley, that the mitochondria (green in the Janus) and the segrega- tion granules which take up the azo red dye, are separate entities. This opinion is not shared by Tschaskin {Fol. Hcem., 1914), by Levy (E. Accad. d. Lmcei, 1916), and by Maximow {Arch. Buss. d'Anat. d. Hist, et d'Embryol., 1916). Trypan blue is also used for demonstrating areas of osteoblastic activity (Shipley and Macklin). See § 780. The Methods of using Benzidine Dyes are as follows : — Trypan blue and pjrrrol-blue of 1 per cent, strength in Einger's solution are injected subcutaneously, intraperitoneally or into the blood vessels. Whereas 1 c.c. of a 1 per cent, solution per 20 grms. of the animal's body weight injected subcutaneously has no ill-effect on the animal, no more than 0-5 c.c. of the same solution should be used for intra- vascular work. In the latter case coloration sets in speedily, increases up to the second day, but rapidly fades after the fourth day, in any case, quicker than when gradual absorption of the stain takes place through the lymphatic channels. It is undoubtedly safest and best for histological study to inject the staining fluids subcutaneously. Injections of 1 c.c. of a 1 per cent, solution per 20 grms. body weight may be repeated many times once a week. 390 BLOOD AND GLANDS. In some cases Goldmann has given fifteen consecutive injections. These remarks refer especially to small animals like the rat ; for bigger animals, such as the rabbit or ape, intraperitoneal injections are preferable to. subcutaneous : use the standard of 1 c.c. of a 1 per cent, solution per 20 grms. of animal's weight. Trypan blue, isamin blue and pyrrol blue allow of fixation in 10 per cent, formalin solution (injected intravenously if possible), but it is only tissues stained in trypan blue that allow ordinary processes of histological technique, but even for trypan-blue, the fixative should contain a little formalin. Sections are cut with a freezing microtome from tissue fixed in 10 per cent, formalin not less than forty-eight hours. Stain as necessary in alum carmine or hsemalum, etc. Pappenheim's pyronin and methyl green are good for connective tissues, Ehrlich's triacid for hsemopoetic tissues (Goldmann, Proc. Roy. Soc, Ixxxv, 1912), G. B. Wislocki and H. Downey (Anat. Record, xii, 1917), after staining, fix in Zenker or formol, upgrade in alcohols, imbed in wax and section. Counter- stain in hsemalum". Goldmann (he. cit.) mentions the following " vital stains," apart from those given above : Trypan violet, benzopurpurin, diamin blue B.B., diamin black B.H., vital " neu rot," vital " neu orange," vital " neu gelb," dianil blue R (Griibler). These are used in 1 per cent, strength in salt solution. Trypan blue, trypan red, Congo red, azo blue, and benzopurpurin can be used on tissue cultures by introducing some of the dye into the culture medium (Shipley, Amer. Journ. Physiol, 1919, p. 287). Apart from the references given above see also, Hoffman {Fol. Hcem., 1911), Eenault {Arch. d'Anat. Min., 1907) ; Loele {Fol. Bcem., 1913) ; Batchelor (Proc. Amer. Assoc. Anat., 1914, Anal. Record, 1914). 791. Mierochemical Tests for the Oxygen Place in Tissues.— Recently certain workers have claimed to be able to locate centres or regions of oxidation in the cell by means of some substances sensitive to free oxy- gen. Unna's method is to use a solution of rongalit white, which is a solution of the leucobase of methylen blue kept in a state of reduction by excess of rongalit, an absorption product of formaldehyde with sodium sulphite. See Unna (Bie EeduUionsorte und Sauerstofforte des tierschen Gewebes, Arch. f. Mikr. Anat., Ixxviii, 1911). A. N. Deuey (Proc. Boy. Soc, 1914) has shown that Unna's claim is inadmissible, and consequently his theory of staining by oxidation and reduction is not proven. Graham (Journ. Med. Research, Boston, xxxv, 1916) claims to have demonstrated by means of PI2O2 and naphthol, that the granules of leucocytes and myelocytes contain a peroxidase of the peroxide type. Schultze in his Oxydase Reaction uses a-naphthol and dimethyl-p-phenylen-diamin (Merck). Blood and marrow smears fixed in formalin vapour are treated firstly in the a-naphthol solution ; pre- CHAPTER XXX. 391 pared by melting 1 grm. of naphthol on the surface of 100 o.c. of aq. dest., and adding potassium hydrate till the naphthol dissolves. After a few minutes in this solution (cooled) the smears are transferred to a 1 per cent, solution of the dimethyl— for the same time, when a blue colour is seen to appear where the oxydases lie. Mount in glycerine jelly, but blue colour fades. Myelocytes and not lymphocytes are said to give a positive reaction. It is doubtful how far these various colour indicators for oxygen place in cells and tissues are reliable. It has been claimed that by means of the last-mentioned method, it is possible to show that the staining (oxygen place) appears especially around the nucleus of the ceU. This has not been confirmed. Glands. 792. Mucin. — Hoyer {Arch. mik. Anat., xxxvi, 1890, p. 310) finds that the mucin of mucus cells and goblet cells stains with basic tar colours and with' alum haematoxylin, but not with acid tar colours. He obtained his best results by means of thionin, and good ones with toluidin blue, both of these giving a metachromatic stain — tissues blue, mucin reddish — and also with methylen blue (which is particularly useful from its power of bringing out the merest traces of mucin), safranin, etc. Tissues should be fixed for two to eight hours in 5 per cent. sublimate solution, and paraffin sections stained for five to fifteen minutes in a very dilute aqueous solution of the dye (2 drops of saturated solution to 5 c.c. of water). Hyaline cartilage, the jelly of Wharton, and the Mastzellen of EhrUch give the same reactions with basic dyes as mucin does. See also Sussdorf, Deutsche. Zeit. Thiermed., xiv, pp. 345, 349 (Zeit. wiss. Mik., vi, 1889, p. 205) ; Bizzozero, Atti. R. Accad. di Sci. di Torino, 1889 to 1892 (reports in Zeit. wiss. Mik., vii, 1890, p. 61 ;■ and ix, 1892, p. 219) ; also Unna, ibid., xiii, 1896, p. 42. The safranin reaction is not obtained with all brands of the dye ; that of Bindschedler and Busch, in Bale, gives it, whilst safranin of Griibler does not. Unna employs chiefly polychrome methylen blue. As regards the thionin stain, see Hari, Arch. Mik. Anat., Iviii, 1901, p. 678. Bruno {Bull. Soc. Nat. Napoli, 1905, p. 220) fixes and stains the skin of the frog in a mixture of 100 c.c. of formol of 1-25 per cent, with 8 c.c. of 1 per cent, solution of thionin. Mucus glands red. KuLTSCHiZKY {Arch. mik. Anat., xlix, 1897, p. 8) fixes in his mixture (§ 57), and stains sections either in safranin with 2 per 392 BLOOD AND GLANDS. cent, acetic acid, or in a similar solution of neutral red (two to three days, washing out with alcohol). Mater {Jtfi«. Zool. Stat. Neci/pel.,xu, 1896, p. 303, or Ust edition) girea the following two formulae for mixtures that stain exclMsively mucus. 793. Mater's Mueiearmine {op. eit, last §). —One gramme of carmine, and 0-6 grm. of aluminium chloride with 2 c.c. of distilled water heated over a small flame for two minutes, and made up to 100 c.c. with 50 per cent, alcohol. This gives a stock solution, which is as a rule to be diluted for use tenfold with distilled or tap water. Mater's Muehaematein (i6id.).— Hsematein 0-2 grm., aluminium chloride 0-1 grm., glycerin 40 c.c, water 60 c.c. An dlcohoHe solution may be made by dissolving in 100 c.c. of 70 per cent, alcohol, with or without the addition of 2 drops of nitric acid. 794. Mueicarminie Acid (Rawitz, Anat. Anz., xv, 1899, p. 439).— One gramme of carminic acid,' 2 of aluminium chloride, and 100 c.c. of 50 per cent, alcohol. 795. Goblet Cells. — So far as these contain mucia they give the reactions above described. See Paneth, Arch. mih. Anat., xxxi, 1888, pp. 113 et seq. ; List, ibid., xxvii, 1886, p. 481 ; and Guyeisse, C. R. Soc. Biol, 1907, p. 1212. For intestinal epithelium, especially the cells of Paneth, see also Martin, Unters. ueb. Oberflachen u. Driisenepithel, Leipzig, 1910 ; and KxJLL, Arch. mik. Anat., Ixxvii, 1911, p. 541 (sections stained with alum, hsematoxylia, treated for twenty to thirty seconds with tincture of iodine, stained a few minutes with Victoria blue, then, with eosin). 796. Salivary Glands. — Solger {Unters. z. Naturlehre d. Menschen XV, 5 and 6, pp. 2 — 15 ; Festschr. f. Gegenbaur, ii, 1896, p. 211^ demonstrates the granules ia serous cells and half-moons of the submaidllary gland by hardening ia a 10 per cent, solution of formol, and then making sections and staining with hsematoxylin of Delafield or of Ehrlich, the granules taking the stain. Krause {Arch. mik. Anat., xlv, 1895, p. 94) stains sections either with Heidenhain's iron hsematoxylin or with Ehrlich-Biondi mixture or thionin. See also Krause, ibid., xlix, 1897, p. 709 ; and Muller, Zeit. wiss. Zool., 1898, p. 640. 797. Gastric Glands. — Kolster {Zeit. wiss. Mik., xii, 1895, p. 314) difEerentiates the two kinds of cells in stomach glands by over-staining with hsematoxylin, washing out with alcohol containing 1 per cent, of HCl, blueing with alcohol containing 1 per cent, of ammonia, and, after washing, staining for one to five minutes in a CHAPTER XXX. 393 weak solution of Saurefuchsin. Peptic cells blue, parietal cells red. Osmic material cannot be employed. Cade {Arch. Anat. Micr., iv, 1901, p. 4) stains material fixed with Bouin's picroformol in Victoria blue of 1 per cent. R. and L. Monti {Rich. Lab. Anat. Roma, ix, 1902) demonstrate ducts and canalicuU of delomorphous cells by Golgi's bichromate and silver impregnation, especially with rejuvenated material (see Saceedotti), leaving it for five or six days in half -saturated sulphate of copper, then for twenty-four hours in the osmic-bichromate mixture. You can embed in paraffin (rapidly). 798. Intestine. — Bensley {Amer. Journ. Anat., v, 1906, p. 323) stains sections of- glands of Lieberkiihn in a mixture of equal parts of saturated solutions of orange G and Saurerubin, and then with toluidin blue, and mounts in balsam. 799. Liver. — Braus {Denkschr. Med. Nat. Ges. Jena, v, 1896, p. 307) demonstrates the bile capillaries by the rapid method of GoLGi, hardening in a mixture of 1 part formol with 3 parts liquid of Miiller or ^ per cent, chromic acid. Bppinger {Beitr. path. Anat., xxxi, 1902, p. 230) studies them by means of a compUcated modification of Weigeet's neuroglia stain, and Ciechanowski {Anat. Anz., xxi., 1902, p. 426) by means of Weigeet's myelin stain (the 1885 method). Oppel {Anat. Anz., v, 1890, p. 144 ; vi, 1891, p. 168) puts pieces of liver or spleen (alcohol material) for twenty-four hours into a solution of neutral chromate of potash (^ to 10 per cent.), then for twenty-four hours into a f per cent, solution of silver nitrate, washes, dehydrates and cuts without embedding. - The lattice fibres are only stained near the surface, so that tangential sections must be made. Similarly Berkley, ibid., 1893, p. 772, fixing in picric acid, then in an osmium bichromate mixture, and then silvering. See also Eanviek, Journ. de Microgr., ix, x, 1885-6 ; Igacuschi, in Arch. path. Anat, xcvii, p. 142, or Zeit wiss. Mik., 1885, p. 243 (gold process for study of fibrous networks) ; Kuppper, Siieh. Qes. f. Morph., etc., Miinclien, Juli, 1889, oi Zeit wiss. Mik., vi, 1889, p. 506 ; Krause (Arch. mik. Anat, xlii, 1893, p. 57) ; and Timopejew, Anat. Anz., xxxv, 1909, p. 296 (sections of frozen tissue stained with methylen blue). 800. Spleen. — For lattice fibres, see Oppel, last §. KuLTSCHiTZKY {Arch. mik. Anat., xlvi, 1895, p. 675) studies the musculature in sections (of material from liquid of MiiUer) stained for a day or more in a solution of lakmoid in ether and mounted in balsam. 3U BLOOD AND GLANDS. For elastic fibres he puts sections for half an hour or a day into a mixture of 800 parts 96 per cent, alcohol, 40 parts 1 per cent, solution of carbonate of potash, 2 parts Magdala red, and 1 part methylen blue. For the blood vessels he puts sections of Miiller material for a few minutes into a solution of 1 or 2 parts of Saurerubin in 400 parts of 3 per cent, acetic acid, washes out in 2 per cent, acetic acid, and after-stains in a similar solution of helianthin or Wasserblau until the red only remains in the erythrocytes. See also Whiting {Trans. Boy. Soe., Edinburgh, xxxviii, 1896, p. 311) ; Schumacher {Areh. mile. Anat, Iv, 1899, p. 151) ; Wbideneeich {ibid., Iviii, 1901, p. 261). 801. Lymphatic Glands. — For lattice-fibres especially, see Eoessle and YosHiDA, Beitr. path. Anat., xlv, 1909, p. 110, or Zeit. wiss. Mik., xxvi, 1909, p. 295. Sections stained with hsematoxylin and eosin, or Weigert's iron hsematoxylin, or Bielschowsky's neurofibril stain as applied by Maebsch, loc. cit., § 752. The sections should not remain for more than fifteen to thirty minutes in the oxide bath. See also for the thymus some very complicated methods of Sal- kind, Anat. Anz., xli, 1912, Nos. 6 and 7. 802. Kidney. — Sauee (Arch. mik. Anat., xlvi, 1895, p. 110) finds that for the renal epithelium the best fixative is Carney's acetic alcohol with chloroform (three to five hours, washing out with absolute alcohol). A mixture of 9 parts alcohol with 1 of nitric acid is also good, as is liquid of Perenyi. He stains with iron hsematoxylin, and after-stains in a very weak solution of Saurerubin in 90 per cent, alcohol, which stains the ciliary plateau. He macerates with iodised serum or one-third alcohol, staining after- wards with dahlia. Aenold (Anat. Anz., xxi, 1902, p. 417) employs intra vitam staining methods for the study of the granules of the epithelium cells. Sections of fresh kidney are cut with a Valentin's knife, and brought into a very dilute solution of neutral red, or methylen blue, in which the granules stain in a few minutes or hours. Or saturated solutions of the dyes, or of indigo carmine, may be injected sub- cutaneously during life, at intervals of fifteen to twenty minutes, and after two to five injections the organ may be excised and sections made and examined (see §§ 208 and 342 to 344). 803. Thyroid.— Bensley (Amer. Journ. Anat., xxix, 1916) uses brazihn and water blue. Fix gland in Zenker-formol. Section in paraffin and fix sections to slide w^ith water alone, or very little CHAPTER XXX. 395 albumen ; pass through toluol, absolute alcohol, water, iodise, and place in this braziUn solution for several hours : — Phosphotungstic acid .... 1-0 grm. Distilled water ..... 100-0 c.c. BrazUia ...... 0-05 grm. The brazilin is first dissolved in a small quantity of distilled water by the aid of heat and added to the phosphotungstic acid solution. This solution goes bad after three days. After staining in the brazilin, wash in water, and place for five minutes in this mixture : — ■ Phosphomolybdic acid .... 1-0 grm. Wasserblau . . . . . . 0-2 „ Water 100-0 c.c. Wash rapidly in water, dehydrate in absolute alcohol, clear in toluol, and mount in balsam. Cytoplasm stains blue to lilac, nuclear chromatin deep red, contents of thyroid vacuoles sky blue, and colloid droplets of Hiirthle deep blue to deep red. 804. Pancreas. — Most of the techniques given under the heading of " Mitochondria " and " Golgi apparatus," etc. (§§ 673—712) give important results with the zymogen granules of the pancreas. The methods of Bensley-Cowdry (§ 686), Regaud (§ 685), Benda (§ 683), and Schridde (§ 687), all apply here. For the Golgi apparatus Cajal's formalin silver nitrate method may be used (§ 847). Bensley's Neutral Red Method (Amer. Journ. Anat., xii, 1911 — ^12). — ^Animal kiUed by bleeding ; a cannula introduced into aorta and a solution of neutral red in isotonic salt solution, containing 1 in 15,000 neutral red, is injected. Immediately after the pancreas has assumed a faint rosy tint a part of the organ is removed — the islets of Langerhans stain intense yellow red, the rest faint rosy-pink. In a short time after mounting the islets remain the only stained elements, owing to bleaching in the acini. Method applicable to the counting of the islets of Langerhans. Janus Green Method. See § 702. — Islets deep blue on a red background. Pyronin Method for Ducts. — Inject a 1 in 1,000 solution of pyronin, as above, for neutral red method. The ducts stain intensely red. Double stains may be made by injecting mixed Janus green and pyronin (Bensley, op. cit.). Methylen blue, 1 in 10,000 may also be used for this purpose. After injection fix in 5 per cent, ammonium molybdate, for which see also Chapter XVI. Grand-Mouesel and Teibondeau (C. R. Soc. Biol., hii, 1901, 396 BLOOD AND GLANDS. p. 187) recommend for pancreas Nicolle's " thionine ph6mqu6e," which stains the insulse of Langerhans hardly at all, the rest strongly. Lane's Methods for Demonstration of A Cells of the Islets of Langerhans. (1) Fix tissue for from two to four hours in equal parts of saturated alcoholic solution of mercury chloride, and 2J per cent, potassium bichromate. Wash in 50 per cent, alcohol, then upgrade and embed ; 3 ix sections are stained in neutral gentian, obtained by precipitation of equivalent solutions of gentian violet (crystal violet) and orange G. If the correct quantity of the latter is added to the former, a practically complete precipitation is obtained. The precipitate is soluble in alcohol or acetone. For staining add the stock alcohol solution to 20 per cent, alcohol until a solution having the colour of good hsemalum is obtained. Allow to stand for twenty-four hours. Stain for twenty-four hours, blot, dehydrate in acetone, toluol, differentiate in absolute alcohol 1 part, oil of cloves 3 parts, wash in toluol, and mount in balsam. (2) Fix in 70 per cent, alcohol, then stain in neutral gentian as above. Lane's Methods for Demonstration ofB Cells of Islets ofLangerhans. Fix for four to twenty-four hours in : — KgCr207 ...... 2'5 grms. HgCla 5-0 „ Aq. dest 100 -0 c.c. Dehydrate, clear, embed, and section ; stain in neutral gentian as above. Formulin Bichromate, Method for Fixation. — This gives a very regular and reliable fixation, and is suitable where one is carrying out observations which necessitate a successful routine method. Bensley {op. cit.) uses 10 c.c. of neutral formalin to 90 c.c. of Zenker's fluid without acetic acid, for twenty-four hours. Stain in neutral gentian, acid fuchsin and toluidin blue, iron hsematoxylin or Mallory (§ 314). HoMANS {Journ. Med.. Research, xxx, 1914) used Bensley's modified Altmann fixative (OSO4 of 4 per cent., 2 c.c. ; potassium bichromate of 2"5 per cent., 8 c.c. ; glacial acetic acid, 1 drop). Lane's methods (vide supra), and ordinary hsematoxylin and eosin. Very pretty results are obtainable by using Mallory's polychrome methylen blue and eosin (§ 314). See also Babkin, Rubaschkin and Ssawitsch, Arch. f. Mikr. Anat., Bd. 74 ; Helly, ibid., Bd. 67 ; Lane, Am^r, Journ. Anat. vii, 1907, Saguchi, ihid.. Vols. 26 and 28, and § 713. CHAPTEE XXXI.* NERVOUS SYSTEM — GENERAL METHODS. 805. Introduction. — Tke microscopic investigation of the nervous system pursues two ends. Either it is desired to elucidate the intimate structure of nerve-cells, nerve-fibres and their supporting tissues, or to study the morphology of nerve-cells, their distribution in the grey matter, their coimections with each other, and with the nerve fibres which chiefly constitute the white matter, and lastly to investigate the architectural arrangement of both nerve-cells and nerve-fibres in the various regions of the central nervous system. The processes employed in the first case form a group of cytological methods, whilst the processes used iu the second instance are spoken of as the anatomical methods. The processes used in the study of nervous tissue in peripheral organs having been described in the chapter on " Methylene Blue," " Impregnation Methods," " Tegumentary Organs," and " Muscle and Tendon," the following chapters are chiefly devoted to the description of methods for the investigation of the central nervous system. Fixation. 806. Fixation by Injection. — Fixation, ia the proper sense of the word, is of course out of the question for the human subject. But in the case of the lower animals it is possible to inject fixiag fluids into their nervous centres when still in an almost liviug state. The practice ensures a very rapid penetration into and even distribution within the tissues of the fixing agents, and has, moreover, the capital advantage of greatly helpiag to prevent distortion of the nerve- tissues during their subsequent treatment. And as in most instances the practice does not meet with special di£6.culties, it should be adopted as far as possible also in the case of human subjects, but particularly for a preliminary fixation and hardening of the very soft cerebral mass of yotmg individuals, which is particularly liable to much injury and distortion in the process of removing it from the brain case. * Revised and in great part rewritten by Dr. C. Da Fano, King's College, University of London. 398 NERVOUS SYSTEM— GENERAL METHODS. The choice of the fluid to be injected depends upon the object in view and the subsequent treatment to which the tissues are to be submitted. In the case of animals it is a good practice to warm the fixing fluid to body- temperature before injecting it, and, whenever possible, to wash out the blood by first injecting physiological solution as suggested by Mann. The injection can be carried out through the carotids if the fixation is to be limited to the encephalon, and through the aorta if it is desired to fix the spinal cord too. The above applies to higher vertebrates and particularly to mammals ; in the case of lower vertebrates, fixation by injection has not, as a rule, the same importance, and one must have recourse to special methods. See on this subject Golgi, op. oii., in § 880 ; Gbeota, § 811 ; De QuEEVAiN, Virohow's Arch., oxxxiii, 1893, p. 481 ; Mann, Ztuchr. wiss. Milcr., xi, 1894, p. 482 ; Strong, Anat. Am., xi, 1886, p. 655 ; Journ. Gomp. Neurol., xiii, 1903, p. 291 ; McFaeland, Journ. App. Micr., ii, 1899, p. 541. Hardening. 807. Hardening by the Freezing Method. — This phrase has often given rise to confusion and should, therefore, be clearly understood. One can harden by freezing either fresh tissues, or material already fixed and consequently also a little hardened. In the first instance small pieces of fresh tissue, immediately after removal and without any previous treatment, are hardened on a freezing microtome. The sections are generally floated on to water, and immediately after- wards treated for a minute on the shde with a 0-25 per cent, solu- tion of osmic acid ; or otherwise treated according to the object of one's investigation. In this case the ether freezing method should be preferred, bearing in mind, however, that there is considerable difficulty in obtaining sufficiently good sections, and that the results attainable are very Umited particularly since Brodmann {Journ. Psychol. Neurol., ii, 1903 — 4, p. 211) has shown thatformalin material can be used even for investigations by polarised fight. (See also p. 361.) The hardening by freezing of already fixed material may be also attended with some difiiculty, but this will be easily overcome if pieces are relatively small, the fixing agent properly washed away, and one has, eventually, recourse to one or other of the processes described in § 183. Material fixed in formalin, however, does not, as a rule, require any soaking in gum, or syrup, or the like, and is easily cut if the formalin has been washed away. In this case the COg freezing microtome is in my opinion to be preferred. CHAPTER XXXI. 399 The hardening and section cutting by the freezing method of very large pieces require special apparatus and special methods, for which see Nageotte, C. R. Soc. Biol., Ixvii, 1909, p. 542. 808. Hardening by Reagents. — If large pieces of nervous tissue are to be hardened, it is necessary to take special precautions in order to prevent them from being deformed by their weight during the process. The spinal cord or small portions of any region of the encephalon may be cut into thin slices, laid out on cotton wool in a vessel into which the hardening fluid is poured. The specimens may also be suspended in the liquid (§ 34). Another good plan consists in adding to the hardening fluid just enough glycerine or sodium chloride to make tissues float. If several pieces are placed in the same vessel, they should never be put on top of each other. Voluminous organs to be hardened in toto should be at least incised as deeply as possible in the less important regions. With the exception of the dura mater, the membranes are not generally removed at first, as they serve to protect the tissues. They can be removed partially or entirely later on when the hardening has made some progress. In the case of material intended for Golgi's methods it is best not to remove them at aU. The spinal cord, medulla oblongata and pons Varolii may be hardened in toto, and the preparation hung up in a cylindrical vessel with a weight attached to its lower end to prevent it from becoming distorted. . The cerebrum should have light plugs of cotton wool in the fissure of Sylvius, and as far as possible between the convolutions. If it is desired not to open the lateral ventricles, the hardening fluid may be injected into them. Unless there are special reasons to the contrary, the brain should be divided into two portions by a middle frontal section, or better into two symmetrical halves by a sagittal cut passing through the median plane of the corpus callosum. The cerebellum should be treated in the same manner. The action of most hardening fluids is greatly enhanced by heat. But in the judgment of most histologists this rapid hardening is not, as a rule, attended by good results, and one should have recourse to it only for particular reasons and special purposes after a tentative experiment, whenever possible, at establishing the degree of tem- perature at. which the desired results may be obtained without otherwise injuring the delicate structure of the nervous tissues. 400 NERVOUS SYSTEM— GENERAL METHODS. On the other hand the hardening action at room temperature of certain reagents, such as solutions of chromic salts, proceeds so slowly that decomposition may set in before the fluid has had time to act effectively. For this reason voluminous preparations which are to be hardened in toto in solutions of chromic salts, and were not injected as described in § 806, should be put away in a very cool place or in an ice-chest. A human cerebral hemisphere may require eight or nine months for hardening in this way. The volume of the fluid should always be very large in proportion to that of the pieces of tissue and to their number. It should be taken in solutions as weak as is consistent with the proper preserva- tion of the tissues. It should be frequently changed and its strength gradually increased. Marie's method of fixing and hardening in situ is highly recom- mended; for its indications and contra-indications, see Sainton and Kattwinkel {Deutsche Arch. Jclin. Med., Ix, 1898, p. 548) and Pfistee {Neurol. Centrbl, xvii, 1898, p. 643). 809, The Reagents to be Employed. — ^As in the case of the fixation by injection one should bear in mind that the preservation of tissues for neuro-histological investigations greatly depends upon the purpose in view. Fixing and hardening fluids which are excellent for cy tological investigations are very often unsuitable for anatomical methods. (See § 805.) On the other hand, material collected and prepared for cyto-architectonic or fibro-architectonic studies can hardly be used to elucidate questions regarding the intimate structure of nerve-cells or nerve-fibres. Alcohol, formalin * and chromic salts are most frequently used because they are generally ready at hand, • and because they are useful for carrying out afterwards either a great number of methods, or certain methods, under constant conditions of hardening and staining. 810. Alcohol. — It is generally employed in the strength of 94 to 96, per cent., penetrates well and hardens quickly ; but as it rapidly * Wrong as it is, I find it expedient to use the term " formalin " or " formol " in the generally accepted sense, viz., as if it were a chemical reagent, while it is only a commercial denomination which ought not to have been introduced in the histological terminology. See § 108. As is well known, the commercial formalin is only a 40 per cent, solution of formaldehyde ; but when in this and the following chapters on the nervous system a 6, 10 or 20 per cent, solution of formalin is mentioned, it is intended to mean 5, 10 or 20 parts of commercial formol, and 95, 90 or 80 parts of water, respectively, while, e.g., a 20 per cent, solution of formaldehyde is the commercial formalin diluted with half its volume of water. — C. d. P. CHAPTER XXXI. 401 absorbs water from the tissues tbe latter sbrink considerably, wbilst the alcohol loses its fixing and hardening properties through hydra- tion. It has consequently to be changed soon for a fresh supply and used in quantities exceptionally large in proportion to the size of the pieces, which ought to be neither too small nor too large. For this reason one seldom hardens in alcohol voluminous organs, and its use has become on the whole very restricted. Alcohol, however, remains the principal fixing and hardening reagent for cytological investiga- tions by Nissl's method (see § 826), and for carrying out some of Ramon y Cajal's reduced silver processes (§ 827), its shrinking influence being counteracted by having recourse for the first fixation to weaker dilutions of alcohol (60 to 70 per cent.) to be raised gradually up to 95 or 96 per cent, within the first nine to twelve hours, and to be changed once or twice or more often in the next few days. 811. Formalin. — Since the time when it was introduced into histological technique by F. Blum (Ztschr. wiss Mikr., x, 1893, p. 314) ; J. Blum (Zool. Anz., xvi, 1893, p. 434) ; Heemann {Anat. Anz., ix, 1893, p. 112) ; Hoyer, jun. {Anat. Anz., ix, 1894, p. 236) ; Lachi (Monit. Zool. Ital, v, 1895, p. 15) and many others, its use has been steadily increasing because of the many advantages it offers. As a matter of fact it penetrates more quickly than solutions of chromic salts, and even than alcohol ; it is not Kkely to over- harden ; it allows of the most various after-treatments and methods of staining, including neurofibril stains and Golgi's impregnation method. Several writers have insisted that for nervous tissue it should not be acid, but some prefer it acid. See "Retina." For neurofibrils it should be preferably neutral. To neutralise it, it is generally suffi- cient to prepare its solutions with spring water, but one may shake it with raagnesium or sodium or calcium carbonate. Some authors prefer to neutralise with ammonia. (See also § 108.) The strength of the formalin solutions generally used for fixing and hardening nervous tissues varies considerably with the quality of the material in hand, but particularly with the age of the subjects. As a rule the more delicate the material and the younger the subject, the weaker should be the formalin solutions to be employed at first. Generally, however, one starts with a 3 or 5 per cent, solution in the case of very soft tissues, gradually increasing the strength up to 10 or 12 per cent. An adult human encephalon can be very well preserved in a 10 or 15 or 20 per cent, solution with two changes of M. 26 402 NERVOUS SYSTEM— GENERAL METHODS. the fluid during the first days of fixation and hardening. See further on this subject, § 108. Formalin can be associated with, or followed by, alcohol (§ 109) or other reagents. Thus Fish {Proc. Am. Micr. Soc, xvii, 1895, p. 319) recommends : — ■ Water 2,000 c.c. Formalin . . . . . . 50 „ Sodium chloride .... 100 grms. Zinc chloride . . . . . 15 „ Braius are left in the mixture eight to ten days or longer, and then transferred into a mixture of water 2,000 c.c. and formahn 50 c.c, in which they may remain indefinitely if the jar is kept tightly stoppered. Parker and Floyd (Anat. Anz., ix, 1896, p. 156) advise for sheep's brains a mixture of 6 volumes of 95 per cent, alcohol and . 4 volumes of 2 per cent, formol. Brains may be kept in the mixture for months. Flatau {Anat. Anz., xiii, 1897, p. 323) finds that brain iacreases in weight shghtly in 10 per cent, formol (spinal cord somewhat more) ; whilst in 1 per cent, solution it may increase as much as 24 per cent. GrEROTA {Int. Monatschr. Anat., xiii, 1896, p. 108) puts human brains into a 5 or 10 per cent, solution of formol, and after twenty- four hours removes the pia mater, and changes the hquid ; this is also further done every five to seven days, and in one or two weeks the hardening is complete. In the case of f cetal brains of Canis, Felis and Homo, he first injects the vascular system with a 10 to 15 per cent, solution of formol in 85 per cent, alcohol, and then brings the heads into the 5 to 10 per cent, watery solution ; after one or two days he removes the brains from the skuU and puts them back for fifteen to twenty days into the formol. Kadyi {Poln. Arch. Biol. Med. Wiss., i, 1901, p. 80) takes 5 parts of formol, 100 of water, and 2 of bicarbonate of soda, for four to ten days. Herdlicka {Proc. U. S. Nat. Mus., xxx, 1906, p. 304) takes 3 parts of formol, 25 to 45 of water, and 72 to 52 of 95 per cent; alcohol. Strecker fixes small pieces for twenty-four to forty-eight hours m equal parts of 10 or 20 per cent, formol and EhrUch-Biondi triacid mixtures, and imbeds in paraffin, thus getting a stain at the same time as a fixation. Similarly with toluidine blue fixing it with ammonium molybdate. See Ztschr. wiss. Mihr., xxviii, 1911, p. 17, and the hterature discussed therein. CHAPTER XXXI. 403 812. Chromic Salts. — The most commonly used is potassium bichromate. The liquid of Erlicki has a more rapid action than other solutions of chromic salts, but it has been generally abandoned because of the alterations it very often produces. Sahli {Ztschr. wiss. Mikr., ii, 1885, p. 1), after investigating the action of the usual solutions, concludes that the best hardening agent for fresh tissues is pure potassium bichromate, in 3 or 4 per cent, solution, the hardening being done in a cold place. He rejects the liquid of Erlicki on account of the precipitates to which it so fre- quently gives rise. Oberstbiner is of the same opinion, and recommends pure bichromate for general hardening purposes ; whilst for the study of delicate structural details he recommends fixing in Fol's modifica- tion of Flemming's fluid (§ 47) for twenty-four hovirs, followed by washing with water and hardening in 80 per cent, alcohol. In view of the slowness of penetration of chromic salts, it is often advisable to treat the preparations for twenty-four hours or more with 80 to 90 per cent, alcohol, or better, for a few days with a formol solution before putting them into the hardening fluid, or to add formol (say 3 per cent.) to it, in order to avoid maceration of the deeper layers of the tissues. In this case, however, the fluid must be changed after twenty-four to forty-eight hours. Potassium bichromate should be employed at first of not more than 2 per cent, strength ; this is then gradually raised to 3 or 4 per cent, for the cord and cerebrum, and as much as 5 per cent, for the cerebellum. Ammonium bichromate should be employed of half the strength reconunended for potassium bichromate at first ; it may be raised to as much as 5 per cent, for cerebellum towards the end of the hardening. NissL (Enzycl. Mile. Technik., ii, 1910, p. 245) uses, for rapid hardening, large quantities of Miiller's fluid 100 parts, formol 3 parts, and enough glycerine to make the tissues float. If the solution is often changed, even entire brains are in a few days sufficiently hardened to be safely transferred into pure MiiUer's fluid, or potassium bichromate of about the same strength. Betz's method (Arch. mik. Anat., 1873, p. 101). Brain and spinal cord are first hardened, for some days or weeks, in 70 to 80 per cent, alcohol containing enough iodine tincture to give it a Ught brown coloration (as soon as the alcohol becomes colourless, more iodine must be added.) They should then be definitely hardened in 3 per cent, potassium bichromate for spinal cord, medulla 26-2 404 NERVOUS SYSTEM- GENERAL METHODS. oblongata, and pons, 5 per cent, for cerebellum, and 4 per cent, for cerebrum. The methods of Bevan Lewis {op. cit.) and Hamilton (Joum. Anat. and Physiol., 1878, p. 254) can be considered as superseded, chiefly because they are based on a fixation with methylated spirit (Bevan Lewis) or mixtures of Miiller's fluid and methylated spirit (Hamilton), which cannot be used any. longer for histological pur- poses, owing to the excess of impurities it now contains. Also the methods for encephala of Duval (Robin's Journ. de I' Anat., 1876, p. 497) and Deeoke {Journ. R. Micr. Soc, 1883, p. 449) can be considered as obsolete. Orth uses formol-Miiller changed every few days. See § 113. BoNVicmi {Ztschr. wiss. Mihr., xxvi, 1909, p. 412) puts entire human brains into 10 per cent, formol (first injected through the' carotids or into the ventricles) for six to eight days, cuts them into 1 cm. thick slices by means of a special macrotome, and transfers them into a mixture of 4 parts of potassium bichromate and 2-5 parts of chromium sulphate in 100 of water, and keeps them therein, in the dark, for two months for cerebral hemispheres, twelve to fourteen days for medulla and pons, five or six days for spinal cord. In the case of slices of brain, the fluid must be changed every week. Ravwtz {ibid., p. 338) puts formol material for exactly five days into alcohol with 10 per cent, tinctura iodi P. Gr., then for eight to ten into saturated solutions of potassium bichromate changed after the first day, and lastly into 95 per cent, alcohol for three days in the dark. 813. Other Reagents. — Osmic acid is hardly useful for specimens of more than 2 or 3 mm. thickness. Chromic acid is rarely used alone. Its action is rapid, but uneven, and causes shrinkage and brittleness. A very little {e.g. 3 to 5 drops of a 1 per cent, solution to every 100 c.c. of fluid), added to bichromate solutions will do no harm and quicken the hardening. Nitric acid has been and still is employed in strengths of 10 to 12 per cent. Neutral acetate of lead affords, according to Kotlaeewski {Ztschr. wiss. Milcr.fiy, 1887, p. 287), an excellent preservation of ganglion cells. Oorrosive sublimate solutions either alone or mixed with other re- agents (see Chapter V.), have been very often used for cytological studies. Similarly acetic alcohol. Mann {op. cit.) for cell studies, puts small pieces for twenty-four hours into a solution of 5 parts of potassium iodide and 25 of iodine in 100 parts of water, and then into 70 per cent, alcohol. Olmacher {§ 65) recommends his mixture. Kodis (Arch, milcr. Anat., lix, 1901, p. 212) fixes tissues in a saturated solution of cyanide CHAPTER XXXI. 405 of mercury, biings them into 10 per cent, formo], and makes sections by the freezing method. Nelis {Bull. Ac. Sc. Belg., 1899-1900) fixes spinal ganglia for twenty- four hours in a solution of 20 grms. of copper sulphate and corrosive sublimate to saturation in a litre of 7 per cent, formol with 5 o.o. of acetic acid. King {Anat. Bee, iv, 1910, p. 213) after trying over twenty-five methods on brains of rats, concludes that the best is Ohlmaoher's. The brain should be put into it for two to three hours, then for one iato 85 per cent, alcohol, then into 70 per cent, with iodine for at least twenty- four hours, then passed through alcohols of ascending strength and alcohol--ether into 2 per cent, celloidin for two to three days, and through chloroform and benzol into paraffin. In her opinion, Bouin's is the best of the formol liquids ; Tellyesnicky's is the only one of the bichromate mixtures that equals it. All sublimate mixtures fix the nuclei well, but vacuolise the cytoplasm. See ftirther particulars on this subject in the original papers of Tkzebinski, Virohow's Arch., ovii, 1887, p. 1 ; Diomidoff, ibid., p. 499 ; Fish, The Wilder Quarter-Oentury Booh, 1893, p. 333 ; Donaldson, Journ. Morphol., ix, 1894, p. 123 ; Maeina, Neurol. Oentrbl., xvi, 1897, p. 166 ; TiMOFBEW, Intern. Monatschr. Anat., xv, 1898, p. 259. 814. Nervous Centres of Reptiles, Fishes and Amphibia. — ^Mason (Central Nervous System of Certain Beptiles, etc. ; WniTMAst's Methods, p. 196) recommends iodised alcohol, six to twelve hours ; then 3 per cent, bichromate, changed once a fortnight until the hardening is sufficient (six to ten weeks). BuECKHAEDT {Das Centrolnervensystem von Protopterus, Berlin, 1892 ; Ztschr. wiss. MiTcr., ix, 1893, p. 347) recommends a liquid composed of 300 parts of 1 per cent, chromic acid, 10 parts of 2 per cent, osmic acid, and 10 parts of concentrated nitric acid, in which brains of Protopterus are hardened in twenty-four to forty-eight hours. Pish {Journ. of Morphol., x, 1895, p. 234) employed for Desmog- nathus a mixture of 100 c.c. of 50 per cent, alcohol, 5 o.c. of glacial acetic acid, 5 grms. of corrosive sublimate, and 1 grm. of picric acid, fixing for twelve to twenty-four hours, and passing through the usual alcohols. Strong {Journ. comp. Neurol., xiii, 1903, p. 296) fixes (and decalcifies at the same time) the heads of young Acanthias in a mixture of 9 parts of 5 per cent, iron alum and 1 part of formol, for about two weeks, makes paraffin sections, stains with hsematoxylin, and differentiates in 1 or 2 per cent, iron alum. Johnston {Morphol. Jahrb., xxxiv, 1905, p. 150) recommends for nerves of Petromyzon to make paraffin section from Zenker material, and stain them with a mixture of saturated solution of nigrosin, saturated solution of picric acid, and 1 per cent, acid fuchsin, in water mixed in proportions arrived at by trial. Sections. 815. Imbedding is by no means always necessary, and is objected to in some cases. Indeed sections can be obtained from any part 406 NERVOUS SYSTEM— GENERAL METHODS. of the central nervous system without it, if the tissues are well hardened. Material hardened in alcohol, or in chromic solutions, or treated according to Golgi's method's may be glued on to a piece of wood or hard cork (or still better to a glass cube) by means of a rather thick solution of gum arable. As soon as it begins to stick to the support the whole is put into 70 to 80 per cent, alcohol to harden the gum, and then fixed in the obj ect-holder of the microtome and cut. Or one can simply make a clean cut at the bottom of the specimen, dry it with blotting paper and stick it on the support with sealing wax or paraffin of high melting point. For section cutting the knife should be wetted with alcohol or water ; if the latter is used some soap may be added to it to prevent it from running into drops on the knife. Formalin material is preferably cut by the freezing method, this being very largely used since the introduction of COg microtomes, by means of which many and relatively very thin sections can be rapidly obtained with great economy of time and imbedding reagents. Imbedding in paraffin is not advised for the nervous system in general, particularly after fixation in alcohol, and bichromate solutions. One should have recourse to it only for special cytological methods, taking care not to use paraffin of too high a melting point. Imbedding in celloidin is very largely used, and to great advantage, for many purposes. If, notwithstanding every precaution, the celloidin has not thoroughly penetrated the tissues, good sections may still be obtaiaed by Duval's method of collodionising the sections. The cut surface of the block is dried by blowing on it, and is covered with a thin layer of collodion laid on it with a brush. As soon as this layer has somewhat dried, which happens very rapidly, a section is cut, and the cut surface collodionised as before, and so on for each section. This process gives very good results, and may be advantageously employed even with material that has been successfully imbedded, as it gives greater consistency to brittle or otherwise deUcate tissues. The above applies to section cutting of small, medium-sized and even relatively large pieces. Also unusually large pieces, entire human hemispheres, and brains of high vertebrates can be cut into thin, and, if necessary, serial sections both without, and after im- bedding either in celloidin or paraffin or by mixed methods. The processes used for the purpose do not differ essentially from those above-mentioned and fully described in Chapters VII., VIII. and CHAPTER XXXI. 407 IX., but (particularly for cyto-architectonic and fibre-architectonic studies) special apparatus and installations are needed, the description of which is outside the province of this book. See Feist, Ztsehr. iviss. Mihr., viii, 1891, p. 492 ; Deegke, op. cit. ; Dejebine, Anat. Centres Nerveux ; Stkassek, Ztsehr. wiss. Mikr., ix, 1892, p. 8 ; Bkodmann, Journ. Psychol, u. Neurol, ii, 1903-4, p. 206 ; Wabnke, ibid., p. 221 ; Liesegang, Ztsehr. wiss. Mikr., xxvii, 1910 p. 369 ; VENDEKOVie, Anat. Am., xxxix, 1911, p. 414. General Stains. 816. Carmines. — Ammonia-carmine is good for general views. Stain very slowly in extremely dilute solutions. Bichromate material should be brought direct into the stain without passing through alcohol (see § 51). Picro-carmine has much the same action, but gives a better demonstration of non-nervous elements. BoUes Lee (see 1913 ed.) prefers carmalum with formol material as giving a more deUcate stain. He finds it better then paracarmine. The best way of staining formol material with ammonia carmine, carmalum, picro-carmine and the like, consists in my opinion in cutting sections by one or the other of the freezing methods, and transferring them for a few hours either in Miiller's fluid, or 0-5 per cent, chromic acid as suggested by Schwalbe (C&ntrbl. allg. Pathol., xii, 1901, p. 881). Sections are then washed for a longer or shorter time according to the amount of mordant one wishes to extract, proceeding afterwards to stain with one of the above-mentioned carmine solutions. On the other hand sections of non-imbedded material fixed and hardened in one or the other of the fluids mentioned in §§ 810 to 812 may be stained not only with carmines, but also with a great variety of dyes if one so desires (see Chapter XI.). The same applies to sections of imbedded material, though the after-treatment to which it has been submitted may render more or less difiicult the carrying out of certain general stains. But one should remember that in any case the results thus obtained are not very instructive, and by no means comparable with those attainable by the rational use of the special methods described in the following chapters. For other carmine processes of staining, see Soi-imaus (Miineh. med. Wochensehr., 1891, p. 147) ; Upson (Neurol. Oentralh., vii, 1888, p. 319) ; Freeborn (Journ. Roy. Mic. Sac, 1889, p. 305) ; Kadti (Neurol. Centralb., xx, 1901, p. 687) ; Chilesotti (ibid., xix, 1902, p. 161, and Gentralb. allg. Pathol, xiii, 1892, p. 19)). 408 NERVOUS SYSTEM— GENERAL METHODS. 817. Nigrosin and Anilin-Blue-Black. — Nigrosin has given useful results in some hands. Anilin-blue-black has been much recommended by Sakket (Lancet, 2, 1875, p. 82) ; Betan Lewis (Human Brain, p. 125, and Quart. Journ. Micr. 8c., 1876, pp. 73-75) ; Vejas (Arch. f. Psych, xvi, 1885, p. 200) ; Martinotti (ii, 1886, p. 478) ; Jelgeksma (Ztschr. wiss. Mihr., 1886, p. 39) ; Schmatjs (Munch, med. Wochenschr., 1891, p. 147), and others. And see also previous editions. 818. Picronigrosin. — Mabtinotti (he. cit., 1885, p. 478) stains for two or three hours or days in a saturated solution of nigrosin in saturated solution of picric acid in alcohol, and washes out in .a ' mixture of 1 part of formic acid with 2 parts of alcohol. 819. Kaisee (Ztschr. wiss. Mikr., vi, 1889, p. 471) stains sections of ■ spinal cord for a few hours in a solution of 1 part otnaphthylamin brown, 200 of water, and 100 of alcohol, washes with alcohol, clears with origanum oil and mounts. 830. Alizarine. — Schkotteb {Neurol. Centrbl., xxi, 1902, p. 338) staias sections for twenty-four hours in a 1 to 2 per cent, solution of sulphalizarinate of soda, differentiates for half to one minute in tap-water, dehydrates, and mounts. This is a general stain, but demonstrates Nissl bodies and other internal details. 821 Mallory's Phosphomolybdic Acid Hsematoxylin and Kodis' modification, see § 271. For Aueebach's modification, see Neurol. Centrbl, xvi, 1897, p. 439. 822. Hsematoxylin and Acid Fuehsin. — ^Finotti (Virchow's Arch., cxliii, 1896, p. 133) stains in hsematoxylin, counterstains for three minutes with 0'5 to 1 per cent, solution of acid fuehsin, and difleren- tiates in 75 per cent, alcohol containing a very little caustic potash. Van GtIESOn's hsematoxylin and pioro-fuchsin (§ 398) may give useful general views of nerve-cells, axis-cylinders, and neuroglia. 823. Other General Stains. — ^Alt (Munch, med. Wochenschr., 1892, No. 4) stains for a couple of hours in a solution of Congo red in absolute alcohol, and washes out with pure alcohol. This is useful for peripheral axis-cylinders and other elements. Scaepatetti (Neurol. OentrM., xvi, 1897, p. 211) stains sections of formol material for iive minutes in 1 per cent, hsematoxylin, treats for five minutes with concentrated solution of neutral copper acetate, differentiates with Weigert's borax-ferricyanide, treats with con- centrated solution of lithium carbonate, washes and moimts. Myelin is not stained. ROTHIG (Folia Neurobiol., ii, 1909, p. 385) fixes and stains for about four weeks in saturated solution of methylenazur I., in 10 per cent, formol, puts for ten to fifteen minutes into acetone, then for twelve hours into chloroform, and imbeds in paraffin. He also has a process with trichloraoetate of lead and methylenazur. CHAPTER XXXI. 409 Ratvitz {Ztsehr. wiss. Mikr., xxvi, 1909, p. 341) has some compli- cated methods with Indulin, Indaminblau, and Azosdureblau, which take twenty-eight days; and {ibid., xxviii, 1911, p. 1) others with fuchsin and azofuohsin which take over thirty-six days. Ariens Kappees {ibid., xxviii, 1911, p. 417) describes a staining method with extract of elderberries for material fixed and hardened in Miiller's fluid or similar solutions. It is very simple and particularly recommended for photographic purposes ; it should be carried out as follows : Stain celloidin or paraffin sections overnight in neutralised elderberries extract ( obtained by fermentation at 20° to 25° C. ), to which 1 per cent, carbolic acid has been added. Wash in water. Difierentiate in 3 per cent. Liquor ferri sesquichlorati P.G., wash, dehydrate, and mount. CHAPTER XXXIL* NERVOUS SYSTEM — SPECIAL METHODS, CHIEFLY CYTOLOGICAL. 824. Introduction. — The .ordinary methods of cytology are, of course, available for nerve cells ; but there are certain constituents of these cells, as well as of nerve fibres, which require, for minute study, special methods, such as the following : — A. Methods for Cells, demonstrating Tigroid Substance and other Granular Materials. 825. Tigroid substance or bodies, chromophilic or chromatophilic substance or material or granules, Nissl's bodies or granules, etc., are all denominations for a markedly basophil substance which appears as blocks, granules or irregular patches within the cytoplasm of nerve cells under certain conditions of fixing and staining. It is now almost universally admitted that this substance exists in the living cells as a fluid or semi-fluid " plasm rich in nutritive value," and that the blocks, granules or patches are appearances chiefly due to the coagulation of this plasm, as brought about by the fixing agents employed for their demonstration. As, however, these bodies or granules appear always the same under constant optical conditions in healthy cells fixed and stained in a constant manner, they are said to be the equivalent of such healthy cells during life. " It follows that if the cells, prepared by the same method and examined under the same conditions, show a difference from the equivalent or symbol of healthy cells, the difference is the measure of some change that has occurred during life." See Halliburton, Handbook of Physiology, London, 1920, p. 194. This is pointed out here to make it quite clear why Nissl has always insisted that his method should be carried out according to his suggestions, and in a constant manner. At first {Neurol. Centrbl., iv, 1885, p. 500) he used to stain sections of material fixed in alcohol with a warmed watery solution of magenta red or dahlia violet or vesuvine, and to differentiate them with alcohol. Later (Allg. Ztschr. Psych., xlviii, 1892, p. 197) he suggested floating sections on a warmed solution of methylene blue (B patent), with * Ke-written by Dr. C. Da Fano, King's College, University of London. CHAPTER XXXII. 411 subsequent differentiation with a 10 per cent, solution of anilin oil in 96 per cent, alcohol. The present form of the method was published in 1894 {Neurol. Centrbl., xiii, p. 507) ; but NrssL con- tinued to introduce into it slight modifications, as one understands from many of his papers, to which due attention was paid when preparing the following account. It must be added here that Nissl's method has been, and still is, extremely useful for the study of nervous tissue under various physiological and pathological conditions, and that it stains, when properly carried out, not only the tigroid substance and the basophil parts of nuclei of nerve cells, but also the nuclei and certain parts of the cytoplasm of neuroglia cells and connective tissue elements normally or abnormally present in the nervous tissue. 826. Nissl's Methylene-blue Method. — Not too small pieces of fresh tissue are fixed in 96 per cent, alcohol and hardened therein for a few days. They should not be allowed to fall to the bottom of the bottle, but kept floating by means of some filter paper or cotton wool. The alcohol must be in large quantities in proportion to the nimiber of pieces, and repeatedly changed. The pieces are cut without embedding and the sections collected in 96 per cent, alcohol, from which they are directly floated on some stain filtered into a watch glass at the moment of using it. The stain should be at least three to four months old, and shaken at the moment of filtering the quantity needed. It is prepared by carefully dissolving 1 -75 grms. of Venetian soap in 1 litre of distilled water and adding to it 3 -75 grms. of methylene blue (B patent). It is a good practice to vigorously shake the bottle from time to time, and to re-filter into the same bottle the amount of stain left in the watch-glass after staining one or more sections. The watch-glass containing the stain with the section floating on it is warmed carefully over a flame until small bubbles rise to the surface. The section, which should not have fallen to the bottom of the watch-glass, is immediately transferred into a mixture of 10 parts of anilin oil and 90 parts of 96 per cent, alcohol, and as soon as no more colour is given off (it often takes only some seconds), it is lifted on to a slide, pressed with smooth filter paper, and cleared with a few drops of pure anhydrous cajeput oil. Care should be taken not to dry the section excessively with the filter paper and to pour the cajeput oil on to the section very quickly. The cajeput oil not only clears the section, but stops the differentiation ; it is, therefore, advisable to renew it after a little 412 NERVOUS SYSTEM— SPECIAL METHODS. while on the section. As soon as this has become quite transparent, the cajeput oil is dried off with filter paper, the section thoroughly washed with benzol and covered with a drop of thick xylol-colo- phonium, rendered more fluid by passing the slide carefully over a flame, and quickly covering the section with a thin cover-glass before the colophonium sets again by cooling. 827. Suggestions regarding the Carrying-out of Nissl's Method (as deduced from Nissl's papers ; see chiefly Enz. d. mikr. Techn., ii, 1910, pp. 252 — 280, and the references therein quoted at p. 287). — For the fixation of tissues alcohol should be ahnost exclusively used. Formalin, mixture of formahn and alcohol, subhmate and mixture of the same with alcohol or picric acid and the like, nitric acid may be occasionally employed, but no particularly good results can be expected from them. These are, however, somewhat better if tissues are placed for some time in alcohol after fixation with one or the other of the above reagents. This applies particularly to formalin material, which can be kept with advantage for many weeks, and even months, in repeatedly changed 96 per cent, alcohol. The bichromates of potassium and ammonium and mixtures con- taining chromic' salts, though useful for other purposes, should be entirely avoided for cytological investigations in Nissl's sense. See on this subject also Burchardt, La Cellule, xii, 1897, p. 337. If tissues are too brittle to be sectioned without embedding, or if embedding is for any other reason desirable, one should have recourse to celloidin, paraffin being used only when unavoidably necessary or for special purposes. Pieces to be embedded in celloidin are not to be passed through alcohol-ether, but directly from absolute alcohol into thin celloidin. Embedding should, in any case, be carried out as quickly as possible. Sections of material which was not fixed in alcohol and of em- bedded tissues, however fixed, stain, as a rule, very poorly by Nissl's soap-methylene-blue method ; but good and even excellent results can be obtained by staining such sections with watery solutions (generally 0-5 to 1 per cent.) of toluidine blue, thionine, Unna's polychrome methylene blue, dahlia violet, vesuvine, neutral red, magenta red, Azur I, Azur II, and the hke. If one of such stains is used, it need not be warmed until bubbles come to the surface, but only until vapour arises. For the differentiation pure 96 per cent, alcohol, viz., without any addition of anilin oil, should be used. In this connection I find that very good results can be obtained from material embedded in CHAPTER XXXn. 413 celloidin if the sections are, whenever possible, freed from the celloidin before staining them, and if they are re-stained a second and a third time after having been each time completely differentiated. See Da Fano, Proc. Physiol. Soc, Journ. Physiol., liv, 1920-1. All preparations stained by Nissl's method keep badly, but they keep a little better and may last almost unchanged even for years : (1) if the anilin-alcohol or alcohol used for the differentiation is properly washed away with pure benzol ; (2) if the xylol-colo- phonium for the mount is prepared with pure xylol, and of the thickness needed ; (3) if sections of material embedded in celloidin are submitted to the above-mentioned succession of staining and differentiation ; (4) if preparations are carefully protected from light. 828. Modifications of Nissl's Method. — Eehm (Miinch. med. Wochenschr., xxxix, 1892, p. 217) floats sections for half a minute to a minute on a hot 0-1 per cent, solution of methylene blue, differentiates them in 96 per cent, alcohol and clears them with origanum oil. Lenhossek (Fein. Bau. d. Nervens., 1895) stains sections of f ormol material in a concentrated aqueous solution of thionine, rinses them with water and mounts them like Mssl. LuxENBUEG (Neurol. Centrbl., xviii, 1899, p. 629) stains paraf&n serial sections either with Nissl's methylene blue or with thionine as Lenhoss6k. JuLiusBUEGER (Neurol. Centrbl., xvi, 1897, p. 259) stains sections of material fixed in Orth's fluid and embedded in celloidin, either with Nissl's methylene blue or with warmed neutral red. EosiN (Deutsche med. Wochenschr., xxiv, 1898, p. 615) treats sections of formol material similarly. Lesthossek (Neurol. Centrbl., xvii, 1898, p. 577) stains parafiin or celloidin sections of spinal ganglia fixed in Carney's fluid with a concentrated watery solution of toluidine blue overnight, rinses with water, differentiates quickly with alcohol and clears with xylol or carbol-xylol. PoLUMOEDWiNOW (Ztschr.'vnss. Mihr., xvi, 1899, p. 371), uses 1 part of 1 per cent, solution of toluidine blue to 119 of distilled water and 1 of sodium carbonate. Van Gehuchten and Nelis (La Cellule, xiv, 1898, p. 374) recom- mend fixing spinal ganglia in Gilson's mixture. Van G-ehuchten (see 1913 ed.) uses paraffin sections mounted on slides by the water method and stains them for five to six 414 NERVOUS SYSTEM— SPECIAL METHODS. hours in Nissl's methylene blue solution in the incubator at 35° to 40° C. GoTHAED (C. R. Soc. Biol, V, 1898, p. 330) stains celloidin sections for twenty-four hours in Unna's polychrome methylene blue, and difierentiates them with a mixture of 5 parts of creosote, 4 of cajeput oil, 5 to 8 of xylol, and 16 of absolute alcohol. The mixture is removed with absolute alcohol and sections mounted in xylol dammar after clearing with cajeput oil. LuiTHLEN and Sorgo (Neurol. Centrbl., xvii, 1898, p. 640) diffe- rentiate in Unna's glycerin-ether mixture, remove this with absolute alcohol, and clear with origanum oil. Similarly Lbnnhopf (ibid., 1910, p. 20) ; or, polychrome methylene blue two minutes, distilled water quickly, carbol-pyronin-methyl green twenty minutes ; distilled water quickly, absolute alcohol, oil, balsam. Lord (Journ. Ment. Sc, xliv, 1898, p. 693) makes sections from frozen fresh tissues, treats them for a few seconds with a mixture of equal parts of 6 per cent, formaldehyde and saturated solution of picric acid, then rinses them with distilled water and stains them in 5 per cent, solution of methylene blue B pat. 829. BiBLSCHOwsKY and Plien's Cresyl Violet Method (Neurol. Centrbl., xix, 1900, p. 1141).^ — Celloidin or paraffin sections of material fixed either in alcohol or formalin, or sections made by the freezing method from formalin material, are stained for twenty-four hours in a very diluted solution of cresyl violet R.R., prepared by adding 6 to 8 drops of a concentrated aqueous solution, to every 50 c.c. of distilled water. After a quick wash in distilled water sections are brought through the ascending series of alcohols, cajeput oil, and xylol, into balsam. The preparations are said to keep better than those stained with thionine or toluidine blue. 830. Picrocarmine has been successfully used by Messnee (Journ. Psychol. Neurol., xviii, 1912, p. 204, and xx, 1913, p. 256). Sections of alcohol material, embedded in celloidin or not, are washed in water and then stained for five minutes in a warmed diluted solution of Ranvier's picrocarmine. After a quick wash, they are differen- tiated in 3 per cent, hydrochloric acid, dehydrated and mounted as usual. In the case of the spinal cord, medulla oblongata and pons the method succeeds also if material was fixed in formalin. 831. Other Methods for Tigroid Substance and Basophil Granules in General. — See Goldscheidee u. Flatau, Norm. u. path. Anat. d. Nervenz, Berlin, 1898, or Ztsehr. wiss. Mikr., xvi, 1899, p. 102, and CHAPTER XXXII. 415 Nissl's remarks thereon, Deutsche Ztschr. Nervenheilk, xiii, 1899, p. 348 ; lisMMQ, Neurol. Centrbl, XV, 1896, p. 831; Cox, Anat. Eefte, x, 1898, p. 75 ; Int. Monatschr. Anat.,xv, 1898, p. 241 ; Aueebach, Monatschr. Psych. Neurol, iv, 1898, p. 31 ; Myers, Anat. Bee, ii, 1908, p. 434 ; Savini, E. u. Th., Oentrbl. BaU., I Ahth., xlviii, 1909, p. 697 ; Mosse (argentamin stain), Arch. mikr. Anat., lix, 1902, p. 403 ; Menxz v. Keogh, Gentrbl. Baht, I Abth., Iviii, 1911, p. 95 ; Johnston, jlmai. Bee, xi, 1916, p. 287. 832. Held's Methylene Blue and Erjrthrosin Method {Arch. Anat. Phys., Anat. Abth., 1895, p. 399 ; 1897, pp. 226—233, 273—385, Supplementband). — Material may be fixed in alcohol, but preferably either in picro-sulphuric acid, or in van Gehuchten's mixture of alcohol, chloroform and acetic acid, or 1 per cent, corrosive sublimate in 40 per cent, acetone. Tissues should be carefully embedded in paraffin and sections stuck to slides by the water method. They are stained with the aid of gentle heat for one to two minutes in a solution of 1 grm. erythrosin in 150 of distilled water acidulated with 2 drops of glacial acetic acid. After washing with water the slides are transferred into a mixture of equal parts of Nissl's methylene blue solution and 5 per cent, acetone, warming until all odour of the latter has disappeared. Differentiation is carried out after cooling by means of a -1 per cent, solution of alum until sections are reddish. Rinse in distilled water, dehydrate as rapidly as possible in absolute alcohol, wash in xylol and mount iu balsam. BoccARDi (Monit. Zool. Ital., x, 1899, p. 141) uses a mixture of erythrosin 0-1 grm., toluidine blue 0-2 grm., and water 100 c.c, and differentiates in -5 per cent, alum solution. By means of Held's method, besides the tigroid substance, other granules — viz., Held's neurosomes — become stained. It may, there- fore, be considered as a typical example of double staining of nerve- cells. Other double stains demonstrating basophil, acidophil and other granules have been repeatedly proposed and may be easily obtained by the combination of an acid and a basophil dye. One generally uses watery solutions, e.g., of acid fuchsia, and methylene or toluidine blue, and one staius first with the acid dye and then with the basophil one, differentiation being carried out with alcohol. One may also have recourse to Ehrlich's triacid (§ 296) as originally proposed by Rosin (Neurol. Centrbl., xii, 1893, p. 803), or to one or other of the methods used for staining blood films (§ 784), such as Pappenheim's panoptic triacid stain, Jenner's mixture, Leishman's Romanowsky stain, Pappenheim's method as described in § 785, and so on. See on this subject Cowdkt, Int. Monatschr. Anat., xxix, 1913, 416 NERVOUS SYSTEM— SPECIAL METHODS. p. 673, and for pathological specimens Alzheimer's methods 5, 6 and 9 in Histol. u. Histopath. Arb. iiber d. Orosshirnr, iii, 1910, pp. 406 — 412, wMch may be useful for the study of nerve-cells though originally pro- posed for the investigation of neuroglia. B. Methods for Cells and Fibres, demonstrating Neurofibrils. 833. Neurofibrils ; General Characters. — Nerve cells and the fibres into which they are prolonged contain, in addition to the chromatic constituents shown by the methods already dealt with, a character- istic so-called achromatic element, consisting chiefly of very fine and fairly refractive fibrils which can only be seen with great difficulty in the unstained state, but may be fixed with osmic acid and made out in thin sections of meduUated nerve fibres observed in diluted glycerin or water, and may be to a certain extent isolated by macera- tion. For their demonstration, however, one or the other of the methods chronologically described in the following paragraphs must be employed. They are all regarded as giving true stains of neuro- fibrils. For the method of Kupfee (8itzb. math. Kl. Alcad. Wias. Munohen, xiii, 1884) see former edition. 834. Apathy's Methods. — The gold method (" Nachvergoldung ") has been given in § 371. The staia is very sharp, but good results are obtained only in certain invertebrates, and even in these with considerable difficulty. The hcemateine method (Mitth. Zool. Stat. Neapel, xii, 1897, p. 712) has the same advantages and disadvantages, and has been little used since the discovery of the Cajal and Bielschowsky processes. Material may be fixed with corrosive sublimate, Zenker's fluid, picro-sulphuric acid, or any other mixture which is not inimical to staining with alum hsematoxylin, and should be preserved in 90 per cent, alcohol. Portions, no more than ^ cm. thick, are stained for at least forty- eight hours in haemateine I.A. (§ 259), and then washed for up to twenty-four hours in absolutely pure distilled water, or preferably suspended therein. Before the stain has become washed out of the neurofibrils entirely, it is fixed by putting the preparations for three to five hours into spring water, after which they are put back for not more than two hours into distiUed water, dehydrated as rapidly as possible by hanging them up in absolute alcohol, and embedded in paraffin or celloidin, after clearing with chloroform, and carefully protecting them from light whilst in chloroform or celloidin. The sections are mounted either in a resin or in neutral glycerin. CHAPTER XXXII. 417 835. Bethe's Molybdenum-Toluidine Blue Method (Ztschr. wiss. MiJcr., xvii, 1900, p. 13).— Pieces of the central nervous system of vertebrates are fixed for twenty-four hours in 3 to 7-5 per cent, nitric acid, and then brought directly into 96 per cent, alcohol for a day or longer. They are afterwards put for twelve to twenty-four hours in a mixture of 1 part of ammonia (of sp. gr. -95) with 3 of distilled water and 8 of 96 per cent, alcohol ; for six to twelve hours into pure alcohol ; for twenty-four hours into a mixture of 1 part of concen- trated hydrochloric acid, 3 of distilled water, and 8 to 12 of alcohol ; for ten to twelve hours into pure alcohol ; for two to six hours into water. They are now mordanted with 4 per cent, ammonium molybdate, washed again, dehydrated and embedded in paraffin. The sections, 8 to 10 /^i thick, are seriated on slides by means of egg albumen, but without water, then passed through xylol and alcohol and " differentiated," viz., covered with water poured on the sections so as to form over them a layer 1 -5 to 2 mm. deep, and put into an incubator at 55 to 60° C. for ten minutes. They are then rinsed with water, covered with a 1 : 3000 solution of toluidine blue, stoved for another ten minutes, rinsed with water, and lastly treated with 96 per cent, alcohol till no more colour comes away. After dehydration with absolute alcohol they are mounted in the usual way. The method is also applicable to invertebrates for which other fixing agents besides nitric acid are admissible, and the impregnation with ammonium molybdate may be done on the sections instead of previously on the uncut tissues. For LuGAKO's modification see Biv. pat. nerv. ment., x, 1905, p. 266. 836. DoNAGGio's Methods {Riv. Sper. Freniatr., xxx, 1904, p. 397, and xxxii, 1906, p. 394). — There are five methods of Donaggio. By the first two, pieces are stained in bulk before embedding, but results are not so good as by the other three, the most important of which is — Method III. — Good for spinal cord, pons, meduUa oblongata, spinal and sympathetic gangha. Thin slices of tissues are fixed for five to six days in pure pyridine changed at least once, and then treated with repeatedly changed distilled water until the pyridine has been entirely eliminated. The surfaces of pieces are smoothed by means of a sharp razor, and the pieces brought for twenty-four hours into 4 per cent, ammonium molybdate to which 4 drops of hydrochloric acid have been added. After a quick wash, they are rapidly dehydrated in 95 per cent, and absolute alcohols, and em- M. 27 tl8 NERVOUS SYSTEM— SPECIAL METHODS. bedded in paraffin. The sections, which must be rather thin (3 to 6 /i), are brought through xylol, absolute and 95 per cent, alcohols into distilled water and here washed. This is the crucial point of the method because, by washing, ammonium molybdate becomes extracted from the sections, and the success of the subsequent staining depends almost entirely on carrying out the extraction up to the right point. I find that the only way of ensuring this consists in proceeding by trials, which must be repeated for every series of sections. Once the right amount of washing has been decided upon, one can proceed to stain even many slides at the same time by means of a 1 : 1 0,000 solution of thionine, to be freshly prepared every time from a less diluted stock solution. The staining is a " progressive " one, and must be controlled under the microscope. It generally takes about twenty minutes to obtain it, at the end of which time the grey substance has a red- piu-ple tone whilst the white substance appears bluish. If the staining is right the preparations can be quickly washed, dehydrated and mounted. But if the neurofibrils are not quite sharply stained, the preparations can be " differentiated "for another fifteen to twenty minutes in the ammonium molybdate solution used for mordanting the pieces, or for ten seconds in a diluted solution (1 : 10 to 1 : 20) of " pink salt " (C. Erba, Milano). Preparations last only a few months, but are sometimes of great interest. See Da Fano, Ziegler's Beitrdge, xliv, 1908, p. 495. Method IV, which is particularly useful for the demonstration of neurofibrils in the cells of the cortex cerebri and cerebeUi, differs from Method III only in regard to a preliminary fixation of pieces for twenty-four hours in a mix;ture of pyridine nitrate 10 grms., and pyridine, 100 c.c. ; they are then transferred for another thirty-six hours into pure pyridine, proceeding as in Method III. Method V may be used for the demonstration of both Nissl's substance and neurofibrils. Pieces are fixed in a saturated solution of corrosive sublimate ; after a day they are treated for twenty-four hours with distilled water to which a few drops of iodine tincture have been added, then for two to three hours with pure distilled water ; and lastly passed for forty-eight hours into pure pyridine, this being changed at least once. The rest as in Method III. Paeavicini {Boll. Mus. Z. Anat. Oomp. Tormo, xx, 1905, p. 1) fixes and mordants in the dark, and differentiates after staining with, extremely weak hydrochloric acid. ToMASELLi (Ztschr. wiss. Mikr., xxiii, 1906, p. 421) fixes spinal ganglia for six to seven hours in absolute alcohol 100 c.c. with four to five drops of ammonia, and then transfers them for two days into pure CHAPTER XXXII. 419 pyridine to be repeatedly changed, the vessel with the pieces being kept at 36° to 37° C. After washing for two to three hours in running tap water, he continues as in Donaggio's method III. For the criticism of Jadbeholm, see Arch. mikr. Anal., Ixvii, 1906, p. 108 ; and for that of Montanaki, Ztschr. wise. Milcr., xxviii, 1911, p. 22. 837. Ramon y Cajal's Methods. Introductory. — It has been said by some authors that Cajal's methods were originally only modifica- tions of the photographic process of Simarro. The criticism is unjust because even the first formula of Cajal differs so profoundly from Simarro's process as to form an entirely new method. One cannot, however, deny the existence of a certain similarity oi con- ception between the two processes in so far as both are based on the silver-reducing power of certain photographic reagents. For this reason it has been thought expedient to briefly describe here Simarro's process, which though uncertain in its results, may still be of some value to elucidate certain histological questions. Simarro's Process (Rev. Trim. Micr., v, 1900, p. 45) consisted in poisoning animals with subcutaneous injections of solutions of sodium or potassium bromide or iodide in order to impregnate their living nervous tissues with one or the other of these salts. As soon as the animals showed that the poisoning had reached its maximum they were killed and their central nervous system removed in the photographic dark room. Small pieces were then immersed in a solution of silver nitrate, which, by combining with the bromine or iodine with which the tissues were impregnated, gave rise to the formation of silver bromide or iodide, which is easily affected by light. Sections were then made (always in the dark room), best by means of a freezing microtome, and exposed for a little while to light. There remained only treating them with a photographic developer, such as hydroquinone, pyrogallol or the like, and fixing them with sodium hyposulphite and so on, as if they were photographic plates ; they were lastly washed, dehydrated and mounted in the usual way. One can easily understand the many drawbacks of such a method and the reason for which it was abandoned as soon as Cajal published in 1903 his first reduced silver methods. From that time onwards, Ramon y Cajal continued improving them and adding new formulae, which he himself summarised in a special article of his Trab. Lab. Invest. Biol., Madrid, viii, 1910, on which the following account is based. The numbering is that of Ramon y Cajal. Formula la. — Small pieces of fresh tissue are directly put into 1-5 per cent, silver nitrate and kept therein for three to four and 27—2 420 NERVOUS SYSTEM— SPECIAL METHODS. even to five days at a temperature of about 35° C. In summer, with a temperature constantly over 22°- C, the stove may be dispensed with, provided the impregnation is prolonged for two to three days more. The tissues are known to be ripe for reduction when a freshly cut surface shows a brownish-yellow colour. They are then washed for one to two minutes in distilled water and put into — Pyrogallol or hydroquinone . . . 1- — 2 grms. Distilled water ..... 100 c.c. Formalin ...... 5 — 10 c.c. The formol is not necessary but useful. One may use pyridine instead (1 to 3 per cent.). The addition of a small quantity of sodium sulphite (0-2 to 0-5 per cent.) has been abandoned by Cajal. The stronger the pyrogallol, the greater the contrast, so that it may be useful to take, sometimes, as much as 3 per cent., but then the over-impregnation of the outer layers will be increased. Hydro- quinone reduces more energetically than pyrogallol. The pieces remain in the reducing fluid for about twenty-four hours and are then quickly washed, hardened in alcohol and embedded in paraffin or celloidin. The sections (15 to 20 /n thick) are mounted in dammar after toning with a solution of gold chloride if the reaction . is rather weak, without toning if the impregnation is a good one. Faintly impregnated sections can be advantageously toned with — Distilled water ..... 100 c.c. Ammonium sulphocyanide ... 3 grms. Sodium hyposulphite . . . . 3 „ 1 per cent, gold chloride ... a few drops. If subsequently found to be too dark they can be bleached by Veratti's potassium permanganate and sulphuric acid mixture (see § 846). The sections from the outer layer are generally too dark for study, those from the innermost too pale, .whilst those from the intermediate layer are good. The over-staining of the outer layer can be diminished by diluting the silver nitrate with 1 volume of water for the last twelve hours. The method has the defect of giving an imperfect fixation of the nervous tissue and of impregnating, almost exclusively, cell bodies and dendrites. It is not good for ganglia and large cells of adult subjects, but excellent for small and medium-sized cells of very young subjects and early embryos. Formula la, A.r— As the last, but pieces are fixed in 3 to 6 per cent. CHAPTER XXXII. 421 silver nitrate. This formula gives better fixation, and was success- fully used by DoaiEL (Anat. Anz., xxv, 1904, p. 558, and Arch, mikr. Anat., Ixvii, 1906, p. 638) for the study of Grandry's corpuscles and other sensory nerve endings, by Kolmer (Anat. Anz., xxvi, 1905, p. 560) for the epidermis of Lumbricus, etc., and by other authors for the ganglionic chain of Hirudinea. Formula la, B. — As above, but taking -75 per cent, silver nitrate and very small pieces, preferably from embryos and new-born sub- jects. Poor fixation, much shrinkage, but vigorous stain of the neuro- fibrils, nucleolar granules and the intranuclear rodlet of Roncoroni. Formula la, C. — As above, but tissues are fixed in 2 per cent, silver nitrate to which one-fourth of absolute alcohol or acetone has been added. Better fixation than with pure silver nitrate. Eesults very similar to those obtauiable by Formula la with dog, cat and rabbit, and better results with human cerebrum and cerebellum. Formula 2a. — Fixation for twenty-four hours in 96 per cent, alcohol. Tissues not washed, but mopped with blotting paper and put into 1-5 per cent, silver nitrate for seven days at 35° C, or six days at 40° C. The rest as Formula la. Good impregnations of nerve centres of adults, of peripheral nerve endings, of regenerating nerves, of early embryos, and of young fishes. It impregnates meduUated and many non-meduUated fibres, large and medium nerve cells, the basket fibres of Purkinje's cells, etc. Results fairly constant, but sometimes showing a granular precipitate of unknown origin. To hinder this precipitate and, at the same time, to hasten the impregnation, it is well to add to the alcohol certain substances which Cajal calls " accelerators." Such are chloral hydrate, veronal, pyridine, nicotine, ethylamine, antipyrine, and others. Hypnotics, particularly veronal'and chloral, and in a less degree pyridine and ammonia, also act as " rejuvenators," reviving the susceptibility of impregnation in tissues which have lain too long in alcohol. Formula 2a, A. — ^Fixation for twenty-four to forty-eight hours in 96 per cent, alcohol with 2 per cent, of chloral hydrate. Silver bath of 1 -5 per cent, for five days in the stove. The rest as usual. Veronal (same proportion) gives the same result, as do also sulphonal, trional, hedonal, etc. The results are very constant. MeduUated fibres well shown. Formula 2a, B. — Fix for twenty-four hours in 96 per cent, alcohol with 10 to 20 per cent, of pyridine ; wash for some hours in pure alcohol and transfer pieces into 1 -5 silver nitrate for five days. 422 NERVOUS SYSTEM— SPECIAL METHODS. I understand from Cajal's pupil, Del Rio Hortega, that this formula may be successfully employed for the study of peripheral nerve endings. In this case material is better fixed for twenty-four hours in pyridine to which one-third its volume of distilled water or 96 per cent, alcohol has been added. Pieces should be washed in running tap-water overnight and then transferred for six hours into pure 96 per cent, alcohol. Impregnation, reduction, embedding, etc., as above. Results are good, but pieces become extremely hard even if dehydrated very quickly, and are consequently difficult to cut. See also Formula 5a. Formula 2a, C. — Fix for twenty-four hours in 50 c.c. of alcohol with 10 drops of nicotine. Mop up with blotting paper, without washing, and silver as usual for five days (or four at 40° C). Good results with adult tissues, especially spinal cord. Good penetration and less shrinkage than with pure alcohol. Formula 2a, D. — Fix for twenty-four hours in allyl alcohol (the industrial product will do). Wash for some hours in several changes of water. Put for a day into 50 c.c. of alcohol with 4 drops of ammonia. Silver for four days at 35° to 38° C, and reduce as usual. Good for human tissues, especially for fibre plexuses of cerebrum and cerebellum. Instead of allyl alcohol one may take acetal or acetone. Put for six hours into acetone with 25 per cent, of water, then for twenty-four into pure acetone, wash in water, etc., as above. Formula 3a. — Fixation in ammoniacal alcohol for twenty to forty- eight hours. The most generally useful formula is 50 c.c. of 96 per cent, alcohol with 4 to 5 drops of ammonia (of 22° strength). But for cerebrum not more than 1 to 3 drops ; for cerebellum, ganglia, spinal cord and regenerating tracts, 4 drops ; for neurofibrils of the large nerve cells of the medulla oblongata and spinal cord, 9 to 10 drops. To avoid shrinkage, it is well to begin by putting the pieces for six hours into 70 per cent, alcohol, then in 85 per cent., without ammonia ; then for the rest of the time into the ammoniacal alcohol. Do not wash, but mop up with blotting paper before putting into the silver. Silver for four to four and a half days (small specimens) at 40° C, or medium to large (3 to 4 mm. thick) for five days at 32° to 35° C. So long as the tissues are only yellowish-white, they are not ripe for reduction ; light grey indicates ripeness ; dark grey over-ripeness. Reduce as by Formula la. Specimens may be decalcified, after reducing and washing, in 96 per cent, alcohol to which a few drops of nitric acid have been added. For the delicate impregnation of the neurofibrils of the large and CHAPTER XXXII. ' 423 medium nerve cells this formula is superior to all others. It gives good results with the majority of nerve centres, and is particularly good for non-meduUated fibres, peri-cellular baskets of cerebellum, buds of Held and Auerbach in the oblongata, for human sympathetic, and for the study of regenerating nerve fibres. Formula 3a, A. — Fix in 50 c.c. of alcohol with 10 grms. of glycerin and 6 to 10 drops of ammonia. Good for retina and non-medullated fibres, but especially for the buds of Held and Auerbach. Formula 3a, B. — -Fix in 50 c.c. of alcohol with 1-5 c.c. of a 33 per cent, alcoholic solution of ethylamine. Results the same as with ammoniacal alcohol. Formula la. — ^Pieces of tissue of not more than 4 mm. in thickness are fixed for six to twelve hours in 15 per cent, formol. Wash for six or more hours in running tap-water. Put for twenty-four hpurs into 50 c.c. of alcohol with 5 drops of ammonia. Wipe with blotting paper, silver for five days (or four if the stove is at 38° to 40° C). The rest as usual. Sharp impregnation of the finer fibres of nerve centres and of the terminal buds of pericellular nests. Adult tissues give better results than young ones. Energetic stain of the arborisa- tions of the moss fibres of the cerebellum. Formula 4a, A. — ^Fix in " a mixture of formol and alcohol." Wash out thoroughly with running tap-water, silver, and reduce as usual. Fixation more rapid and better ; results similar to those of 3a. Formula 5a. — This is characterised by a preliminary fixation in pyridine as originally suggested by Held {Arch. Anat. Physiol., Anat. Abth., 1905, p. 77 ; Anat. Anz., xxix., 1906, p. 186). He used to fix tissues in pure pyridine, but Cajal finds that this is likely to cause much shrinkage, and he recommends fixing small pieces first for six to eight hours in a mixture of equal parts of distilled water and pyridine, then for eighteen to twenty-four hours into pure p)rridine. Wash for several hours in running water, and put for a day into 90 per cent, alcohol. Wipe, and put for four to five days into 1 -5 per cent, silver nitrate at 35° to 38° C, and reduce as usual. Not very good for adult organs, but superior to all others for the earliest phases of neurogenesis, and good for regenerative processes, as well as for peripheral nerve endings. Formula 6a. — Put for twenty-four hours into 50 c.c. of water with 5 grms. of chloral hydrate, rinse, and put into 50 c.c. of 96 per cent, alcohol with 5 drops of ammonia (time not stated). Wipe with blotting paper ; put for four to five days at 35° to 38° C. into 424 NERVOUS SYSTEM— SPECIAL METHODS. 1-5 per cent, silver nitrate, -and reduce as usual. Results very- constant, without shrinkage. Good for the fine plexuses of cere- brum, bulb and cord, the baskets of Purkinje's cells, and moss fibres ; also for motor plates and for regenerating nerves. Formula 6a, A. — Fix for twenty-four hours in 10 per cent, chloral hydrate, wash for six, and put direct into the silver. Stove for four days. Eesults similar to those of Formula la. Medullated fibres well stained. Formula 7a. — Fix for twenty-four hours in" Merck's fihrolysine, wash for six, put for twenty-four into 50 c.c. of alcohol with 5 drops of ammonia. The rest as by other formulae. Instead of fibrolysin, lysidine may be taken. 838. Application of Cajal's Methods to different Objects. — (1) For the study of the evolution of neuroblasts and nerve fibres in very early embryos it is necessary to avoid fixing with formol, or alcohol with an accelerator, or ammoniacal liquids. The best formulse are 2a and 5a which are applicable to all vertebrates, but preferably to embryos of birds and fishes. (2) For late embryos and foetus of mammals. Besides the above formulse, 3a, 6a and alcohol with an accelerator. Best subjects, embryos of chick from the fifth day, and of rabbit from the tenth to the twelfth day; or new-born birds, with ammoniacal alcohol, or 5a. (3) For sympathetic ganglia. Formula 3a, or pure alcohol, or 4a and 5a. Best with man. Dog, cat, and rabbit give mostly weak reactions. The visceral ganglia are the most difficult. (4) Sensory ganglia. Formula 2a or 3a. Easy. (5) Cerebellum. For Purkinje cells, la or 3a. For the baskets, climbing fibres, and medium and small dendrites, 2a or its varig-nts. For terminal rosettes and collaterals of moss fibres and for the plexuses of the granular layer, 4a or sometimes 5a or 6a. For the stellate cells of the molecular layer, 2a and 3a. The best subject for the latter is the dog. (6) Cerebrum. In general, the same formulse as for the cere- bellum, especially la for pyramids of young dogs and cats (of eight to twenty days). In Formula 3a the proportion of ammonia should be diminished. For fine plexuses, 4a, 5a, and 6a. (7) Spinal cord and bulh. All the formulse are applicable. For neurofibrils of motor cells the best subject is the dog of four to fifteen days, with Formula 3a, with a large dose of ammonia (10 drops) ; also the alcoholic fixatives with an accelerator. For medullated CHAPTER XXXII. 425 fibres, large and small, 2a or 6a. For buds of Held and Auerbach and for fine plexuses, 4a, 3a, A, or 5a. (8) Ganglia of invertebrates. For the medicinal leech (not for other leeches), la, A. For Hcemopis, Aulostomum, Pontobdella and Glossiphonia, 2a or, better, 3a, with not more than 2 to 5 drops of ammonia and 3 per cent, silver nitrate, stoving three or three and a half days. For further details see Sanchez, Trab. Lab. Invest. Biol., Madrid, vii, 1909, pp. 42—47. Lumbricus is generally refractory to Cajal's methods. Boule (Le Nevraxe, x, 1908, p. 15) obtained good impregnations by acidi- fying the fixatives. He takes : (a) 25 per cent, formol with 5 per cent, of acetic acid ; or (6) the same with -5 per cent, of ammonia ; or (c) 100 c.c. of alcohol, 25 c.c. of formol, 5 c.c. of acetic acid, and 0-5 c.c. of ammonia. For the impregnation he uses 3 per cent, silver nitrate with 15 per cent, of alcohol, and reduces in the usual hydroquinone-formol solution, with the addition of 15 c.c. of alcohol. These results are confirmed by Kowalski (La Cellule, xxv, 1909, p. 292, and by Schutz (Anat. Anz., xlii, 1912, p. 262). Kowalski gets impregnations also by simply starving worms for several days, or exposing them to cold ( — 5° C.) for a quarter of an hour. (9) Regenerating nerve tissue. For nerves operated a month or more previously. Formula 2a or 3a, with not more than 3 drops of ammonia, will stain equally the old and the new fibres ; for nerves operated not more than two to ten days previously, Formulse 3a with 4 to 6 drops of ammonia, 5a with pjn^idine, and 4a, also some- times 6a ; for regeneration in cord, cerebrum, and cerebellum, 3a with 3 drops of ammonia, or 5a, or pure alcohol. 839. Modifleations of Eamon y Cajal's Methods. — ^Da Fano {Ziegler's Beitr., xliv, 1908, p. 495) recommends using solutions of silver nitrate and hydroquinone in 1 : 10,000 gelatin in order to obtain a deeper and sharper impregnation. Kat6 (Folia neurobiol. ii, 1908) fixes in 10 to 16 per cent, formol, and silvers for one to five days at 35° C. in 5 per cent, argentamin to which 3 per cent, of silver nitrate has been added in such a way as to have an impregnating fluid with a little argentamin in excess ; or argentamin eight to ten parts, with 3 per cent, potassium bichromate thirty parts and distilled water 100 parts. For the reduction he uses 10 per cent, formol with 1 per cent, hydroquinone. PusATEEi (see Amato, Virchow's Arch., olxxxv, 1908, p. 547) fixes for three to six days at 35° to 38° C. in a mixture of 45 c.c. of taehiol ( 10 per cent, silver fluoride) and 155 of distilled water. Besta (Eiv. pat. nerv. mewl. Firenge, xv, 1910, p. 333) fixes for forty- eight hours in alcohol with 5 per cent, nitric acid, neutralised in alcohol with ammonia. 426 NERVOUS SYSTEM— SPECIAL METHODS. hl&S^GAlilG (Kolloidchemie, Beihefte, iii, 1911, H. 7 ; Ztschr. wiss. Mikr., xsviii, 1912, p. 369) makes sections of formol material by the freezing process, and silvers them until yellow. He then adds to the silver bath an equal volume of 50 per cent, solution of gum arabic and the same amount of saturated solution of hydroquinone. After one or two minutes the sections are brought into 10 per cent, solution of sodium hyposulphite, washed and mounted. Results said to be the same as by the usual process. AscoLi (Boll. Soc. med. chir., Pavia, 1911, p. 177) recommends for the sympathetic nervous system of Hirudinea the following : The animals cut open at the back are stretched on a piece of cork and fixed in a solution prepared by dissolving over a flame 5 gr. of pulverised crystals of silver nitrate in 100 o.c. of 95 per cent, alcohol. After a few minutes the animals may be detached from the cork and put back in the same fixative for twenty-four to forty -eight hours in an incubating stove. They are then transferred for another twenty -four to forty-eight hours into a 10 per cent, watery solution of silver nitrate, to be kept also in the incubator. After a quick wash they are reduced for five to eight hours in Amidol-Hauff 0-5 gr., sodium sulphite oryst. 10 grms., distilled water 100 c.c, and lastly passed into glycerin. Preparations are made by teasing, the thinner ones being toned and counterstained as usual. For mounting he prefers Ap4thy's syrup. Ranson (Anal. Am., xlvi, 1914, p. 522) has the following for the demonstration of non-meduUated nerve fibres in cranial and peripheral nerves : Fix in absolute alcohol containing 1 per cent, of strong ammonia for forty -eight hours ; rinse in distilled water, put in pyridine for twenty -four hours, wash in many changes of distilled water for twenty-four hours, place in 2 per cent, silver nitrate at 35° C. in the dark for three days, rinse in water, and place for one day in a 4 per cent, solution of pyrogallic acid in 5 per cent, formalin. As suggested by Haber and Guild (Anat. Bee, vii, 1913, p. 253) the results can be improved by a preliminary injection of 95 per cent, alcohol, containing 1 per cent, of ammonia, through the arteries tiU tissues are thoroughly saturated, after which they are dissected out and placed in a similar ammoniated alcohol solution for from two to three days. Huber and Guild have found this method of use for the study of cranial nerves of small animals and embryos, since the entire heads can, after fixation, be decalcified by means of 7 per cent, nitric acid, brought through 80, 90, and 95 per cent, alcohols, each containing 1 per cent, of ammonia, and finally treated as above. 840. Bielschowsky's Methods. Introductory. — It is well known that, if ammonia be poured into a solution of silver nitrate, a pre- cipitate is formed which is redissolved by the addition of some more ammonia. If an alkaline solution of formaldehyde be slowly added to this easily reducible di-ammoniacal silver nitrate (N(NIl4)AgH2N03), metaUic silver is immediately precipitated and deposited on the walls of the test tube. Both Fajeestajn (Neurol. Centrbl., xx, 1901, p. 98) and Bielschowsky (ibid., xxi, 1902, p. 579) CHAPTER XXXII. 427 thought of taking advantage of this reaction for histological purposes with the object of finding out a silver impregnation of the nervous tissue similar to that which characterises Golgi's method. The results of their attempts were different : Fajerstajn was able to obtain only a difficult method for staining axis-cylinders which is now superseded; Bielschowsky also published, at first, a compKcated silver method for impregnating axis-cylinders very similar to that of Fajerstajn, but, through successive modifications of his first process, was led to the discovery of a new method, which, is as important as Cajal's reduced silver methods from an histological point of view, but is of still greater advantage than the latter for ■ histopathological investigations. Moreover, Bielschowsky' s method is applicable to any formol material, even if very old. Bayon (Die Vntersuchungsmeth, etc.) succeeded with four-year-old material, and I with brains which had been left in formalin for. more than eleven years. There are at present three Bielschowsky methods : one for sections, one for peripheral nerve-fibres and axis-cylinders, and one for pieces. It seerns better to describe them separately in the following account which is based on the original papers of Bielschowsky, as well as on some personal experience I gained through a visit paid to him when in Berlin. Bielschowsky's Method for Sections (Journ. Psychol. Neurol, iii, 1904, p. 169 ; and xii, 1909, p. 135). — Pieces from central nervous organs, fixed in 15 to 20 per cent, formalin, are washed for some hours in running tap-water and then cut by means of a COj freezing microtome. The sections are collected in distilled water, thoroughly washed therein and passed in a 2 or 3 per cent, solution of silver nitrate where they are left for twenty-four hours in a dark place, and at room temperature. The sections can also be passed first into pure pyridine for twenty-four to forty-eight hours, washed in many changes of distilled water until the pyridine has been completely eliminated and then transferred into 2 to 3 per cent, silver nitrate as above. The pyridine bath is optional and has the advantage of ensuring a sharper stain of axis-cylinders whilst neuroglia, which is more or less coloured when the pyridine bath is dispensed with, remains unstained. Also connective tissue and nuclei are generally very faintly stained after the pyridine treatment. Intracellular neuro- fibrils, however, are not always so well shown as by the direct passage of sections into the silver nitrate solution. Before proceeding further, one should prepare the Bielschowsky 428 NERVOUS SYSTEM— SPECIAL METHODS. ammoniacal silver nitrate-and-oxide bath as follows :■ — ^Pour 5 c.c. of a 20 per cent, solution of silver nitrate into a measuring cylinder and add to it first 5 drops of a 40 per cent, solution of NaOH, and then ammonia, drop by drop, until the brown precipitate formed disappears ; dilute to 25 c.c. with distilled water, and filter through paper washed with the same water. For staining take sections one by one from the silver nitrate bath, quickly wash them in distilled water and transfer them into the • ammoniacal silver bath. Here they remain for about ten minutes when they become yellowish-brown and should be, once more, quickly washed in distilled water and placed in 20 per cent, formalin prepared with spring water. The reduction takes place immediately, and if one works with a number of sections it is advisable to re- transfer them into a fresh bath of 20 per cent, formalin. At the end of half an hour and even less, the reduction can be considered as accomplished and sections can be washed in distilled water and toned with a diluted (0-2 per cent.) solution of gold chloride. This may be slightly acidified with acetic acid if one wishes to obtain a faintly purple background, or neutralised with a few drops of a diluted solution of sodium or lithium carbonate if one prefers greyish- white backgrounds. Instead of gold chloride one can use a slightly acid solution of chloroplatinic acid. After toning there remains only the washing once more of sections in distilled water, and the passing of them for a few minutes in a 5 per cent, solution of sodium hyposulphite, or any diluted fixing bath for photographic plates. Wash again, dehydrate in alcohols of increasing strength up to 95 per cent., clear in carbol-xylol, and mount in balsam. For other details about the toning and fixing of sections see the < original papers of Bielsci-iowskt {op. eit. and Journ. Psychol. Neurol., iv, 1904—5, p. 227), as well as Wolff {Biol. Oentrhl., xxv, 1905, p. 683), and Da Fano {Froc. Physiol. Soo. Journ. Physiol., liii, 1920). Bielschowsky states that this method is also suitable for sections of celloidin or paraffin blocks of formol material, but he does not recommend the practice, and I have no experience of it. Bielschowsky's Method for Peripheral Nerve-fibres {Journ. Psychol. Neurol., iv, 1904—5, p. 227). — This method can be applied to the study of spinal and sympathetic ganglia, peripheral nerve- endings, and end-organs in normal conditions, but its chief appUca- tions belong to the domain of histopathology. According to my experience good results are rarely obtained, and the method requires important modifications to become as useful as the above and following ones. CHAPTER XXXII. 429 The staining is carried out on sections of formol material in the same way as described above. There is only this diiJerence that the staining in the ammoniacal silver bath is carried on a few minutes longer, viz., until the sections have taken a decidedly brown colour, after which they are washed in 10 c.c. of distilled water acidified with 5 drops of iicetic acid, when they acquire (sometimes in a few seconds) a yellowish tinge. They should then be immediately transferred into the usual 20 per cent, solution of formalin. For the toning a neutral gold bath is necessary : sections should be left therein until red-violet. In the finished preparations axis-cylinders are black, myelin red-violet, connective tissue violet or blue-violet. The washing in acidified water and the prolonged toning both answer for the purpose of creating a sharp contrast between nerve fibres and connective tissue fibres, which might otherwise become stained almost as black as the axis-cylinders. Bielschowsky has also a method for central nerve-fibres. Sections made by freezing from formol material are placed for twenty-four hours or longer in a 4 per cent, solution of copper sulphate or Weigert's mordant for neuroglia stain (§ 910). After washing they are placed for a few seconds in the usual ammoniacal silver bath and then washed, reduced, toned and fixed as above. The preparations are similar to those obtainable by the methods of Fajerstajn, Strahiiber and Kaplan. Bielschowsky's Method for Pieces {op. cit.). — Good for peripheral nerve-endings and embryonic material, and also for small specimens of adult subjects. This method has been described by Bielschowsky in various ways, probably because of the difficulty of giving fixed rules in a case in which the greatest freedom had to be left to histo- logists to adapt the method to the quality of their material and the purpose of their investigations. In what follows two forms of the method are described : one without and one with pyridine treatment of pieces. A. Method for Pieces without Pyridine Treatment. — Thin slices or small pieces of formol material are washed for some hours, first in ruiming tap-water and afterwards in distilled water. They are then placed in a 2 per cent, solution of silver nitrate for from one to eight days in the dark. The use of an incubator at 35° to 37° C. is optional. After a wash in several changes of distilled water (to be prolonged for some minutes up to some hours according to the length of time during which pieces have been kept in the silver bath, and if in an incubator or not) they are transferred into an ammoniacal solution of silver nitrate prepared as in the method for sections, but diluted 430 NERVOUS SYSTEM— SPECIAL METHODS. up to 100 c.c. They are kept therein for from an hour up to six, washed once more in distilled water, passed for twelve to twenty-four hours into the usual 20 per cent, solution of formalin. Wash, dehydrate quickly, embed, preferably in paraffin, tone sections as described above, counterstain, if necessary, mount in balsam. B. Method for Pieces with Pyridine Treatment. — Pieces of formol material, up to 1 cm. thick for adult tissue, and up to 5 cm. long for embryos, is put for two, three or four days into pure pyridine, washed for some hours in several changes of distilled water and put for three to five days into 3 per cent, silver nitrate at 36° C. Wash in distilled water and transfer into the diluted ammoniacal silver bath as above, but leaving pieces therein for twenty-four hours. Wash for about two hours in several changes of distilled water, reduce in 20 per cent, formalin. The rest as above. 841. Modifications of Bielschowsky's Methods. — Favoesky (Journ. Psychol. Neurol., vi, 1906, p. 260) uses 10 per cent, silver nitrate for the first silver bath instead of 2 or 3 per cent. Paton (Mitth. Zool. Stat. Neapel, xviii, 1907, p. 576) fixes fish embryos in 4 per cent, formaldehyde neutraUsed with carbonate of magnesia. For the first silver bath he uses -75 to 1 per cent, silver nitrate and keeps material therein four days in summer, five to seven in cooler weather. To make the ammoniacal silver nitrate-and- oxide bath he takes 20 c.c. of -75 to 1 per cent, silver nitrate, adds to it 4 drops of 40 per cent, caustic soda and then ammonia drop by drop in the usual way. The embryos are first washed in distilled water, then kept for five to fifteen minutes in 10 c.c. of water acidified with 5 drops of acetic acid, washed once more in pure water, and trans- ferred for twelve hours into a reducing fiuid consisting of 1 per cent, hydroquinone 20 c.c, neutralised formalia, 2 c.c. After embedding in paraffin, the sections are toned as usual and counterstained with 1 per cent, eosin in absolute alcohol. ScHUTZ (Neurol. Centrbl., xxvii, 1908, p. 909) finds that.the times given by Bielschowsky are too short and washes sections for twenty- four hours after the 2 per cent, silver nitrate bath, leaves them thirty to forty minutes in the ammoniacal silver bath, and twenty-four homrs in the 20 per cent, formalin. For toning he puts them for ten minutes into 10 c.c. of water with 2 drops of acetic acid, then for thirty to forty-five minutes into 10 c.c. of water with 3 drops of a 1 per cent, gold chloride solution (until blackish-grey). BoEKE (Anat. Anz., xxxv, 1910, p. 193) has obtained excellent results by the use of Bielschowsky's method for pieces when applied CHAPTER XXXn. 431 to the study of peripheral nerve endings. He fixes in 10 per cent, formalin prepared with 60 per cent, alcohol, changes the fluid two or three times, and then either leaves material therein until wanted or keeps it in 70 to 80 per cent, alcohol. For staining, pieces are brought into 10 to 12 per cent, formalin, and left in it until they are quite free from alcohol. The rest as by Bielschowsky's method for pieces, with or without pyridin treatment. Boeke finds that the method succeeds also after other kinds of fixation. ScHLEMMEE {Ztschr. wiss Mikr., xxvii, 1910, p. 22) makes the ammoniacal silver nitrate-and-oxide bath by adding to any silver nitrate solution, 40 per cent, caustic spda, drop by drop, until no more precipitate is formed. He then washes the precipitate by repeated decantation until the wash water no longer gives an alkaline reaction, takes it up with the smallest possible quantity of ammonia, and filters through glass wool. This concentrated solution keeps for many days unaltered, and should be diluted ten times its volume before using it. Del Eio-Hoetega (Trah. Lab. Invest. Biol., Madrid, xiv, 1916, p. 181) has made known a similar method used in those laboratories for preparing the ammoniacal silver nitrate bath. Forty drops of 40 per cent, caustic soda are added to 30 c.c. of 10 per cent, silver nitrate, and the precipitate washed ten to twelve times by means of about a litre of distilled water. Fifty cubic centimetres of water are then added to it, and ammonia, drop by drop, until the precipitate is dissolved. The solution, brought finally to 150 c.c. and filtered into a dark brown bottle, keeps well for many months. I find that the ammoniacal silver ^ath thus prepared can be further diluted with one, two, up to five times its volume of water, and usefully employed for Bielschowsky's method for pieces, particularly for the study of peripheral nerve endings. Agduhr {Ztschr. wiss. Mikr.j xxxiv, 1917, pp. 1-99), who has exhaustively investigated almost all questions relatiag to the results obtainable by Bielschowsky's method for pieces, has come to the conclusion that material is best fixed in neutral or slightly acid 20 per cent, formaldehyde (50 per cent, formalin). Pieces should then be washed in distilled water for many days until the wash- water is free from substances reducible by an ammoniacal silver nitrate solution used as test. For the first silver bath he uses 3 per cent, silver nitrate, and for the second a solution obtained by adding to 10 c.c. of 10 per cent, silver nitrate, first 20 drops of 25 per cent. NaOH, then from 200 up to 600 c.c. of distilled water, and lastly ammonia enough to dissolve the precipitate. For the reduction he 432 NERVOUS SYSTEM—SPECIAL METHODS. uses again 20 per cent, formaldehyde. To avoid an excessive im- pregnation of the connective tissue lie also finds it useful to wash pieces in acidified distilled water (see the Bielschowsky method for peripheral nerve-fibres), but he uses as much as five times the amount suggested by Bielschowsky. 842. Da Pano's Modifications. — An important point of this series of modifications of Bielschowsky's method for sections is the use of distilled water, re-distilled on potassium permanganate, with the object of ensuring elimination of any trace of organic matter from the ordinary distilled water and of avoiding the forma- tion of precipitates. Da Fano's first modification (Mod. 1) (Atti. Soc. Lomb. Sc. Med. Biol., Milano, iii, 1914) was meant for the study of recticular tissue of spleen, lymph glands, and other organs, and is to be carried out as follows : — (1) Fix small pieces of fresh tissue in 10 to 20 per cent, formalin or in Kayserling's first fluid (forty-eight hours at least), or in Orth's fluid (twenty-four to forty-eight hours). (2) Wash pieces in rurming tap-water for twenty-four to thirty hours, and then in distilled water for another twenty-four hours. (3) Wash sections made by the freezing method in re-distilled water (twenty-four hours), and then place them in filtered 2 per cent, silver nitrate (prepared with redistilled water) in a Petri dish, taking care that they do not touch each other. Here they are kept in the dark and at room temperature from six hours to three days. (4) Treat sections for twenty to thirty minutes with Bielschowsky's ammoniacal silver nitrate solution prepared with only 2 drops of 40 per cent, caustic soda and diluted with redistilled water to 40 to 70 c.c. (5) Reduce, tone; counterstain, and mount as by Biel- schowsky's method for sections. Mod. 2 (Proc. Physiol. Soc. Journ. Physiol., Iii, 1919) consists in an application to nervous tissues of Mod. 1. The use of redistilled water and the mode of preparing the ammoniacal silver bath are the same, but Da Fano lays stress on the following points : — (1) Nervous tissue must be fixed in 10 up to 20 per cent, formalin for at least three weeks, better still for two months. Attempts to obtain a rapid fixation with 10 to 20 per cent, formalin at 37° C. gave bad results. (2) Sections of nervous tissues may be placed, after washing in redistilled water, in anhydrous pyridine (six to twelve hours), then repeatedly washed and left overnight in redis- tilled water, to get rid of all pyridine. This treatment appears to render neurofibrils a little thinner and, consequently, a little CHAPTER XXXII. 433 sharper, but increases the length and cost of the method, and may cause precipitates to form, especially where much myelin is present. (3) It is possible to keep sections, which cannot be stained imme- diately, for some days or even a fortnight, in redistilled water to which a few drops of formalin have been added. Thorough washing with redistilled water is then imperative before they are transferred into the 2 per cent, silver nitrate solution. (4) Sections of nervous tissues must not remain in the 2 per cent, silver nitrate more than forty-eight hours, or precipitates may form. The longer their stay there, the longer must be the washing before staining ; this, however, must not, as a rule, exceed five minutes. (5) The volume to which the ammoniacal silver nitrate is diluted should be 35 to 45 c.c, and the sections remain in it fifteen to twenty minutes. The subsequent washing before transferring the sections into 20 per cent, formalin should not occupy more than ten to fifteen seconds, and their stay in the final formalin solution (especially for cerebral cortex) should not exceed two to three hours. The other eight Da Fano modifications [Proc. Physiol. Soc, Journ. Physiol., liii, 1919-20) were all proposed for the study of cortex cerebelli, and -are characterised by a special treatment of the sections (cut by the freezing method) with various reagents before transferring them into the 2 per cent, silver nitrate solution, nothing having been changed, however, in regard to the long fixation of material in formalin and the use of redistilled water. They may be summarised as follows : — Mod. 3. Place sections, after washing in redistilled water, in 2 to 3 per cent, silver nitrate at 36° to J7° C. for about twenty-four hours ; wash quickly ; stain in ammoniacal silver nitrate solution diluted to 40 c.c. for thirty minutes. Wash, reduce, tone, and mount as usual. Mod. 4. Place sections in 50 per cent, pyridine for six to eighteen hours; wash in redistilled water for twenty-four to forty-eight hours ; 2 per cent, silver nitrate at 37° C. for twenty-four hours, etc., - as in Mod. 3. Mod. 5. Place sections in pure pyridine for four to twelve hours. Wash in redistilled water overnight. Transfer sections into 20 per cent, formalin prepared with redistilled water for about twenty-four hours. Wash again in redistilled water overnight ; 2 per cent, silver nitrate at 37° C, etc., as before. Mod. 6. Sections are treated first with 20 per cent, formalin, and then with pure pyridine, in the reverse order of Mod. 5. Mods. 7 and 8. The same as Mods. 5 and 6, but replacing the M. 28 434 NEEVOUS SYSTEM— SPECIAL METHODS. pyridine with a mixture of 3 parts of metliyl-alcohol and 2 parts of water. Mod. 9. Place sections in a mixture of equal parts of 20 per cent, formalin and methyl alcohol for twenty-four hours ; wash in redistilled water for six to twenty-four hours ; 2 per cent, silver nitrate at 37° C. for twenty-four hours, etc., as before. Mod. 10. Place sections into 20 per cent, formalin for twenty- four hours, transfer them, without washing, into a mixture of equal spart of 20 per cent, formalin and methyl alcohol, etc., as in Mod. 9. Mod. 3 is particularly suitable for human material of young individuals : Mod. 4 for adult subjects. Mods. 5 and 6 are useful for the study of neurofibrils in the various elements of the cortex cerebelli and for the staining of the granules. Mods. 7, 8 and 9 are to be preferred for the demonstration of pericellular baskets and nervous processes. Mod. 10 gives very complete stainings, and is the most certain of all ; preparations are, however, fairly dark, and, therefore, more suitable for general view. Peedeau {Journ. Pathol. Bact., xxiv, 1921) has worked out a modification which appears to be particularly suitable for the study of the connective tissue in nervous organs. *He washes pieces and sections as in Da Fano's modifications, but without having recourse to redistilled water. He then places sections for about ten minutes in -25 per cent, potassium permanganate, washes, and treats them as by Pal's modification of Weigert's myelin stain (see § 857). After another wash in distilled water, he transfers sections into 2 per cent, silver nitrate, and continues as in Da Fano's Mod. 1. Nerve cells, nerve fibres, neuroglia, etc., unstained ; connective tissue and elastic fibres stained in various shades ©f purple-grey to black. 843. Neurofibrils ; Other Methods. — Cox's Method for fibrils of spinal ganglion cells ; see Ztschr. wiss. Mikr. xiii, 1896, p. 498, and Anat. Hefte, X, 1898, p. 98. S. Meter's Berlin blue, see Anat. Anz., xx, 1902, p. 535. Lttqako's collargol (colloidal silver) method, see Monit. Zool. Ital., XV, 1904, p. 353. JoRis' colloidal gold method has not been received with favour ; see Bull. B. Acad. Med. Belg., xviii (S. iv), 1904, p. 293. Sand (C.R. Ass. Anat. Bruxelles, 1910 ; Bibliogr. Anat. Supp., 1910, p. 128, or Ztschr. wiss. Mikr., xxviii, 1911, p. 500) gives the following as entirely certain for man, dog, cat, and rabbit. Speci- mens of not more than 5 mm. in thickness are fixed for forty-eight hours in a freshly prepared mixture of 90 parts of acetone and 10 of nitric acid, to be changed for fresh after half an hour, and once CHAPTER XXXII. 435 again within twenty-four hours. Wash out for at least six hours in pure acetone, changed two or three times. Make paraffin sections and bring them through xylol and acetone into distilled water ; silver for three days at about 37° C. in 20 per cent, solution of silver nitrate. Put for ten minutes into a mixture (at least three days old) of 1000 parts of water, 10 of sodium acetate, 5 of gallic acid, and 3 of tannin (to be changed if it becomes turbid). Mount at once or tone until grey (five minutes) in 80 parts of water with 17 of 2 per cent, ammonium sulphocyanide and 3 of 2 per cent, gold chloride ; fix for a few seconds in 5 per cent, sodium hyposulphite. Neurofibrils grey-violet, shown in cells, dendrites, and axons. Terminal buds of Held also clearly shown, and nothing else stained. One may counterstain in any way, even by Weigert's or Benda's methods for neuroglia staia. The methylene blue intra vitam method is important, and may be usefully employed for the study of neurofibrils. See the processes of Apathy, Dogiel, and Bethe in Chapter XVI. C. Methods for the Demonstration of Golgi's Internal Apparatus. 844. Introduction. — The discovery of the " apparato reticolare interna " was made by Golgi in 1898 by means of his rapid process (see Chapter XXXIV). Soon afterwards he had recourse to a mixture due to Veratti (see next paragraph), and Negri, Pensa, and others of Golgi's pupils found that the internal apparatus is not a peculiarity of nerve cells. In 1902 Kopsch showed that the apparatus can be staiued by a simple immersion of nervous tissues (spinal ganglia) into 2 per cent, osmic acid for eight to ten days. Since then the apparatus was shown to exist in almost every kind of cells, and new processes proposed for its demonstration in nervous and other tissues by Sjovall, Golgi, Eamon y Cajal, Gatenby, Da Fano. As the methods of Kopsch and Sjovall, the Mann-Kopsch method, and Gatenby' s Mann-Kopsch- Altmann combination have already been fully discussed in Chapter XXVI, and particularly described in §§ 692 — 694 and 696, there remain to be described here only those methods which are particularly suitable for the study of the internal apparatus in nervous tissues, with exception of Golgi's rapid process, for which see Chapter XXXIV, § 882. 845. Golgi- Veratti's Method (see Golgi, Anat. Anz. Verh. Anat, Ges., xiv, 1900, p. 174). — Small pieces are hardened for a time 28—2 436 NERVOUS SYSTEM— SPECIAL METHODS. varying from a few hours to ten days or longer in Veratti's mixture, consisting of — 5 per cent, potassium bichromate . . 30 parts. 0-1 per cent, chloroplatinic acid . . 30 ,, 1 per cent, osmic acid . . . 15 to 30 „ From time to time pieces are put in one or other of Golgi's rejuvenating fluids (as described in § 888), and thence into -8 to 1 per cent, silver nitrate. Sections are cut and mounted as by Golgi's bichromate and nitrate of silver method (see § 882). 846. GoLai's Arsenious Acid and SUver Nitrate Method {Arch. Ital. Biol., xlix, 1908, p. 272). — Small pieces of quite fresh tissues are fixed for three, six, eight or twelve hours in equal parts of 20 per cent, formalin, saturated solution of arsenious acid, and 96 per cent, alcohol. After a quick wash with distilled water, they are passed for some hours (or days) into 1 per cent, silver nitrate, and then treated with a reducing fluid, usually Cajal's hydroquinone mixture (hydroquinone 20 grms., sodium sulphite 5 grms., formalin 50 c.c, water 1000 c.c). Wash quickly, dehydrate, and embed either in celloidin or paraffin. The sections are toned with equal parts of 1 per cent, gold chloride and a mixture consisting of water 1000 c.c, with 30 grms. each of sodium hyposulphite and ammonium sulphocyanide, and then rapidly bleached by the following method, due to Veeatti : — Wash the toned sections in distilled water and transfer them for one, two or three minutes into potassium per- manganate 0-5 grm., distilled water 1000 c.c, sulphuric acid 1 c.c. ; wash again ; transfer into 1 per cent, oxalic acid until the yellowish colour imparted to the sections by the potassium permanganate has disappeared ; wash thoroughly in repeatedly changed distilled ' water ; counterstain, dehydrate, and mount as usual. 847. Ram6n y Cajal's Uranium Nitrate and Silver Nitrate Method '{Trab. Lab. Invest. Biol, Madrid, xii, 1914, p. 127).— (1) Small pieces of quite fresh tissues are fixed for ten to fourteen hours in a mixture of neutralised formalin 15 c.c, distilled water 85 c.c, uranium nitrate 1 grm. Instead of uranium nitrate, uranium acetate, as suggested by Del Rio-Hortega, may be sometimes used. Should a very fine reaction be desirable, the following formula may be employed : — Uranium nitrate 1 grm., ethyl or methyl alcohol 30 c.c, distilled water 80 c.c, neutralised formalin 15 to 20 c.c. (2) After a quick wash in distilled water, pieces are transferred into 1 -5 per cent, silver nitrate and kept therein for thirty-six to forty- eight hours at room temperature. If the pieces are only a few and CHAPTER XXXII. 437 small, 1 per cent, silver nitrate will be sufficient. (3) Wash quickly and reduce for eight to twenty-four hours in hydroquinone 1 to 2 grms., formalin 15 c.c, distilled water 100 c.c, sodium sulphite 0-5 grm. (4) Wash quickly, embed in paraffin or celloidin, or make sections by the freezing method. (5) Tone and counterstain sections if desirable. Dehydrate and mount as usual. Best results are obtained from vertebrates, preferably kittens and young rabbits. The method may be applied to human material, if available in a sufficiently fresh condition. From invertebrates results are not so good, and rather uncertain, so that Cajal advises a simple fixation in formalin or formalin-acetone, followed by impregnation with silver nitrate, as by his reduced silver methods for neurofibrils. 848. Da Fano's Cobalt Nitrate Modification (Proc. Physiol. Soc, Journ. Physiol, liii, 1920 ; Journ. R. Micr. Soc, 1920, p. 157).— Small pieces of quite fresh tissues are fixed for six to eight hours at room temperature in cobalt nitrate 1 grm., distilled water 100 c.c, formalin 15 c.c. The solution can be prepared beforehand, and keeps unaltered for months. The formalin need not be neutralised unless strongly acid or containing free sulphuric acid, in which case it is necessary to neutralise it by one of the usual methods. For the fixation of embryonic organs and in all cases in which a shrinkage of delicate tissues is to be feared, the quantity of the formalin may be reduced to 10, 8, or 6 c.c. for every 100 c.c. of distilled water. The time of fixation should be shortened to three to four hours or even less in the cas3 of very small pieces, such as spinal ganglia of mice and rats, the pituitary body of the same animals, etc. Pieces of spinal cord, cerebrum, cerebellum of adult animals give better results if fixed for about eight to ten hours. The fixation may be prolonged in special cases to twelve to twenty hours, but should not exceed twenty-four hours. The fixation in an incubator at a temperature varying between 25° and 37° C. has been attempted with success in the case of tissues of adult subjects, but it leads to a staining of both the internal apparatus and intracellular formations, which, according to their morphology, are to be considered as mitochondria. For the impregnation, Da Fano quickly washes the pieces in distilled water, makes their surfaces smooth if necessary, and then places them into 1 -5 per cent, silver nitrate in the dark for twenty- four to forty-eight hours at room temperature. For very small fragments, 1 per cent, silver nitrate may be used, whilst for pieces of 438 NERVOUS SYSTEM— SPECIAL METHODS. spinal cord of adult subjects, 2 per cent, should be preferred. For the reduction he uses Cajal's hydroquinone-formalin mixture, taking care in further recuttiug the pieces, before transferring them into the reducing fluid, so that their thickness should not exceed 2 mm. He dehydrates and embeds pieces, preferably in parafiin, or he makes sections by the freezing method. He usually tones these by means of 0-2 per cent, gold chloride, fixes with 5 per cent, sodium hypo- sulphite, counterstains and mounts as usual. The method gives good results also with material from lower vertebrates and invertebrates.* 849. Other Methods and Modifications. — Besta (Anat. Anz., xxxvi, 1910, p. 477) fixes for two days in 20 parts of formol with 2 of acetic aldehyde and 80 of water, washes for twenty-four hours in distilled water changed seven or eight times, and puts for two days in 4 per cent, solution of ammonium molybdate, makes paraffin sections, stains in a 1 : 1000 solution of thionin, differentiates in 3 parts of creosote to 1 of absolute alcohol, and passes through pure creosote and xylol into neutral balsam. Recommended for Purkinje cells and spinal ganglia of young animals. SucHANOW {Neurol. Centrbl., xxi, 1902, p. 777) has obtained good results by the use of Golgi-Veratti mixture, keeping pieces of spinal cord and spinal ganglia for twenty to thirty days in the mixture and for two to three days in the rejuvenating fluid. Legendee (Anat. Anz., xxxvi, 1910, p. 209) omits the toning and bleaching by Grolgi's arsenious acid method, and embeds in parafBin. Similarly Collin et Lucien, BiUiogr. Anat. Supp., 1909, p. 238. Savagnone {Pathologica, i, 1909) silvers pieces fixed in Golgi's arse- nious acid mixture with 30 o.c. of tachiol (10 per cent, silver fluoride) in 100 of water. Caeleton {Journ. B. Mior. Soc, 1919, p. 321) reduces pieces treated according to Cajal's uranium nitrate method for only two hours in the usual hydroquinone mixture. Penfield {Brain, xHii, 1920) has successfully employed Cajal's uranium nitrate method for his experimental investigations on the alterations of Golgi's apparatus in nerve cells of spinal cord and spinal ganglia of young cats. He adds 20 c.c. (instead of 15) to Cajal's fixing fluid and as much as 1-5 grms. of sodium sulphite to the hydroquinone-formalin solution. He finds it imperative to dehydrate pieces very quickly before embedding them in paraffin. In order to obtain perfect fixation of the spinal cord he sometimes performs a laminectomy in the lower lumbar region of the aneesthe- * Da Fano's method has been used by me with great success for the study of gametogenesis of many invertebrata (J. B. G.). CHAPTER XXXII. 439 tised animal, passes a needle in the subarachnoid space, and allows the fixative to flow in "under a gravity pressure of 75 cm." The heart stops about a minute after the beginning of the injection, which is continued for twenty hours. At the end of this time the cord is removed, pieces cut and dropped directly into the silver bath. For counterstauiing Penfield finds it particularly useful to immerse untoned sections into a diluted solution of Unna's polychrome- methylene blue for one to four hours, this being followed by passage through alcohols of increasing strength and differentiation in absolute alcohol. By this method, also Holmgren's, trophospongium is sometimes stained. But for the study of the relationship between the latter and Golgi's apparatus, Penfield (in litteris) prefers to make drawings of the apparatus from certain selected cells, subsequently removing the coverslip and bringing the slides through graded alcohols into 5 per cent, iron alum for twelve to twenty-four hours. This removes all silver fiom the cells as well as the counterstain, and at the same time mordants the tissues for further staining by Heiden- hain's iron-hsematoxylin method. If the proper amount of diffe- rentiation has been secured of the particular cells already drawn, the trophospongium wiU be found stained with great detail. Addendum. Holmgren's Method for Trophospongium.^Fix small pieces or ganglia in trichlorolactic acid for twenty-four hours. Dehydrate and embed in paraffin as usual. Stain thin sections for twenty-four hours in Weigert's resorcin fuchsin solution for elastic fibres (see § 758) recently prepared and a little diluted. Dehydrate and mount as usual. - D. Methods for the Demonstration of the Sustaining Apparatus of Medullary Sheaths, Neurokeratin, etc. 850. Methods demonstrating Funnels and Spiral Filaments.— GrOLGi (see Rbzzonico, Arch. p. I. Sc. Med., iv, 1880, p. 78 ; Golgi, Opera Omnia I, p. 163) puts small pieces of spinal cord in 2 per cent, potassium bichromate for eight to fifteen days in summer, or a month in winter. After a quick wash he transfers them into -50 to -75 per cent, silver nitrate for two or three days in summer, or eight, ten or more in winter. The pieces are then washed in 95 per cent, alcohol, dehydrated in absolute alcohol, cleared ia oil of turpentine and teased therein. The preparations, mounted in dammar, must be exposed to sunlight for eight to ten days ; or to diffused daylight for twenty to ioitj days. 440 NERVOUS SYSTEM—SPECIAL METHOi)S. For peripheral nerve fibres, Golgi (Op. Omnia I, p. 162) has proposed two methods. Of these the first is a modification of his rapid process (see § 882), and should be carried out as follows : — Tracts of peripheral nerves are cut with care not to stretch them, and put in a mixture of 10 parts of 2 per cent, potassium bichromate and 2 of 1 per cent, osmic acid. After about one hour the tract or tracts of nerves are sufficiently hardened to be further recut in pieces of about ^ cm. in length, which are put back in the same mixture. After another three hours, and successively at intervals of three hours during twenty-four hours, pieces are transferred into -5 per cent, silver nitrate where they may remain for any time, but no less than eight hours. Preparations are made and mounted as above. The other method is a modification of that used for central nerve fibres, the only - difference consisting in keeping the pieces in the bichromate for a much shorter period, i.e., 'for from four hours to at most two days, and in transferring specimens into the silver bath at intervals of about three hours. After twelve to twenty-four hours preparations can be made as described above. The preparations made by the first method show the spiral filaments very clearly, but do not keep well. The preparations made by the second method do not show the spiral filaments so completely, but are more useful for the demonstration of the funnels and last longer. Cattani {Arch. Ital. Biol, vii, 1886, p. 345) either fixes in Flem- ming's fluid and teases and mounts in glycerine, or puts pieces into Grolgi's bichromate and osmic acid mixture, dehydrates and passes into oil of turpentine to be changed until it remains colourless. The turpentine dissolves the myelin and leaves funnels and spiral fila- ments visible. Cattani also has a modified Golgi method, now superseded. Sala {Verh. Anat. Ges. Anat. Anz., 1900, p. 176) employs the Golgi- Veratti method for the intracellular network (see § 845). See also concerning these methods, Mondino, Arch. p. I. Sc. Med., viii, p. 45. Galli {Ztschr. wiss. Mikr., iii, 1886, p. 467) hardens peripheral nerves for eighteen to twenty days into Miiller's fluid, cuts out pieces 5 to 6 mm. long, and keeps these in Miiller's fluid diluted with 2 parts of water for another two days. He then stains for fifteen to twenty minutes in aqueous solution of China blue, washes out in alcohol, clears in essence of turpentine, and mounts in damar. %_ Ram6n y Cajal has successfully employed some modifications of CHAPTER XXXII. 441 his reduced silver and uranium nitrate methods, for which see Trab. Lab. Invest. Biol, Madrid, x, 1912, p. 221. 851. Methods demonstrating Neurokeratin Network. — Flatner (Ztschr. wiss. MiJcr., vi, 1889, p. 186) fixes for several days in a mixture of 1 part of Liq. Ferri Perchlor. (Ph. G., ed. 2) and 3 to 4 parts of water or alcohol, washes out well in water and stains for several days or weeks in a concentrated solution of " Echtgriin'' (dinitroresorcin) in 75 per cent, alcohol. See also Beer, Jahrb. Psychiatrie, ii, 1893. Cox (Anat. Hefte, i, 1898, p. 102, note) fixes nerves in 2 per cent, osmic acid (rabbit) or 1 per cent, (frog), washes, dehydrates, clears with bergamot oil, and mounts in balsam. The bergamot oil dissolves out the myelin, and leaves the neurokeratin visible. It may be necessary to leave the nerves for forty-eight hours in the oil. Corning (Anat. Anz., xvii, 1900, p. 309) studies the neurokeratin ■network in the sciatic of the frog by means of sections of sublimate material strongly stained with iron hsematoxylin. Kaplan {Arch. Psychiatr., xxxv, 1902, p. 825) stains sections with acid fuchsin and differentiates them by Fal's method. Gedoelst {La Cellule, v, 1889, p. 136) has the following : (a) A nerve is treated with liquid of Ferenyi, either pure or with addition of a trace of osmic acid, and examined in glycerin. By this treat- ment the myelin loses its excessive refractivity and the neurokeratin network comes out clearly. (6) Silver nitrate. Good images, but uncertain, (c) Treatment with a mixture of 1 per cent, osmic acid and absolute alcohol. The network comes out black. CHAPTEE XXXIII.* MYELIN STAINS. 852. Iron Hsematoxylin. — According to A. BoUes Lee (see 1913 Ed.) the simplest way of staining myelin is to make paraffin sections of formol material and stain them with iron hsematoxylin exactly as for central corpuscles (say, twelve to fourteen hours in the mordant, six in the hsematoxylin, and a few minutes for the differentiation). Sections best not over 15 (j.. One may counterstain the cells with carmalum, but not for more than half an hour, or the hsematoxylin wiU be attacked. The stain is not so sesthetic as Weigert's, but quite as sharp. Axis cylinders are not shown. Similarly Eegaxtd {C. R. Acad. Sc, cxlviii, 1909, p. 861), but adding a chrome mordantage either concurrently with the formol fixation, or subsequently. Also Nageotte {C. R. Soc. Biol., Ixvii, 1909, p. 542), with sections of formol material by the freezing method ; HousER {Journ. Gomp. Neurol., x, 1901, p. 65), and Brookover (ibid., XX, 1910, No. 2) ; Spielmeyee (Neurol. Centrbl., xxix, 1910, p. 348) ; and his Technih d. mikrosl. Untersuch. d. Nervensy stems, 1911, p. 87, with sections of 25 to 35 /^ by the freezing method ; LoYEZ (C. R. Soc. Biol., Ixix, 1910, p. 511), who differentiates first lightly, till the grey matter begins to appear, in the iron alum, then washes, and differentiates further in Weigert's borax f erricyanide ; Gilbert (Ztsch. mss. Mikr., xxviii, 1911, p. 279), who mordants with iron alum, stains with molyhdic acid hcematoxylin, and differentiates with the borax ferricyanide ; Stoeltzner (ibid., xxiii, 1906, p. 329), who mordants celloidin sections of formol material for five minutes in Liq. ferri sesquichlorati, stains in -5 per cent, hsematoxylin, and differentiates in the mordant or in borax ferricyanide ; and Kodis (Arch. mik. Anat., Ux, 1902, p. 211), who fixes for one or two days in saturated solution of mercury cyanide, hardens in 10 per cent, formol, and stains sections, made by the freezing method, with Heidenhaia's iron hcematoxylin. 853. Weigert's Methods^ — There have been in all three methods of Weigert :— the 1884 method, the 1885 method, and the 1891 method. * Eevised by Dr. C. Da Fano, King's CoUege, University of London. CHAPTER XXXIII. 443 The 1884 method {FoHschr. d. Med., ii, 1884, pp. 120, 190 ; Ztschr. mss. Mikr., i, 1884, pp. 290, 564), which depends on the formation of a chrome lake of hsematoxylin, may be considered as superseded. Not so the two others, which depend on the formation of a copper lake in addition to the chrome lake. 854. Weigbrt's 1885 Method (Fortschr. d. Med., iii, 1885, p. 236 ; Ztschr. wiss. Mikr., 1885, pp. 399, 484; Ergebn. Anat., vi, 1896 (1897), p. 10). — The tissues are hardened in potassium bichro- mate. Weigeet takes {Ergebn., p. 10) a 5 per cent, solution, and if time is an object hardens in a stove. (Other bichromate mixtures will do, e.g., MiiUer's, Kultschitzky's, Zenker's ; Erlicki's is not to be recommended.) The tissues are " ripe " for staining when the hardenrag has been carried to a certain point. They are first yellow, without differentiation of the grey matter from the white ; these are xmripe. Later they show the grey matter light brown, the white matter dark brown ; and these are ripe. More recently (Ergebn., p. 14) he added to the bichromate solution 2 per cent, of chrome alum or of chromium fluoride, which hastens the hardening, so that small specimens become brown and ripe in four to five days, without stoving. After hardening, tissues are generally embedded in celloidiu and the blocks hardened in the usual way. They are then put for one or two days, in an incubating stove, into a saturated solution of neutral copper acetate diluted with 1 volume of water. By this treatment the tissues become green and the celloidin bluish-green. They may then be kept, till wanted for sectioning, in 80 per cent, alcohol. Sections are made, weU washed in water, and brought into a stain composed of : — HsematoxyUn -75 to 1 part. Alcohol 10 parts. Water . . ■■ . . . 90 „ Saturated solution of lithium carbonate . 1 part. They remain there, for spinal cord, two hours ; for medullary layers of brain, two hours ; for cortical layers, twenty-four hours. They are then again weU washed with water, and brought into a decolorising solution composed of : — Borax 2-0 parts. Ferricyanide of potassium . . . 2-5 „ Water 200-0 „ They remain there until complete differentiation (half an hour to 444 MYELIN STAINS. several hours), and are then well washed with water (running, or changed several times), dehydrated, and mounted in balsam. They may be previously counterstained, if desired, with alum-carmine. The method is applicable to the study of peripheral nerves as well as to nerve centres, and also the study of lymphatic glands, skin (see Schieffeedbcker, Anat. Anz., ii, 1887, p. 680), bile capillaries, and other objects. The process is applicable to tissues that have been hardened in alcohol or in any other way, provided that they be put into a solution of a chromic salt until they become brown before mordanting them in the copper solution. It is not necessary that the mordanting be done in bulk. Max Flesch {Ztschr. wiss. Mihr., iii, 1886, p. 50) prefers (following Lich- theim) to make the sections first, and to mordant them separately. Vassale (iJw. sperim. Freniatr.,xv, 1889, p. 102) _^rsi stains the sections in 1 per cent, heematoxylin for three to five minutes, then puts them for three to five minutes into saturated solution of copper acetate, and differentiates as Weigert. 855. Weigert's 1891 Method {Deutsche med. Wochenschr., xvii, 1891, p. 1184). — The material is hardened in bichromate and embedded in celloidin (see last §). It is then (according to the latest form of the process (Enzycl. mik. Technik., 1903, p. 942) ), put for twenty -four hours in a stove into a solution of 2^ parts of chromium fluoride, 5 of copper acetate, and 5 of acetic acid in 100 of water.* Sections are then made and stained for from four to twenty- four hours at room temperature in a freshly prepared mixture of 9 volumes of (A), a mixture of 7 c.c. of saturated aqueous solution of lithium carbonate with 93 c.c. of water, and 1 volume of (B), a solution of 1 grm. of hsematoxylin in 10 c.c. of alcohol (A and B may be kept in stock, but A must not be too old). The sections should be loose ones, and not thicker than 25 fx. They are then washed in several changes of water, and treated with 90 per cent, alcohol, followed by carbol-xylol, or by a mixture of 2 parts of anilin oil with 1 of xylol, then pure xylol and xylol balsam (not chloroform balsam). It was, however, found that preparations thus made, without differentiation, did not keep well, and Weigert (Ergebn. Anat., iii, 1894, p. 21) reverted to the practice of differentiating with the borax-ferricyanide mixture. * Instead of the chromium iiuoride one may use chrome alum, as Weigert did at one time, and as some still do. But then one must boil, as directed for Weigert's Neuroglia stain. CHAPTER XXXIII. 445 Later still (Enzycl mik. Technik., 1903, p. 942) he employed a stain composed of equal parts of (A), a mixture of 4 c.c. of tte officinal Liquor ferri sesquichlorati'P . G. with 96 of water, and (B), a mixture of 10 c.c. of a 10 per cent, alcoholic solution of haematoxylin with 90 of 96 per cent, alcohol. The two (A and B) must be mixed immediately before use, and the sections should remain in the stain overnight or longer, then rinsed and differentiated as usual. This has the advantage of demonstrating very fine fibres, and of giving a colourless back ground. For difficult objects the differentiating liquid may be diluted with water, and gives better results than dilute acetic or hydrochloric acids or the like, which were formerly recommended. By means of Weigert's methods only the myelin sheaths of normal nerve-fibres are stained, whilst those of degenerated tracts are of a paler colour and, if the degeneration is sufficiently old, they may even be stainless. See also § 870. 856. Formol Material {Ergebn. Anat., vi, 1896, p. 14) may be employed if mordanted till brown (four or five days) in 5 per cent, solution of potassium bichromate with 2 per cent, of chromium fluoride. I understand from Dr. Perdrau that this method is the most satisfactory of aU for rouDine work, and relatively small pieces ; but particularly for the histopathological investigation of parts of the human spinal cord, medulla oblongata, pons, and midbrain. He generally cuts from material fixed in formalin, for no less than ten days, slices J to | cm. thick, and places them direct in the mordant (potassium bichromate 5grms., chromium fluoride, 2-5 grms., water, 100 c.c.) for five to six days. Relatively large pieces may be left in the mordant four or five days longer. After a thorough wash in ruiming tap water, he dehydrates and embeds in celloidin. The sections are stained overnight in Kultschitzky's hsematoxylin (§ 859) several months old, washed in water, and placed in a bowl of distilled water to which about 2 c.c. of a saturated solution of lithium carbonate have been added. They are stirred about several times and transferred into a fresh bath of the same solution if necessary, until the celloidin is all but colourless. He lastly differentiates, as by Pal's method (§ 857), washes, and counterstains either in alum carmine for ordinary work or in an alcoholic solution of eosin if the preparations are to be photographed. P. Meter {Neurol. Gentrbl., xxviii, 1909, p. 353) embeds formalin material in celloidin and outs before putting into Weigert's copper fluid. 446 MYELIN STAINS. For Sheldon's modifloation, wMch is also based on a formalin fixation, see Folia Neurobiol, viii, 1914, p. 1. Modifications of Weigert's Method. 857. Pal's Method (Wien. med. Jahrb., N.F. i, 1886, p. 619 ; Ztschr. wiss. Mikr., iv, 1887, p. 92 ; Med. Jahrb., N.F. ii, 1887, p. 589).- — One proceeds as in Weigeet's process, but omitting the copper bath. After staining in the lisematoxylin solution the sections are washed in water (if they are not stained of a deep blue a trace of lithium carbonate must be added to the water). They are then brought for twenty to thirty seconds into 0-25 per cent, solution of potassium permanganate, rinsed in water, and brought into a decolorisiag solution composed of : — Oxalic acid 1-0 grm. Potassium sulphite (SO3K2) . . . 1 '0 „ Dist. water . . . . . .200-0 c.c. In a few seconds the grey substance of the sections is decolorised, the white matter remaining blue. If the differentiation is not complete the whole process can be repeated a second time, and so on. The sectipns should now be well washed out, and may be counter- stained with Magdala red or eosia, or (better) with picrocarmine or acetic acid carmine. • Pal's process gives brilliant results, the ground of the preparations being totally colourless. Weigert (Ergebn. Anat., vi, 1896, p. 21) considered it superior to his own for thick sections, but not so safe for very fine fibres. Makcus stains by the Pal method sections of material hardened in formalin. GuDDEN {Neurol. Oeni/rhl., xvi, 1897, p. 24) makes celloidin sections of material hardened in 5 to 10 per cent, formol followed by alcohol, treats them for ten hours with 0-55 per cent, chromic acid, rinses with water, and treats with 80 per cent, alcohol ; then stains by the method of Pal, adding to the haematoxylin a few drops of dilute nitric acid (Minnich). TsOHEENTSCHEW and Kaeusin (ZtseJir. wiss. Milcr., xiii, 1896, p. 354) stain for twenty-four hours in Kultschitzky's haematoxylin. Pavlow {ibid., xxi, 1904, p. 14) uses the permanganate twice as strong as Pal. KozowSKT {Newrol. OenPrbl., xxiii, 1904, p. 1041) stains as Weigert, and differentiates the sections first with 1 per cent, permanganate, till the grey matter comes out brown, and finishes the differentiation with Liq. ferri sesquichloraU. PoTTEE {Ztschr. wiss. Mikr., xxvli, 1910, p. 238) stains as Weigert, last §, and difEerentiates first in 0-25 per cent, permanganate, then in borax ferricyanide. CHAPTER XXXIII. 447 858. Kaiseb (Nemol. Oentrbl., xii, 1893, pp. 364) hardens first in Miiller's fluid, then for eight days in Marchi's fluid (§ 870), mordants sections for five minutes with sesquichloride of iron (1 part to one of water and 3 of 70 per cent, alcohol), stains and differentiates with Pal's liquid. For details see early ediUons. BoiTON (Jowrn. Anat. & Phys., xxxii, 1898, p. 247) makes sections of formalin material, and mordants them for a few minutes in 1 per cent, osmic acid, or for a few hours in iron-alum or ammonium molybdate, stains in Kultschitzkt's hsematoxyUn (next §), and differentiates by Pal's process. Similarly Wynn, ibid., xxxiv, 1900, p. 381. Laslett {Lancet, 1898, p. 321) mordants in Marchi's fluid (1 week), makes sections, stains by Kultschitzkt's method, and differentiates by Pal's. 859. Kultschitzky's Method {Anat. Anz., iv, 1889, p. 223 ; and v, 1890, p. 519). — Specimens are hardened for one or two months in Erlicki'^ fluid, imbedded in celloidin or photoxyhn, and cut. Sections are stained for from one to three hours, or as much as twenty-four, in a staia made by adding 1 grm. of hsematoxylin dissolved in a little alcohol to 100 c.c. of 2 per cent, acetic acid. They are washed out in saturated solution of Hthium or sodium carbonate. Differentiation is not necessary, but by adding to the lithium carbonate solution 10 per cent, of a 1 per cent, solution of potassium red prussiate, and decolorising therein for two or three hours .or more, a sharper stain is obtained. After this the sections are weU washed in water and mounted in balsam. Myelin dark blue. WoLTEBS (Ztschr. wiss. Mihr., vii, 1890, p. 466) proceeds as Kultschitzky, except that he stains at 45° C. for twenty-four hours, after which the sections are dipped in Miiller's fluid, and differentiated by Pal's method. Similarly Kaes {Neurol.- Centrbl., x, 1891, p. 456). Myelin dark blue, cells yellow-brown. 860. MiTKOPHANOw {Ztschr. wiss. Mikr., xiii, 1896, p. 470) mordants photoxyhn sections for at least twenty-four hours at 40° C. in a mixture of equal parts of saturated aqueous solution of copper acetate and 90 per cent, alcohol, stains for ten minutes in Kultschitzkt's htematoxy- lin, and differentiates with Weigert's ferricyanide fluid. 861. Berkley's Rapid Method {Neurol. Centrbl, xi, 1892, p. 270). — Slices of tissue of not more than 2 J mm. in thickness are hardened for twenty-four to thirty hours in Flemming's fluid, at a temperature of 25° C, then in absolute alcohol, then imbedded in celloidin and cut. After washing in water the sections are put overnight into a saturated solution of copper acetate (or simply warmed therein to 35° to 40° C. for half an hour). They are then 448 MYELIN STAINS. washed, and stained for fifteen to twenty minutes in a lithium carbonate hsematoxylin similar to Weigert's, warmed to 40° C, allowed to cool, and differentiated for one to three minutes in Weigert's ferricyanide liquid, which may be diluted if desired with one third of water. 862. Hill {Brain, xix, 1896, p. 1 ; Phil. Trans., 184, b, 1894, p. 399) stains well-washed Miiller material in bulk in alum carmine, cuts and mordants sections for twenty-four hours in half-saturated solution of copper acetate, stains and differentiates as Weigert, taking the differentiating fluid only half as strong. 863. Benda's Rapid Method {Berlin klin. Woohensohr., xl, 1903, p. 748). Sections otformol material by tlie freezing process (alcohol being avoided) are stained (without any mordanting) for twenty-four hotirs in Boehmer's hsematoxylin, differentiated with Weigert's ferricyanide, and mounted in balsam. Only recommended for peripheral nerves, or for preliminary examination of the central nervous system. Similarly, Nageotte, O.R. Soc. Biol., ii, 1908, p. 408, staining with hsemalum. Similarly the Enzycl. mik. Technik., 1910, ii, p. 239, ■with fresh material cut by the freezing process, and the sections mounted in Isevulose (as alcohol somewhat extracts the stain). 864. Streeter (Arch. mik. Anat., Ixii, 1903, p. 734) stains small nerve-centres in bulk (after mordanting in Weigert's bichromate and fluoride mixture, § 845) with Weigert's hsematoxylin (four' to six days), washes for a couple of days in 70 per cent, alcohol, makes paraffin sections, and differentiates them by the method of Weigert or Pal. 865. Bbsta's Ammonio-Chloride o! Tin Methods (Riv. Sperim. Freniatr., xxxi, 1905, p. 569). — Pieces of peripheral nerves are fixed for one to three days in 100 c.c. of water with 25 of formol, and 4 grms. of Merk's ammonio-chloride of tin, and then dehydrated and em- bedded as usual. The sections may be stained in different ways : (a) For twenty-four hours in Mallory's phosphomolybdic-carbolic- acid haematoxylin with subsequent differentiation in Lugol's solu- tion ; (b) for thirty to sixty minutes in a very diluted solution of Delafield's hsematoxylin and then for a minute in Held's acetic solution of erythrosin ; (c) for five to ten minutes in erythrosin, and then for two hours in a mixture of equal parts of 1 per cent, haematoxylin and 4 per cent, ammonium molybdate with 3 drops of acetic acid to every 50 c.c. of the mixture. 866. Gallein. — Akonson {CertPrbl. med. Wiss., xxviii, 1890, p. 577) stains sections of material, hardened in liquid of Erlieki or Miiller and mor- CHA PTER XXXIII. 449 danted with copper acetate, for twelve to twenty-four hours in a solution of 3 to. 4 c.c. of Oallein in 100 c.c. of water with 20 of alcohol and three drops of a concentrated solution of sodium carbonate. Sections are differentiated by the method of Weigert, or Pal. Nerve-fibres red. A second stain with methylene blue may follow (best after differentiating with potassium permanganate). Similarly Scheottbr {Oentrabl. allg. Path., xiii, 1902, p. 299). 867. Scheottbr {Nemol. Centrhl, xxi, 1902, p. 338) also stains sections for two to three hours in a 5 per cent, solution of sodium, sulphalisarinate, to which a few drops of 6 per cent, oxalic acid (enough to give an orange tint) are added, then differentiates until no more colom- comes away in sodium carbonate solution of ^-^^^^ strength, and mounts in balsam. Myelin red, on a colourless ground. 868. Toluidine Blue and Methylene Blue. — Harris {PMladelpMa Med. Journ., i, 1898, p. 897) stains sections (of material hardened as for Weigert's stain) for several hours in a 1 per cent, solution of toluidine blue in 1 per cent, borax solution, and differentiates in saturated aqueous solution of tannic acid. Similarly, but with methylene blue, in a com- plicated way Frabnkel {Neurol. Oentrbl., xxii, 1903, p. 766). BiNG and Ellermann (Arch. Anat. Phys., Phys. Abth., 1901, p. 260) harden in 9 parts of acetone to 1 of formol, cut without imbedding, stain for five to ten minutes in saturated methylene blue solution, and put for one or two into saturated solution of picric acid. 869. Other Modifications or Similar Methods. — Flechsig, Arch. Anat. Phys., Phys. Abth., 1889, p. 537 ; Brbglia, Ztschr. wiss. Mihr., vii, 1890, p. 236; Rossi, ibid., vi, 1889, p. 182; Mercier, ibid., vii, 1891, p. 480 ; Haug, ibid., p. 153 ; Walsem, ibid., xi, 1894, p. 236 ; Eobert- SON, Brit. Med. Journ., 1897 (1), p. 651. Strong {Journ. Oomp. Neur., xiii, 1903, p. 291) finds copper bichro- mate (of 2 to 3 per cent.) the best mordant ; and that the mordanting is best done before bringing into ceUoidin. After staining, he treats for half a minute with 0-25 per cent, osmic acid and differentiates as Pal. K. Koch {Berl. Klin. Wochenschr., li, 1914, p. 422) makes sections by the freezing method of formalin material imbedded in gelatine, and after staining with Weigert's iron hsematoxylin, differentiates by Pal's method, and mounts in glycerin jelly. 870. Maechi's Method (for Degenerate Nerves) {Riv. sperim. Fren., xii, 1886, p. 50). — Small pieces of nervous tissue are hardened for a week in Miiller's solution, and then put for a few days into a mixture of 2 parts of Miiller's solution and 1 part of 1 per cent, osmic acid. Sections are cut, best without imbedding, and mounted in balsam. The myelin sheaths of normal nerve fibres take a yellowish-brown colour, those of degenerated fibres a black one. This process, therefore, gives positive images of the degenerated elements, Weigert's process only giving negative ones. For a critical review of this method and its modifications, see M. 29 450 MYELIN STAINS. Weigert (Ergebn. Anat., vii, 1897 (1898), pp. 1—8) ; Matuszewski (Arch. path. Anat., clxxix, 1905, p. 12) ; De Lai^ge (Le Nevraxe, X, 1908, p. 83) ; and Lewy (Fol. Nemobiol, ii, 1909, p. 471). FiNOTTi (Virchow's Arch., cxliii, 1896, p. 133) makes sections of material that has been in Miiller's fluid for not more than a few- weeks or months, and puts them for four to ten hours (in the dark) into a freshly prepared mixture of 1 or 2 parts of 1 per cent, osmic acid, and 1 part of a concentrated solution of picric acid in one-third alcohol. For peripheral nerves ; myelin (normal), black. Ore {Journ. Path, and Bact., vi, 1900, p. 387) treats small pieces of fresh tissue with a mixture of 8 c.c. of 2 per cent, osmic acid, and 2 c.c. of 1 per cent, acetic acid, which increases the penetration. Should the mixture be darkened at the end of twenty-four hours, then it ought to be renewed. After forty-eight hours, the pieces are placed in 10 per cent, formalin for three days to complete reduction and hardening. Vassale (Arch. Ital. Biol, xxvii, 1897, p. 131) takes 75 c.c. of Miiller's solution, 25 c.c. of 1 per cent, osmic acid, and 20 drops of nitric acid. NissL (Encycl. mik. Technik., ii, p. 248), holding that alcohol attacks the myelin, cuts without imbedding, and hurries sections through alcohol and bergamot oil into balsam. Eamon t Cajal (Trah. lab. Biol. Madrid, ii, 1903, p. 93) has a com- plicated method of treating Marchi material. BuscH {Neurol. Gentralb., xvii, 1898, p. 476) puts formol material for five to seven days into a solution of 1 part osmic acid, 3 of iodate of sodium, and 300 of water. Same stain as Marchi's, but more penetrating and sharper. See also Venderovi6 (Anat. Anz., xxxix, 1911, p. 414) who cuts slices of formol material -5 cm. thick, and treats these, with Marchi's fluid, thus getting increased depth of reaction. Steensland {Anat. Rec., viii, 1914,. p. 123) recommends clearing sections of Marchi material with oleum origani cretici, and mounting in chloroform-balsam. Osmic Acid (Exnee, Sife6. Alcad. Wiss. Wien, Ixxxiii, 1881, Abth. 3, p. 151 ; Bevan Lewis, The Human Brain, 1882, p, 105). — A portion of brain, not exceeding a cubic centimetre in size, is placed in 1 per cent, osmic acid, and after five to ten days is cut (best without imbedding). The sections are treated with caustic ammonia (20 drops to 60 o.o. of water), which clears up the general mass of the brain substance, leaving the meduUated fibres black. The preparations are not permanent, unless (Ranvtek, Tradte, 1 ed., p. 1086) they are fixed for a quarter of an hour in osmic acid vapour. CHAPTER XXXIII. 451 871. Azoulay's Osmic Acid Methods (Anat. Anz., x, 1894, p. 25). — (A) Sections of Muller material are put for five to fifteen minutes into a solution of 1 : 500 or 1 to 1,000 of oxmic acid, rinsed with water, and put for two to five minutes into a 5 or 10 per cent, solution of tannin, warming them therein over a flame till vapour arises, or in a stove at 50° to 55° C. Wash for five minutes in water, counter- stain with carmine or eosin, and mount in balsam. If the sections are too thick it will be necessary to differentiate by Pal's process, or with eau de Javelle diluted with 50 volumes of water. (B) Material that has been in an osmic mixture (fluids of Flemming, or Marchi, or Grolgi). Sections as before, then taimin bath, warming for three to ten minutes, the rest as before. 872. Heller and Gumpeetz (Ztschr. tviss. Mikr., xii, 1895, p. 385) give for peripheral nerves, and Heller {ihid., xv, 1898, p. 495) for central nervous system, the following method. Sections of Miiller material are put into 1 per cent, osmic acid (twenty-four hours at 37° C. for peripheral nerves ; ten to thirty minutes, at room temperature, for central nerve fibres). They are treated with pyro- gallic acid (a photographic developer will do) tiQ the nerve fibres are black, then with a violet-coloured solution of potassium per- manganate till the sections become brown, then with 2 per cent, oxalic acid till they become yellow-green. Wash out well between each operation. 1 Similarly, Teljatnik (Newol. Centrbl., xvi, 1897, p. 621) ; Eobebtson (Brit. Med. Jowrn., 1897 (1), p. 651), the material beiag previously mor- danted with Weigert's chrome alum-copper fluid for neuroglia ; and Oke, Journ. Path, and Baet, vi, 1900, p. 387. See also Eossolimo and BuscH, Ztschr. wiss. Mikr., xiv, 1897, p. 55. WiTTMAACK {Arch. Ohrenheilh., Ixi, 1904, p. 18) mordants till green (temporal bones) in 90 parts of Miiller's fluid with 10 of formol and 3 to ,5 of acetic acid, decalcifies with nitric acid and formol, treats sections (para^n or celloidin) for a few minutes with 2 per cent, osmic acid, and reduces in 5 per cent. pyrogaUol. 873. Iron. — ^Allerhand (Nemol. Oentrhl., xvi, 1897, p. 727) puts sections of MiiUer material for fifteen minutes iato warm 50 per cent, solution of Liquor ferri sesqwichloraU, then for an hour or two into 20 per cent, tannin solution (old and brown). They are then diflferentiated by Pal's method, using, however, the liquids twice as strong. An iron-alum process is described by Strong in Journ. Oomp. Neurol., xiii, 1903, p. 291. 874. Silver Nitrate. — Vbstabini-Ceesi (AU. Acead. Med. Ohw. Napoli, 1, 1896) hardens in formol, cuts thick sections, washes them with 29—2 452 MYELIN STAINS. 40 per cent, alcohol, puts them in the dark into 1 per cent, solution of silver nitrate in 40 to 70 per cent, alcohol, then washes thoroughly. Similarly, Mosse {Arch. mik. Anat, lix, 1902, p. 401), impregnating bichromic material with 1 per cent, solution of argentamin, and reducing in 10 per cent. pyrogaUio acid, and difierentiating by the method of Pal. Myelin-and-axis-cylinder Stains. 875. Methylene Blue. — Sab.i.i {ZtscJir. wiss. Mikr., ii, 1885, p. 1) stains sections of tissue hardened in bichromate for several hours, in con- centrated aqueous solution of methylene blue, rinses with water, and stains for five minutes in saturated aqueous solution of acid fuchsin. If now the sections are rinsed first with water, then for a few seconds in a 1 : 1,000 alcohoUo solution of caustic potash, and lastly brought into a large quantity of water, the stain becomes differentiated, axis -cylinders being shown coloured red and the myelin sheaths blue. Or (ibid., p. 49), the sections are stained for a few minutes or hours in : — Water ....... 40 parts. Saturated aqueous solution of methylene blue 24 „ 5 per cent, solution of borax . . . 16 „ then washed either in water or alcohol until the grey matter is distinctly difEerentiated from the white substance, cleared with cedar wood oil, and mounted in balsam. Preparations similar to those obtainable by Weigert's method. 876. Acid Fuchsin. — Pinotti {Virchow's Archiv., cxliii, 1896, p. 133) stains strongly in Delafleld's hsematoxylin, then for a few seconds in concentrated solution of picric acid, then in 0'5 per cent, acid fuchsin, and treats lastly with alkaline alcohol. Ohlmachek (Journ. Exper. Med., ii, 1897, p. 675) stains sections for one minute with gentian violet in anUin-water, then for a few seconds in a 0-5 per cent, solution of acid fuchsin in saturated solution of picric acid diluted with 1 volume of water, and differentiates with alcohol and clove oil. Kaplan (Arch. Psychiafr., xxxv, 1902, p. 825) mordants for months in Miiller, stains sections for a day or more in ^ per cent, aqueous solution of acid fuchsin, rinses in water acidulated with HCl, and differentiates by the method of Pal. 877. Salranine. — ^Adamkiewicz (SiWb. Akad. Wiss. Wien. Math. Naturw. Kl., Ixxxix, 1884, Abth. 3, p. 245) stains sections of Miiller material in concentrated solution of safranine, differentiates in alcohol and clove oil, brings back again into water, washes in water acidified with acetic acid, and stains in methylene bhie. Myelin red, nuclei violet. Similarly, Ciaglinski (Ztschr. wiss. Milcr., viii, 1891, p. 19) and Steoebe (ibid., x, 1893, p. 384), the former employing safranine followed by anilin blue, whilst the latter first stains with anilin blue, then differentiates with alcohol containing a very little caustic potash, and counterstains with safranine. CHAPTER XXXIII. 453 878. Congo Red. — Nissl (Ztsdhr. wiss. Mikr., iii, 1886, p. 398) stains for three days in Congo red (6 parts to 400 of water) and difEerentiates in alcohol with 3 per cent, of nitric acid. Other Methods. Eothig's Vital-Scharlach vm Counteistain {Newrol. Centrbl., xxxiii, 1914, p. 219, and xxxiv, 1915, p. 265). — Sections stained and difEerentiated by Weigert-Pal's method are kept for twenty-four hours at room temperature in a counterstaining fluid consisting of 90 c.c. of distilled water and 10 to 20 c.c. of a solution of Vital-Scharlach VIII, saturated at room temperature. They are then washed in distilled water for fifteen minutes and difEerentiated in 70 per cent, alcohol for from one or two hours up to twenty-four, when the celloidin will be found to be colourless. After another wash in 96 per cent, alcohol, s.ections are mounted as usual. Nerve cells and their processes, as well as axis-cylinders red, the latter being visible within the deep blue myelin sheaths. The method does not succeed if the sections were previously treated with an osmic acid solution. Vital-Scharlach VIII may also be used as a general stain, in which case the finished preparations are similar to thpse obtainable by the usual carmine stains. Paladino's palladium chloride methods ; see Bendic B. Accad. Scienze, Napoli, iv, 1891, p. 14 ; Arch. Ital. Biol., xvii, 1892, p. 145, and xix, 1893, p. 26. For Woltek's vanadium chloride process, see next chapter. Zosin's magenta red method ; see Neurol. Gehtrlb., xxi, 1902, p. 207. Peeusini's remarks and methods for the study of the white substance of the spinal cord : see Journ. Psychol. Neurol., xix, 1912, p. 61. CHAPTER XXXIV.* AXIS-CYLINDER AND DENDRITE STAINS (GOLGI AND OTHERS). 8'?9. Introduction. — There are three chief methods for the anatomical (§ 805) study of axis-cylinders and nerve-cell processes, viz., the methylene blue intra vitam method, the bichromate-and- nitrate of silver, and the bichromate-and-sublimate methods of GoLGi. The methylene blue method has already been described in Chapter XVI. (§§ 337 — 346), and only a few points remain to be dealt with here. These, together with some other methods suitable for similar purposes, will be given at the end of this chapter, the principal object of which is the description of the Golgi methods. 880. The Methods of Golgi. — There are two methods of Golgi, viz., the Bichromate and Nitrate of Silver Method and the Corrosive Sublimate Method. The bichromate and nitrate of silver method has been worked out by Golgi in three forms — the slow process, the rapid process, and the mixed process. The rapid process is the one mostly used at the present time, and it may be regarded as the classical method of inquiry into the general morphology and distribution of nerve cells and their pro- cesses in hardened tissues. One must, however, remember that * extremely delicate results may be obtained by both the mixed process and the corrosive sublimate method, and that use should be made of them also, particularly for the study of the finer relations of the nervous elements. General Characters of the Impregnation.- — The preparations have not in the least the appearance of the usual stains, and are even very different in aspect from those obtained by the ordinary methods of impregnating with silver or gold. The impregnation is a partial one, by which is meant that of all the elements, whether nervous or not, that are present in a preparation, only some are coloured. This is one of the great advantages of the method, for, if all the elements present were coloured equally, one would hardly be able to follow * Revised and in great part re- written by Dr. C. Da Fano, King's College, University of London. CHAPTER XXXIV. 455 any one of them for more than a very short distance. Golgi's method selects from among the elements present a small number which it stains with great intensity and very completely ; that is to say, they are very clearly separated throughout a great distance from those elements which have remained uncoloured. Axis-cylinders are generally impregnated only as long as they are non-medullated. In the adult the method stains nerve cells and their processes so far as these are not myelinated ; but if it be wished to impregnate throughout a great length the axis-cylinders, their arborisations and collaterals, the method is best applied to embryos or new-born animals, at a time when nerve fibres have not yet become surrounded by their myelin sheath. Nervous tissue is not the only thing that is impregnated in these preparations : neuroglia, connective tissue, fibrils, etc., also become stained, and the method has been applied with success to the study of bile capillaries, gland ducts, and the like. Both on account of this peculiarity and of the fact that the impregnation may be limited sometimes to certain elements, sometimes to others, care should be exercised in the interpretation of the results, obtained. A further source of possible error is found in the formation of precipitates which may, up to a point, simulate dendrites and other structures. The Golgi methods have been applied with success, also, to tissues of invertebrates — insects, Lumbricus, Tubifex, Helix, Limax, Distomum, Astacus, Actinida, etc. The methods have been described at length by Golgi in Biv. iSperim, Freniatr. I, 1875 ; Arch. p. le 8c. Med., iii, 1878 ; Arch. Ital Biol, vii. 1886, p. 15 et seq. ; Opera Omnia, Milano; I and II, 1903, and many other publications. A valuable account of the rapid process has been given by v. LENHOSsiiK in his Feinere Bau d. Nervensy stems, 2nd ed., 1895, and of both Golgi's methods and their modifications by Kallius in the art. " Golgische Methode," in the Brnsyh. d. mih. Teohnih I, 1910. 881. Golgi's Bichromate and Nitrate of Silver Method. Slow Process. — (a) The Hardening. — The tissues must be hardened in a bichromate solution. Either pure potassium bichromate may be employed or MiiUer's fluid. (The reaction can be obtained with Erlicki's fluid, but this is not to be recommended.) The normal practice is to use potassium bichromate, beginning with a strength of 2 per cent, and changing this frequently for fresh solutions of gradually increasing strength— 2|-, 3, 4, and 5 per cent. The tissue should he as fresh as possible, though satisfactory results may sometimes be obtained from human material collected at the 456 AXIS-CYLINDER AND DENDRITE STAINS. P.M. table even twenty-four to forty-eight hours after death. It should be divided into pieces of not more than 1 cm. or 1|- cm, in size. The most difficult point of the method consists in finding out the exact degree of hardening after which the material can be success- fully submitted to the further treatment. In summer good results may be obtained after fifteen to twenty days of hardening, and the material may continue to be in a state suitable for the silver impregnation up to thirty, forty or fifty days. In cold weather good results can seldom be obtained under a month ; when this is the case, the material may continue to give good impregnalions for two, three or even four months. The only way to make sure is to pass, at intervals, trial portions of the tissue into the silver nitrate solution — in summer frequently, in winter every eight or^ ten days — and observe whether and when the reaction has been obtained. It is a good practice to inject the organs (see § 806) with the hardening fluid, generally 2-5 per cent, potassium bichromate, to which, according to Golgi, 5 to 6 per cent, of gelatine may be added, in which case, however, the fluid must be injected after warming it to body temperature. Stoving at a temperature of 20° to 25° C. is useful for abridging the hardening, but there is a risk of over- hardening ; and Golgi thinks that the results are never quite so delicate as after hardening at room temperature. (6) Impregnation.- — As soon as the pieces of tissue have attained the proper degree of hardening, they are brought into a large quantity of silver nitrate solution, the usual strength of which is 075 per cent., but 0'50 per cent, may be used for material which has not been quite enough hardened, and 1 per cent, for material that has been slightly over-hardened. The moment the pieces are put into the silver bath an abundant precipitate is formed. This, of course, weakens the bath pro tantb. It is, therefore, advisable first to wash them well in a weaker silver solution until, on being put into a fresh quantity of it, no further precipitate is formed. Used solutions wiU do for this purpose. The final silver bath needs, generally, no further attention ; but it should be changed for a fresh one if it becomes yellowish, as it some- times does, particularly in the case of tissues which have taken up a great deal of bichromate. It is not necessary to keep the material in the dark during the impregnation ; in winter it is well to keep it in a warmed room. The time generally necessary for the impregnation is from twenty- CHAPTER XXXIV. 457 four to forty-eight hours ; but tissues may remain in the bath without hurt for days, weeks or months. (c) Preservation. — As soon as a trial has shown that a suiSciently satisfactory impregnation has been obtained, the pieces are brought into 80 to 90 per cent, alcohol.- The alcohol is changed two, three or more times, until it remains transparent, even after specimens have been two or three days in it ; for, in view of good preservation, it is necessary that the excess of silver nitrate should be washed out from them thoroughly. Sections are now made (see § 892). These are to be washed thoroughly in three or four changes of absolute alcohol and cleared, first in creosote, in which they should remain only a few minutes, then in oil of turpentine, in which they are usually left for three to fifteen minutes, though they may be kept in it even for some days without being spoiled. They are then mounted in thick xylol- damar (rather than in balsam), without coverslip. Preparations mounted with coverslips in the usual way always go bad sooner or later, whilst those mounted without a cover keep well for years, especially if they are protected from dust and light. Instead of creosote and oil of turpentine, fluid cedar-wood oil is now used in Golgi's laboratory for clearing the sections, which are then mounted, without cover, in thick cedar-wood oil. But care must be taken to leave the sections in fluid cedar-wood oil no longer than one hour or so, as otherwise they become brittle and difficult to mount. To make sure of complete dehydration and that no curling of the sections should take place in the fluid cedar- wood oil, they are quickly passed through liquid absolute guaiacol, the whole procedure being carried out as follows : A small qaiantity of absolute guaiacol is poured in a watch-glass and some fluid cedar-wood oil in two other small glass dishes. Two or three sections are carried from the absolute alcohol into the guaiacol by means of a perforated spatula, which is to be used for all the other passages, and cleaned at every passage. After a few seconds the sections are transferred into the first dish of fluid cedar- wood oil and there left for the time necessary to pass another two or three sections from the absolute alcohol into the guaiacol. The first batch of sections is now transferred into the second dish of cedar- wood oil, the second batch into the first cedar-wood oil and a fresh batch into guaiacol, and so on until all sections are collected in the second dish of cedar-wood oil. For mounting the sections are lifted, one by one, by means of the same small spatula, and arranged in the order and number one may 458 AXIS-CYLINDER AND DENDRITE STAINS. wish, either on ordinary slides, or on coverslips if the Golgi hollowed- out wooden slides are preferred for definite preservation. The excess of cedar-wood oil carried with the spattda is removed hy covering the sections, after having definitely arranged them on the slides, first with a sheath of cigarette paper and then with a folded piece of filter paper, to be held by the left hand while the right is passed over it so as to press down the sections and absorb the oil. The whole mancEuvre may be repeated a second time, and then a drop of thick cedar-wood oil put on each section. On the next day the oil which may have run from the sections is cleaned from the edges of the slides and a fresh drop of the thick cedar-wood oil put on the sections, to be protected from dust and light at least until the oil has become quite dry. Preparations mounted in this way last for years unaltered ; in fact, I have some which were made in Golgi's laboratory over fifteen years ago and I find that they have kept without change. 1 have no experience of the use of creosote or of the mixture, originally proposed by Andriezen, of equal parts of pyridine and xylol instead of the guaiacol, but they should equally well serve the purpose. As a general rule one makes sections of 20 to 40 ;>, ; thicker sections of 50 to 60 ju., or more, show more than thin ones but do not seem to keep so well. The order in which the elements of nervous tissues impregnate is generally — first, axis-cyUnders, then nerve cells, and lastly, neuroglia cells. 882. Golgi's Bichiomate and Nitrate of Silver Method. Rapid. Process. — Small pieces of very fresh tissues are hardened in a mixture of 2 to 2-5 per cent, potassium bichromate 8 parts, and 1 per cent, osmic acid 2 parts. Or, if a very quick hardening is desirable, 2 parts of 3 per cent, bichromate to one of 1 per cent, osmic acid. In Golgi's laboratory mixtures of 3 parts of 3 per cent, bichromate and 1 of 1 per cent, osmic acid are now generally used. The tissues begin to be in a state suitable for the silver impregnation from the second or third day ; in the next following days they are in a still more favourable state, but this soon declines, and is generally quite lost by the tenth or twelfth day. The silver impregnation is conducted exactly in the same way as in the slow process, and sections are prepared and mounted in the same manner, but they should not be left in alcohol for more than an hour or so before mounting. CHAPTER XXXIV. 459 There is this difference, that the impregnated material cannot be preserved for any length of time in alcohol and must not remain in it for more than one or two days. But it may be kept in the silver solution until wanted for sectioning. According to V. Gehuchten (La Cellule, vi, 1890, p. 405) pieces may be kept with advantage for many days, weeks and months in the silver nitrate solution. An abundant impregnation was found by him after many days up to six months where almost none had been seen after twenty-four to forty-eight hours only. But the material must be kept in the dark. As to the proper duration of the hardening process in difEerent cases, it must be pointed out that definite rules can hardly be given, while investigators can easily find out the right moment for success- fully transferring the pieces into the silver bath by means of attempts made in accordance with the purpose in view and the quality of the material with which they are working. However, the following points should be borne in mind : — Spinal cord of cMok from the sixth to the tenth day of incubation — twelve to forty-eight hours in the hardening mixture (up to the fifth day the embryos may be treated whole, later the vertebral column should be dissected out and ciit into two or three segments ; it need not be opened). The spinal column of newborn rats and mice should be treated in the same way, and remain in the mixture for twenty-four hours for spinal ganglia, or for two to six days for the cord itself. The encephalon of these subjects may be treated in just the same way, without being dissected out. V. Lenhossek (op. cit.) recommends for human fmtal cord two to three days for neuroglia, three to five for nerve-ceUs, and five to seven for nerve-fibres and collaterals. Oerehellum of new-born subjects three to five days in the hardening mixture. Cerebral cortex of young subjects two to three days (mice), or as long as five (rabbit, oat) ; cortex of adults, eight to fifteen days. The most favourable region of the brain is the Ammon's horn, especially in the rabbit. JSe^iwa— twenty-four to forty-eight hours in the mixture, then " double " impregnation (§ 923). SympatUeUc. — Sala, L. (Mon. Zool. Ital, iii, 1892) found the inferior cervical ganglion particularly suitable for staining by Golgi's rapid process. He proceeds thus: osmium bichromate mixture, three days ; quick wash in distilled water ; silver bath, two to three days ; further wash in distilled water and passage into the same osmium- bichromate mixture for about four days ; a third impregnation can be ' resorted to, in which case pieces should remain in the hardening fluid for five to seven days. Spinal cord of larvw of AmpMbia.—T^he entire larvae (best 2 to 2-6 cm. 460 AXIS-CYLINDER AND DENDRITE STAINS. long) should be put for two to five days into the hardening mixture, and for one to two into sUver nitrate. Epidermis of Lumhricus. — Three to six days in the mixture, and two in the sUver, or double impregnation if necessary. Nervous system of Helix (glia-oells). The above mixture for eight to ten days, then silver of 0-75 to 1 per cent. As a general rule, the younger the subject the shorter should be the hardening. If it has been too short, sections wiU have a brownish-red opaque aspect, with precipitates, and irregular impregnation of cells and fibres. If it has been too long, the ground will be yellow, without precipitates, but with no impregnated elements, or hardly any. This process has the advantage of great rapidity, and of sureness and delicacy of results, and it is the one that has found most favour with other workers. But for the methodical study of any given part of the nervous system Golgi himself prefers the following : — 883. GoLGi's Bichromate and Nitrate of Silver Method. Mixed Process. — ^Fresh pieces of tissues are put for periods varying from two to twenty-five or thirty days into the usual bichromate solution (§ 881). Every two or three or four days some of them are passed into the osmio-bichromate mixture of the rapid process, hardened therein for from three or four to eight Or ten days, and finally impregnated with silver nitrate and subsequently treated exactly as by the rapid process. The reasons for which Golgi prefers this process are : The certainty of obtaining samples of the reaction in many stages of intensity, if a sufficient number of pieces of tissues have been used for the purpose. The advantage of having at one's disposal a considerable time — some twenty-five days — during which the tissues are in a suitable state for taking the silver. The possibility of greatly hastening the process whenever desired by simply bringing all the pieces over at once into the osmic mixture. Lastly, a stiU greater delicacy of results, particularly noticeable in the staining of axons and their collaterals. 884. Theory of Impregnation. — It was once held that the reaction depends on the formation in the tissues of a precipitate of some salt of silver. And Kallius has put forward the suggestion that this precipitate may consist of a protein-silver-chromate combination. But this seems to B. Lee incorrect (see 1913 Ed.). In agreement with V. Lenhoss6k, he finds that the colouration is not due to a visible precipitate, but is a true stain accompanied, particularly in unsuccessful impregnations, by precipitates which not only do not CHAPTER XXXIV. 461 help the stain, but are injurious to it. It has been maintained that the stain is merely superficial, and the method has been called an " incrustation method." But it is easy to realise that it generally extends throughout the whole thickness of the impregnated elements, though in special cases or by slight modifications of the original method, the stain may be limited to certain constituents of the nerve-ceU body, such as Golgi's pericellular investment and intracellular network. The chemical nature of the stain has not as yet been discovered. A critical review of the Golgi method by Weigbkt may be found in Ergebn. d. Anat, v, 1895, p. 7. Seealso Hill (Braim, xix, 1896, p. l),and Kallius {op. cit.). Modifications of Golgi's Bichromate and Silver Nitrate Method concerning the Impregnation of Tissues. 885. Instead of potassium bichromate, ammonium bichromate has been recommended by Golgi and sodium bichromate by Kallius. Both these salts appear to penetrate more quickly into the tissues than potassium bichromate. According to Strong {N.Y. Acad. Sc. Proc. xiii, 1894) lithium bichromate hardens more rapidly than potassium bichromate. The influence on the reaction of the bichro- mates of ammonium, sodium, calcium, magnesium, rubidium, lithium, zinc and copper, has been investigated by L. Sala {see Kallius, op. cit., i, p. 564), but he came to the conclusion that they do not offer any particular advantage, with the exception of calcium bichromate, this last to be preferred for the staining of the tangential fibres of the cerebral cortex. Eamon y Cajal (Ztschr. wiss. Mikr., vii, 1890, p. 332) gives 3 per cent, as the strength of the bichromate in the mixture for the rapid process, but in numerous other places has given it as 3-5 per cent. This latter strength has been adopted by many workers for the rapid process, and the mixture containing this proportion of bichromate is generally known as the Ramon y Cajal mixture. 886. Ramon y Cajal's Double-Impregnation Process {La Cellule, vii, 1891, p. 130). — Sometimes the usual rapid method fails to give a good impregnation. This, however, may frequently be obtained by putting the tissues back for a day or two into the osmium- bichromate mixture used for the first hardening, or into a fresh but weaker one containing 2 parts of 1 per cent, osmic acid and 20 parts of 3 per cent, potassium bichromate. Tissues are then washed quickly with distilled water or with a weak solutionof silver nitrate, 462 AXIS-CYLINDER AND DENDRITE STAINS. and put for a second time into the silver bath, where they should remain from thirty-six to forty-eight hours. It is important to find out the proper duration of the first hardening. If it has been too long (four days) or too short (one day) the second impregnation wiU not succeed. In this case a third impregnation may be resorted to, the objects being again treated with the weak osmium-bichromate mixture and then again with the silver nitrate solution. I find that this modification, which is the most important that has hitherto been made, gives excellent results if one proceeds by tests, viz., re-transferriug into the weak osmium-bichromate mixture those pieces in which the reaction has been found to have succeeded to some extent. 887. KoLOSSOw's Modification (see Zuschtschenco, Arch. Mikr. Anal., xlix, 1897). — Tissues are hardened for one to seven days in 3 to 5 per cent, potassium bichromate containing 0-25 per cent, of osmic acid. They are then washed quickly in. distiUed water, dried with flltej paper and transferred for two to three days into a bath of 2 to 3 per cent, silver nitrate to which 0'25 to 0-5 per cent, of osmic acid has been added. This is a good modification for sympathetic ganglia. 888. GoLGi's Processes for the Rejuvenation of Over-hardened Tissues. — Tissues which have been too long in the osmium-bichro- mate mixture will no longer take on the silver impregnation. They can, however, be made to impregnate by one or the other of Golgi's so-called processes of rejuvenation. These can be carried out in various ways given here with sufficient detail, as they may he of great use not only for rejuvenating ordinary pieces of central nervous system, but also, and particularly, for the staining of nerve-endings in glandular and other tissues, internal apparatus, spiral filaments , of peripheral nerve-fibres, etc. Golgi at first suggested washing the over-hardened pieces in a half-saturated solution of copper acetate until they no longer give a precipitate, afterwards putting them back again for five or six days into the osmium-bichromate mixture, and subsequently transferring them into the silver nitrate solution. Later he advised leaving tissues in 3 to 4 per cent, copper sulphate or 1 to 2 per cent, arsenic acid. After one, two and three days some pieces are brought back into the osmium-bichromate mixture in which they had been hardened, or into a weaker one, proceeding further as in the rapid process, viz., as if the pieces had been freshly fixed in the osmium-bichromate mixture. More recently Golgi appears to have preferred mixtures of equal parts of 2 or 3 or 4 per cent, copper sulphate or acetate and 4 to 5 per CHAPTER XXXIV. 463 cent, potassium bichromate, filtering them if copper acetate was used, and treating the pieces as stated above. As a rule these copper acetate and potassium bichromate mixtures ought to be tried first and in preference to others. As with other points of Golgi's methods, so also in this case, one must proceed by tentative experiments, according to the purpose of one's investigation and the quality of the material in hand, but chiefly according to the length of time during which the tissues have been left in the osmio-bichromate solution. See on this subject Saceedotti, Intern. Monatsehr. Anat., xi, 1894, p. 326 ; GOLGi, Cinquant. 8oe. Biol., 1899, p. 514, and Opera Omnia II, 1903, p. 677 ; Fusabi, TraU. Elem. Istol. Teen. Istol, Torino, 1909 ; Sala Gr., Anat. Ans., xviii, 1900, p. 176 ; Gemmelli, Anat. Anz., 1913, p. 444. 889. Formaldehyde Modifications o/ Golgi's Bichromate and Nitrate of Silver Method. — Many investigators have found that formaldehyde can take the place of the osmic acid in the osmio-bichromate mixture of the rapid process. This has certain advantages : A cheap reagent is employed instead of the expensive osmic acid. Pieces much larger than by Golgi's original process may be used. The stage of hardening favourable for a good impregnation lasts longer, i.e., formalin-bichromate mixtures do not over-harden. Moreover, the formaldehyde modifications can be usefully resorted to for impregnating nervous tissues of adult or young subjects, as well as for material which after repeated attempts has been found imperAdous to the osmic mixtures. However, it should be remem- bered that many investigators have failed to obtain good results by the formaldehyde methods and that they are unsuitable for embryonic specimens. HoYEE, Jun. {Anat. Anz., ix, 1894, p. 236) was the first to point out that material fixed in formalin could be used for carrying out Golgi's method. Lachi (Monit. Zool. Ital., v, 1895, p. 15) used, at first, to harden tissues for five to nine days in equal parts of 20 per cent, formalin and 6 per cent, potassium bichromate. Afterwards [Anat. Anz., X, 1895, p. 790) he adopted the mixture proposed by his pupil Dell'Isola {Boll. Ace. Med. Genova, 1895, No. 2) of equal parts of 10 per cent, formalin and 10 per cent, potassium bichromate, with the addition of 1 part of 1 per cent, osmic acid to every 10 of the mixture, this last formula being particularly suitable for quick work, as forty-eight hours afterwards pieces can be already transferred into the silver bath. Stkong {Anat. Anz., x, 1895, p. 494) suggested fixing pieces of 464 AXIS-CYLINDER AND DENDRITE STAINS. brain of adult specimens in mixtures of 100 volumes of 3 -5 per cent, potassium bichromate and from 2^ to 5 volumes of formalin. One or more pieces are, during several days, daily transferred into 1 per cent, silver nitrate. Or the tissues are left for one to two days in the above formalin-bichromate mixture and then passed into a fresh one consisting of 2 volumes of 5 per cent, potassium bichromate and 1 volume of formalin ; after another twelve to twenty-four hours all the pieces are transferred into the silver bath. DuRiG {ibid., p. 659) obtained good results by fixing ^ cm. thick pieces in 3 per cent, bichromate containing 4 to 6 per cent, of formalin, and hardening therein for three days. After silvering for two days, the pieces are brought back into the fixing mixture and one proceeds as in Eamon y Cajal's double impregnation process. Fish {Proc. Amer. Micr. Soc, xvii, 1895, p. 319) uses 2 c.c. of formalin for every 100 c.c. of 3 per cent, potassium bichromate, and leaves tissues three days in this fluid, and another three days in -75 per cent, silver nitrate ; or, with advantage, Miiller's fluid, 100 c.c. ; 10 per cent, formalin, 2 c.c. ; 1 per cent, osmic acid, 1 c.c. ; silvering as above. KoPSCH (Anat. Anz., xi, 1896, p. 727) uses 4 parts of 3-5 per cent, potassium bichromate and one of formalin ; after twenty -four hours he transfers all pieces to pure 3-5 per cent, bichromate for at least two days (retina) or three to six (central organs). He finds that by this means, precipitates are almost entirely avoided. B. Lee (1913 ed.) confirms this, but points out that the method gives a too abundant impregnation of capillaries. Geeota (Intern. Monatsch. Anat., xiii, 1896, p. 108) first hardens brains for a week or two in 5 to 10 per cent, formalin, then puts, small pieces for three to five days into 4 per cent, bichromate, and lastly transfers these into the silver bath, where they are left with advantage for ten to twenty days. Bolton {Lancet, 1898 (1), p. 218 ; Journ. R. Micr. Soc, 1898, p. 244) has obtained good results from brains of cats and half-grown kittens placed whole in 5 per cent, formalin and from human brains hardened whole in formalin of the same strength for two to twelve months. Small pieces are then cut out, and placed into 1 per cent. ammonium bichromate and left therein for from a few hours up to five days, some being transferred at intervals into 1 per cent, silver nitrate. ScHEEiBEE {Anat. Am., xiv, 1898, p. 275) obtained good results from appendages of Crustacea impervious to the osmic mixture, with 5 parts of 2-5 per cent, potassium bichromate to 1 of 4 per cent. CHAPTER XXXIV. 465 formalin, or 1 part of 2-5 per cent, bichromate to 2 of 5 per cent, formalin, the specimens remaining for one day in the first mixture and for two in the second. Similarly Dubosq (Arch, de Zool. exp., vi, 1898-90). Smirnow (Arch. mikr. Anat., lii, 1898, p. 201) fixes the cerebellum of a freshly-killed animal in 5 per cent, potassium bichromate 4 parts', and formalin 1 part, for one to eight weeks. He then divides the organ in two halves and places them into pure 3-5 per cent, bichromate, where they are left for another two to five weeks. Of the two halves one may be used for Weigert's myelin stain ; the other is divided in pieces 1 to 2 cm. thick, and these put for one to one and a half weeks into a mixture of 5 per cent, potassium bichromate, 5 parts, and 2 per cent, ositnic acid, 1 part. Pieces are then transferred, at first into a weak silver bath and then into the usual 1 per cent, silver nitrate solution. The method gives good results also in human brains of adult subjects. Odier {La Rachicocainisation, Geneve, 1903, p. 27) takes 100 c.c. of Miiller's fluid with 2 c.c. of undiluted formalin and 1 c.c. of 1 per cent, osmic acid. The mixture should be kept in the dark and made up at the instant of using it. Odier finds that formalin-bichromate mixtures generally afford a more abundant impregnation with fewer precipitates than the osmio-bichromic ones. Brookover (Journ. comp. neurol., xx, 1910, p. 49) finds useful for adult specimens a preliminary fixation in 4 per cent. " formalde- hyde," neutralised with lithium carbonate or ammonia, before carrying out Golgi's rapid process. 890. Other Modifications. — Vassalb and Donaggio (Monit. Zool. Ital., vi, 1895, p. 82) harden pieces of at most 1 cm. in thickness for fifteen to twenty days in a mixture of 5 parts of acetic aldehyde and 100 of 3 to 4 per cent, potassium bichromate, changing the fluid as soon as it has become dark. The rest as Golgi. Eamon t Cajal (Bev. Trim. Ristol., No. 2, 1888, note) found that the addition of a very little formic acid to the silver bath facilitated reduction. According to van Gehuchten (La CelMe, vii, 1891, p. 83) 1 drop of the acid shodd be added to every 100 c.c. of the silver nitrate solution. But the practice is now generally abandoned. Maetinotti (Bif. med., 1887 ; Ztsehr. wiss. Mikr., v, 1888, p. 88) pointed out that Golgi's method can be successfully carried oat on relatively large pieces by using unusually large quantities of silver nitrate solution with 6 per cent, glycerin added to it, and by keeping this for thirty days at a temperature of 25° C. to impregnate nerve-cells, and of 35° to 40° C. to stain the neuroglia. Andbiezen (Brit. Med. Journ., i, 1894, p. 909) found useful for human brain to suspend thin shoes of 2 to 4 mm. in diameter in 95 c.c. 30 466 AXIS-CYLINDER AND DENDRITE STAINS. of 2 per cent, potassium bichromate to -which after ten to fifteen minutes 5 c.c. of 1 per cent, osmic acid are added. The mixture is kept in the dark and after twenty-four hours changed for a fresh one made up with 90 c.c. of 2i per cent, bichromate and 10 c.c. of 1 per cent, osmic acid. After another two days the mixture is changed over again for one made according to the proportions given by Golgi (3 per cent, potassium bichromate, 80 c.c. ; 1 per cent, osmic acid, 20 c.c. ). Pieces are transferred into the silver bath after three and a half days (for nerve cells and neuroglia) up to six days. They are washed for five to fifteen minutes in f per cent, silver nitrate, and then put into a solution of silver nitrate of the same strength, but to which 1 drop of formic acid to every 100 or 120 c.c. of solution has been added. The whole is kept in an incubator at 25° to 27° C. for about three days, changing the silver bath after the first twenty -four houi-s. The same author advised, for the impregnation of neuroglia (Intern. Monatsehr. Anat. x, 1893, p. 533), adding 1 drop of a saturated solution of chromic acid and 1 drop of formic acid to the first hardening bath. Bekkelet (Johns HopMns Hosp. Sep., vi, 1897, p. 1) hardens tissues in MiiUer's fluid until they are of sufficient consistency to admit of fairly thin sections (about two weeks at room temperature). The portions of the brain selected are cut into slices 3 mm. thick and immersed for about three days in a mixture of 3 per cent, potassium bichromate, 100 parts, and 1 per cent, osmic acid 30 parts. For the impregnation, tissues are removed from the hardening fluid, dried a little with filter paper, washed in a weak solution of silver nitrate, and put for no less than two to three days into a freshly prepared solution of 2 drops of 10 per cent, phosphomolybdio acid and 60 c.c. of 1 per cent, silver nitrate, which in winter should be kept at a temperature of about 26° C. Hill {op. cit. § 884) uses, instead of silver nitrate, a | per cent, solu- tion of silver nitrite, with 0-1 per cent, formic acid added. GrUDDBN (NeuTol. Centrhl., xx, 1901, p. 151) uses the lactate of silver (sold as " actol "), and finds it more penetrating. 891, Avoidance of Precipitates. — Golgi's method frequently gives • rise to the formation at the surface of the pieces of irregular and sometimes voluminous precipitates, which destroy the clearness of preparations. To minimise this, Sehrwald (Ztschr. wiss. Mihr., vi, 1889, p. 456) pours 10 per cent, gelatin, which is just liquid, into a paper box, embeds the tissues in it with the aid of a little heat, and brings them therein into the silver bath ; or the tissues are coated with gelatin by dipping and cooling several times. After the impregnation is completed the gelatin is removed, before cutting, by means of warm water saturated with silver chromate. Mann {Physiol. Histol., 1902, p. 276) finds that the method gives good results provided the gelatin is not rendered insoluble by the action of light. To prevent this he proceeds thus : — Either in the photo- graphic dark room or in the evening, by artificial light, tissues, tied loosely to a thread, are immersed three times into liquefied CHAPTER XXXIV. 467 10 per cent, gelatin, and, as soon as this has set, they are put into the silver bath, keeping the latter in some dark place. It appears that surrounding a tissue with gelatin makes the impregnation slower, and for this reason Mann allows a day longer for the silver bath. Maetinotti {op. cit.) covers pieces with a layer of a pap of filter paper and distilled water. Athias wraps tissues in wafer papers. Ramon y Cajal covers them with a layer of congealed blood, which need not be removed before cutting, or with celloidin or peritoneal membrane. See " Retina." Modifications concerning the Preservation of the Preparations. 892. Cutting. — As pointed out in § 880, one of the chief qualities of Golgi's method consists in allowing one to foUow nerve-cell processes for a great distance. Evidently this cannot be done with very thin sections ; and as sufficiently thin ones can be obtained without embedding, the general practice is simply to wash the pieces taken from the silver-bath with distilled water, fix them with gum to a cork or wooden cube, put the whole into alcohol for a little while to harden the gum, and cut by means of a sliding microtome without embedding. But quick embedding, particularly in celloidin, is quite possible, and should be resorted to for material either brittle or otherwise difficult to cut. Pieces of tissue as small as possible are brought in the course of about two hours through the ascending series of alcohols into absolute alcohol ; after having changed this a couple of times, pieces are transferred for another one or two hours in thin celloidin, then coated with thick celloidin, and by means of this fixed to a wooden cube, the celloidin being a, little hardened by means of chloroform vapour, as usual. The whole is left for a little while in 70 per cent, alcohol, and sections made in the usual way. If these operations are started in the morning, when going into the laboratory, pieces are ready for cutting at about 2 p.m., sufficient time remaining for the further treatment of the sections according to the directions given above (§ 881). Care should be taken, of course, not to transfer the sections into absolute alcohol if it is not considered safe to dissolve the celloidin. In this case dehydration can be carried out as usual up to 98 per cent, alcohol, and the sections transferred into fluid absolute guaiacol and cedar-wood oil as already described in § 881. 30—2 468 AXIS-CYLINDER AND DENDRITE STAINS. Embedding in paraffin is also possible, but results are usually rather poor, and one should have recourse to it only for special objects, such as muscles (see Veratti, Mem. R. Inst. Lomb. Sc, xix, 1902, p. 87). In any case tissues should be passed quickly through the lower grades of alcohol, and remain only a few hours in 95 per cent, and absolute alcohols. They should be cleared with cedar- wood oil, as xylol and similar reagents may be injurious to the silver impregnation. One should transfer pieces directly into paraffin of as low a melting point as possible. According to Beookover (op. cit.), cedar-wood oil should be used over and over again, as it becomes saturated with silver nitrate. 893. Mounting. — As pointed out in § 881, Golgi preparations do not keep well if mounted under a cover-glass in the usual way. How and why this happens it is very difficult to say. Though an elaborate discussion between Sehrwald {Ztschr. iviss. Mikr., vi, 1889, p. 443), Samassa (ibid., vii, 1890, p. 26), and Fish (ibid., viii, 1891, p. 168) has furnished the net practical result that watery fluids should be avoided as much as possible during the after- treatment, it is not clear why preparations should deteriorate, when mounted under a cover-glass in thick cedar-wood oil or neutral balsam ; while Mann (op. cit., p. 277) states, on the other hand, that sections keep well if mounted under a cover-glass in Price's No. 1 pure neutral glycerin. For these reasons the general practice is to mount sections without a cover, either on ordinary slides or on cover-glasses to be inverted for study over the aperture of a hoUowed-out wooden slide. If mounting under a cover is desirable, this should either be raised ' free of contact with the slide by means of wax feet or the like, or the balsam of the mounl; should be rendered perfectly anhydrous by care- ful heating it on the slide with the section in it, until it immediately sets hard on cooling, when a slightly-warmed cover can be applied. This last method is also recommended by Huber (Anat. Am., vii, 1892, p. 587). B. Lee (see previous editions) advises keeping the preparations uncovered until the sections have become quite dry and the balsam, applied from time to time in thin layers, quite hard, and then to cover them with a warmed cover-glass, this being slightly pressed down on the sections. Variods processes have been devised for mounting Golgi's prepara- tions at once under a cover, but none of them give really satisfactory results. .One should have recourse to them either for special objects, or if coimterstaining with carmine or hsematoxylin, or by Weigert -Pal's CHAPTER XXXIV 469 method, or the like, is particularly desirable. In this case one of the following methods may be employed : — G-KEPPiN {Areh. Anat. u. Entwich., Anat. Ahth., Supp., 1889, p. 55) treats sections for thirty to forty seconds (tintU whitish) with 10 per cent, hydrobromic acid, washes them in several changes of water, dehydrates, clears with clove oil and exposes them for ten to fifteen minutes to sunhght. Obeegia (Virchow's Arch., cxxii, 1890, p. 387) transfers sections into a mixture of absolate alcohol, 10 c.c, and 1 per cent, gold chloride, 10 drops, to be previously exposed to diffuse daylight for half an hour. Sections are then passed into it and put in a dark place. After fifteen to thirty minutes they are washed successively in 50 per cent, alcohol, distilled water, 10 per cent, sodium hyposulphite (five to ten minutes), and repeatedly changed distilled water. They may be then counter- stained, dehydrated and mounted in balsam under a cover. Kallius (Anat. Refle., ii, 1893, p. 271) uses 230 c.c. of distilled water and 20 c.c. of commercial hydrpquinone solution (hydroqninone 5 grms., sodium sulphite 40 gfms., potassium carbonate 75 grms., dis- tUled water 250 c.c). The solution is further diluted before using with one-third to one-half its volume of absolute alcohol and the sections (freed from unreduced silver by washing them in many changes of alcohol) left in it for several minutes. Here they become dark-grey to black, and are then transferred for ten to fifteen minutes into 70 per cent, alcohol, for five minutes into 20 per cent, sodium hyposulphite, and for twenty-four hours into a large quantity of distiUed water. Counter- stain, dehydrate, clear and mount as usual. Eberth and Eunge (Arch. mikr. Anat., xlvi, 1896, p. 370) have successfolly used a process similar to that of Greppin. They convert the silver impregnation into silver chloride by keeping sections in chlorine water for fifteen to twenty minutes, and they then reduce the white silver chloride, either through exhibition to sunlight just before mounting, or by means of Kallius' process. Bolton (op. cit.) has obtained good results with Kallius' process applied to his formol-bichromate modification. CuEKEEi (Anat. Anz., xxxii, 1908, p. 432), after fixing by Kallius' method, tones in 0-7 grm. of gold chloride, 3 grm. of sodium acetate and 100 c.c. of water. Zimmeemann's process (Arch. mikr. Anat, lii, 1898, p. 554). Paraffin sections of formol-Golgi material are brought from alcohol into a large quantity of a mixture of 1 part of physiological salt solution and 2 parts of 96 per cent, alcohol. They are kept in motion therein for ten to fifteen minutes, after which they are brought into 75 to 96 per cent, alcohol in a bright light until they have become dark (about half a day) ; or sections are left for half to one hour in 100 c.c. of absolute alcohol to which a few drops of ammonium hydrosulphide have been added. In the first case the silver deposit becomes converted into silver chloride, in the second into silver sulphide. Later (Arch. mikr. Anat., Ixxviii, 1911, p. 199) he reduces for several hours in 20 c.c. of saturated solution of sodium carbonate (made up with 60 per cent, alcohol) to which 0-5 grm. of adurol are added. These processes are useful for 470 AXIS-CYLINDER AND DENDRITE STAINS. studying the inter-relationship between gland -dUcts and gland-cells (stomach, liver) if the silver chloride sections are afterwards stained with thionin or toluidine blue or safranin, the sulphide sections with Delafleld's hsematoxylin, and the adurol ones with hsemalum or alum cochineal. For toning, fixing and counterstaining sections of tissues treated by the sublimate method and the like, see next paragraph. The Sublimate Method. 894. GoLGi's Bichromate and Sublimate Method (Arch. Sc. Med.., iii, 1878 ; Rend. R. Inst. Lomb. Sc. (2), xii, 1879, p. 205 and (2), xxiv, 1891 ; Arch. Ital. Biol, op. cit., § 880 ; Rif. Med., 1891 ; Opera Omnia, I, p. 143, and II, pp. 505 and 607). — For hardening, use either a solution of potassium bichromate progressively raised from 1 to 3 per cent., or Miiller's fluid. It is best to take small pieces of tissue, large quantities of hardening fluid, and change the latter frequently. But the reaction can be obtained with much larger pieces, even entire hemispheres. In this case the brain should at flrst be treated with "repeated" injections of. the fixing agent, or this should be injected from the carotid or the aorta. Pieces, particularly if small, begin to be ready for the subsequent treatment eight to ten days afterwards, but it is advisable to wait until the twentieth or thirtieth day of immersion, this being not injurious if prolonged for several months ; it is, on the contrary, to be recommended if the pieces are uncommonly large. When it is thought that the tissues have been hardened enough, they are passed directly from the bichromate into -5 to 1 per cent, mercury bichloride. One generally prefers weak solutions (0 -5 per * cent.) if pieces have been left in the fixing fluid for a relatively short period, having recourse to the stronger ones (1 per cent.) for materials which have been hardened for many weeks or months. The subli- mate solution must be changed at first every day, and later as often as it becomes yellowish. At the end of the reaction pieces will be found decolourised and almost with the aspect of fresh tissue. To obtaia a good reaction, about ten days of immersion in the mercury bichloride are necessary if pieces are small, longer periods, and even months, being required for large pieces and entire hemispheres. Particularly fine results were obtained by Golgi from brains which had been kept in 1 per cent, sublimate for as long as two years. The reaction may be said to have begun by the time tissues are nearly decolourised. From that time onwards sections may be made and mounted if successful. CHAPTER XXXIV. 471 Embedding is not necessary, but in many cases desirable. It can be easily carried out by washing pieces in many changes of alcohol of ascending strengths and embedding them in celloidin. Sections, however made, must be repeatedly washed with distilled water, otherwise they will soon be spoilt by the formation of opaque granules and needle-like crystals which very much hinder proper observation. After dehydrating, sections can be passed through creosote and turpentine and mounted, preferably without a cover- glass, in dammar or balsam. It is, however, preferable to treat sections by the following £Lxing-and-toning process which was suggested by Golgi for trans- forming the whitish mercury impregnation (to which the reaction is due) into a full-black stain, much more suitable for observation under high power. Moreover, the process helps in preventing the formation of opaque precipitates, and allows of moimting in the usual way without any danger of spoiling the specimens. One proceeds thus : — Sections of pieces embedded in celloidin are thoroughly washed in many changes of water, and then transferred for a few minutes into a photographic fixing and toning bath to be prepared at the moment of using, as follows : — Solution A. Distilled water 1,000 c.c. Sodiam hyposulphite Potassium alum . Ammonium thiocyanate Sodium chloride . 155 gr. 20 „ 10 „ 40 „ Allow to stand for eight days and then filter. Solution B. Gold chloride 1 gr. Distilled water 100 c.c. For use take 60 c.c. of sol. A, 7 c.c. of sol. B, and 40 c.c. of old combined bath. From the fixing and toning bath sections are transferred into distilled water and again thoroughly washed ; they are then slightly counterstained with an acid solution of carmine diluted with some alcohol, dehydrated, cleared, and mounted in the usual way. The elements stained by the method are :— (1) Nerve cells with all their processes and ramifications. (2) Nuclei, which is not the case with the silver process. (3) Neuroglia cells. But the reaction in this case is far less precise and complete than that obtained by the silver method. (4) Blood vessels, and particularly their muscle fibre-cells. 472 AXIS-CYLINDER AND DENDRITE STAINS. The method gives particularly good results with cerebral cortex and Ammon's horn, very poor ones with the cerebellum and spinal cord. It is superior to the silver method in so far that the reaction can always be obtained with certaiuty in a certain time ; that the preparation can be preserved by the usual methods ; that large pieces of tissue can be impregnated. Moreover, it is cheaper and may give a more abimdant and finer impregnation than even the rapid process. 895. Modifications of Golgi's Bichromate and Sublimate Method. — MoNDiNO (Ztsch. wiss. Mikr., ii, 1885, p. 157) has obtained good results from even whole human brain treated according to Golgi's original method. Flatau {Arch. mikr. Anat., xlv, 1895, p. 158) fixes whole human brain in 3 to 4 per cent, potassium bichromate. After two or three months slices i cm. thick and 1 to 2 cm. wide are brought into 0-1 per cent, mercury bichloride to be changed every two to three days for the first three weeks or so. Pieces are ripe for cutting after nine to twelve months, at which time they are washed and embedded in celloidin. Sections are passed through alcohols, cleared in oarbol- xylol and mounted in balsam. Pal {Erratim " Tal ") (Ztschr. wiss. Mikr., iv, 1887, p. 497) converts the whitish mercury impregnation into a black one by treating sections with 1 per cent, sodium sulphide. They may then be oounterstained with Magdala red. Golgi's sublimate method may be combined with Weigert's myelin stain (see Pal, Wiener med. Jahrb., N.F. 1, 1886, p. 619, and the abstract of this paper in Ztschr. wiss. Mikr., iv, 1887, p. 92, in which Edingek pointed out that the mercury impregnation can be turned black by treating sections with diluted ammonia). Plechsig [Arch. Anat. Phys., Physiol. Ahth., 1889, p. 537) has pub- lished a rather complicated combination of Brama's Guinea red-wood process for meduUated nerve-fibres and Golgi's sublimate method, as slightly modified by Held. 896. Cox's Process (Arch. mikr. Anat., xxxvii, 1891, p. 16). — This is the most important of all modifications of Golgi's bichromate and sublimate method. Cox found that the sublimate and bichromate can be used together, and that potassium chromate can be usefully added to the mixture in order to reduce the normally acid reaction of the bichromate, as otherwise axis-cylinders are not impregnated. He used a fluid consisting of 20 parts of 5 per cent, potassium bichromate, 20 parts of 5 per cent, corrosive sublimate, 16 parts of 5 per cent, potassium chromate, and 30 to 40 parts of distilled water. To prepare it, the bichromate and sublimate are mixed together, the chromate diluted with the water and added to the mixture. One generally uses small pieces of tissues, but also relatively large CHAPTER XXXIV. 473 ones can be employed, and whole brains of small animals, particu- larly if some of the fluid has been previously injected through the carotid or aorta. The duration of the impregnation is from two to three months, but material can be left in the mixture for much longer, certainly without danger and, very likely, with advantage. • Mann {op. cit.) recommends warming the mixture to the tem- perature of the incubator and diluting it to one-half the strength advocated by Cox, particularly for material of adult subjects. Portions of the brain measuring 1 cm. in thickness or entire brains of young animals are placed by him on cotton-wool in this solution and left in the incubator for twenty-four hours, when the solution is changed. After a second change on the third day the vessel (which should contain the mixture in proportion of 30 : 1 of the brain) is sealed with vaseline and left in the incubator for at least a month, but preferably for two. I find this way of carrying out the Golgi- Cox method very good, but, after incubating for a month or so, I prefer keeping the vessel at room temperature, and cutting after another two or three months or longer. There is considerable difiiculty in making and preserving sections which ought to be made either by free hand or by means of a freezing microtome after slight preliminary washing of the pieces with water, and impregnating them with 20 per cent, dextrin for one to three days as suggested by Mann. To convert the white mercury impregnation into a black one, Cox suggested treating the sections for an hour or two with 5 per cent, sodium carbonate, but 5 to 10 per cent, ammonia is now generally used. They are then thoroughly washed in distilled water, carefully dehydrated, cleared by one of the usual ways, and mounted, without a cover, either in thick xylol balsam or in the original medium suggested by Cox and composed of : — Gum sandarac 75 grms., camphor 15, oil of turpentine 30, oil of lavender 22-5, alcohol 75, castor oil 5 to 10 drops. For examination, add a drop of castor oil, and cover. 897. Methods for rendering Golgi-Cox Preparations more per- manent. — ^Various authors (see Sanders, 1898, in litt. to A. B. Lee, Vade-Mecum, 1913 ed., p. 433 ; Bremee, Anat. Rec, iv, 1910, p. 263) have proposed washing tissues treated according to Cox's process in many changes of alcohol, and embedding them in celloidin — this'chiefly with the object of overcoming the difficulty of cutting brittle pieces by means of the freezing microtome, and also of render- ing preparations more permanent by removing the excess of corrosive 474 AXIS-CYLINDER AND DENDRITE STAINS. sublimate not utilised by the reaction, and which still permeates the tissues. As a matter of fact sections of pieces thus treated are very easily cut and can be transferred from one to another fluid without danger of injuring them. Moreover, they can be counterstained, and the impregnation keeps sufficiently well, particularly if sections are mounted without a cover-glass. But in such preparations, sometimes quickly, sometimes slowly, opaque granules and minute needle-like crystals become almost always developed. To avoid this I recently proposed (see Da Fano, Proc. Physiol. Soc. Journ. Physiol., liv, 1921) to treat sections much in the same way as by the so-called process of toning and fixing Bielschowsky preparations and the hke. (See Da Fano, ibid., liii, 1920.) I proceed thus : — Pieces which, by a trial section, have been found well impregnated, are washed for some hours in distilled water and then brought, through many changes of alcohol of ascending strengths, into absolute alcohol, and then embedded in celloidin in the usual way. The celloidin blocks are hardened in 70 per cent, alcohol, where they can be safely left for many days and weeks. Sections of the desired thickness are collected in 60 per cent, alcohol, transferred into distilled water and here thoroughly washed. They are then treated for five to ten minutes with 5 per cent, ammonia and washed over again in two or three changes of distilled water. At this point toning is carried out by means of a slightly acidified 0-2 per cent, gold chloride solution, in which sections are left for ten or fifteen or twenty minutes, according to their thickness. After a quick washing in distilled water they are passed for three to five minutes in 5 per cent, sodium hyposulphite and washed once more in distilled water. From this they are transferred successively into. 30, 50, and 70 per cent, alcohols, to each of which 1 drop of saturated iodine tincture to every 5 c.c. of alcohol has been added. Sections remain in each alcohol ten to fifteen minutes and are lastly transferred into pure 70 per cent, alcohol. At this point the process is ended, and one can proceed to mount the sections in the usual way, or re-transfer them into distilled water, counterstain them lightly with a carmine solution, dehydrate with alcohols of ascending strength up to 95 per cent., pass them through two changes of carbol-xylol and mount them under a thin cover- glass in xylol-colophonium or balsam. If desirable and safe, the celloidin can be removed before definite mounting by passing sections through absolute alcohol, and alcohol-ether if necessary. The process is simpler than the rather complicated platinum substitutions of Eobertson and Maodonald (Journ. Merit. Sc, CHAPTER XXXIV. 475 xlvii, 1901, p. 327) and is so quickly and easily carried out that many sections can be manipulated at the same time. Processes similar to Golgi's Methods or suitable for the same Purposes. 898. Ziehen's Gold and Sublimate Method (Neurol. Centrbl, x, 1891, p. 65). — Small pieces of fresh tissues are put into a large quantity of a mixture of equal parts of 1 per cent, corrosive sublimate and 1 per cent, gold chloride, and left therein for at least three weeks, preferably for several months up to five, by which time they will have become of a metallic red-brown colour. They are then gummed to a cork or wooden cube and cut without embedding. Sections are treated either with Lugol's solution diluted with 4 volumes of water, or with diluted tincture of iodine, until duly differentiated, then washed, dehydrated, and mounted in balsam. Both meduUated and non-meduUated nerve-fibres, as well as nerve-cells and neuroglia cells are stained. 899. Keohnthal's Lead Sulphide Impregnation (Neurol. Centrbl. xviii, 1889 ; Ztschr. wiss. MiJcr., xvi, 1899, p. 235). — Pure formic acid is slowly added to a saturated solution of lead acetate till white crystals of lead formiate are abundantly formed. The mother liquid is filtered off, and the crystals are dissolved to saturation in distilled water. Equal volumes of this saturated solution of lead formiate and 10 per cent, formalin form the fixing fluid in which pieces of brain or spinal cord are left for five days. Tissues are then directly brought into a mixture of equal parts of 10 per cent, formalin and sulphuretted hydrogen. After a few minutes the first discoloured portion of this mixture is poured off and replaced with fresh solution, in which pieces remain for another five days. They are then gradually dehydrated and embedded in celloidin. Sections are cleared in carbol-xylol (1:1) and mounted in balsam under a cover. Nerve-cells and nerve-fibres are extensively impregnated. Corning (Anat. Anz., xvii, 1900, p. 108) hardens the tissues in 10 per cent, formalin and then brings them into the lead formiate, which he buys from Merk. He prefers to cut without embedding. 900. Wolter's Chloride of Vanadium Process (Ztschr. wiss. Mikr., vii, 1891, p. 471). — Central or peripheral nervous tissues are fixed in Kultschitzky's solution, followed by alcohol as described in § 55. Celloidin sections, 5 to 10 /^ thick, are mordanted for twenty-four hours in a mixture of 2 parts of 10 per cent, vanadium chloride and 8 parts of 8 per cent, aluminium acetate. They are then washed 476 AXIS-CYLINDER AND DENDRITE STAINS. for ten minutes in water, staiaed for twenty-four hours in an incu- bator in Kultschitzky's haematoxyliu, and differentiated in 80 per cent, alcohol acidified with -5 per cent, of hydrochloric acid until slightly blue-red. The acid is then removed by washing with pure alcohol, and the sections dehydrated, cleared with origanum oil, and mounted in balsam. Axis-cylinders, nerve-cells and glia. cells are stained, the myelin being coloured only when the differentiation in the acid alcohol has been insufficient. 901. Azoulay's Ammonium Vanadate Process {Bull. Soc. Anat., Paris, Ixix, 1894, 5th S., p. 924). — Wash in water thin sections of material fixed in a bichromate solution and embedded in celloidin. Lift a section on a slide and pour on it a few drops of -5 per cent, ammonium vanadate, wait a moment, pour off the stain, wash with a little distilled water and pour on the section a few drops of 2 -5 per cent, tannin. After a few minutes pour off the tannin solution, wash, and start all over again, and so on until axis-cylinder and nerve-cells are stained dark green. Wash quickly, dehydrate and mount. These preparations photograph well. 902. Fajeestajn's Hsematoxylin {Poln. Arch. Biol. Med. Wiss., i, 1901, p. 189). — Make sections, by means of the freezing microtome, of material fixed for two to seven days in 5 to 10 per cent, formalin. Transfer them into 0-25 to 0-5 per cent, chromic acid, and after twenty-four hours wash them well, and put them to stain for another twenty-four hours in 1 per cent, aqueous solution of heematoxylin Differentiate by Pal's method. 903. Nabias' Method (C. R. Soc. Biol, Ivi, 1904, p. 426).— Sections of material fixed in alcohol-corrosive sublimate or any other fixing agent easily allowing the penetration of iodine are treated until yellow with Lugol's solution (Gram's formula). They are then quickly washed, and treated for a few minutes with 1 per cent, gold chloride, quickly washed once more, and reduced in 1 per cent, watery solution of anilin oil or resorcin. Dehydrate and mount in balsam. 904. Lennhoff's Processes {Neurol. Centrbl, xxix, 1910, p. 20). — (1) Folychrome-meihylene blue and potassium sulpJioeyanide metfiod for axis -cylinders : Fixation not stated. Stain sections in polychrome methylene blae for two to five minutes, wash them in distilled water and transfer them for half to twenty-four hours into potassium sulpho- cyanide (strength not stated). Wash, dehydrate, clear, and mount in balsam. (2) PolycTirome-methylene blue and potassium ferricyandde method for axis-cylinders and nerve cells. Sections of material fixed in alcohol are treated as above, using potassium ferricyanide instead of CHAPTER XXXIV. 477 the sulphocyaiiide. (3) Iron method. Sections are kept for thirty seconds in 2 o.c. of a 15 per cent, solution of tannin to which 3 drops of a 5 per cent, solution of oxalic acid have been added. Einse them first in distilled water and then for a few seconds in 1 per cent, solution of iron chloride until no further blackening occui-s. "Wash, dehydrate and mount in balsam. Axis-cylinders black, nerve-ceUs grey. Apathy's Gold Method. See § 371. Geelach's Bichromate and Gold Process. See § 369. Ramon t Cajal's Gold Method. See Bev. trim. Micr., v, 1900, p. 95. Upson's Gold and Iron andSVanadium Methods. See Meecibr, Ztsdhr. wiss. Mikr., vii, 1891, p. 474. Magini's Zinc Chloride Process. See Boll. Ace. med. Soma, 1886, or ZtseJir. wiss. Mikr., v, 1888, p. 87. Monti's Copper Process. See Bend. B. Ace. Lincei, Boma, v, 1889, p. 705. Steahuber's Anilin Blue Method. See Centrbl. allg. Path., xii, 1901, p. 422. Chilbsotti's Carmin Stain. See Oentrbl. allg. Path., xiii, 1902, p. 191 ; Ztsohr. wiss. Mikr., xix, 1902, p. 161, and xx, 1903, p. 87. Kaplan's Anthracen Ink Method. See Arch. Psych., xxxv, 1902, p. 825. Malloet's Phosphomolybdic Haematoxylin. See § 271. DoNAGGio's Tin Stain. See § 273. Methylene-blue Methods not considered in Chapter XVI. 905. S. Meyee's Method for the Central Nervous System {Arch, mikr. Anat., xlvi, 1895, p. 282, and xlvii, 1896, p. 734).— The method consists essentially in injecting animals subcutaneously with large quantities of a solution of methylene-blue B.X., and in treating the central organs (brains) with Bethe's fixing bath. S. Meyer used, at first, a 1 per cent, solution ; later, a solution of methylene-blue B.X. saturated at the body temperature of the animal to be injected (viz., about 5 to 6 per cent.). The injections are to be made at short intervals and in such a way that the animal receives the total quantity it can support in about one to two hours. A cat can support even 150 c.c. ; half-grown rabbits, 30 to 50 c.c. ; fuUy developed guinea-pigs, 30 to 50 c.c. ; new-born kittens, 15 to 25 c.c. As soon as the animal used is dead, the braiu is removed, divided into two to four pieces, and these plunged in 10 per cent, ammonium molybdate to which 1 drop of HCl for every gram of ammonium molybdate is added. Here they remain for about twenty-four hours at 0° C. Pieces are then washed for two hours in running tap-water, passed quickly through the ascending series of alcohols into absolute alcohol, and, lastly, embedded in para£&n in the usual way. 478 AXIS-CYLINDER AND DENDRITE STAINS. 906. Eamon y Cajal's Diffusion Process {Rev. Trim. Micr., i, 1896, p. 123). — The brain is exposed, and by means of a sharp razor the cortex is divided into slices about 2 mm. thick. The slices are then covered on both sides, either with finely powdered methylene blue or impregnated with a saturated solution of the same and replaced in their natural situation. The brain is covered over again with its case for about half an hour, after which the slices are removed and fixed for a couple of hours in Bethe's ammonium molybdate solution. They are then washed and hardened for three or four hours in a mixture of 5 parts of chloroplatinic acid, 40 parts of formalin, and 60 parts of distilled water. After another quick wash and a brief treatment (? a few minutes) with a 1 : 300 alcoholic solution of chloroplatinic acid, they are dehydrated and embedded in paraffin. The sections may also be quickly treated with the same weak alcoholic solution of chloro-platinic acid, cleared with xylol or bergamot oil, and mounted in the usual way. 907. Catois' Method for Fishes (C. R. Ac. Sc, cxxiv, 1897, p. 204). — Small quantities (2 to 3 c.c.) of a concentrated solution of methylene blue, prepared with physiological salt solution, is injected into the branchial vessels or intramuscularly. The brain is removed after half an hour, divided into slices, and then left for another half an hour in the same concentrated solution used for injecting the animal. The slices are then fixed in the usual ammonium molybdate solution, or in Cajal's chloroplatinic acid mixture. 908. See also the valuable account of Dogiel Methylen-blau zur Nervenfarbung in the Enzykl. mikr. Teehn., 2nd ed., 1910, and the article of GrOEDON in Anat. Bee, iv, 1910, p. 267 ; and that of Michailow in Ztschr. wiss. Milcr., xxvii, 1910, p. 1, in which the literature of the subject is critically discussed. CHAPTER XXXV. NEUROGLIA AND SENSE ORGANS, Neuroglia* 909. Introduction. — Neuroglia cells may be isolated by teasing after maceration in weak solutions of potassium bichromate or 33 per cent, alcohol, and then stained, preferably by means of dilute picrocarmine or other carmine solutions. They may be studied, also, in sections made from non-embedded material fixed in solutions of chromic salts and stained with carmine, nigrosin, orcein and so on. Sections made from either fresh material hardened by the ether freezing method and treated with a weak solution of osmic acid (§ 807), or from tissues hardened in potassium bichromate, can be advantageously stained with watery solutions of anilin-blue-black or nigrosin. Also, sections cut from material fixed, hardened and embedded by the usual methods may, up to a point, be employed for getting a general, though incomplete, view of the amount and arrangement of the neuroglia in a given nervous organ. Iron hsematoxylin, particularly after fixation in corrosive sublimate or other fluids containing it, gives good results with sections of central nervous organs of lower vertebrates, chiefly of fishes. See GrOLGi, Opera Omnia, i, pp. 1 and 3 to 70 ; ii, p. 61 ; Eanviee, Traite, etc. ; Bevan Lewis, op. oit. ; E. Mullek, Arch. mikr. Anal., Iv, 1900, p. 17 ; Studnicka, Anat. Hefte, xv, 1900, p. .316, and the literature quoted therein. But the best method for the study of the morphology and relation- ship of ependyma cells and astrocytes has been for many years, and in a sense stiU is, Golgi's rapid process (§ 882), the best material being that which has been placed for about two or three days in the osmio-bichromic mixture. This method, however, does not allow of any tinctorial differentia- tion, either between neuroglia cells and nerve ceUs, or between neuroglia cells and neuroglia fibres. One might even say that it is unsuitable for the demonstration of the latter, the existence of which was clearly established only after the publication of ■ * Rewritten by Dr. C. Da Fano, King's College, University of London. 480 NEUROGLIA AND SENSE ORGANS. Weigekt's method (see next §), the first and, perhaps even now, most important of all so-called specific processes for staining neuroglia fibres. But the Weigert method, whilst staining neurogUa fibres and nuclei of neuroglia cells intensely and, up to a point, specifically, leaves the cell-bodies of the latter entirely unstained. It conse- quently led to the erroneous conclusion that the processes of nevirogUa cells were one and the same thing as the neuroglia fibres shown by the new method, and that the latter were, in the adult state, only contiguous to — viz., independent of — the former. Efforts were, therefore, made to discover new methods suitable for the study of neurogUa fibres and neuroglia cells and their reci- procal relations. Many modifications of Weigert's neuroglia stain, the methods of Benda, Mallory, Anglade and Morel, Held, EuBASCHKiN, Da Fano, etc., may be considered as the direct outcome of such efforts. None of these methods, however, was sufficient to entirely solve the problems resulting from Weigert's discovery, and from the comparison between the results attainable by the new neuroglia stain and Golgi's process. Hence the publication of the methods of Ram6n y Cajal, Achucarro, Del Rio-Hortega, and the modern conception that the neuroglia consists essentially of cells provided with variously ramified processes {protoplasmic neuroglia), and of fibres which, though a product of differentiation of the former, remain, very likely, throughout life continuously connected with the protoplasmic bodies and processes of neuroglia cells {fibrous neuroglia). With all that, the very meaning of the word " neuroglia " and the » methods for its study are just at present the subject of fresh discussions and investigations. It is, consequently, expedient to fully describe in the following paragraphs only the principal methods in use for the demonstration of neuroglia, taking this term to mean the whole of the sustaining tissue of the central nervous organs, which is plainly not connective tissue. For minute technical details and methods almost exclusively used in histopathology, the original papers quoted in the following paragraphs should be consulted, as well as Alzheimer, Histol. u. Histopathol. Arb., iii, 1910, pp. 406 to 412 ; NissL, Enzyld. mikr. Techn., ii, 1910, pp. 280 to 283 ; Bonome, Atti R. Inst. Veneto Sc. Ixvii, 1909. 910. Weigert's Neuroglia Stain (Weigert's Beitr. zur Kenntniss d. norm, mensch. Neuroglia, Frankfurt-a-Main, 1895 ; and the CHAPTER XXXV. 481 article " Neurogliafarbung " in Enzyhl. mik. Technik, ii, 1910).— Pieces of very fresh tissue of not more than ^ cm. in thickness are put, for at least four days, into 10 per cent, formol. They are then mordanted for four or five days at 36° to 37° C. (or for at least eight days at the temperature of the laboratory) in a solution containing 5 per cent, of neutral copper acetate, 5 per cent, of acetic acid, and 2J per cent, of chrome alum, in water. (Add the alum to the water, raise to boiling point, and add the acetic acid and the acetate, powdered, or, instead of chrome alum, take chromium fluoride, which obviates the necessity of boiling.) If preferred, the mordant may be dissolved in the formol solution, so that the hardening and mordanting are done at the same time. After mordanting, the tissues are washed, dehydrated, embedded in celloidin, and cut. The sections (not too thick) are treated for ten minutes with a -| per cent, solution of potassium permanganate and well washed in water. They are then treated for two to four hours with a solution of " chromogen." This is a naphthaline compound prepared by the Hoechst dye manufactory. The solution to be used is prepared as follows : 5 per cent, of " chromogen " and 5 per cent, of formic acid (of 1-20 sp. gr., about four times as strong as the officinal) are dissolved in water, and the solution carefully filtered." To 90 c.c. of the filtrate, 10 c.c. of a 10 per cent, solution of sodium sulphite are added. After this the sections are put till the next day into a saturated (about 5 per cent.) solution of " chromogen." (According to BoUes Lee, Pal's potassiimi sulphite may be used instead of the "chromogen.") They are next carefuUy washed and stained. This is best done on the slide. The stain is a warm-saturated solution of methyl Adolet in 70 to 80 per cent, alcohol (to which, after cooling and decanting, there may be added, if desired, 5 per cent, of a 5 per cent, aqueous solution of oxalic acid). The sections are treated with this for from a few seconds to one minute, and mopped up with blotting- paper, then treated for an instant with saturated solution of iodine in 5 per cent, potassium iodide. They are then differentiated till clear and Ught blue with a mixture of equal parts of aniline oil and xylol, washed thoroughly with pure xylol, and mounted in balsam or, preferably, in turpentine-colophonivmi. Glia fibres and nuclei blue, cytoplasm stainless. This method only gives good results with the human subject. 911. Modifications of "Wbigebt's Method. — M.ajJjOb.y {Journ. Exper. Med., 1897, p. 532) fixes tissues for four days in 10 per cent, solution of M. 31 482 NEUROGLIA AND SENSE ORGANS. formalin, then for four to eight in saturated solution of picric acid (or for the same time in a mixture of the two), then mordants for four to six days at 37° C. in 6 per cent, solution of ammonium hichromate, makes sections (celioidin) and stains them in Weigert's fibrin stain. Stokch (Virohow's Archiv., clvii, 1899, p. 127), instead of mordanting the material in bulk with the copper fluid, first makes celioidin sections and then mordants them. Baetel (Ztschr. wiss. Mihr., xxi, 1904, p. 18) first makes paraffin sections and treats them with all the reagents used by Weigert without removing the paraf&n, until they have passed the aniline xylol mixture which should consist of 1 part of aniline to 10 of xylol (or more), and be allowed to act for twelve to twenty -four hours. Sand uses material fixed as for his neurofibril stain (§ 843), and stains it according to Weigert. See also Aguerke, Arch. mik. Anat., Ivi, 1900, p. 509 ; Kkause, AbTi. h. Akad. Wissench. Berlin. Anhang, 1899; Wimmbe , Oentrhl. allg. Pathol u. pathol. Anat, xvii, 1906, p. 566 ; G-ALESESCtJ, 0. B. 8oc. Biol, Ixv, 1908, p. 429. EuBASCHKiN {Arch. mik. Anat., Ixiv, 1904, p. 577) recommends injecting centres of small mammals with the ftxitig liquid. To make this, take 100 parts of 2 -5 per cent, solution of potassium bichromate and 0-5 to 1 of copper acetate, boil, and add 2 -5 to 5 of glacial acetic acid. To this (which may be kept in stock) add, just before use, 10 per cent, of formol. Inject warm, and after ten minutes dissect out and harden in the same fluid for five to seven days at 35° to 40° C. Dry superficially, put for six to twelve hours in 95 per cent, alcohol ■ and embed in celioidin or paraffin. Stain sections on the slide for six to twelve hours in saturated aqueous solution of methyl- violet B ; - treat for half a minute to a minute with Gram's iodine in iodide of potassium ; differentiate in aniline or clove oil, and pass through xylol into balsam. The method gives very sharp results with* small mammals. 918. Benda's Method (Neurol. Centrbl, xix, 1900, p. 796 ; and his article " Neurogliafarbung," Enzyhl. mik. Technik, ii, 1910, p. 308) is as follows : — The material is to be fixed in 90 or 93 per cent, alcohol for no less than two days. Pieces, not thicker than J cm. are put for twenty-four hours in ofiicinal nitric acid 1 part, and distilled water 10 parts ; for another twenty-four hours in 2 per cent, potassiimi bichromate ; for forty-eight hours in 1 per cent, chromic acid. After washing for twenty-four hours, they are dehydrated in alcohols of ascending strength, cleared first in creosote (twenty-four hoiirs), then in benzol (twenty-four hours), and lastly embedded slowly in paraffin, this being dissolved in benzol to saturation first at room temperature, then successively at 38°, 42° CHAPTER XXXV. 483 and 45° C, so that pure paraffin, melting at 58° C, is used only for the embedding proper. The sections, stuck to slides, are mordanted for twenty-four hours in 4 per cent, iron alum or in 50 per cent. Liquor ferri sulfurici oxydati P.G., thoroughly washed, put for two hours into an amber- yeUow aqueous solution of sodium sulfalizarinate as directed in § 683, rinsed with tap water, and put to stain in O'l per cent, toluidine blue either for fifteen minutes by warming until vapour arises, or for twenty -four hours at room temperature. After rinsing in 1 per cent, acetic acid or in a very dilute solution of picric acid, the sections are dried with filter paper, passed through absolute alcohol, and differentiated for about ten minutes with creosote. They are then dried once more with filter paper, washed with xylol and mounted in balsam. Besides this, Benda recommends hardening and making paraffin sections as above, then staining by Weigert's method (§ 910), but without passing the sections through the saturated solution of " chromogen," and using instead of Weigert's methyl violet solution a freshly prepared mixture of 1 volume of saturated solution of crystal violet, 1 volume of 1 per cent, acid alcohol, and 2 volumes of aniUne water. Benda also uses Heidenhain's iron haematoxylin to stain paraffin sections of pieces treated as described, differentiating either with 2 per cent, iron alum or with Weigert's borax-ferricyanide mixture. 913. Malloey's Hsematoxylin Stains (Journ. Exper. Med., v, 1900, p. 19). — -Tissues to be fixed, mordanted and cut as directed under Mailoey, § 911. The sections are put for a quarter of an hour into -5 per cent, solution of potassium permanganate, washed and put .for another quarter of an hour into 1 per cent, solution of oxalic acid, well washed and stained for twelve to twenty-four hours or more in Malloey's phosfhotungstic hcematoxylin. Wash, dehydrate in 95 per cent, alcohol, clear with origanum oil, mount in xylol-balsam. Axis cylinders and nerve cells pink, neurogha blue. To get a more isolated stain of neuroglia, the sections should be brought for five to twenty minutes, after staining, into a 30 per cent, alcoholic solution of iron sesquichloride. Neuroglia and fibrin blue, the rest colourless. Malloey's phospho-molybdic hcematoxylin may also be used for the stain, but it is less elective. 914. Ajstglade and Moeel's Victoria Blue Method {Reo. Neurol, ix, 1901, p. 157).— Harden in a mixture of 3 parts of liquid of Fol. 31 — 2 484 NEUROGLIA AND SENSE ORGANS. (§ 47) with 1 of 7 per cent, sublimate solution, dehydrate with alcohol followed by acetone, make parafiin sections, and stain in saturated aqueous solution of Victoria blue heated till it steams ; rinse with Gram's fluid, differentiate with xylol 1 part, aniline 2 parts, and mount in balsam. Simple, applicable to lower animals, and gives very sharp pictures. 915. Da Fano's Methods {Ricerche Lab. Anat. Roma ed altri Lab. Biol, xii, 1906). ^Method I. is a modification of Malloey's phospho- tungstic haematoxylin process (§ 913). Small pieces of fresh tissue are fixed for twenty-four to forty-eight hours in a mixture of 72 volumes of pyridine and 28 of 50 per cent, nitric acid. After washing for about six hours, the pieces are dehydrated and embedded in paraffin. The sections, stuck to slides by the albumin method, are treated as by Malloey's method, and stained with an old solution of Malloey's phosphotimgstic hsematoxylin, but prepared without the addition of hydrogen peroxide. In order to increase the contrast between neuroglia fibres (blue-violet) and the protoplasm of neuroglia cells (pink) Da Fano dehydrates the stained sections in 95 per cent, alcohol to which a small quantity of an alcoholic solution of eosin has been added. Method II. is a modification of Benda's process (§ 912). Very small pieces are fixed for thirty-six to seventy-two hours in a mixture of 2 volumes of the fixing fluid used for Method I. and 1 volume of 1 per cent, osmic acid. After washing for six to twelve hours, the pieces are embedded in paraffin. The sections, stuck to slides, are successively mordanted for twenty-four hours each with Weigert's copper acetate-chromium fluoride fluid (§ 910), 2 per cent, chromic acid, and 2 per cent, iron alum rinsing in water before passing " them from one into the other mordant. They are lastly either treated and stained as by Benda's alizarine-toluidine blue process, or as by Hbidenhain's iron haematoxylin method. Method III. was arrived at in an endeavour to make use of unsuccessful preparations made by Cajal's reduced silver method. Pieces treated as by Cajal's formula la or one of its modifications (§ 837), or simply fixed in 2 or 3 per cent, silver nitrate at 36° to 37° C, are embedded in paraffin. The sections, stuck to slides, are bleached by Pal's differentiation method for myelin stain, and then mordanted and stained as by Method II. 916. Held's Method for Marginal Neuroglia (Monatschr. Psych. Neurol., xxvi, 1909 ; Ergdnzungsh., p. 360). — Tissues are preferably fixed by means of a modified Zenker's fluid consisting of Miiller's CHAPTER XXXV. 485 fluid 100 c.c. and sublimate 3 gnns., with the addition at the moment of use of acetic acid 3 c.c, formalin 0-5 c.c. The fluid should be warmed at 35° to 40° C. and iajected through the blood-vessels, the blood being first washed away by means of Einger's solution to which 1 : 1000 of amylnitrite was added. The tissues are treated in the usual way and embedded in celloidin. The sections are first treated for five minutes with a 1 per cent, solution of caustic soda in 80 per cent, alcohol and washed in distilled water, and then mordanted for a few minutes in 5 per cent, iron alum and washed once more. For staining, Held adds to some distilled water a few drops of a very old molybdic acid hsematoxylin, enough to impart to the water a bluish- violet tone, and stains therein for twelve to twenty-four hours at 50° C. The stain is prepared by dissolving 1 grm. of heematoxyhn in 100 c.c. of 70 per cent, alcohol and adding an excess of molybdic acid. Differentiation is carried out by means of the same iron alum solution used for mordanting ; wash well ; counterstain with v. Gieson picro-fuchsin solution ; wash in 96 per cent, alcohol, dehydrate and mount as usual. Neuroglia cells and fibres greyish-black ; marginal neuroglia {Tnembrana Umitans marginalis and membrana limitans perivascularis) sharply differentiated ; connective tissue pink-red. 917. Other similar Methods. — ^LniiRMiTTE and Guccione (Semaine Jfe(i.,xxix, 1909, p. 205) have the following modification of An glade and Morel's method : Sections, made by the freezing method from formalin material, are collected in distilled water and then kept for two hours in a cold-satuiated solution of sublimate and for two days in a mixture consisting of 3 parts of 1 per cent, osmic acid, 35 of 1 per cent, chromic acid, 7 of 2 per cent, acetic acid, 55 of distilled water. The rest as Anglade and Morel. Similarly Meezbackee {Journ. Psyehol. Neurol., xii, 1909, p. 1). De Albertis {Pathologica, xii, 1920, p. 240) has recently proposed the following combination of the methods of Weigert, Malloet, and Anglade and Moeel. Sections are made by means of a freezing microtome from pieces fixed in 15 to 30 per cent, formalin for about twenty -four hours, but not longer than three days. They are transferred into a bath of 2 per cent, acetic acid in 1 per cent, chromic acid (time not stated), and then washed for some hours in repeatedly changed distilled water, oxidised for ten to fifteen minutes in J per cent, potassium permanganate, washed again in distilled water, reduced for fifteen to twenty minutes in 1 per cent, oxalic acid and lastly put to stain for twelve to twenty -four hours in a saturated solution of Victoria blue. For the further treatment sections are washed in distilled water and from this lifted, one by one, by means of a thin glass or platimjm spatula, this to be used to plunge each section for an instant first into concentrated Lugol's solution, then into absolute alcohol, and lastly into equal parts of xylol and aniline oil, where the differentiation is accom- 486 NEUROGLIA AND SENSE ORGANS. plished in a few seconds. Sections are finally collected and washed in sliglitly warmed and repeatedly changed xylol, and mounted in xylol- damar without a cover. KuLTSCHiTZKT Rubin's Method {Anat. Anz., viii, 1893, p. 357) is no longer used. For the slight modification of this method of Popow, see Ztschr. wiss. Mihr., xiii, 1896, p. 358, and for that of Bttkchardt, La Cellule, xii, 1897, p. 364. The method of Yamagiwa {Virchow's Arch., clx, 1900, p. 358) is also no longer used. 918. Methods for Protoplasmic Neuroglia and Neuroglia Granules. — Oppbnheim (Neurol. Centrhl., xxvii, 1908, p. 643) mordants sections made from frozen formalin material with Weigert's copper acetate- chromium fluoride mixture and then stains them with Weigert's iron haematoxylin prepared without hydrochloric acid. An important point of this method is that the material and the sections should not have been treated with alcohol before staining. EiSATH (Monatschr. Psych. Neurol., xx, 1906, p. 3 ; Arch. Psych. Nervenkrankh., xlviii, 1911, p. 897) fixes large pieces in a modified Orth's formol-Miiller mixture consisting of water 1000 c.c, potas- sium bichromate 25 grms., sodium sulphate 15 grms., and formalin 150 c.c, to be added at the moment of using the mixture. After about four weeks the tissues are ready for being cut without em- bedding, but can be kept for many months, and even years, in 4 per cent, formalin. The sections are collected in 4 per cent, formalin, in which they may be kept until wanted. For the staining the sections are put for thirty seconds in a -2 per cent, solution of sub- limate, well washed in water, and lifted on to the slide, a dilution of an old Mallory's phosphomolybdic-carbolic acid hsematoxylin being poured on them. After a few minutes they are washed with water, ' differentiated with a mixture of equal parts of 40 per cent, tannic acid, 50 per cent, alcohol, and 20 per cent, pyrogallic acid in 80 per cent, alcohol. Wash in alcohol, dehydrate, clear and mount. FiEANDT (Arch. mikr. Anat., Ixxvi, 1910 — 11, p. 125) fixes in Heidenhain's sublimate-trichloracetic mixture, and treats pieces for five to seven days with 96 per cent, alcohol, to be changed three times during the first twenty-four hours and daily in the following days. After dehydration the pieces are embedded in paraffin as directed by Prantner. The sections, 3 to 5 /i^ thick, are stuck to slides, freed from sublimate by the usual iodine treatment, and then stained for twelve to twenty-four hours with Mallory's phospho- tijngstic hsematoxylin. Dry with filter paper, differentiate for a few hours in 10 per cent, iron perchloride in absolute alcohol,'blot once more with filter paper, wash, dehydrate and mount. CHAPTER XXXV. 487 Neuioglia fibres, cytoplasm of neuroglia cells, and glia granules stained in various shades of blue and greyish-blue ; all other elements yellowish-grey or yeUowish-brown. Ranke (Ztschr. ges. Neurol, u. Psych., vii, 1911, p. 355) uses for similar purposes either ceUoidin sections of festal tissues fixed in picric acid-alcohol or sections made by freezing from formalin (pathological) material. In the first case the sections are stuck to slides by pressing with filter paper and then pourin'g on them methyl- alcohol until all celloidin is dissolved. He next stains them for a few minutes with his acid eosin-thionin solution (see further on), washes with water, and re-stains them, with the help of gentle heat, with 5 : 1000 Giemsa's " Methylenazur I " ; quick differen- tiation with distilled water ; 96 per cent, alcohol, cajeput oil, xylol, balsam. In the case of pathological material the sections are first treated with 1 per cent, osmic acid in order to stain fatty products of degeneration, etc., then pressed on to slides and stained as above. To prepare the acid eosin-thionin mixture, mix and shake repeatedly 1000 c.c. of each 1 : 1000 watery solution of eosin W.G. and 1 : 1000 watery solution of thionin. Leave for forty-eight hours, pour out the fluid part, and wash the sediment into a paper filter with distilled water until the wash water is only a little stained. Dry what remains in the filter, and dissolve it in methyl-alcohol in the proportion of -3 to -5 per cent. ■ 919. Ram6n y Cajal's Gold Chloride and Sublimate Method (Trab. Lah. Invest. Biol., Madrid, xi, 1913, pp. 219 and 255 ; xiv, 1916, p.. 155). — At first Cajal used to harden pieces of quite fresh tissues in 14 per cent, formalin, but in his successive papers he recommended fixing from two to ten days in — Formol 15 c.c. Ammonium bromide .... 1 -5 — 2 grms. Distilled water ...... 85 c.c. Relatively thick sections (20 to 25 /x) are made by the freezing method, and collected in distilled water to which a few drops of formalin have been added. After a quick wash, batches of four to six sections are each transferred into glass dishes of about 6 cms. in diameter, and each containing 15 c.c. of a mixture of — DistUled water ...... 60 c.c. Corrosive sublimate . . . . . -5 grm. 1 per cent, gold chloride (Merk, 6rj Calcium chloride 00012 y> Basic sodium phosphate (Na^HPO^) 00001 )»' Acid potassium phosphate (KH2PO4) 00001 ♦» Magnesium sulphate . . . . 0001 )) Sodium bicarbonate 0002 ff Glucose . 003 Histidine . 001 »j Arginin . 001 j» Leucin 001 5S Ammonium lactate 0003 Ferric chloride , Trace Potassium iodide Trace Manganous chloride Trace The substances are made up with glass distilled water. The con- stituents are autoclaved separately, and the final mixture sterilised by heating to 80° C. on three successive days. The organisms were cultivated first in sterile media in depression slides, experiment showing the most suitable concentration for division. The first divisions were apt to be slow. After a nimiber of individuals have been obtained in this way, they are sub-cultured in test tubes, using all sterile precautions, and when a satisfactory test tube culture has been obtained, it can be used for culture pur- poses as required. A culture has now been liept going by weekly sub-culture upon this medium for three months. (Temperature from 15° to 20° C.) From successful cultures grown in this way with the paramcecium in question, it was impossible to obtain any bacterial growth (or other 538 METHODS FOR INVERTEBRATES. growth) by sowing a drop of the paramcecial culture with a platinum loop, either upon (I) nutrient broth, (2) nutrient agar, (3) glucose agar (anserobio) and (4) litmus milk at room temperature or at 36° C. It was also not possible to obtain growth upon the medium itself stiffened with a trace of agar. Examination of a growing culture under the jij oU immersion lens, however, showed the presence of peculiar rod- shaped bodies. These were about 10 /j. long and 2 /x broad. They were motUe, but appeared as a rule to be anchored at one end to the slide. They were never observed to divide. After a varying time they would cease to move in the moist drop preparation. When stained they were found to He in rows, varying in shape from curved to straight. The absence of any sign of parasite or symbiotic organism in the paramoe- cium and the general resemblance of these bodies to split off cilia has led to the belief that the cultures in question really contain no other organism than the paramoeoium. 1015. Method for Examination of Faeces for Protozoa (H. M. Woodcock, B. M. J., November, 1915). — A very small quantity of the faeces is taken up on a platinum loop and well mixed with a drop of -5 per cent, salt solution sufficient in amount to run under a coverslip. The faeces must be well diluted, otherwise cysts are apt to be overlooked. The faeces should be examined as freshly as possible, as after four or five hours most of the active flagellates become motionless and die. A convenient and rapid way of making a permanent preparation is as follows : — A thin smear of the diluted faeces is made on a slide in the same manner as a blood film, and the slide is immediately placed in a stain tube containing at the bottom a small quantity of 4 per cent, osmic acid plus 1 drop of glacial acetic acid for fixation, and is left in for about ten seconds. Allow the slide to dry in air and then place in absolute alcohol for fifteen minutes. Wash with tap-water and stain in Giemsa, 1 • drop to * 1 c.c. neutral distilled water for twenty minutes or so. Rinse with tap -water. 1016. Donaldson's Method of Detecting Protozoal Cysts in Faeces by Means of Wet Stained Preparations {Lancet, 1917). — Donaldson recommends the use of two solutions, A and B. A. (1) Five per cent, aqueous potassium iodide saturated with iodine to which is added an equal volume of ether. B. (1) A saturated aqueous solution of Rubin S. ; or (2) A saturated aqueous solution of eosin ; or (3) Stephen's scarlet writing fluid. Equal parts of stains A and B are mixed just before use. A few loopfuls of one of the above stain combinations are placed on a clean slide, a loopful of faeces is taken and rubbed up with the stain CHAPTER XXXVI. 539 to form a fairly smooth emulsion, and a clean coverslip gently lowered on to the drop. In order to get the best possible definition, it cannot be emphasised too strongly that the film so made should be spread out under the cover -glass by capillarity so as to form the thinnest layer possi))le, preferably a layer which is no thicker than the diameter of an Entamoeba coli cyst. The exact amount of stain to be used will soon be learned after one or two attempts, and depends upon the size of the loop employed and the size of the coverslip. It is a matter of considerable importance the way in which the film is made, especially where Eubin S., or, to a less extent, eosin has been used in the staining combination, for if the layer of fluid between the sUde and coverslip be too thick the super- fluous film of fluid overlying the cysts tends to render the latter less bright. In the case of cysts of the size and shape of Entamoeba coli or histolytica, this makes little difference, but if one is hunting specially for the Lambha, or, still more, Tetramitus mesnili, the colour contrast is not so marked, owing to the smaller size of the cysts and the conse quent thicker red layer of fluid overlying them. Where the worker has not acquired sufScient dexterity in making such a film, the difficulty may be got over by using Rubin S. or eosin of only half saturation in the stain combination, or by employing the scarlet writing fluid mentioned. In this way the effect of the super-imposed deeper red is to some extent obviated. In a wet preparation stained by this method there is a more or less homogeneous red background, from which the cysts stand out as brilUant yellow or greenish-yellow spheres which even the tyro cannot miss seeing. 1017. Method for the Tsetse Files (M. Eobbetson, Tmns. Boy. Soe., Series B , vol. 203, p. 1 6 1 ). — The newly hatched flies are starved for twenty- four to thirty -six hours and are then fed on the infecting monkey once, or in some cases twice. The infecting feed is the first blood ingested by the flies. After the infecting feed the cage is starved for one or two days and thereafter fed on clean monkey's blood every second or third day. Daily feeding is not essential to the welfare of glossina, and does not appear to occur in nature. Dissections are made in a drop of physio- logical salt solution. The trypanosomes are studied both in the Uve state and in fixed and stained preparations. Preserved material is fixed whUe wet by dropping the coverslip film side downwards into Schaudinn's solution ; the preparations are subseqiiently stained by Heidenhain's iron hsematoxylin. 1018. Immobilisation, — See the narcotisation methods §§ 20 to 25. According to Schurmaybe {Jen. Zeit., xxiv, 1890, p. 402), nitrate of strychnin, of 0-01 per cent, or less, gives good results with some forms, amongst which are Stentor and Carchesium. Antipyrin (0 -1 per cent.), or cocaine of -01 per cent., seems only to have given good results as regards the extension of the stalk in stalked forms. EiSMOND (Zool. Am., xiii, 1890, p. 723) slows the movements of 540 METHODS. FOR INVERTEBRATES. small organisms (small worms and Crustacea as well as Ciliata) by means of a drop of thick aqueous solution of cherry-tree gum added to the water containing them (gum arabic and the like, it is stated, will not do). The objects remain fixed in their places, with cilia actively moving, and all vital processes retaining their full activity. Cbrtes (Bull. Soc. Zool. France, xvi, 1891, p. 93) has found that an intra vitain stain may be obtained by adding methyl blue or " violet dahlia, No. 170 " to the gum solution. Jensen (after Stahl ; see Biol. Centralbl., xii, 1892, p. 558) makes a solution of 3 grms. of gelatin in 100 c.c. of ordinary water by the aid of heat. This makes a jeUy at the normal temperature. It is slightly warmed, and a drop of it is mixed in a watch-glass with a drop of water containing the organisms. See also Volk, ante, § 886 ; Statkewitsch, Arch. Protistenk., v, 1904, p. 17 ; Lyon, Amer. Journ. Phys., xiv, 1905, p. 427 (neutralised gum). 1019. Staining intra vitam, — See hereon Beandt (Verh. physiol. Ges. Berlin, 1878) ; Certes (Bull. Soc. Zool., 25 janv., 1881) ; and Hennbguy (Soc. Philom., 12 fev., 1881). See also § 208. Brandt recommends a 1 : 3000 solution of Bismarck brown ; also (Biol. Centralb., i, 1881, p. ^02) " a dilute solution of hsemato- xylin." Certes (op. cit., pp. 21, 226, 264, and Zool. Anz., iv, 1881, pp. 208, 287) found that living Infusoria stain in weak solutions of cyanin, Bismarck brown, dahlia, violet 5 B, chrysoidin, nigrosin, methylen blue, malachite green, iodine green, and other tar colours, and hsematoxylin. The solutions should be made with the liquid that constitutes the natural habitat of the organisms. They should be very weak, that is, of strengths varying between 1 : 10,000 and 1 : 100,000. For cyanin, 1 : 500,000 is strong enough. As to the staining of the Nucleus, see Przesmycki, Biol. Centralb., vii, 1897, p. 321 ; and as to that of the Granula, the same author, Zeit. wiss. Mik., xiii, 1896, p. 478. Also Loisel, § 208. Examination in a coloured medium in which the organisms do not stain, but show up on a coloured background, is sometimes helpful. Certes (Bull. Soc. Zool. de France, xiii, 1888, p. 230) recommends solution of anilin black — Infusoria will live in it for weeks ; Fabre- DoMBRGUE (Ann. de Microgr., ii, 1889, p. 545) concentrated solution of diphenylamin blue. For the mitochondria and other granules, see Fauri^i-Fremibt (Arch. d'Anat. micr., xi, p. 457). Dahlia in salt solution, Pictet's fluid, Janus green, or crystal violet being useful. CHAPTER XXXVI. 541 For mitochondria and Golgi apparatus of a sporozoon, J. Hirschler (Anat. Anz., xlvii, 1914 — 15) used the Mann-Kopsch metliod, § 693. 1020. Demonstration of Cilia (Waddington, Jmirn,. Roy. Mic. Soc, 1883, p. 185). — -A drop of solution of tannin, or a trace of alcoholic solution of sulphurous acid, added to the water containing the living organisms is efficacious. 1021. Fixing and Preserving. — Protozoa may be killed by heat, by toxic vapours or by toxic liquids. Almost instantaneous fixation can sometimes be obtained by steam or by iodine (Kent) or iodine vapour (Overton) : see § 83. E. S. Goodrich (Quart. Journ. Micr. Science, Ixiv, 1919) modifies Kent's method in a way which we have found useful for amoebse. A strong solution of iodine in potassium iodide is diluted to about the coloiur of sherry with normal saline for terrestrial and fresh water animals, and with sea water for marine organisms. Such a solution is run under the covershp and followed by the definitive fixing agent, e.g., Bouin's fluid, etc., and the preparation proceeded with in the usual way. The iodine does not appear to destroy any of the cell contents. With regard to fixatioji, read §§ 29, 30, § 655, § 663, and especially §§ 673 to 713. See also the important section from §§ 768 to 772, dealing with fats. Woodcock and Wilson's Modification of Schaudiim's Fixative (Phil. Trans. Eoy. Soc. B., ccvii, 1916, p. 379 ; and Univ. Calif. Pub., xvij 1916, p. 244). — Woodcock gives saturated aqueous sublimate, 2 parts ; absolute alcohol, 1 part ; and acetic acid, 5 per cent. Wilson uses alcoholic sublimate + 5 per cent, acetic. See also Gilson and Petrunkewitsch fluids, § 69, and acetic alcohol, § 86. For Schaudinn's original fixative, refer to § 1031. Lucidol or Peroxide of Benzol. See §§ 107 and 783. For killing by heat, see § 13. . Pfitzner (Morph. Jahrb., xi, 1885, p. 454) used concentrated solution of picric acid run in under the cover. Bntz (Zool. Anz., iv, 1881, p. 575) adds liquid of Kleinenberg to the water containing the organisms in a watch glass. KoRSCHELT (ibid., V, 1882, p. 217) employs in the same way 1 per cent, osmic acid, or, for Amoebee, 2 per cent, chromic acid. Lansberg (ibid., p. 336) advises the same reagents, but recom- mends bringing the organisms into the fixing liquid with a pipette. For sulphurous acid, § 62. Cattaneo (Bollettino Scientifico, iii and iv ; Journ. Roy. Mic. 542 METHODS FOR INVERTEBRATES. Soc, 1885, p. 538) fixes for a few minutes with. J per cent, solution of chloride of palladium. Brass (Zeit. wiss. Mih., 1884, p. 39) employs a mixture of 1 part each of chromic acid, platinum chloride, and acetic acid with 400 to 1,000 parts of water. Certes {Comptes rend., Ixxxviii, 1879, p. 433) fixes with 2 per cent, osmic acid, or its vapours (ten to thirty minutes). For details, see previous editions. LoNGHi {Bull. Mus. Zool. Univ. Genova, 1892, No. 4) kills in 10 CO. of 1 per cent, sulphate of eserin with 1 drop of 1 per cent, sublimate. ScALA {Rev. Mils. La Plata, xv, 1908, p. 57) fixes for five or ten minutes in a mixture of 2 mg. of atropin, 10 drops of formol, 10 grms. of glycerin and 50 c.c. of water. See also Puschkarbw, Zeit. wiss. Mik., xxviii, 1911, p. 145 (agar process for fixing and staining Amoebae). FoL {Lehrh., p. 102) fixes delicate marine Infusoria {Tintinnodea) with the perchloride of iron solution (§ 80), added to the water containing them, and stains with gallic acid. Lo Bianco {loc. cit., p. 444) fixes Gregarinse with picro-sulphuric acid (one hour), Vorticellse with hot sublimate, Aoinetse with subli- mate in sea water,- or with osmic acid, Thalassicola with -5 per cent, chromic acid (one hour), Acanthometrae and Aulacanthse with 50 per cent, alcohol or with concentrated sublimate, or by adding a little osmic acid to the water. For Sphserozoa he proceeds as Brandt, § 1019. ZoGRAF fixes Rhizopoda and Infusoria as Rotatoria, § 886, but without narcotisation. See also Pabre-Dombkgue, Ann. de Mierogr., ii, 1889, p. 545, and 1890, p. 50 ; ScHEWlAKOFF, Biblioth. Zool., v, 1889, p. 5 ; Journ. Boy. Mic. Soe., 1889, pp. 832, 833 ; ZOJA, Boll. Soi. Pavia, 1892 ; Zeit. wiss. Mih., ix, 1893, p. 485 ; Latjtebbobn, Zeit. wiss. Zool, lix, 1895, p. 170 ; ScnATJDiNN, ibid., p. 193; Bai.biani, Zool. Ans., xiii, 1890, p. 133; Kaeawaibw, ibid., xviii, 1895, p. 286. 1022. Embedding of Protozoa and other Small Objects (Minchin, Q. J. M. S., Ix, 1915, p. 508).— A thin slice of a block of amyloid liver preserved in alcohol is floated into a shallow glass vessel with a flat bottom; containing alcohol. The dish is placed on the stage of a dissecting microscope. The objects to be embedded are taken up in a pipette and placed on the slice of liver and orientated as desired. A tiny drop of glycerine and albumen solution is taken up on the CHAPTER XXXVI. 543 point of a needle and caused to touch the surface of the alcohol immediately above the small objects. The dense albumen solution falls at once through the alcohol and spreads out over the objects on the liver ; at the same time the glycerine is extracted and the albumen coagulated by the alcohol, with the result that the objects are stuck on to the liver. The liver is now trimmed with a scalpel into a rectangular shape and embedded in the usual way. Minchin used this method for the stomachs of fleas. I have found it most successful for Bchinoderm and other small eggs. Or the following method may be used : — The organisms should be strongly fixed, then dehydrated and cleared, and brought into melted paraffin in a small watch glass. After a few minutes therein they are brought on a cataract needle on to a small block of paraffin, and arranged there with a heated needle and sectioned. They may be stained after fixation, or the sections may be stained on the slide, § 186 or 187. Entz {Arch. Protistenk., xv, 1909, p. 98) brings the objects from clove oil into clove oil collodion of the consistency of honey, then brings them in this into a funnel made of parafiin, and when they have collected at the bottom of this puts it into chloroform, which dissolves the paraffin and hardens the collodion. Some Current Stains for Protozoa. — Below are given a number of special staining methods suitable for routine protozoological work. 1023. Heidenhain's Alcoholic Iron Hsematoxylin.— (1) Fix in Schaudinn's solution for fifteen minutes. (2) Place at once in 70 per cent, alcohol for at least one hour. (3) Mordant for five hours or more in 50 per cent, alcohol 10 parts. 4 per cent, aqueous iron alum solution . 1 part. (4) Stain for twelve to twenty-four hours in Heidenhain's hsematoxylin ... 1 part. 70 per cent, alcohol 10 parts. Heidenhain's hsematoxylin — Haematoxylin 1 grm- Absolute alcohol . . . • . 10 c.c. Distilled water . . . . • . 90 ,, Thymol 1 crystal. Dissolve the heematoxylin in the absolute alcohol and then add the distilled water. (5) Differentiate in the same solution as was used in (3) for mordanting. 544 METHODS FOR INVERTEBRATES. (6) Wash, thoroughly in several changes of 70 per cent, alcohol to remove mordant. (7) Dehydrate and mount in Canada balsam. 1024. Dobell's Hsematein Method (see Arch. f. Protistinkunde, 1914, p. 144.) — ^Pilms or sections are transferred from 70 per cent, alcohol into 1 per cent, solution of iron alum in 70 per cent, alcohol. This is most easily made in the way described by Hickson [Qimi't. Journ. Micr. Sci. 44, 1901, p. 470), 1 grm. of salt dissolved in 23 c.c. of warm water, then add 77 c.c. of 90 per cent, alcohol after cooling. They are mordanted in this for ten minutes, rinsed in 7.0 per cent, alcohol and transferred to a 1 per cent, solution of hsematein in 70 per cent, alcohol. They are left in this for ten minutes and then difierentiated, either in the iron alum or in acidified 70 per cent, alcohol (0-6 per cent. HCl in 70 per cent, alcohol). After differentiation wash in several changes of 70 per cent, alcohol and then pass up through the alcohols into any of the usual mounting media. Any alcoholic counterstain can be used, light green in 90 per cent, alcohol being very good. By this method the organism is stained a purple grey, and hard black and white contrasts are not obtained. The various nuclear and cytoplasmic constituents may be stained with individual intensity. (The haematein method is good, when it works, but often it is a complete failure ; certain amoebae, for instance, are not stained at aU by it.) 1025. Dobell's Modifleation of Mann's Methyl Blue, Eosin Stain. — (1) Stain with Mann's methyl blue eosin mixture (see Lancet, p. 196) in distilled water till everything is overstained (two to eight hours). (2) Einse in distilled water and differentiate in 70 per cent, alcohol dried from water, containing a small quantity of Orange G — ^just enough to colour it. It is best to keep Orange G in concentrated solu- tion in 90 per cent, alcohol ; add sufficient of this with a glass rod to clear 70 per cent, before differentiating. This weak Orange G cannot be used often as it gets discoloured very quickly. (3) Dehydrate in absolute alcohol (not too long) and transfer to xylol (also not too long, as eosin comes out in time if left). (4) Mount in balsam. By this method permanent and pretty results are obtained, often quite as good or better than those got with Giemsa. The method is especially good after Benin fixation, but it is important that aU the picric acid is removed before staining. 1026. Giemsa Stain (Minchin's Method, Q. J. M. S., Ix, 1915, p. 510). — Slides are washed in tap water and put in dilute Lugol solution (1 c.c. Lugol to 25 c.c. distilled HgO) for ten minutes. After this, rinsed in tap water and put into a -5 per cent, watery solution of hyposulphite of soda for ten minutes. Next wash in a current of water for five minutes and then put into the stain. The distilled water used to dilute the Giemsa stain has to be neutralised in the way prescribed by Giemsa. CHAPTER XXXVI. 545 A measured volume of the distilled water is taken and to it are added a few drops of hsematoxylin solution (5 per cent, in dist. H2O) sufficient |;o tint it. Then a weak solution (1 per cent, in dist'. water) of potassium carbonate is added drop by drop until the colour of the tinted water changes from yellow-red to reddish purple. In this way the number of drops of the carbonate solution required to .neutralise a given volume' of the distilled water is found. The slides are now placed in the stain, 1 drop Giemsa to 1 c.c. neutralised distilled water for one hour. Transfer to a weaker solution (1 drop stain plus 4 or 5 c.c. water) and leave overnight. Eemove excess of stain by washing in water. Differentiate stain by carrjdng slides through different strengths of acetone mixed with xylol, beginning with 95 per cent, acetone and ending with pure xylol. Mount in dammar or Canada balsam. (If these instructions are carefully followed the results are excellent, but care must be used.) 1027. Giemsa Method for Staining Gut Flagellates (J. G. Thomson). — { 1 ) Fix wet sat. percMoride, 2 parts ; alcohol, 1 part. (2) Wash in weak alcohol (circa, 60 per cent.). (3) Wash in water (aq. dist.). (4) Wash in water, to which is added a few drops of Gram's iodin. (5) Wash in 1 per cent, solution of hypo. (6) Wash in running water. (7) Stain in Giemsa (1 drop, 1 c.c.) twelve hours. (8) Differentiate in (a) acetone, 95 per cent. ; xylol, 5 per cent., for about five minutes ; then (6) acetone, 70 per cent. ; xylol, 30 per cent. ; (c) pure xylol to clear. (9) Mount in Canada balsam. 1028. Iron Brazilin Stain (Hickson). — (1) Fix in Sohaudinn's solution for fifteen minutes. (2) Seventy per cent, alcohol for one hour. (3) Mordant for four hours in a 1 per «ent. solution of iron alum in 70 per cent, alcohol. (4) Stain twelve to sixteen hours in a 1 per cent, solution of brazihn in 70 per cent, alcohol. (5) Wash thoroughly in several changes of 70 per cent, alcohol. (6) Dehydrate and mount in balsam. 1029. PONTANA'S Stain.— (1) Fix in Hugo's fluid for one minute. Acetic acid (B.P.) .... 1 c.c. Formalin . . ■ • • ■ 20 ,, Distilled water 100 „ (2) Wash thoroughly in tap water. (3) Mordant. Slide should be gently warmed. Tannic acid 5 grms. Carbolic acid ...••! c.c. Distilled water 100 c.c. M. 35 546 METHODS FOR INVERTEBRATES. (4) Wash thoroughly in tap-water. (5) Stain and warm gently : (a) 5 per cent, solution of silver nitrate in water ; (6) ammonia. Add the ammonia until the precipitate redis- solves in excess of ammonia. Then add more silver nitrate drop by drop until the precipitate reappears and remains constant. (6) Wash thoroughly in tap-water. (7) Dry rapidly in air. 1030, Sphserozoa.— Brandt {Fauna u. Flora Golf. Neapel, xiii, 1885, p. 7) fixes with chromic acid of 0-5 per cent, to 1 per cent, (half an hour to an hour), or with a mixture of equal volumes of sea water and 70 per cent, alcohol with a little tincture of iodine for a quarter to haU an hour, or with a 5 to 15 per cent, solution of sublimate in sea water. Kaeawaibw {Zool. Anz., xviii, 1895, p. 286) fixes Aulacantha for twenty-four hours in equal parts of strong liquid of Flemming and acetic acid, and hardens for several days in pure liquid of Flemming. See also Lo Bianco, § 1021. 1031. Spoiozoa. — Wasielewski {Sporozoenkunde, Jena, 1896, p. 153) studies them living in their natural medium, or in normal salt solution, or in a medium composed of 20 parts white of egg, 200 of water, and 1 of common salt. He fixes Gregarinee and Coccidia with osmic acid, sublimate, or picro-sulphuric acid, and Myxosporidia with liquid of Flemming. Schaudinn {Zool. Jahrh., Ahih. Anat., xiii, 1900, p. 197) fixes Coccidia with a mixture of 2 parts of saturated aqueous sublimate and 1 of absolute alcohol, with, if desired, a trace of acetic acid. Stempell {Arch. Protistenk., xvi, 1909, p. 389) fixes caterpillars infected with Nosema in 2 parts of saturated sublimate with 1 of alcohol and a little acetic acid, and stains sections for as much aS four days in Giemsa's mixture, rinses with alcohol and passes through xylol into balsam. Leger {ibid., iii, 1904, p. 311) fixes cysts for a minute in " acetic sublimate," puts for a minute into absolute alcohol, and stains as a smear with heemalum or iron heematoxylin. Beasil {Arch. Zool. Exper., 4, iv, 1905, p. 74) fixes them for twenty-four hours in a mixture of 1 grm. picric acid, 15 c.c. acetic acid, 60 c.c. formol and 150 c.c. alcohol of 80 per cent., and stains paraffin sections in iron heematoxylin followed by eosin and orange G. , ' or Lichtgriin and picric acid. 1032. Haematozoa. — Geassi {Att. Accad. Lincei, iii, 1900, p. 357) demonstrates the Malaria-parasites in the intestine, body-cavity and salivary glands of Anopheles by treating them with normal salt CHAPTER XXXVI. 547 solution containing 2 per cent, of formol (pure formol produces swellings), or in a mixture of 1-5 grm. of salt and 250 c.c. of water with the white of an egg. He fixes with sublimate, makes parafiin sections, and stains with hsemalum or iron hsematoxylin. He stains the Sporozoites by making cover-glass preparations, which are allowed to dry, put for twenty-five minutes into absolute alcohol, and stained by the process of Eomanowsky, §§ 784, 1008. For minute instructions for the application of this process to sections, see G-iemsa, Deutsch. med. Wochenschr., xxxvi. No. 12, 1910 ; and Schubeeg, ibid., xxxv. No. 40, 1909 {Zeit. wiss. Mik., xxvii, 1910, pp. 160, 161 and 513). For clinical methods, see Coles, The Diseases of the Blood, London, J. and A. Churchill, 1905. Beadford and Plimmer {Quart. Journ. Micr. Sci., xlv, 1902, p. 452) fix Trypanosomes in vapour of equal parts of acetic acid and 2 per cent, osmic acid, or with Gulland's formol and absolute alcohol, and stain with methylen blue and eosin, and mount in turpentine colophonium. HiNDLE {Univ. Calif. Pub. Zool., vi, 1909, p. 129) makes smears on cover-glasses coated with albumen, fixes for five minutes in liquid of Flemming, passes through water up to absolute alcohol, then for ten minutes into alcohol of 80 per cent, with a good propor- tion of iodine in potassic iodide, then into 30 per cent, alcohol, and staias with iron hsematoxylin or safranin, then with polychrome methylen blue, and lastly with Unna's orange with tannin, and gets quickly through alcohol into xylol and balsam. MiNCHiN {Quart. Journ. Micr. Sci., hii, 1909, p. 762) makes cover- glass smears, fixes them with vapours of osmic acid (with or without acetic acid), and mounts them dry, or in balsam after fixing in liquids and various stains, amongst these that of Twoet. Half- saturated solutions of neutral red and Lichtgriin are mixed, the precipitate dried and dissolved to about 0-1 per cent, in methyl alcohol with 5 per cent, of glycerin. Three parts of this are diluted with 1 of water, the smears stained for an hour, differentiated with Unna's glycerin-ether, and mounted in balsam. This stain works best after fixation with sublimate. Policaed {C. R. Soc. Biol, Ixviii, 1910, p. 505) stains Trypano- somes intra vitam by adding a drop of concentrated solution of neutral red to th^ edge of a drop of blood spread between slide and cover. 1033. Flagellata. — Lauterborn {Zeit. unss. Zool., lix, 1895 p. 170) fixes Ceratium for about ten minutes in liquid of Flemming, puts iuto alcohol for twenty-four hours, brings back into water, 35—2 548 METHODS FOR INVERTEBRATES. bleaches if necessary with hydrogen peroxide, and stains with picrocarmine or Delafield's heematoxylin. He also embeds in paraffin, § 1022, and stains sections with iron heematoxylin. Zachaeias (Zool. Am., xxii, 1899, p. 72) fixes Uroglena, etc., with a mixture of 2 volumes saturated aqueous solution of boracic acid and 3 of saturated sublimate. 1034, Stains for Flagella. — The Eomanowsky stain will give a red stain of the flagella of some forms. The method of Lofflee {Centralbl. Bakteriol., vi, 1889, p. 209 ; vii, 1890, p. 625 ; Zeit. wiss. Mik., vi, 1889, p. 359 ; vii, 3, 1890, p. 368 ; Journ. Roy. Mic. Soc, 1889, p. 711 ; 1890, p. 678) is as follows. To 10 c.c. of a 20 per cent, solution of tannin are added • 5 c.c. of cold saturated solution of ferrous sulphate and 1 c.c. of (either aqueous or alcoholic) solution of fuchsin, methyl violet, or " WoUschwarz." Cover-glass preparations are made and fixed in a flame in the usual way, special care being taken not to over-heat. Whilst still warm the preparation is treated with mordant (i.e. the above- described mixture), and is heated for half a minute, until the liquid begins to vaporise, after which it is washed in distilled water and then in alcohol. It is then treated in a similar manner with the stain, which consists of a saturated solution of fuchsin in anilin water (p. 177), the solution being preferably neutralised to the point of precipitation by cautious addition of 0-1 per cent, soda solution. See also Liebetanz, Arch. Protistenk., xix, 1910, p. 23. BuNGE (Journ. Roy. Mic. Soc, 1894, p. 640 ; Zeit. wiss. Mik., xiii, 1896, p. 96) makes the mordant by mixing 3 parts of the tannin solution with 1 of liquor Jerri sesquichlorati diluted twentyiold with water, and lets the mixture ripen for some days exposed to the air, or (Journ., 1895, pp. 129, 248) adds to it a few drops of hydrogen peroxide, until it becomes red-brown, when it is shaken up and filtered on to the cover-glass and allowed to act for, a minute. The cover-glass is then mopped up and dried, and stained with carbol gentian. KoERNER and Fischer (quoted from Encycl. mik. Techn., p. 514) make the mordant with 2 parts of tannin, 20 of water, 4 of ferrous sulphate solution of 1 : 2 strength, and 1 of saturated alcoholic solution of fuchsin. Warm, let it act for a minute, rinse and stain with aiulin-water-fuchsin, or carbol fuchsin. Similarly Ellis (Centralh. Bakt., xxi, 1903, p. 241 ; Journ. Roy. Mic. Soc, 1904, p. 249), but staining with Saureviolett, 1 part to 75 of alcohol and 75 of water. Peppler (Centralb. Bakt., xxix, 1901, p. 376 ; Zeit. wiss. Mik., CHAPTER XXXVI. 549 xviii, 1901, p. 222) makes the mordant with 20 parts of tamiin in 80 of water, and 15 parts of 2-5 per cent, chromic acid added gradually. This mordant will keep for months. Eossi {Arch, per le 8c. med., xxiv, 1900, p. 297 ; Zeit. wiss. Mik., xviii, 1901, p. 226) takes for the mordant a solution of 25 grms. of tannic acid in 100 of caustic potash of -1 per cent., which wiU keep indefinitely. The stain is Ziehl's carbol fuchsin, § 289. Cover- glasses are prepared with a drop of culture, dried, and treated with 1 drop of the mordant and at the same time 4 to 5 of the stain, allowed to remain for fifteen to twenty minutes, washed, and mounted. See also Centr'alb. Bakt., xxxiii, 1903, p. 572 (Zeit. wiss. Mik., xix, 1903, p. 517). ■Gemelli {Central})., xxxiii, 1903, p. 316 ; Zeit. wiss. Mik., xix, 1903, p. 516) mordants for ten to twenty minutes in 0-025 per cent, permanganate of potash, rinses and stains for fifteen to thirty minutes in a mixture of 20 parts -75 per cent, aqueous solution of calcium chloride and 1 part of 1 per cent, neutral red solution. A method of Pitfield is described by Kendall, Journ. ajyp. Mic, V, 1902, p. 1836 {Journ. Roy. Mic. Soc, 1902, p. 502). The mordant consists of 10 parts of 10 per cent, tannin solution, 5 parts of saturated sublimate solution, 5 of saturated solution of alum, and 5 of carbol fuchsin. Mordant for a miuute with heat, and stain with a mixture of 2 parts saturated aqueous solution of gentian violet with 10 of saturated solution of alum. Van Ermengem {Journ., 1894, p. 405) fixes for a few minutes with a mixture of 1 part 2 per cent, osmic acid, and 2 parts 10 to 25 per cent, solution of tannin, washes, treats with 0-25 to 0-5 per cent, solution of nitrate of silver, then for a few seconds with a mixture of 5 parts gallic acid, 3 of tannin, 10 of acetate of soda, and -350 of water, then puts back again into the silver for a short time, then washes and moimts. See also Stephens, ife/d;., 1898; p. 685, and GrOB,DO^, ibid., 1899, p. 236, and the methods of Tkenkmann (Oentralb., vi, 1889, p. 433 ; Zeit. wiss. Mik., vii, 1890, p. 79) ; Bkown {Journ. Boy. Mic. Soc, 1893, p. 268) JuLiEN {ibid., 1894, p. 403) ; Sclavo {Zeit. wiss. Mik., xiii, 1896, p. 96) Hesseet {ibid., p. 96) ; Mum {Journ. Boy. Mic. Soe.j 1899, p. 235) McCeoeie {ibid., 1897, p. 251 ; he stains for two minutes in a mixture of equal parts of concentrated solution of night-blue, 10 per cent, solution of alum, and 10 per cent, solution of tannic acid) ; Zettnow {ibid., 1899, pp. 662, 664); Moeton {ibid., 1900, p. 131); Weloke {ibid., p. 132) ; Levaditi, C. B. Soc. Biol., lix, 1905, p. 326 (for Spiro- chcete pallida, Ramon's neurofibril stain) ; Meikowsky, Munch, med. Wochenschr., Ivii, 1910, No. 27 ; Kalb, ibid.. No. 26 {Zeit. wiss. Mik., xxix, 1912, pp. 123, 124 ; both for Spiroohcete). CHAPTER XXXVII.* THE CULTIVATION OP TISSUE " IN VITRO " AND ITS TECHNIQUE. 1035. A culture of tissue consists of a special medium, natural or artificial, such as lymph or plasma, inoculated with small fragments of living tissues, and is characterised by an active growth of the cells of the fragment into the nutrient plasmatic or lymph medium. Cells wander out into the latter, and may live up to twenty days without any signs of necrobiosis. "* The cultivation of tissues outside the body was first aecomplished successfully by Ross Harrison, of Johns Hopkins University in the year 1907. This brilliant observer has demonstrated by a series of experi- ments that fragments of nervous tissue of the frog embryo, covered with fluid from the lymph sac of an adidt frog, show growth of long nerve fibres (Haerison, Proc. Soc. Exper. Biol, and Med., iv, 1907, p. 140). Alexis Carrel, at about the same time, had been studying the laws of redintegration of tissues, and adapted Harrison's technique to mam- malian tissues. M. Burrows, a pupU of Ross Harrison, at this period began to work on tissue- culture, and first used blood plasma instead of lymph. Subsequently Burrows adapted the technique of Harrison to the cultivation of tissues of the chick. In September, 1910, Carrel and Burrows, working in conjunction at the Rockefeller Institute, succeeded in cultivating, in vitro, the adult tissues of mammals, and thus began a series of contributions which have taught us many valuable facts regarding senesence and rejuvenescence and the pathology of tissues (Careel and Burrows, Jour. Exper. Med., xiii. No. 3, 1911). Two methods of tissue-culture may be distinguished : — (1) Hanging-drop or smear cultures (Harrison). (2) Large plate cultures (Carrel and Burrows). The former are useful for direct observation of living growing cells, the latter can be studied when fixed and cut into sections. There has been a great deal of work done on tissue-culture, but most of it has been carried out by vertebrate pathologists and histologists. There seems little doubt that this field is a most promising one for zoologists as well as histologists. Many problems of gametogenesis and general cytology might be settled by recourse to tissue-culture, especially by the application of such methods to the cells of invertebrate animals, whose cytology had previously been examined in detail with the aid of modern techniques. * By J. B. G. CHAPTER XXXYII. 551 1036. Precautions to Insure Complete Sterilisation o£ Apparatus. — It cannot be too strongly emphasised that the utmost precautions must be taken to insure complete sterility of all apparatus. Bacterial infections of the cultures are fatal. The worker who is not familiar with the minute precautions taken by surgeons and bacteriologists in sterilising instruments, apparatus, etc., is advised to become so before undertaking tissue-culture work. A rigid asepsis is necessary for the preparation of any tissue-culture. These words are addressed especially to the zoologist who may undertake tissue-culture work ; dirty floors and benches, dirty waUs and garments, and casual methods will all contribute towards failure. A clean, warm room should be set aside for making the cultures, another for making the various plasmas, and, if possible, another for incubators and incubator microscopes. The ordinary zoological or botanical laboratory is generally unsuitable for such delicate work. Dr. Drew informs me that whether in vitro culture be carried out in a special laboratory or not, it is advantageous to use a specially constructed glass chamber to shield the cultiires from chance con- tamination. Such a chamber is supplied by Hearson's (§ 11), or can be made by any carpenter. The apparatus consists essentially of a glass box in a wooden or metal framework measuring about 2 feet 6 inches in length, 2 feet in width, and about 1 foot in height. The top is hinged so as to allow ready access to the interior for cleaning, etc. The side facing the worker is made of wood, with either two small wooden doors or a piece of thick sheet rubber pierced so as to allow the easy entrance of the hands. The box should have glass racks to contain the hollow ground slides and a glass table for the cover-glasses. The slides are cleaned and stored in absolute alcohol ; they are removed from this by means of forceps and are flamed in a Bunsen and quickly transferred to the glass racks ; cover-glasses are cleaned in acid bichromate, washed in water till free from all trace of the acid, rinsed in distilled water, then in absolute alcohol, and stored in ether. They are removed by means of forceps and flamed and placed on the previously sterilised glass table. Here both slides and cover-glasses are protected from all falling dust, and can be manipulated easily with the hands through the openings in the case. The majority of failures occur through infection taking place, and the glass chamber reduces such a possibility very greatly. 1037. Simple Culture Technique by means of Frog Lymph (Harri- son, op. cit., 1907). — Pieces of embryonic tissues of frog embryos 552 THE CULTIVATION OF TISSUE "IN VITRO." about 3 mm. long are dissected out with clean instruments, removed to a coverslip, and covered by a drop of lymph freshly drawn from one of the lymph sacs of an adult frog. The coverslip is inverted over a hollow slide, and the rim sealed with paraffin wax. When reasonable aseptic precautions are taken, tissues wiU live under these conditions for from a week up to four weeks. GoLDSCHMiDT (Arch. f. Zellf., 1916) has investigated the sperm cells of Lepidoptera by tissue culture methods. 1038. Technique for Culture of Mammalian Tissues. — Preparation of the Animal for Procuring Plasma. — The animal is anaesthetised with ether, and must be kept just at the correct depth of aneesthesia. A. J. Walton (Journ. -Path, and Bad., xviii, 1914), from whose article the following paragraphs are partly culled, recommends for this purpose a wide-necked bottle, with a closely-fitting cork pierced with two tubes of wide bore, both of which pass down to within |-inch of the surface of the ether placed in the bottle ; one tube communicates with a tin funnel having a mackintosh flange ; this tube also has a side tube, and the other tube is open to the air. The animal's head is placed in the funnel, and, when the side tube is clamped, breathes air and ether vapour. By clamping either the side or the short tube the amount of air or ether can be suitably controlled. The hair of the throat, is either shaved off, or removed by the application of a solution of sodium sulphide jii ad Oi, which rapidly dissolves it. The skia is then sterilised by painting with a 2 per cent, solution of iodine in spirit. 1039. Preparation of Instruments, etc. — Previous to the operation . the following apparatus is steriUsed : — Short test tubes, 2| inches in length ; corks kept in stoppered bottles, to fit these tubes ; small glass cannulee in olive oil ; three glass tubes, 3 inches by 1 inch ; several narrow-bore pipettes which are kept corked in the last- mentioned tubes, and which just before the operation on the animal, are removed from the tube by means of sterile forceps, dipped in a deep tin of molten paraffin, everted to allow the paraffin to run out, and when cool placed in another sterile tube. Two small sterile test tubes, as mentioned above, are similarly treated in paraffin, and should be corked with sterile corks as soon as cool: These two tubes are placed in two other larger tubes made to fit the centrifuge, and ice is packed between. Just before the operation, the instruments and some rubber teats to fit the pipettes are boiled in water for ten minutes. CHAPTER XXXVII. 553 1040. Removal of Plasma. — When dogs, rabbits, cats, cbickens, guinea pigs and rats are. used, the carotid artery is ordinarily selected ; an incision is made in the inid-liue in the neck, and as soon as the skin is divided the edges are clipped to sterile towels. The carotid is exposed, its distal end ligatured and its proximal end clamped. A little sterile oil is placed on the artery, which is opened, and one of the cannule from the sterile oil is taken, inserted and tied in position ; on releasing the clamp the blood flows freely. This is collected in the paraffined test tubes for centrifuging. The tubes should be in their ice-jackets ; they are corked at once and im- mediately centrifuged for about five minutes ; they are then removed and placed in an ice box at 0° C. For human plasma one may remove blood from a vein by means of a needle pipette steriMsed in olive oil. After the centrifugiUsation the supernatant plasma may be removed with pipettes coated in paraffin (§ 1039). It should be used immediately for making the cultures, but can be preserved for some time in a fluid condition if kept very cool. Chicken plasma can be so preserved for more than a week, human and dog plasma for a few days, while rat plasma always coagulates after a few hours (Caerel and Burrows, Journ. Exp. Med., 1911) ; when coagulation takes place the plasma is no longer of use. Careel and Burrows {Jour. Exper. Med., xiii, 1911) found that dilution of the plasma had a marked influence on the rate of growth of splenic tissue ; normal plasma is not the optimum medium for growth of tissue ; the most favourahle plasma for spleen culture contains two- flfths distilled water, and slightly less for liver and heart, and generally for skin, too. 1041. Preparation of Tissues. — The tissues for cultures should be in normal condition, and are best when taken from the living animal or immediately after death. Positive results can still be obtained, however, when the tissues have been deprived of circulation for more than thirty minutes. With a cataract knife and a fine needle, a small fragment of tissue is dissected from the animal and placed on a glass plate ; the piece is rapidly cut into smaller pieces about the size of a millet seed and transferred to a perfectly clean sterile coverslip. This process must be carried out rapidly because some tissues die in even as short a time as ten seconds when exposed to the air {e.g., thyroid). To prevent this the tissue may be dissected in serum or Ringer. 1042. Preparation of Cultures.— For cultures of the hanging-drop type one uses a hollow ground slide of a sufficient depth to prevent 554 THE CULTIVATION OF TISSUE "IN VITRO." the drop of plasma from touching the bottom. The tissue is quickly- placed on a coverslip, 2 drops of plasma from the paraffined pipette (§ 1039) are added and evenly and thinly spread around the tissue ; this must be done before coagulation occurs. If the plasma is not spread evenly the tissue-culture will grow in many planes, and will be less easy to observe, manipulate, and to fix and staia. When the plasma is spread out the cover glass is inverted over a hollow slide of suitable depth ; a little sterile vaseline may be placed at each side of the cell to assist adhesion preparatory to waxing down. The latter process is done by brushing molten parafiin around the edge of the coverslip, and on the slide, to prevent drying. Im- mediately this has been done the preparation is transferred to an incubator. Carrell and Burrows use a small portable electric incubator which is used for carrying the finished cultures to a bigger incubator in the observation room. For the large plate cultures the same technique is used. Tissue may be rapidly removed, cut into very small fragments, suspended in Ringer, and then spread on the cover of a flat glass-covered (Gahrits- chewski) box, and covered with plasma. The Grabritschewski boxes after several days' incubation (three to five days), are opened, and the plasmatic jelly cut out into blocks and preserved. Or, instead of using Grabritschewski boxes, one may make the culture on large black plates, which must then be placed in glass boxes with cotton sponges soaked in water, in order to preserve the proper hu- midity. The boxes are then carefully sealed with paraffin and kept in such a position that the fluid products of the oultiu-e may drain to the bottom. 1043. Subcultnring is generally difficult, the technique of culti- vating of tissue cells in series being far from perfect. One extirpates a piece of the primary culture at its most active period, and transfers it to a fresh medium, growth often, but not always, beginning anew. Tertiary cultures are made in the same way. Careel and Burrows (Journ. Exper. Med., xiii, 1911) find that a very good way is to cut out the middle of the old culture around the original piece of tissue, and then fill up the space with new medium. The old cells grow into the new plasma. 1044. Fixation and Staining of Cultures.— Carrel and Burrows (Journ. Exper. Med., 1911) remove the cover-glass to which the culture is adherent, and immerse in corrosive sublimate, acetic acid, or formalin, or the various preparations of chrome salts. After- wards the culture is stained in heematoxylin of Benda, Heidengain or Weigert. Dr. A. Drew informs me that he has found that the best fixatives CHAPTER XXXVII. 555 for in vitro cultures are, 70 per cent, alcohol and 5 per cent, acetic, Flemming, and Bouin. In all cases the cultures on the slip should be first detached from the slide and placed in warm Ringer's solu- tion 37° C. for five minutes. They are then placed in the fixative. Alcohol-acetic gives the cleanest pictures. Staining is best done by Ehrlich's hsematoxylin, Delafield's hsematoxylin, iron hsema- toxylin, carmine or Giemsa. As counterstain either eosin or orange G. may be used. ♦ 1045. Artificial Culture Media. — Margaret R. Lewis and W. H. Lewis {Anat. Beeord, v, 1911, p. 277) have investigated tissue cultures of ohiols: embryo cells made in artificial media. Eighty combinations of NaCl, CaCIa, KCl and NaHCOa and water, to form culture media have been proposed. It was possible to obtain growth in such media, in which either the CaClj or the KCl or the NaHCOj was omitted, but not when the NaCl was left out. Such growths continue only for several days, and are never as extensive as those grown in plasma media. More recently Mabgaret R. Lewis {Oontrib. to Embryology, ix, 1920, Nos. 27-46) for tissues of chick embryos of foiir to twelve days' incubation uses " Locke-Lewis " solution (90 c.o. of NaCl 0-9 per cent. + KCl 0042 per cent., + CaClj 0025 per cent., -|- NaHCOs 0-02 per cent., + 10 c.c. of chicken bouiUon -f 0-25 per cent, dextrose). The embryo was removed from the egg and placed in a petri dish con- taining 20 c.o. of the warmed solution. Pieces of tissue .to be explanted were removed, washed through one or more changes of warm medium, and cut with sharp scissors into pieces about 0-5 mm. in diameter ; each piece was then placed in the centre of a covershp, part of the drop drawn off, and the coverslip sealed on to a vaseline ring around the well of a hollow slide. Cultures thus prepared were kept in an incu- bator at 39° C, and observations made in a warm box at 39° C. CHAPTER XXXVIII. A GUIDE FOR STUDENTS OF MICROTOMY. 1046. Three Exwmples for Begirmers : — (1) Tlie preparation of whole Hained mounts of some small object (Daplmids). (2) The preparation of sections of the muscle or an organ of a- vertebrate. (3) The preparation of an embryo (or tadpole) for the making of serial sections. Example I. — From a pond or ditch obtain some water -fleas {Baphnia or Simocephalus) ; allow the jar to stand for several hours tUI the suspended material has settled. Capture some of the organisms as follows : — Take a piece of glass tubing some 8 inches in length ; place ■ a finger over one end, dip the other end under the water and by taking away the finger, suck up some of the Daphnids into the tube ; put your finger over the end of the tube, remove the latter and transfer the organisms to a capsule or watch-glass about 2 inches in diameter. With a clean pipette carefully suck up most of the water, hardly allowing the animals enough to swim in ; now add a fixative to kill the organisms (see § 2), and to coagulate their protoplasm (§ 29) as rapidly as possible so as to leave the groups of cells forming the organs intact and in situ. Use corrosive acetic acid (§ 63), 2 per cent, acetic acid in saturated aqueous corrosive. Pour the fixative into the watch-glass or capsule, till it is full (the watch-glass or capsule contains about 15 to 20 c.c). Place a glass square or plate over the capsule, and leave it for thirty minutes. The organisms become opaque, indicating the coagulation of the proteids of their cells. With a pipette carefully remove as much of the fixing fluid as possible. Now that the organisms are killed, the mercury salt must be removed ; unless the fixative is thoroughly removed, it will form masses of pin-shaped crystals at a later stage when the animals are being mounted in balsam. To remove the corrosive sublimate, it is necessary to convert it into another substance which may. be more easily washed away ; this is effected by immersing the animals in some 70 per cent, alcohol which has been coloured light port-wine shade with tincture of iodine (§ 63), whereupon the mercury bichloride becomes mercury iodide, which is very soluble in 70 per cent, alcohol. The iodine and alcohol mixture CHAPTER XXXVIII. 557 should be used until it no longer loses its colour, which indicates excess of iodine. The whole process should last several hours and may be carried on overnight. The iodine and 70 per cent, alcohol are poured away, and the animals washed for several hours (a minimum of two) in at least two changes of 70 per cent, alcohol to remove as much of the iodine as possible. The objects are then transferred to 50 per cent, alcohol for one half- hour, then into 30 per cent., for the same time. They are brought down these grades in order that shrinkage may not occur when they are being transferred to stains containing little alcohol, or none at aU. ' Two stains may be tried, Mayer's acid haemalum (§§ 248 and 249), and Grenacher's alcoholic borax carmine (§§ 213 and 233). The time that both these stains should be used depends almost entirely upon the accessibility of the cells of the object to the stain. Daphnids are covered by a chitinous shell, which though delicate tends to prevent instant penetration. It is a good thing to leave the animals in the stain for about five hours at least, and overnight preferably. Take two clean capsules, pour into one about 10 c.o. of borax carmine, into the other a similar quantity of the hsemalum. With a camel-hair brush or a pipette transfer some of the organisms to the stains and leave as directed above. See that the capsules are securely After some hours in the stain, the latter is poured away, and the process of difierentiation (§ 203) is begun. The object of differentiation is to wash away superfluous stain from certain organs or parts of organs, in order that a contrast in depth of colour may be obtained in the various other organs and tissues. Both borax carmine and Mayer's acid hsemalum may be differentiated in acid alcohol (4 to 6 drops of HCl to 100 c.o. of 70 per cent, alcohol), which should generally be allowed to act at least for as long as the stain has been used, and, if necessary, longer. In both cases when differentiation has reached the right stage, the objects examined under a microscope have a trans- parent appearance, an^ such parts as the viscera and muscles should he well contrasted. The borax carmine specimens are washed out for several hours in neutral 70 per cent, alcohol. They are then upgraded to 90 per cent, and absolute alcohol, two hours in each, or overnight in absolute alcohol, and cleared in cedar wood or clove oil for at least two hours, and then mounted in xylol balsam. The hsemalum specimens have to be brought to an alkaline solution in order to " blue " the stain, and to get rid of all acid. Some workers " blue " the stain in 70 per cent, alcohol made slightly alkaline with ammonia or bicarbonate of soda, but the best results are obtained by downgrading the objects to tap-water, which is allowed to run over them gently tiU they go quite blue, which shoidd occur for small objects within an hour. The animals are then gradually upgraded through 30, 50, 70 and 90 per cent., to absolute alcohol, and cleared as above described for borax carmine specimens. 558 A GUIDE FOR STUDENTS OF MICROTOMY. In order to obviate the differentiation stage, one may dilute both the borax carmine and the acid hsemalum till they are about one-third or one-half as strong ; dilution of the borax carmine may be carried out with 50 per cent, alcohol (not methylated spirit) and with distilled water in the case of hsemalum. In these solutions the animals remain till sufficiently stained. But the best results are got by the overstaining and differentiation method. 1047. Example II. — From a frog remove a large leg or thigh muscle, and out it into two pieces about as big as the naU of the little finger. If desired, the liver, a halved testis, or a kidney msiy also be used. Transfer the material to a capsule containing at least 20 o.c. of Zenker's or Helly's fluids (§§ 73, 684). Leave till next morning, and wash in running water under the tap for at least three hours, preferably over- night, then transfer to 50 per cent, alcohol for an hour ; then to 70 per cent, alcohol containing enough tincture of iodine to give the solution a light port-wine shade. Add more iodine as the colour disappears, prolonging the treatment overnight for large pieces. Pour away the alcohol, and add pure 70 per cent., in which the material is washed at least three hours. Transfer to 90 per cent, for several hours and leave in absolute alcohol overnight. Next morning it is safest to give the material another hour in a fresh change of alcohol absolute. Pour away a good deal of the alcohol and add about the same quantity of xylol or cedar oil. Shake, leave half an hour, and then transfer the material to pure xylol or cedar oil ; leave half an hour. Pour away some of the xylol, either add chips of hard wax to cover the tissue, or add some of the stock xylol and wax mixture. Leave an hour in thermostat on the upper shelf, pour off, and add molten pure wax ; leave one or two hours on the bottom shelf. Embed blocks (§§ 142, 143). 1048. Example III. Preparation of am, Embryo for Serial Sections.— Fix in Bouin's fluid corrosive acetic or picro-nitric, overnight (§§ 110, 63, 97). In the case of the first and last mentioned fixatives, the embryo is afterwards transferred to 30 per cent, alcohol (half-hour), 50 per cent, (two hours), and then washed for a day in several changes of 70 per cent. The corrosive acetic fixed specimens are similarly treated except that at this stage iodine solution is added to the 70 per cent, (or this may be done in 90 per ^ent.) alcohol tU] the corrosive sublimate is removed. Leave overnight in 90 per cent, alcohol (or at least three or four hours), and at least six hours in two changes of absolute alcohol (preferably overnight). De-alcoholisation and clearing must be done carefully as directed in § 591, p. 269. It is a good plan to bring embryos from absolute alcohol, through several gradually strengthening mixtures of alcohol and cedarwood oil — to pure cedar-wood oil, and then wash out in benzole. Embed in wax as described in § 591, generally about one hour in benzol and wax, and two hours in pure wax. Embed blocks (§§ 142, 143). Now read §§ 144 to 151. CHAPTER XXXVni. 559 1049. General Plan of Procedure Applicable to Histological Specimens. Anseathetiae animal, kill it, quickly take out organ, cut pieces 1 cm x 1 cm x J cm. i' (■ 2i % K2 Cr-2 O7 Fix for 24 houia in Zenker's fluid s' 5 % Hg CI2 * (, 5 % Gl. Acetic acid. Wash in running water 24 Iiours. Freezing method. Parajgin method. Preserve in 5% Formalin Wash in water Impregnate with gum Cut sections with freezing microtome Float in water on to slide stain with Picro-carmine 15 minutes Drain o£S and wipe away the stain around ''' Mount in Farrant s medium. Stain Pass through increasing strengths o( ale. 70% alcohol t Remove Hg deposit with iodine in 80% alcohol i (Preserve in 80% alcohol) t Pass through 90% alcohol 1 day Dehydrate in abs. ale 1 night Clear in xylol or chloroform...Froml to2hour3 ■ Pass through xyl ol satd. with paraffin wax 1 hour t Impregnate with paraffin at 52° C 2 hours and Embed and make blocks Cut sections with microtome Celloidin method, Alc-ether aa 1 day t Thin celloidin 1 week (15% in alc-ether) t rhick celloidin 1 wBek (30% in alc-ether) After evaporation, mount on block of vulcanised fibre t Harden celloidin in chloroform 1-2 hours andthenin80% ale 1-6 hours f Cut with razor (oblique) wetted with 80 % ale. Stain without removing celloidin t (Eemove celloidin with oil of cloves) t Mount in balsatb. [1° Apply fixative to slide or glycerine and alhumen water] t 2° Float section on drop of water on slide Warm gently to open out the section Wipe away excess of water and dry in air 3° Eemove paraffin with xylol 4" Remove xylol with abs. ale. 5° Pass through 90 and 70 % alcohol to water t 6° Stain in Haematoxylin, etc., 5-15 minutes, etc. t 7° Wash in water, 5 minutes — 1 hour t 8° Counter-stain in Bosin — 1 minute, etc. 9" Remove excess with 90% alcohol 10° Dehydrate with abs. ale. 11° Clear in clove oil or xylol 12° Mount in balsam. (Modified from D. T. Harris' "Practical Histology.' 560 A GUIDE FOR STUDENTS OF MICROTOMY. 1050. General Rules and Hints for Students.— (1) Keep all your bottles and capsules as clean as possible. (2) Try to keep your bench in order (it is difficult, J. B. G.). (3) Keep notes of the time necessary for changing reagents. (4) Thoroughly clean your slides and coverslips in acid alcohol before using. See addendum. (5) Note that corrosive sublimate tends to harden material. (6) Corrosive sublimate is difficult to remove from tissue unless you use iodine. If not properly removed you will find numerous pin-shaped crystals in the finished sections. § 63. (7) Corrosive sublimate attacks the surface of steel and other metals. Use quiUs, or wooden needles for manipulating tissue in sublimate. (8) Watery stains after picric acid fixation wiU. cause maceration if prolonged. § 93. (9) Unless very well washed out, picric acid should not be used in conjunction with thionin or toluidin blue. Precipitates form. Certain other dyes do likewise. (10) Osmic acid crystals should be dissolved in the purest distilled water. Wash the tube with distilled water before you break it, removing label. Wash out capsules and bottles for osmic acid solutions in distilled water. Keep solutions in shade or dark. § 27. (11) Osmic acid tends to harden yolk and certain other cell materials. The vapour of osmic acid is injurious to the eyes and nose. (12) Osmic acid and fixatives containing it inhibit staining, but if necessary you can induce osmicated material to stain in delicate dyes by bringing sections down to distilled water and treating in a ■ 25 per cent, solution of permanganate of potash for a short time. Permanganate also decolorises sections. See page 31. (13) Nitric acid tends to soften ohitin and yolk, but it may inhibit staining a little. § 97. (14) Imbed material in paraffin in the shortest time possible, for materials left in the thermostat longer than necessary go hard, especially from xylol ; this refers especially to vertebrate material and yolky embryos. (15) Alcohol and chloroform dissolve fats and lipoids, acetic acid dissolves away lipins. Vegetable oils dissolve fats less readily than xylol or chloroform. Read §§ 120 et seq. (16) Strong alcohol is bad for the finger nails and skin. (17) When diluting stains with alcohol, use solutions made up by breaking down pure absolute alcohol. Do not use methylated spirit, as this generally precipitates the stain. (18) You can soon learn to teU roughly the strength of alcohols by the smell. (19) Don't use the dregs of the absolute alcohol bottle for dehydrating anything. The dregs are no longer absolute. Keep a waste alcohol bottle for used liquid. (20) Some workers add a little bag of fused copper sulphate to their store bottles of absolute. This keeps the alcohol dehydrated. (21) After fixation, when dehydrating and embedding a piece of tissue, an egg or an embryo, it is at its softest when in weak alcohol, and its CHAPTER XXXVIII 561 hardest when in xylol or a clearing oil. PJatten or otherwise manipu- late a fixed object, while it is stUl in weak alcohol, or it will break up ; but some objects may be dissected successfully in clove oil. § 122. (22) Cells alter soon after death : formalin fixation is the best for corpse material. Carefully note § 31. (23) The organs of animals over-anaesthetised by chloroform or ether are often spoilt (especially in the vicinity of large blood vessels) and are sometimes useless even for general purposes. § 12. (24) Keep balsam or colophonium jar in the dark, or paint it black outside. Acid balsam soon removes stains from tissue ; acid balsam is the micrologists' bite noire. § 443a. (25) After Zenker fixation sections may overstain in eosin. (26) If finished sections have crystals in them this is due to improper washing out of fixative, or stain. (27) Formaldehyde gas dissolves in water up to 40 per cent. The commercial formalin is acid and must be neutralised with magnesium or sodium carbonate kept in a little bag in the stock bottle. § 1,08. (28) Formaldehyde gas is injurious to the skin and mucous membrane of nose. (29) If after staining in delicate dyes {e.g., methyl gTeen), all the colour keeps coming out of the sections during passage through alcohols, try the following method : — Wipe superfluous water from around the sections, and dehydrate by dropping acetone on sections : then plimge into a jar of half acetone, half xylol, then pure xylol. (30) For clearing embryos or pieces of tissue for whole mounts, cedar- wood oil is better than xylol. § 120. (31) If bubbles get under the coverslip they can often be removed by gently warming, or by placing slide under beU jar of an exhaust pump. (32) If after mounting an object in balsam white or black lines and blotchy areas appear, this means that dehydration was not complete. Bring back through xylol to absolute alcohol. (33) When, after embedding, the block is set aside for a time and it is found that the object is surrounded by a halo of white wax, this means that all the clearing oil was not removed and is now exuding from the object. Ee-imbed in pure wax. (34) When, after embedding, the material seems soft and tends to fall out of the wax, this indicates that dehydration was not complete, and possibly also that the time in pure wax was not long enough. Without efficient dehydration it is impossible to make good sections. (35) If when cutting the sections curl up, it means that either the knife is bluht or the material has been overhardened during imbedding. Occasionally an incorrect slope of the knife may be the cause of curling. (36) When the sections will not form a ribbon, this means that either the wax is too hard or the slope of the knife is not correct. If the wax is hard, place 1 drop of soft wax on each side of the block and flatten it out with a warm knife. Eead carefully pp. 83 to^90. (37) The broad side of a block should be parallel to the knife. (38) Some people use miniature drums for rolling up the wax ribbon. Laying them on a piece of foolscap does quite well. Avoid sticky ^ 36 562 A GUIDE FOR STUDENTS OF MICROTOMY. paper. If sections accidentally adhere you can often release them by cautiously wetting the paper with absolute alcohol. ( 39) Before placing sections on a slide, write with a diamond pencil the number of the slide and the material used. At a pinch, a glass wax-pencil may be used instead- (40) If you have not used a diamond, it is always possible to tell on which side of the slide the section lies, simply by slightly tilting the slide and observing the shadow thrown on the other side of the glass. (41) Finally, if your first attempts are failures, do not be discouraged — even the most skilful miorotomists generally produce atrocities at their first attempts. Try again ! APPENDIX. 1051. Chemicals, Stains, and Apparatus. — Addresses of British firms from wMch it is recommended that these be obtained are given in § 11. 1052. Cleaning SMes and Covers. — New ones should first be soaked in one of the following liquids : strong sulphuric, hydro- chloric or nitric acid, or aqua regia, or a mixture of an ounce each of sulphuric acid and bichromate of potash with from 8 to 12 ounces of water, then washed first with water and lastly with alcohol, and dried with a clean cloth. For iised ones, if a balsam moxmt, warm, push the cover into a vessel with xylol or other solvent of the mount, and put the slide into another vessel with the same, leave for a few days, and then put into strong alcohol. If this is not sufficient, treat as for new ones. Some persons boil in lysol, which I do not find efiicacious. For the final treatment, see p. 121. 1053. Gum for Labels. — Labels stuck on glass often strip off. This may be avoided (Maepmann, Zeit. Angew. Mik., ii, 1896, p.. 151 ; Journ. Roy. Mic. Soc, 1897, p. 84) by means of the following adhesive : 120 grms. of gum arable are dissolved in a quarter of a litre of water, and 30 grms. of gum tragacanth in a similar quantity. After a few hours the tragacanth solution is shaken until it froths, and mixed with the gum arable solution. Strain through linen and add 150 grms. of glycerin previously mixed with 2^ grms. of oil of thyme. Peiece (Journ. app. Mic., ii, 1899, p. 627 ; Journ. Roy. Mic. Soc, 1900, p. 404) finds that if the end of the slide be painted with a thin solution of balsam, it may be written on with ink when dry, and the record preserved by a second coat painted over it. For other receipts see emly editions. 36—2 INDEX. Names like Lo Bianco are given under the latter half of the name. Abderhalden, 357 Acalephse, 523 Acanthocepbali, 513 Acephala, injection, 503 Acetate of lead, brain, 404 of potash, medium, 219 Acetic acid, fixation, 51 alcohol and sublimate, 53 and alcohol, 52 bichromate, 41 decalcification, 252 alum, carmine, 137 Aceto-carmine, 138 Acetone, 168 for dehydration, 4 fixation (Lucidol), 47, 59 and formol, 64, 494 and sublimate, 47 -chloroform, for narcotisation, 14 Achucarro, neuroglia, 490 Acid Juchsin, 171 for mitochondria, 320, 321 and malachite green (Pianese), 175 and methyl green, 324 myelin, 452 and orange G, 172 Acid haemalum, 153 hsematoxylin, 155 magenta, 171 rubin, 171 Acidic dyes, 120 Acidophilous tissue, 128 Actinians, narcotisation, 13, 521 Adamkiewicz, myelin, 452 Adenoid tissue, 249 Adsorption, 120, 122 Adurol, 469 Agar, sections between coverslips, 307 Agar-Agar, growing amoebaB on, 527 Agassiz and Whitman, 281 Agduhe, Bielschowsky, 431 Aguerre, 482 De Albettis, neuroglia, 485 Albumen, method of mounting sections, 113 mercurial mounting medium, 220 removal of from eggs, 260 et seq., 283, 284 Alcohol, for dehydration, preservation, 4 narcotisation, 13 fixation, 56, 68 for maceration, 244 Alcohol — contd. absolute, 58 table for dilutions, 57 and nitric acid for decalcifying, 253 Alcoholic, mercury bichloride, 46 hsematoxylin. Apathy and de Groot, 156 Heidenhain, 543 Dobell, 544 cochineal, 144 Alcyonaria, 522 Alcyonidium, 500 Aloyonium, 12, 521, 522 Alexander, 262 Alfieri, bleaching, 256 Alizarin, 184 and crystal violet, 322 nervous system, 408 Alkanna, 368 Allen, methylene blue, 192 Allen, Ezra, 304 chromic Bouin's fluid with urea, 306 on clearing, 307 Allen, E. J., menthol, 12 Allen and Browne, 27, 523, 524, 525 AUerhand, iron myelin method, 451 Alt, 408 Altmann, 20 et seq., 38 acid fuohsin picric acid method, 320 corrosion, 249 fat, 366 fluid, 37 Alum, aniline, 355 carmine, 136 and picric acid, 138 -nitric acid for decalcifying, 253 hsematoxylin, generalities, 151 Aluminium hsematein, 151 Alzheimer, 416, 480 Amann, lactophenol, 222 Amato, 425 Ammonia, carmine, 140 chromate, 43 Ammonio-chloride of tin for mvelin, Besta, 448 Ammonium, bichromate, brains, 403 sulphooyanide for maceration, 245 vanadate, Golgi method, 476 Amoebse, cultures, 527 Amphibia, 276 et seq. brains, 405 Ohampy's fluid, 37, 318 INDEX. 565 Amphioxus, 281 Amphipoda, embiyology, 288 Amyl uitiite, 233 Amyloid, 133 Andeer, 254 Andres, actinida, 12, 13, 521 Andrews, avian embryology, 272 Andriezen, Golgi method, 465 Anemones, narcotisation, 12 See Actinians. Anglade and Morel, Victoria blue neuro- glia stain, 483, 485 Anilin dyes, 1 59 et seq. blue, 182 blue-black, 183, 408 blue and carmine, 213 red, 169 oil, 69 anilin oil water, 166 Anitschkow, 110 Annelids, 39 blood vessels, 510 Champy-KuU, 322 killing, 12 nerves, 510 Antedon, 519 Ap4thy, 97, 98, 511 alcoholic corrosive, 46 hsematoxylin, 156 bergamot oil method for celloidin sections, 117 . embedding in oil of cedar, 78 Canada balsam, 226 cemeilt for glycerine mounts, 231 gold, 198, 206, 207 glycerine gelatin embedding, 93 gum sjrrup medium, 221 haematin mixture, 155 methylen blue, 189, 192, 193 • on knife tilt, 85 neurofibril methods, 416 nitric acetic for maceration, 247 picro-saurefuchsin, 177 series-on-knife method for celloidin sections, 118 theory of gold impregnation, 203 Apel, 512 Aqueous humour, 219 Aianeida, 287 Arctiscoida, 509 Argentamin, 415, 452 Argyroneta ova, 287 Arndt, bone saw, 370 Arnold, 178, 297, 517 chondriosomes, 323 staining kidney, 394 Arnstein, 190, 193, 342 method for corpuscles, 342 Aronson, myelin, 448 Arsenic acid, decaicifier, 254 Arthropoda, 504 et seq. embryology, 284 et seq. fixation, 44, 47, 55 mounting whole, 504 Artifacts, 20, 304 Artificial fecundation, 258 et seq. Artificial iodised serum, 219 Ariom, ascaris ova, 290 Ascaris ova, 52, 289 Aschofe-Eiyono, 388 Ascidia, narcotisation, 12 buds, 282 general, 499 Ascoli, Cajal'a method, 426 Asphalt varnish, 230 Asphyxiation, by boiled water, carbonic acid gas, 16 Assmann, blood stain, 384 Astacus, eye, 508 methylen blue, 192 rierve-endings, 344 Asteroidea, 519 Astrocytes, 479 Aihanasiu and Dragoiu, 352 Atheson, 511 Athias, 467 Atta, ova, 287 Auerbach, 415 buds, 425 stain, 312 Augstein, 514 Anricularia, 520 Aves, embryology, 271 et seq. Gerlach's window method, 271 Axis cylinder, 452 (methylene blue) other stains, 454 et seq. and dendrite, advice on special forms, 459 processes similar to Golgi methods, 475 rapid process, 458 Azoeosin, 313 Azoulay, 209 ammonium, vanadate process, 476 osmic acid methods (myelin), 451 Babcock, 102 Babes (safranin), 166, 167 Babkin, 396 Bacteria, in amoeba cultures, 528 in tissue, 326, 354 Ballowitz, 348 electric organ, 346 mammals, 264 reptiles, 275 Balsams, 225 et seq. cedar wood, alcoholic, 226 method, section-grinding, 109 neutral, 226 Barnes, 514 Barrois, echinoderm larvae, 520 Bartel, 482 Baiyta-water, for maceration, 245 Basic dyes, 120 Basophil granules, nerve tissue, 410, 414 " Basophilous " tissue, 128 Bastian, gold, 205 566 INDEX. Bataillon and Koehler, 308 ascaris ova, 289 Batchelor, 390 Bath (paraffin), 77 Baumgaiteu, 183, 213 Bayerl, deoalcifler, 254 ossifying cartilage, 377 Bayliss, 125, 309 on dyes, 120 on specificity of stains, 134 Taso-dilators, 233 Beale, 232 digestion by pepsin, 248 Beard, raja embryos, 280 Beauchamp, 513 Beckwith, 309 Bedot, 524 Bees, brain, 508 Behrens, 182, 221, 344 mounting medium, 224 salmon embryos, 280 Bell, cement, 229, 366 fat, 367, 369 Benario, blood, 381 Benda, 495 alizarin method, 322 copper haematoxylin, 157 crystal violet, 184 fatty acids, 368 " Flemming fluid," 319 iron hsBmatoxylin, 148 neuroglia stain, 482 pioro-saurefuohsin, 177 rapid myelin method, 448 safranin and light green, 181 secretion, granules, 315 Beneoke, fibrils, 351, 388 Van Beneden, acetic acid, fixation, 51 method against contraction, 12 acetic alcohol, 52 live mammal embryo in serum, 267 mammals, 265 taenia eggs, 289 and Neyt, ascaris ova, 290 Bengal rose, 180 Bengtsson, eggs of diptera, 285 Bensley, braziliu and water blue, (thyroid), 394 pancreas, 395 intestine, 393 Bensley-Cowdry, acid fuchsin methyl green stain, 324 Benzidine dyes, 388 et seq. teeth, 373 Benzoazurin, 170, 184 cartilage, 376 Benzol, 70 embedding, 78 peroxide, 59, 382 Benzoputpucin, 179, 390 Benzoyl, green, 181 Bergamot oil, 68, 323 for oelloidin sections, 117 Becgh, annelids, 510 Bergonzini, 356 Berkeley, Golgi method, 466 liver, 393 rapid myelin method, 447 Berlese, Aoarina, 504 Berlin blue, aqueous masses, 240 of Mayer, 240 injecting teeth, 372 mass, Bruclce, 236 Bernard, mollusc, 504 Berner, 368 Bernheim, 206 Beroe, 524 Bertarellis, protozoa, 532 Best's carmine stain for glycogen, 295 Besta, ammonio-chloride of tin, 448 Cajal's method, 425 Golgi apparatus, method, 438 Bethe, 507 chitin, 506 methylen blue, 194 molybdenum-toluidin blue, neurofi- brils, 417 Bettendorf, 616 Betz, hardening nerve, 403 Bevau Lewis, 183, 404 , Lo Bianco, 14, 15, 37, 510, 514, 515, 516, 518, 519, 524 acetic fixation, 51 ascidia, 499 moUuscoida, 500 • chromo-sublimate, 48 corrosive acetic, 44 gephyrea, 512 mixture for narcotisation, 13 protozoa, 542 Bichloride of mercury. See under Mercury. BichoS, mammals, 264 Bichromate of ammonia, 43 of calcium, 43 o£ potash, 41 decoloration of, 41 i, maceration, 245 Muller's fluid, 42 and alcohol, 43 fixation, nervous system, 403 et seq. -oamio, 37 -platinic, 37 ohromic-osmio, 37 and mercury, 48 -sublimate, Golgi method, 470 BickSalvi, digestion, 249 Biebrich scarlet, 180, 313 Biedermann, methylen blue, 344 Bielaszewics, 150 Bielsehowsky methods, 426 et seq. Da Tano modifications, 432 et seq. other modifications, 430 et seq. silver method, for connective tissue, 352 and Bruehl, ear, 497 and Plien, cresyl violet, 414 Bigelow, Medusae, 524 INDEX. 567 Bile capillaries, 393 Bilbaizia, 516 Binet, 31 Bing, 6i, 449 Biniodide of mercury mounting liquid, Binnennetz, 316 Biondi, blood, 380 Bipinnaria, 520 Bismarck brown, 161, 169 for cartilaginous skeletons, 377 Bizzozeco, 391 blood-platelets, 386 and Torre, blood, 382 Bjeloussow, gum arabic mass, 241 Bladder, frog, nerves, 349 Blastoderms, general, 260 of mammals, 267, 268 Bleaching, Mayer's chlorine method, 255 sulphurous acid, hydrogen peroxide, chlorine, 31 Bles, frog embryology, 278 Bleu de Lyon, 183 Blochmann, 115, 500 oestodes, 516 frog embryology, 276 Blood, 379 et seq. cells, mitochondria, 333 elective stain for reds, 388 fiiation, 379 et seq. fixing in bulk, 380 new Golgi body, 387 and iron salts, 300 platelets, 386 -serum media, 219 stains like methyl green, etc., 380 Blue gelatine mass, 236 lumiere, 183 Blum, 401 Bobretzky, 286 Boccardi, 206 erythrosin and toluidin blue, 415 Bodecker, decalcification, 251, 373 Boeke, Bielsohowsky, 430 — 431 Bohm, 206 and Oppel, 41, 219 reptile blastoderms, 274 Bohmer, hsematoxylin, 154 Bohmig, 515, 517 Bolina, 524 Bolton, 469, 447 Golgi method, 464 Bombyx mori eggs, 285 Bone, 369 et seq. decalcification. 251 dry sections, 369 et seq mounting, 370 non-decalcified, 369 soft parts, 371 et seq. saw, 370 Bonnet, 269 Bonney, 178 Bonome, 480 Bonvicini, hardening human brain, 404 Borax carmine, 141 Bordeaux R., 178 Borgert, 75 Boring, ascaris ova, 290 Born, 262, 278 Borrel, 213 -method, 532 Borrel's blue, 534 Bouffard, Benzidine dyes, 388 Bouin, picro-formol, 62 frog larvae, 278 salmon embryos, 280 Boule, Gajal's method for lumbricus, 425 Bouma, 376 Boveri, ascaris ova, 290 embedding of echinoderm ova, 260 picro-acetio, 55 Boyce and Herdman, copper, 300 Boycott, 507 Brachiopoda, 500 Bradford and Plimmer, 547 Braem, Bryozoa, 282 Brain, cat, dog, neurofibrils, 421. See under Nervous System. cat, man, preliminary fixation, 402 insect, 508 preliminary treatment, 397 — 399 Branca, 168 sublimate foi'mol, 63 Brandt, 546 glycerine jelly, 223 protozoa, 540 Brasil, 546 Brass, on embedding, 76 protozoa, 542 Braun, 514, 517 Braus, 26, 281 bile capillaries, 393 Brazilin, 211 for sponges, 526 Breglia, 449 Bremer, 181, 473 Bresslau, mesostomid ova, 288 Brilhant kresylblau, blood, 383 Bristol, 511 Brittle object, cutting of, 88 Brock, maceration, 245 Brodmann, 398 Bromide of soda or potash, Simarro, 41 9 Brookover, 442, 468 Golgi method, 465 Brown, 549 Briicke, digestion, 249 Bruel, dipterous eggs, 285 Briihl, corrosion, 249 Bruno, mucin, 391 Brunotti, gold gelatin, 93 gelatin embedding, 108 Brunswick black, 230 Bryozoa, 282, 500 heat-killing, 12 Buehner, glycogen, 290 nucleoli, 314 568 INDEX. Budge, injection, 241 Bugula, 500 Bujor, 523 Bulb, 424 Bullard, 367 Bumpus, 103 Bunge, fluid for iron stain, 299 flagella stain, 548 Burchardt, 139 jSj paraflin, 92 chrome fixation, 40 protopterus brain, 405 Burzyn^i, 94 Buigei, nemertiaa, 515 Burrows, 550 Busch, 29, 32, 214, 252 decalcification, 251 Marchi, 450 Butschli, 154 Buzzi, eleidia, 341 Cade, gastric glands, 393 Cajal, 435, 465 double-impregnation, Golgi process, 461 avoidance of silver precipitates, 467 gold chloride-sublimate method of neuroglia, 487 Golgi apparatus method, 436 methylene blue diffusion process, 478 myelin, 450 nucleolini, 311 retina, 495 spirals and funnels, 440 Cajeput, oil of, 68, 103, 411 Calberla, 161, 181, 182 Bismarck brown, 161 liquid, 222 Calcium chloride medium, 219 Calyptoblastea, 523 Camsal balsam, 227 Canada balsam, 225 et seq. Caoutchouc, cement of jNQUer, 230 Capitellidse, narcotisation, 13, 510 Capsicum berries, 368 Carazzi, 256 Carbol-pyronin-methyl green, 172, 355 Carbolic acid (clearing), 69 fuchsin, 169 thionin (King), 168 Carbon, bisulphide for embedding, 77 Carter on, 372 insects, 507 tetrachloride, 77 Carleton, Golgi apparatus, 438 nucleolini, 311 Carmalum, 137 and indigo-carmine, 212 Carmine-generalities, 136 alcoholic stains, 141 aluminium chloride solution, 138 and anilin blue, 213 ammonia, soda, lithium, magnesia, 140 Carmine-generalities — contd. and cochineal stains, 135 -gelatine masses, 234 Hoyer, 235 Pol, 235 Krause, 235 glycerine mass (cold), Beale, 238 Robin, 233 Grenacher's alum-carmine, 136 and malachite green, 213 for nervous system, 407 neutral alkaline, 140 blue, 183 Carminic acid, 135 Carnoy, 151, 178 acetic alcohol, 52 and Lebrun, frog eggs, 276 iron, 300 Cacothers, Bouin and urea for insect chromosomes, 306 Carrel, 550 et seq. Carter, J. Thornton, Tbone, 369 et seq. on post-mortem changes, 25 teeth sections, 371 Cartilage and bone, 376 silver, 200 skeletons, 377 Caryophyllia, 521, 522 Cassiopeia, 524 Castellarnau, 519 Castle, Ciona, 281 Castor oil, mounting medium, 228 Catois, methylene blue method for fishes, 478 Cattaneo, 541 Cattani, funnels and spiral filaments, 410 Caullery, 499 Causard, 509 Caustic soda bleacher, 257 potash or soda, corrosion, 250 maceration with, 244 CavaliS, electric organs, 346 Cavazzani, 215 Cedar wood oil, 66 et seq., 77 for minute dissections, 7 Cell inclusions, 316 eJ seq. granules, lymph and blood cells, 315 CeUoidin embedding, 95 et seq. See also imder Collodion, knife smeared in vaseline, 102 for injection, 241 sections. Apathy's method, 118 BoUes Lee's method, 116 Summer's method, 116 staining, 102 Celloidinum inelasticum, 96 Cements and varnishes, 229 et seq. Centrifuge, for oogenesis studies, 332 Centrosomes, 315 C6pede, 307 Cephalopoda, 282 ' eyes, 502 Ceratium, 547 Cercaria, 517 INDEX. 569 Cerebellum, 424. See under Nervous System. preliminary treatment, 399 Cerebium, hardening, 399 Cajal's methods, 424 Cerfontaine, amphioxus, 281 Ascaris ova, 290 worm, 509 Geites, protozoa, 540 Cesaris-Demel, 383 Gestoda, 515 ova, 289 Chseiopoda, 509 marine, 510 Chalicodoma, eggs, 286 Chambers, intra vitam stain, 310 Champy, 314 fluid, 37 iodide of osmium method, 331 trichloracetic, 53 Champy-Kull,mitochondrialmethod,321 fixation, Gatenby, 322 Chenzinsky, 181 blood stain, 383 Chick, embryology, 271 et seq. axis cylinder and dendrite, 459 Child, fish embryos, 279 Chilesottl, 407, 477 Chilopoda, blood, 381 China blue, spirals, funnels, 440 Chiiin, 506 et seq. bleaching, 256 tests for, 507 Chiton, eggs, 284 Chitonidae, 503 Chloral hydrate, for narcotisation, 14 jelly, 224 maceration, 248 mounting medium, 220 Chlorcarmine, 139 Chloreton, for narcotisation, 14 Chloroform, for killing, injurious in cytology, 11 clearing, 70 embedding, 76, 78 vapour, collodion embedding, 98 Cholesterin, 356 et seq. Chondriokonts, 316 Chondriome, 316 Chondriosomes, 316 Chorion, removal, 284 et seq- Chromates, 40 Chromate masses, 237 Chromatin, microchemistry, 294, 308 et seq. and enzymes, 308, 309 digestive fluids, 294 methyl green test, 293 Chrome salts, Burckhardt on, 40 Chromic acid, decalcification, 252, 254 fixation, 32, 33 washhig out, 33 formol, 63 for hardening, 34 Chromic acid — contd. ' and hydrochloric acid, decalcifier, 254 maceration, 246 nerve tissue, 404 and platinum chloride (Merkel), 39 Chromidia, 308, 309, 310, 316 Chromo-acetic acid, 35 ChromO'^aceto-osmic decalcifier, 254 Chromo-formic, 35 Chromo-nitric acid (Per^nyi), 39 decalcifier, 254 Chromophility tests, 309, 310 Chromosomes, techniques for, Gatenby, 303 et seq. Chromo-sublimate, 48 Chrysoidin, 364, 369 Ciacco, 206, 343 method, 369 . (fat) tendon, 347 . Ciaglinski, myelin, 452 Ciechanowski, bile capillaries, 393 Cilia, mollusc, 503 protozoa, 541 Ciliated epithelium, 339 Ciliates, general staining, 533 Cilimbaris, 353 Cinnamon (cassia) oil, 67 Cladocera, Haker, 288 Clasmatocytes, 356 Cleaning slides, 112, Appendix Clearing, 5, 65 Clove oil, for minute dissections, 7, 67 Coal gas, for killing, 11 tar dyes (plasma), 171 Cobb, differentiator, 3 Cocaine, for narcotisation, 14 Coccidia, staining, 532 Cochineal, 135 alum carmine, 137 Cochlea, 375, 496 et seq. Coe, Distomum, 289 Cceleuterata, 521 maceration, 524 nervous system, 522 Coerulein S., 182 Cohnheim, gold, 204 Cold-blooded animals, injection, 241 Cole, gum mass, 110 Coleoptera, eggs, 286, 287 CoUargol, 434 Collin, 610 and Lucien, 438 CoUinge, 281 Collodion, embedding, 95 et seq. alcohol hardening, 100 bone sections, 372 bath, 96 for celloidin sections (Weigert), 118 blocks, clearing in cedar oil, 104 clearing and mounting, 102 cutting, 101 dry cutting, 104 hardening, 98 newer method, 103 570 INDEX. Collodion — contd. Obregia's syrup method of mounting sections, 115 older method, 96 preservation of blocks, 100 rapid process, 104 section mounting with albumen, 116 method of mounting paraffin sections, 115 and paraffin, 105 solution of, 97 CoUodionisation, brittle objects, 88 Colloidal gold, 133 Colloidal-complex, 120 Colloids, 123 CoUoxylin, 96 Colophonium, 226 and wax method, section-grinding, 109 Colueci, 226 Comatula larva, 520 Congelation masses, 109 Congo-Corinth, 179 Congo red, 121, 122, 124, 178 et seg. nervous system, 408 myelin, 453 Conklin, Crepidula, 284 Connective tissue, 350 et seq. Bielsohowsky, 434 Conser, 500 Contraction, prevention of, 11 acetic acid in, 12 corrosive sublimate in, 12 Cooling paraiifin, 82 Copal method, 108 Copepoda, 288, 505 Copper, in tissue, 300 bichromate, 64 chloride and acetate fixation, 53 mounting fluids, 220 ferrocyanide injection mass, 234 formol, 64 nitrate, 54 sulphate and corrosive for nerve tissue, 405 fluid for frog eggs, 279 Corallin, 169 Corals, decalcification, 251 Cori, narcotisation mixture, 14, 15 osmic solutions, 29 Cornea, 342 et seq. maceration, 247 fibres, maceration, 246 silver method, 198 Corning, 475 neurokeratin, 441 Corpuscles, Golgi, 347 ^leissner, Krause, 342 Nissl, 180 tactile, 341 Herbst and Grandry, 342 Corrosion. 249 et seq. Corrosive sublimate. See also under Mercury bichloride. preventative of contraction, 12 Corrosive sublimate — contd washing out, 26 Cowdry, 304, 415 acid fuchsin stain, 324 Janus green, 332 Mitochondria, 338 Cox, 415, 434 Golgi process, 472 et seq. neurokeratin, 441 Cramer, 30 fatty substances, 356 osmic vapour method, 330 Peiss and Bullock, 366 Creases (paraffin sections), 88 Creighton, 297 Creosote (clearing), 69 Crepidula, 284 Cresyl violet, nerve, 414 Crinoidea, 519 Cristatella, 282, 500 Crustacea, 505 et seq. Crystal violet, 184 for mitochondria, 322 Crystalline lens, 247, 343 Csokor, 230 bone saw, 371 Ctenophora, 524 Cucumaria, 518 Cultures, manipulation of amoebsa, 527 Cunningham, micro -injection, 270 Curare, 292 for narcotisation, 16 Curling of sections, 86 Curreri, 469 Cutting, parafiin, 86 tissue, 319 Cyolas ova, 284 Cytological methods, 292 et seq. Cytoplasmic inclusions, 316 et seq. Czokor, 137 Daddi, fat, 367 Dahlia, 162, 169, 333, 340 Dakin, 503 Dallinger, 530 Damar, 226 Davidoff, 524 Tunicata, 281 Dead cells, 131 Dealcoholisation, 65 et seq. or clearing, 5 Death, 25, 131 Decalcification, 251 et seq. teeth, 251 et seq. and 372 et seq. Decapod, eyes, 508 Decapoda, ova, 288 Deecke, 404, 407 Deetjen, 387 Deflandre, fat, 369 Degenerate nerve, Marchi, 449 Dehydration, 2 by alcohol, acetone, methylal, aniline oil, 4 Dejeiine, 407 INDEX. 571 Dekhuyzen, 218, 386 liquids, 43 osmaoet, 380 Delafield. haematoxylin, 154 Delage, Turbellaria, 517 sponge, 526 Delamare, 351 Dell'Isola, Golgi method, 463 Delia Rosa, Indian ink mass, 240 Delia Valle, Orohestia, 288 Deltapurpuiin, 179 Demoor, 214 Dendrite stains, 454 et seq. Dendrocoelum, 517 Dendy, Geonemertes, 515 sponges, 526 Denne, orientation of objects, 81 embedding method, 78 et seq. Dentine, 370 et seq. Dependorf, 374 Depigmentation, 256. See Bleaching. Descemet's membrane, 343 Desiccation method for paraffin sections, 111 Desilification, 251 et seq., 256 Dewey, 373 Dewitz, 503 Oictyosome, 316 Dietrich, 366, 509 Digestion, 248 et seq., 308 and dissociation, 243 et seq. Dimmer, 115, 119 Diomidofi, 405 Diptera eggs, 285 Disse, 377 Dissections-minute, cedar wood oil for, clove oil for, 7 glycerine for, 8 Distomum ova, 289 Dobell, modification of Heidenhain stain for protozoa, 544 Mann's stain, 544 Dodeilein, 519 Dog brain, 402 Dogiel, 342 corpuscles of Herbst, 342 Meisser and Krause, 342 Grandry's corpuscles, 421 iris, 348 methylen blue, 189, 190 et seq. for epithelia, 195 tendon organs, 347 Donacia eggs, 286 Donaggio, 158 neurofibril methods, 417 el seq. Donaldson, 405 faeces, 538 Doncaster, chromosome fixation, 305 Double-embedding collodion paraffin, 105 Double-staining in haematoxylin and acid fuchsin, 326 Downey, benzidine dyes, 388 Dtasch, 204 Dreuw, 341 Drew, formol-chrome method, 325 manipulation of amoeba cultures, 527 protozoa, 526 et seq. staining amoebae, etc., 531 tissue culture, 551, 554 Drew-Griffln live.-slide, 530 Drew-Murray, connective tissue stain, 354 Driessen, 297 Drost, 504 Driiner, 26 Duboscu, blood, 381, 465 Dubreuil, connective tissue, 350 Duerden, coelenterates, 521 Duerk, 354 Dunham's mixture (oelloidin sections), 103 Durig, Golgi method, 464 Duval, 404 carmine and anilin blue, 213 collodion embedding, 95 orientation method for blastoderms, 273 silver, 200 Dyes, nature of, 120 electric charges, 122 Ear, inner, 496 et seq. Eau de Javelle and Eau de Labarraque bleachers, 256 corrosion, 250 for eggs, 284, 285 for frog embryology, 276 Eberth and Rimge, 469 Echinodermata, 518 et seq. larvae, 520 Echinoderms, decalcification, 251 Echinoidea, 518 spines, 518 Edinger, 34 Edington, blood, 381 Egg-capsules, removal, 284 orthoptera, 286 Egg, white of, injection mass, 240 Eggs, Unio, 284 Ehrenbaum, grinding, 109 Ehrlich, 155 blood, 379 haematoxylin and eosin, etc., 214 Indulin-Aurautia-Eosin, 180 mast cell method, 356 methylen blue, 188 et seq. neutral red, 179 triacid mixture, 175 Ehrlich-Biondl, 161, 173 and Lazarus, 297 Ehrmann, 340 Eichler, 498 Eisath, neuroglia granules, 486 Eisenberg, Nile blue, 368 Eisig, 13, 510 fluid, 40 maceration, 245 572 INDEX. Eismond, 639 Ekman, 500 Elastheematein, 353 Elastic tissue, 352 et seq, fibres, of spleen, 394 Elderberries, Kappers, 409 Electric organs, 345 Eleidin, 341 Ellis, 548 Elschnig, 97 Embedding, gelatine masses, 92 et seq. collodion, 95 paraffin, 76 et seq. lead-gum, 106 boxes, 73 brass squares, 74 thimbles, 73 trays, 72 in vacuo, 80 Embryoiogical methods, 258 et seq. ' Embryonic cartilage, 376 et seq. Embryos, Bielschowsky methods, 429 Cajal's methods, 424 fixation, 260, 268, 278, etc. Emery, aqueous carmine mass, 240 Encephala, 401, 404 Endothelium, silver, 200 Engelmann, 218 Enriques, 501 Entamoeba, 538 Entire objects, preparation of, fixing agents for, best stains for, 7 Entz, 81, 543 protozoa, 541 Enzymes and chromatin, 308 Eosinophilous cells, 180, 383, 386 Eosins, 180 et seq. methyl green, 181 methylen blue, 383 et seq. Epeira eggs, 287 Ependyma cells, 479 Epidermis, digestion, 249 neurofibrils, 421 Epiploon, silver method, 199 Epithelia, silver method for, 198 Epithelium, maceration, 244, 245 Eppinger, 393 Erhard, 297 Erlanger, ascaris ova, 290 Erlicki's fluid, 42 Van Ermengem, 549 Ernst, 341 Erythrosin, 180 with toluidin or methylen blue for nerve tissue, 415 Eternod, 83 Ether method, for celloidin sections, 116 for narcotisation, 13 Eucaine, for narcotisation, 15 Euler, 309 Euparal, 227 Evans, benzidine dyes, 388 micro-injection, 270 Everard, 214 Ewald. blood, 380 section washing apparatus, 3 Examination media, 216 et seq. Eycleshymer, 98, 103 Eye, 493 et seq. arthropod, 508 et seq, bleaching, 255, 257 mollusca, 502 Fabre-Domergue, 220 protozoa, 540 Faded sections, treatment of, 6 Faeces, examination for protozoa, 538 smears, lucidol, 382 Fairchild, 3 Fajerstain, 342 hsematoxylin, Golgi method, 476 Da Fano, 428, 434, 435 nervous system, 397 et seq. axis cylinder stains, 454 et seq. Bielschowsky method, generalities, 426—427 modifications, 432 et seq. Cajal's methods, 419 et seq. advice on Cajal's methods, 424 modification of Cajal's method, 425 cobalt nitrate, Golgi apparatus method, 437 formaldehyde, Golgi methods, 463 Golgi preparations, on cutting, 467 on mounting, 468 Golgi-Cox method, 473 special treatment for Golgi-Cox preps., 474 neurofibrils, 416, 417, 418 neuroglia methods, 479, 484 Golgi's sublimate method, 470 Fanz, grinding bone, 370 Farrant's medium, 221 Fat and glycogen stain, 296 Fatty substances, Cramer and Gatenby, 356 et seq. % Faure-Fremiet, centrifuge, 332 protozoa, 540 Faussek, cephalopoda, 283 Feist, 407 Felizat, 168 Ferreri, decalcifying, 255 Ferria, 352 Ferric and ferrous salts, 297 et seq. Fettponceau, 367 Fibres of Sharpey, 375 Fibrils, connective tissue, 350 et seq. Fibrin, Weigert stain, 388 Fick, 184 kerato-hyalin, 340 Siredon, 277 Fieandt, neuroglia granules, 486 Fiedler, 525 Field and Martin, 105 Fiessinger, 297 FUms, blood, 379 fixation, 381 et seq. INDEX. 573 Finotti, 408 Marchi method, 450 myelin, 452 Eisohel, 505 chick embryos, 273 Fischer, 223, 344, 353 on coagulation, 20 et seq. nucleoli, 314 trematodes, 516 Fischler, 368 Fish, 405 embryos, Bielsohowsky (Paton), 430 eggs, 279 et seq. methylen blue nerve method, 478 Fish, on clearing celloidin sections, 103 brain of Desmognathus, 405 decalcification, 253 Golgi method, 464 Fixation, in embryology, 259 et' seq. by immersion, 25 by injection, 26, 397 by salts, 40 Fixing agents, cytological, 301 theory of, 18, 19, 131 Flagella. stains for, 548 Flagellata, general stains, 533 Flatau, 402 . Golgi, corrosive method, 472 Flattening sections, 90 Flechsig, 206, 449, 472 Flamming, 339 ohromo -acetic, 35 chromo-aceto-osmic, 35 Dahlia, 169 decalcified bone sections, 375 orange method, 171, 177 pioro-osmic, 56 safranin, 166 Flesch, 444, 498 blood, 380 Floyd, 508 Plustra, 500 Fo&, 214 sublimate and bichromate, 49 Foettinger, 14, 515 Fol, 16, 542 mixture, 39 mounting watery sections, 116 carmine mass, 235 picro-chromic, 56 Fontana, tannin-silver protozoon stain, 545 Foot and Strobell, smears, 308 Formaldehyde, 60 et seq. andalcohol, 62 modifications of Golgi method, 463 mordant, 165 Formalin, blood, 381 for nervous system, 401 and picric, 62 chrome techniques, 323 and sodium chloride, for maceration, 244 Formalin — contd . Formol, chromic, 63 rormol-Mulier, 63 nitric acid for decalcifying, 253 sublimate, 63 vapour, blood, etc., 381 Formic acid, gold method, 204 Foster and Balfour, embryology, 271 Frankel, 297, 449 Francotte, 150, 289 Freeborn, 407 picro-nigrosin, 350 Freezing methods, 109 Frenkel, palladium and osmio, 50 Frenzel's mercuro-nitric, 47 Fresh cells, 293 Frey, 219 Friedenthal, hardening mass, 237 FriedBinder, 524 Frog embryology, 275 eggs, removal of mucin, 276 et seq. Gatenby's fluid for eggs, 277 methylen blue, 191 skin, 391 Frohlioh, 176 Frozen sections, 109 brain, 406 Fuchsin (basic), 169 See Acid Fuchsin. carbolic, 169 Furst, bleaching, 256 Fusari, 377 Gage, 101, 297 albumen and mercury mounting medium, 220 alum, nitric acid decalcifier, 253 celloidin sections, 117 clearing mixture, 69 maceration, 248 with formalin, 244 Galesesou, 482 Gallein, myelin, 448 Galli, China blue, 440 Garbini, 522 Gardiner, ova of polychoerus, 288 Garlic water, 115 Gaskell, 94 Gastric glands, 392 et seq. Gastropoda, 283, 500 embryology, 283 eyes, 502 Gatenby, 308, 435 amoeba culture, 536 benzidine dyes, 388 et seq. centrifuge, 332 Champy-Kull fixation, 322 Cramer's osmio vapour method, 330 differentiation between cytoplasmic inclusions, 334 et seq. Donacia eggs, 287 double stain for mitochondria, 326 (and Cramer) fatty substances, 356 on Fischer's theory, 20, 21 574 INDEX. Gatenby — contd. on fixing agents, 22, 23, 24, 25 Flemming modification, 36 fluid for frog eggs, 277 on killing, 11 Limnaea embryology, 284 Mann-Kopsoh method, 328 Mann-Kopsch-Altmann combination stain, 329 on methods of mammalian embryo- logy, 263 mitochondria, etc., 316 et acq. mitochondrial fluid, 319 new advances in embryological tech- nique, 258 plan for cytological research, 337, 338 sponges, 526 technique for chromosomes, 303 et seq. tissue culture methods, 550 et seq. on Unna's oxypolarity theory, 21 and Woodger, 365 Gaule, desiccation method for paraffin sections, 111 solution, 46 Gaultheria, oil of, 68 Gavazzeni, 341 Geberg, 206 corpuscles, 342 Gedoelst, 249 neurokeratin, 441 Gee and Harrison, 124 Gehardt, reptile blastoderms, 274 lens, 343 Van Gehuohten, 413, 463 axis cylinder and dendrite, 459 fixing fluid, 415 ear, 498 Gelatin, embedding masses, 92 injection masses, 232 blue, 236 green, etc., 237 red, 234 yellow, 237 cement, 229 freezing mass, 110 and glycerine, 93 Gemelli, flagella, 549 Gentian blue, 183 violet, 162, 167 et seq. neutral, 396 for fibrin, 388 carbol, for flagella, 548 GeoSroy, mounting medium, 224 Gephyrea, 511 Gerlaoh (gold), 207 Gerota, 202, 398 brains, 402 Golgi method, 464 Gerould, 518 Giemsa, blood stain, 384 et seq. for neuroglia, 487 for protozoa, 536, 544, 545 Gierke, 197, 214 maceration, 245 Giesbrecht, 505 embedding, 78 Van Gieson (pioro-saurefuschin), 176, 215 GigUo-Tos, blood, 382 Gilbert, 442 Gilson, bleaching, 266 chloral hydrate jelly, 224 copper formol, 64 mercurial mounting medium, 220 mercuro -nitric, 47 Sandarac mounting media, 227 rapid celloidin method, 104 Glands, 391 et seq. Glaue, 514 Glia, 479 et seq. Glochidia, 284 Glucose mounting medium, 221 Glycerine, gelatin and glycerine injec- tion media, 233 gelatin, 93 and HCl. decaloifier, 254 jelly, 223 for maceration, 247 for minute dissections, 8 mounting media, 222 et seq. Glycerised blood-serum, 219 Glychsemalum, Mayer's, 153 Glycogen, 294 et seq. and cell inclusions, 338 Goadby, 220 Goblet cells, 392 Gold, colloidal forms, 133 gelatin embedding, 93 gelatin mass, Tandler, Pearl, Mayer, Mozejko, 238 impregnation, generalities, 202 et seq. preservation of specimens, 208 Gold-size, 230 Gold-sublimate, Golgi method, 475 Goldmann, 131 benzidine dyes, 388 et seq. Goldscheider and ITlatau, 414 Goldschmidt, 552 Goigi, 26, 435 axis cylinder and dendrite stains, 454 et seq. sublimate method, 470 et seq. mixed process, 460 avoidance of precipitates, 466 cutting and manipulation, 467 mounting, 468 body in red cells, 387 Golgi method, modifications, 461 formaldehyde process, 463 theory of impregnation, 460 metho(^ for funnels and spiral fila- ments, 439 bichromate-sublimate modifications, 472 gold, 207 osmio-biohromate mixture, 458, 516 INDEX. 575 Golgi—^cowid. processes for rejuvenation of over- hardened tissue, 462 apparatus, generalities, 316 bone and teeth, 376 Kopaoh techniques, 327 et seq. Mann-Kopsoh-Altmann method, 329 other methods (silver), 438 plants, 325 silver methods of Golgi, Veratti, Da Fano, Cajal, 435 U seq. Sjovall method, 331 corpuscles of, 347 Golgi-Cox, 472 Da Fano's special mounting method, 474 making permanent preparations, 473 Golgi-Eopsch apparatus, 316 Golgi-Veratti, Golgi apparatus method, 435 Golodetz, 8, 210, 366 ana Unna, cholesterin, 341 Golovine, 179 Goodrich, 308, 377 modification of Kent's method, 541 Goodsiria, 282 Gordon, 180, 549 Gorgonia, 521, 522 Goionoivitsch, 280 Gothard, methylen blue, 414 Giae&e, paraf^ solvents, 76 Giaff, Hirudiuea, 511 Turbellaria, 517 Graham, 390, 514 Gram, gentian violet, 167 Grandis and Mainini, 300 Giand-Mouisel and Tribondeau, pan- creas, 395 Grandly, corpuscles, 342 Grassi, hsematozoa, 546 Gray. 498 Greef, 493 Green, B., 14 Green leucocytosis, oysters, 300 Gregory, micro-injection apparatus, 270 Grenacher, borax carmine, 136, 141 bleaching mixture, 257 eyes of mollusca, 502 Greppin, 469 Griesbach, 178, 182, 352 blood, 380 Grinding sections, 108 de Groot, alcoholic hsemalum, 156 iron carmalum, 139 Groselj, 522 Grosser, partially aqueous ink mass, 241 Griinpulver, 159 Griinstein, bladder, nerves, 349 Grynleitt and Mestrezat, 256 Guanin, 301 Gudden, 465 myelin, 446 Gudger, salmon embryology, 280 Gulick, Ascaris ova, 290 Gulland, 379 blood fixing fluid, 381 Gum, and chloral hydrate mount, 221 glycerine, Allen, Langerhans, 221 mounting medium, 221 syrup masses, 110 Thus, 228 Gurwitsch, 150 Guyeisse, 392 Gymnoblastea, 522 Gymnotus, electric organ, 346 Haber and Guild, Oajal's method, 426 Hadzi, 523 Haecker, 301 Haemacalcium, Mayer, 155 Hsemalum, 152 de Groot, 156 Haemastrontium, Mayer, 156 Hsematein, 145 e,t seq. neurofibrils, 416 Hsematoxylin, 145 et seq. Benda and Heidenhain, 148, 149 Bohmer, 164 Delafield, 154 chemical nature of, 145 chrome, Hansen, 157 Heidenhain, 156 Schultze, 157 oombiaation stains, 213 copper, Benda, 157 elastin, 353 Golgi-method, 476 iron, 148 Kleinenberg, 155 for iron in tissue, 298 mounting in glycerine, 152 osmium, SchiJtze, 158 phospho-tungstie, Mallory, 158 and picro-Saurefuohsin, 215 ripening, 146 and safranin, 214 and Saurefuchsin, 2l4 stock solutions, 146 tin, Donnaggio, 158 vanadium, 157 Hsematoxyline noire, 151 Hsemoglobin, 300 Heemosiderin, 300 Hair, 341 Halle and Bom, orientation metliod, 98 Bela Haller's mixture, maceration, 247 Halliburton, 410 Hamann, 506, 513 Asteroidea, 519 Hamburger, 218 Argyroneta ova, 287 Hamilton, 404 congelation method, 110 Hammarsten, 357 Hanazawa, dentine, 371 Hance, fixation of mammalian chromo- somes, 305 576 INDEX. Hansen, 140, 151, 176, 180 chrome-hEematoxylin, 157 Hantsch, 223 Hardening, 27 injeotion mass, 237 nerve tissue, 398 Hardy, 123, 512 Had, 391 Harmer, 202 Harris, 15, 154 hsematoxylin, for elastin, 353 myelin, 449 methylen blue, 195 Harrison, Ross, tissue culture, 550 el seq. Hart, 353 Harting, 219 gamboge glycerine mass, 239 Hartmann, 269 mammalian embryology, 265 opossum embryology, 267 Haswell, Temnocephala ova, 289 Hatschek, Amphioxus, 281 Haug, 251, 449 decalcifier, 254 Havet, 517 coelenterates, 522 Hayem, blood fluid, 380 Heat, for killing, 12 Heckert, eggs of Distomum, 289 Heidenhain, 163, 173 alcoholic hsematoxylin for protozoa, 543 carbon bisulphide embedding, 507 centrosomes, 315 ohrome-hsematoxylin, 156 theory of dyeing, 125 Ehrlich-Biondi, 173 gelatin glycerine jelly, 223 iron hsematoxylin, 147 et sag. vanadium hsematoxylin, 157 Heinke and Ehrenbaum, 281 Heinrich, 352 Held, 150, 326 • formol Muller, 63 method for marginal neuroglia, 484 methylen blue and erythrosin method,' 415 Helix, 501 ova, 283 Heller, 209 and Gumpertz myelin, 451 Helly, sublimate and bichromate, 49 Henchman, gastropoda, 283 Henking, examination medium, 293 methods for arthropod eggs, 285 ova of Phalangida, 287 Henneguy, 38 acetic alum carmine, 137 albumen water method for section mounting, 114 chick, 273 fish embryology, 279 gastropod embryology, 283 Henneguy — contd. mammal blastoderm, 268 permanganate method, 165 treatment of faded sections, 6 Hennings, 505 eyes of arthropoda, 509 Insecta, 505 Hfinocaue, gold, 205 Herbst, 506 corpuscles, 342 Herdlicka, brain, 402 Hermann, 401 fluid, 38 pyrogallol method, 209 safranin and gentian, 168 L'HSrmitte and Gucoione, 485 Herrick, Astacus ova, 288 Hertwig, silver for marine animals, 202 frog eggs, 278 maceration mixture, 246 Triton eggs, 277 Van Herwerden, nuclease, 309 Herxheimer, 184, 340 Soharlaoh R., 367 Hesse, eyes of heteropoda, 502 Hessert, 549 Heteropoda, 500 eyes, 502 Heyder, Arion embryos, 284 Heymons, 501 eggs of Orthoptera, 286 Hiokson, 508 Brazilin, 211 eosin and hsematoxylin, 214 maceration, 248 High refractive mounting liquids, 224 Hill, J. P., 274 clearing and embedding, 262, 269 manipulation of ova, 266 " marsupial mixture," 265 paper for reconstructions, 262 modification of picro-nitric, 55, 284 treatment and isolation of eggs of mammals, 264 HUl, 461, 466 myelin method, 448 Hindle, 647 Hippel, 495 Hirota, orientation, 274 Hirsohfelder, 512, 513 Hirschler, 328 Donaoia eggs, 286 protozoa, 541 Hirudinea, 511 killing, 12 methylen blue, 192 nervous system, 511 His, 38, 197 Histiocytes, 388 Histriobdella, 511 Hochstetter, injeotion, 241 Hoehl, 37, 249 Holer, 16 INDEX. 577 Hoffmann, 81, 390, 508, fil7 chick embryos, 273 green, 182 Hogben, chromosomes, 303 Hoggans, histological rings, 199 perohloride of iron, 209 Hollande's chloroarmine, 139 Holmes, Planorbis ova, 284 Holmgien, trophospougium, 439 Holothuria narcotisation, 12 Holothurioidea, 518 Homans, pancreas, 396 Homarus, 288 Honing, 84 Hopewell-Smith, teeth, 373 Hopkins, maceration, 246 Horn, hair, nails, 341 Hornowski, 351 Hoiny structures, maceration, 247 Hoskins, cliick, 273 Hot water, for sudden Icilling, 1 2 Houser, 442 Hoyer, 75, 401 carmine mass, 235 gold, 205 Golgi method, 463 mounting medium, 221 mucin, 391 shellac mass, 242 silver method, 200 nitrate yellow gelatin, mass, 237 Huber, 468 Hudson, 512 Human brain, 402, 404 neurofibrils, 421 embryos, 42 Hyatt, 108 Hydatlna, 512, 513 ova, 288 Hydra, 521 methylen blue, 189 Hydrochloric acid, carmine, 143 for decalcification, 251 et seq. for maceration, 247 Hydrogen peroxide bleacher, 256 Hydroidea, general, 523 heat killing, 12 Hydroxylamin, for narcotisation, 15 Hymenoptera, eggs, 286 Hypochlorite of potash, corrosion, 250 of soda, corrosion, 250 Ida, 105 Idiozome, 316 IgacQSchi, 393 Ilberg, 415 Imbedding, 71 et seq. See also Embeddrag. for amphibia, 276 in paraffin or collodion, 5 Imms, 504 ,^„ , Impregnation methods, 197 et seq. Impregnations other than gold, silver, or osmium, see page 210. iron sulphate, 210 palladium chloi-ide, 210 perohloride of iron, 209 mass of gelatin, 237 Indian ink, injection of insects, 509 embryos, 270 mass, 240 India-rubber and paraffin, 92 IndifEetent liquids, 217 media, 292 Indigo, 212 Indigo-carmine, 182, 212 with oxalic acid, 212 Indophenol, 367 Indulin, 409 aurantia-eosin, 180 and nigrosin, 182 Injection masses, purely aqueous, 240 partially aqueous, 240 et seq. celloidin, etc., 241 Fol, Tandler, Beale, 238 gamboge glycerinej 239 gum arable, 241 indigo carmine, Thoma, 239 milk, 241 warm, 232 cold, 238 methods, 232 et seq. arthropods, 508 Hirudinea, 511 mammalian and other embryos, 270 mollusca, 503 "naturalj" 242 Innervation, bladder, 349 Insabato, 352 Insects, 504 et seq. double embedding, 500 carbon bisulphide, 507 mounting whole, 504 — 505 Instruments, microscopes, microtomes, 8 Intercellular bridges, 339 Intestine, 393 Intranuclear rodlet of Roncoroni, 421 Intra vitam, methods, 292 stains, mitochondrial, 332 .staining, alizarin, 130 Bismarck brown, 130, 162 BoUes Lee on, 129, 130 Congo red, 130 Fischel, 130 kidney, 394 Loisel, 130 methylen blue, 180 et seq. neutral red, 130, 179 protozoa, 540 sulphorhodamin, 130 theoretical, 129 et seq. In vacuo, embedding, 80 Inversion plasma stains, 184 Invertebrates, general methods, 499 «? seq. dendrites and axis cylinders, 454 37 578 INDEX. Invertebrates — conid. ganglia, Cajal, 425 nervous system, 416, 417 Iodide of osmium, 331 of potash and biniodide, liquid, 224 for maceration, 244 Iodine, extraction of corrosive, 45 faeces examination, 538 fixation, 51 green, 182 method for glycogen, 295 vapour fixation, 51 Iodised serum, 219 for maceration, 243 Iridium chloride, 50 Iris, 348 Iron, 297 et seq. alum fixation, 50 Brazilin, 211 for protozoa, 545 oannalum, 139 carmine, 139 cochineal, 140 hematoxylin, 147 et seq. Benda, 148 Butschli, 150 rapid method, 150 Weigert, 150 perchloride fixation, 50 sesquichlorate, myelin, 451 Isamin blue, 390 Islets of Langerhans, 395, 396 Isolation of single cyst, etc., 527 Israel, 180, 212 Iwanzofl, 518 electric organ, 346 Hatehett Jackson, 66 Jaooby, 183 Jaoquet, leeches, 511 Jadassohn, 340 Jaderholm, 419 Jaenichen, 517 Jager, 223 Jakimovitch, 201 Jander, 502 bleaching, 257 Janssens, 151, 183 Janus green, 181, 332, 333 pancreas, 395 " Japanese " method of section mount- ing, 114 Jaguet, 510 Jelinek, 101 Jenner, blood stainy 383 Jensen, protozoa, 540 . Joest, 509 Lindsay Johnson, 206 aceto-osmic, 51 .. collodion method, 101 eye, 494 fluid, 37 on metallic stains, 198 Johnston, 262, 415 nerves of Petromyzon, 405 Johnstone-Lavis and Vosmaer, section grinding, 109 Joliet, gum glycerine embedding, 107 Jones, 300 Jonescu, 508 Jordan, 66 Jorgensen, 309 nucleoli, 314 Joris, 434 Joseph, 197, 341 white-of-egg mass, 240 Juliusburger, Nissl, 413 Kadyi, 407 brain, 402 Kaes, myelin, 447 Kaiser, 170, 223, 513 glycerine gelatin, embedding, 93 myelin, 447 solution, 44 spinal cord, 408 Kalb, 549 Eallius, 455, 469 embryonic cartilage, 376 Golgi modification, 461 Kaplan, 477 myelin, 452 neurokeratin, 441 Kappers, elderberries, 409 Karawaiew, 546 anobium eggs, 287 Karger, 493 Karyosome, 310 et seq. Kastschenko, 262 Selachian embryos, 280 Katx3, Cajal's method, 425 Kattwiukel, 400 Katz, ear, 497 Kawamura, 366 Keibel, 270 Kent, iodine fixation, 51 Kenyon, 508 Keratohyalin, 340 Eernschwarz, 211 Graham Eeir, 119 reconstruction method, 261 Kerschner, 205 Kidney, 394 Killing, amphibians, pithing, 1 1 birds, lizards, newts, 11 by sudden heat, 12 by hot water, 12 large mammals, ether and chloro- form for, coal gas for, 11 King, brain hardening, 405 Bufo eggs, 278 carbolic thionin, 168 Kingsbury, 368 Kingsley, Limulus ova, 288 Kionka, orientation method, 274 Kishinouye, spider eggs, 287 INDEX. 579 Eizer, blood, 381 Klein, cornea, 343 Kleinenberg, hasmatoxylin, 155 pioro-STilphurio, 55 Knife position, paraffin cutting, 83 slope, 85 Knowen, 81 Koch, 449 Von Koch, copal method, section grind- ing, 108 Kockel, 388 Eodis, 404 myelin, 442 Koerner and Fischer, tamiin fuohsin flagella stain, 548 Kofoid, gastropod embryology, 283 Kobler, 516 Kolliker, mammal blastoderm, 268 embryology of mammals, 264 indigo-carmine for bone, 376 Kollman's fixative for fish eggs, 280 Kolmer, 26, 495 Kolossow, 32, 206, 209 modifieation of Cajal's axis cylinder stain, 462 prickle cells, 339 Eolster, stomach, 392 Kopsch, 386 osmic method, 327 Golgi method, 464 mollusc eyes, 502 teleost embryology, 280 Korotneff, 13 Korscheli, 541 cephalopoda, 283 Kostanecki, mercuro-nitric, 48 and Siedlecki, Ascaris ova, 290 and Wierzejski, mollusc eggs, 284 Kotlarewski, 404 Kowalewsky, teleost eggs, 280 KowBlski, 425 Kozowsky, myelin, 446 Krause, 173, 182, 393, 482, 496 carmine mass, 235 salivary glands, 392 Krauss, 201 Krecker, 14 Kresofuchsin, 169 Kresyl blue, 383 Kresyl-eohtviolett, 184 Kresyl violet, 184 Krogh, 415 Krohntbal, lead sulphide impregnation, 475 KiomayeT, 184, 388 plasma fibrils, 339 Kronecker's serum, 218, 264 Kriiger, 354 Harpaotida ova, 288 KOhne, maceration, 247 Kuhnt, 496 Eiikenthal, 14, 5J.0 Lumbricus, 509 Kull, 392 Kultschizky, 341 bichromate and sublimate, 4ij double embedding, 105 mucin, 391 myelin method, 447 preservation of tissue in ether or xylol, 4 rubin for neuroglia, 486 solution, 42 spleen, 393 Kupffer, 393 Kuskow, digestion, 249 Lachi, 401 Golgi method, 463 Lactic acid, 233, 254 Lactophenol, 222 Lsevulose, for myelin preparations, 448 LaSont, 341 LahUle, 499 Lake, 132 Lakmoid, 393 Lambert, Epeira eggs, 287 Lamellibranchiata, 284, 500 et seq. cilia, 503 eyes, 502 maceration, 247 pigment, 257 Lams, 265 Landois, maceration solution, 245 Landolt, 496 Lane, pancreas, 396 Lang, corrosive liquid, 46 moUusca, 501 Langdon, 510 Oe Lange, 450 Langerhans, 341 gum glycerine, 221 » islets of, 395 Langeron, 59 Lanis, fixation of mammal eggs, 265 Lankester and Bourne, 508 Lansberg, 541 Lantbanin, 302 Larvae, echinoderm, 520 Laslett, 447 Lattice fibres, 393, 394 Laurent, 181 Lauierborn, 75, 547 Lavdowsky, 192, 228, 498 maceration, 248 Laveran's solution, 534 Law, nerve-endings, 374 Lawrence, 223 Lead-gum embedding, 106 Lead sulphide, Golgi method, Krohn- thai, 475 Leber, 495 Lebrun, Anuran embryology, 276 Lecithin. See under Fatty Substances, 356 et seq. Bolles Lee, 407 decalcification, 251 37—2 580 INDEX. BoUes Lef — contd. oelloidin section mounting, 116 chick embryos, 272 chromosome stain, 303 on choice of stain, 134 dry cutting process, celloidin, 104 ear and eye, 493 — 498 fish embryos, 279 fixation of ground cytoplasm, 301, 302 gold, 206 iron carmine, 139 Kemschwarz, 211 mounting fluid, 222 mounting Golgi preparations, 468 myelin stain, 442 osmic-pyrogallol method, 208 paper cell mounting method, 230 on staining nucleus intra vitam, 310 Tunicates, 499 sponges, 525 Leeches, 511 maceration, 247 nervous system, 611 Legal, alum-carmine, picric, 138 Legendre, Golgi apparatus, 438 L6ger, sporozoa, 546 Legros, 201 Amphioxus, 281 Leiper, 514 Leishman, blood stain, 385 protozoa, 635 Lemon-juice gold method, 204 Von Lendenfeld, 525 Von Lenhossek, 342, 455 Nissl bodies, 413 mollusc eyes, 502 platinum chloride, 50 Lennhoff, methylen blue, nerve, 414 processes for nerve cells, etc., 476 Lennox, 496 Lens, eye, 343 Lenssen, 513 Hydatina, 288 Leontowitsch, 195 Lepidoptera ova, 286 Lepkowsky, 375 vessels in teeth, 373 Leuckhart, embedding boxes, 73 . Leutert, 300 Levaditi, 549 blood smears, 383 Levi, fixation of mammal eggs, 265 mitochondrial method, 325 Levulose, 221 Levy, 389 Bevan Lewis, 408 Lewis, 389, 511 tissue culture, 555 Lewy, 450 Lichtgriin, 159 Liebetanz, 548 Liesegang, 407 Cajal's method, 426 Ligamentum nuchse, digestion, 249 Light green, 181 Lillie, 332 Unio eggs, 284 Limax, 283, 501 Lime salts, 300 ' Limneea ova, 284 Limulus, 288 Liuville, mollusc ova, 283 Lipin, 357 Lipoids, 357 List, 214, 314 Ooocids, 506 Mytilus, 600 Sagartia, 522 Lithia, for picric fixed material, 54 Lithium carmine, 140 Little, Hydra, 521 Live slide, Drew and Griffin, 530 Liver, 393 mollusc, 501 Lizard blastoderms, 275 Locke's solution, 218 Locy, spider eggs, 287 Loele, 390 Loewy, method for integument, 339 Loffler, stain for flagella, 648 Loisel, 367 fat, 369 sponge, 526 Long, constant temperature box for mammal eggs, 266 and Mark, fixing fluid for mammals, 266 mouse embryology, 263 Longhi, 542 Longworth, 342 Lonnberg, 616 Looss, 514, 516 corrosion of chitin, 260 Nematodes, 513 Lopbomonas, 633 Lord, methylen blue, nerve, 414 LBwit, blood fluid, 380 gold, 204 Loyez, 442 Lucidol, 308 'in acetone, 59 blood, 382 Ludford, 308 Lugaro, 417 coUargol, 434 Lugol's solution, 45 Liihe, 516 Luithlen and Sorgo, 414 Lumbricus epidermis, nerve, 421 nerve, 425 sections, 509 Lundvall, 377 Lustgarten, 169 Luxenburg, Nissl bodies, 413 Lymphatics, in teeth, 373 glands, 394 Lymph-spaces, methylen blue, J 95 Lysol, for maceration, 248 INDEX. 581 Maas, 213, 249 sponges, 526 Macallum, 300, 301 iron methods, 297 MacBride, larvse of Echinoderms, 520 Maceration, 243 et $eq. B61a HaUer's mixture, 247 epithelium of molluscs, 503 Ranvier, 244 Molesohott, 244 Landois, 245 Macklin, 271, 389 Maclean, 357 Macrophages, 388 Von Maehrenthal, 209 Magdala red, 169 for spleen fibres, 394 Magenta, 169, 171 Magini, 477 Magnesia carmine, 140 Magnesium chloride or sulphate, for narcotisation, 15 Malachite green, 181 Malaria, 546 pigment, 300 Malassey, 218 Mall, 378 Mallory, 354 eosin and methylen blue, 181 neuroglia, 483 phospho-molybdic acid hajmatoxylin, 157 phospho-tungstic, 158 saure-fuohsin and phospho-molybdic acid, 351 Weigert stain, 481 Mammalia, Cajal's methods, 424 eggs, 266. See also under Ova. Mammals, chromosomes, 305 et seq. clearing and embedding, 269 embryological methods, 263 fixation of whole tubes, 268 isolation of eggs, 263 et seq. Manchester brown, 161 Manfredi, 206 Mann, 26, 31, 114, 155, 398, 468 chromo-sublimate, 48 fat, 366 extraction of corrosive sublimate, 45 Golgi-Cox, 473 Golgi method precipitates, 466 methyl blue eosin, 183 osmio-sublimate, 48 stain, for protozoa, 544 sublimate formol, 63 Mann-Eopsch method, 328 Marcacci, maceration, 247 Marcano, blood, 381 Marchi, degenerate nerve, 449 et seq. mollusca, 501 tendon organs, 347 Marcus, 446 Macesch, 352, 394 Maifori, 300 Marie, 400 Marina, 405 Marine animals, fixation of, 27 cell inclusions, 332 Mark, coUodionisation, 88 reconstruction, 262 Marsh, 229, 230 Martin, 392 traohese, 508 Martinotti, 167, 196, 354, 369, 386, 407, 408 Golgi method, 465 elastic tissue, 352 Mason, brains, 405 Massart, 214 Masson, connective tissue, 351 Mast cells, 354 et seq. Unna's method, 356 Matschinsky, bone sections, 371 Matuszewski, 450 Maurice and Schulgin, 213 Mawas, 496 Maximow, 356, 389 Altmann method, 322 May-Grunwald, 384 Mayer, 15, 33, 155, 184, 297, 350 acetate of potash, 151 albumen water method for sections, 113 alcoholic cochineal, 144 aluminium chloride carmine, 138 benzol embedding, 78 bleaching, 256 carmalum, 137, 212 cartilage, 377 chitin, 608 cochineal stain, 135 decalcification, 253 desilification, 255 eye, 339 glychsemalum, 153 hssmacalcium, 155 haemalum, 152 et seq. hsemastrontium, 156 on hsematoxylin staining, 145 iron staining, 298 methylen blue, 193, 196 mucin stains, 392 paracarmine, 142 picro-hydroohlorio, 56 pioro-magnesia carmine, 141 picro-nitric, 55 Plutei, 520 section stretcher, 87 triacid, 175 Maysel, 161 McClung, urea in fixing, 305 McClure, 502 McCrorie, 549 Medium of Farrant, 221 Medulla, hardening, 399 Medusee, 523, 524 killmg, 12 maceration, 246' 582 INDEX. Medusee — cmtd. narcotisation, 13 sections, 524 Mehnert, 275 Meirowsky, 549 Meiseoheimei, gastropoda, 283 Melanin, 300 Melting point of paraffin, 91 Membrana, nictitans, 339 limitans, marginal neuroglia, 485 Membranes of eggs, 284, 285 Mendel and Bradley, 300 Menthol, for narcotisation, 12 Mercier, 449 Mercury, bichloride, 44 alcoholic liquids, 46 et seq. and acetic acid, 44 extraction, 45 sodium thiosulphate, extraction by, 46 washing out, 45 and bichromate, 48 and f ormol, 63 mounting media, 220 nitric mixtures, 47 sublimate and salt, 46 Merkel, indigo-carmine, 212 Merkel's fluid, 39 Merton, 502 Merzbacker, 485 Mesostomidse ova, 288 Messner, picrocarmine, 414 Metachromasy, 133 Metachromatic dyes, 133 Metagelatin vehicle, Fol, 238 Metallic stains, 197 et seq. theory of, 197 et seq. osmio acid, pyrogallol, 208 Metcalf, Chiton ova, 284 Methyl, aniline green, 159 hive, 183 with eosin (Mann), 183 nephridia, 511 f/cccB, 159 et seq. for chromatin, 293 with eosin, 181 mixture for maceration, 247 salicylate oil, 68 violet, 162 for flbrin, 388 fluid for blood, 382 plasma fibrils, 340 B., 184 Methylen blue, 170, 186 et seq. blood, 383 Cajal's diffusion process, 478 Distomum, 516 and eosin (Mallory), 181 eosin stains for blood, 383 et seq. and erythrosin, Nissl granules, 415 fixation of stain, 192 et seq. frog bladder, 349 intra vitam staining, 187, 344, 477 Methylen — contd. for epithelia, lymph-spacea, 195 insect eyes, 508 method, cornea, 343 methods for sections, 194 modes of staining, 190 myelin, 449, 452 nerve endings, 344 methods, 477 et seq. Nissl bodies, 411 polychrome, 186 Saurefuchsin, 351 for skeletons, 377 Methylenazur, 186, 187 Meves-Flemming modification, 36 Victoria green stain, 333 Meyer, Berlin blue neurofibril method, 434 methylen blue nerve method, 477 myelin, 445 new celloidin method, 103 Mibelli, 352 Mica sheets, for mounting sections, 119 Michaelis, 133, 181 blood stain, 383 fat, 367 polychrome methylen blue, 187 Triton eggs, 277 Michailow, 190, 195 Microchemistry, 293 et seq. Microglia, 493 Micro-injection, 270 Microscopes, S Microtome, 8 sliding, freezing, rotary, Jung, Minot, Tetrander, rocking, 9 Migula, 219 Miller, cement, 229 Minchin, albumen method for mammal eggs, 266 embedding protozoa, 542 hsematozoa, 547 sponges, 525 Minervini, 353 Mitochondria, fresh examination, 332, 333, 334 Gatenby on, 316 protozoa, 540 teeth and bone, 375 vital stains, 332 Mitrophanow, 183, 342 chick embryos, 273 integument, 339 myelin, 447 Mitsukuri, reptile blastoderms, 275 Mixed process of Golgi,'460 Mobius, 348 maceration medium, 246 Moerner, 377 Moleschott and Piso Borme, salt alcohol for maceration, 244 Molge, embryology, 277 Moll, oartUage, 376 Mollison, Alkanna, 367 INDEX. 583 MoUusca, 282, 600 et seq. embryology, 282 et seq. to kill extended, 500 maceration of epithelium, 503 mucus glands, 504 narcotisation, 12 nervous systein maceration, 247 MoUuscoida, 500 Molybdenum-toluidin blue, neurofibrils, 417 Monckeberg and Bethe, 31 bleacher, 256 Mondino, 472 Montanari, 419 Montgomery, 515 nucleoli, 314 ova of Theridium, 287 Monti, 477 gastric glands, 393 Mordant, 131 Mordanting, for coal tar dyes, 165 Moreaux, f ormol - picric - trichloracetic, 62 Morel, 175 Morgan, Ascidia, 282 cockroach eggs, 285, 286 frog embryology, 276, 278 Morphia, for narcotisation, 16 Moseley, 503 Mosse, 415 argentamin, 452 Mosso. 380 Mott, 129 Mounting media, glycerine, 222 resinous, 225 Farrant, Allen, Brun, 221 sections, collodion paper method, 116 Mayer's albumen method, 113 watery, 116 Mozeiko, 503 vasodilators, 233 Muchsematein, 392 Mucicarmine, 392 Mucilage syrup mass, 110 Mucus, 391 et seq. glands, raoUusca, 504 Muir, 549 blood, 381 Mullenix, 497 Miiller, 392 Berlin blue mass, 240 fluid, 42 with formol, 63 for maceration, 245 silver method, 201 Mummery, embedding tooth germs, 372 nerve tissue of teeth, 373 J. A. Murray, Altmann's method, 321, 365 on glycogen stain, 296 connective tissue stain, 354 chrome- osmic method, 326 lead gum, 107 Muscle-fibres, maceration, 246 and tendon, 344, 347 spindles, 345 Myelin, ammonio -chloride of tin, Besta, 448 and axis cylinder stains, 452 Berkley rapid method, 447 bulk stain, 448 iron method, 451 Kultschitzky, 447 osmic acid, 450 et seq stains, 442 silver nitrate, 451 Weigert stains, 442 Myers, 415 Mytilus, 500 Myxosporidia, 546 Nabias, gold chloride Golgi method, 476 mollusca, 501 Nageotte, 399, 448 Nails, 341 Nakahara, urea fixation, 305 Nakanishi, blood, 383 Nansen, 245 Naphtha, clearing, 77 Naphthalin, monobromide, 224 rose, 169 Naphthol, 390 Naphthylamin brown, 408 Naples water bath, 79 Narcotisation, anemones, Holothuria, Ascidia, Mollusca (menthol), 12 Medusae, starfishes (chloroform). Actiniae, Capitellidse (ether alcohol), 13 Cristatella, 14 larvae (chloretone), 14 Alcyonella, Bryozoa, Annellida, Mol- lusca, Nemertians, Actiniae (chloral), 14 hydroxylamin, 15 magnesium chloride or sulphate, 15 morphia, curare, stiychnin, prussic acid, 16 asphyxiation : tobacco, carbonic acid gas, soda water, 16 hydrogen peroxide, 17 Hydra (cocaine), 14 Vorticellidse, Rotatoria, Vermes (eu- caine), 15 Ascidia, Ehopalsea, 15 Ciona, 16 Medusae, 16 snails, 16 menthol, nicotine, 12 chloroform, cocaine, eucaine, ether, alcohol, 13 methyl alcohol, chloreton, chloral hy- drate, 14 Nasal mucosa, maceration, 247 Nathusius, 341 I Nealey, bone sections, 372 584 INDEX. Nebenkern, 316 Nelis, brain fixation, 405 Nematodes, 513 et seg. corrosion of chitin, 250 ova, 289 Nemertina, 514 et seq. heat killing, 12 Nephridia, Histriobdella, 511 Nerve endings, beetle, 344 Bielschowsky, 426 et seq. frog, 344 gold method, 344 insect, 345 methylen blue method, 344 silver method, 345 bichromate of silver, 345 of teeth, 373 et seq. staining, methylen blue, 189, 191 Nervous system, Achucarro's tannin neuroglia method, 490 Apathy's neurofibril method, 416 axis cylinder, 452 axis cylinder and dendrite stains (Golgi and others), 454 Cajal's double-impregnation process, 461 Golgi bichromate - sublimate method, 470 processes similar to Golgi methods, 475 Cajal's method, advice as to choice of formula, 424 (Golgi), axis cylinder and dendrite, treatment of larvae, 459 — 460 axis cylinder and dendrite, form- aldehyde modifications, 463 Berkley rapid myelin method, 447 Bielscliowsky methods, introduc- tory, 426 for sections, 427 for peripheral nerve fibres, 428 for pieces, 429 modifications, 430 el seq. Cajal's gold chloride neuroglia method, 487 methods, special objects, 424 neurofibril methods, 419 et seq. eollodionising sections, 406 Da Fano's Bielschowsky methods, 432 Donaggio's neurofibril methods, 417 Fish's fluid, 402 fi-ralion, 397 alcohol, 400 chromic salts, 403 et seq. formalin, 401 general methods, 397 stains, 407 Golgi method, avoidance of preci- pitates, 466 cutting and mounting, 467 — 469 Qolgi internal apparatva, 435 et seq. Nervous system — contd. Golgi-Cox modification, 472 hardening, 398 et seq. Held's method for marginal neuro- glia, 484 injection fixation, 397 Kultschitzky, myelin, 447 maceration, 245, 246, 247 Marchi method, 449 neurofibrils, 416 et seq. other methods, 434 neuroglia, 479 et seq. Nissl substance, 410 et seq. Pal method, 446 phosphomolybdic acid hsemato- xylin, 157. 158 polarised light, 398 preliminary fixation for Sauro- psida, 406 Sand's neurofibril method, 434 sections, 405 et seq. special cyfohgical methods, 410 et seq. sustaining apparatus of medullary sheaths, neurokeratin, 439 Simarro's process, 419 Weigert's myelin stains, 442 et seq. Nesteroffsky, 206 Nettovitch, 505 Neuberger, decalcifier, 254 Neubert, 297 Neukirch, 297 Neumayer, 262, 270 Neurofibril methods, 416 et seq. advice on, 424 Neuroglia; 479 Achucarro's tannin method, 496 Cajal's gold chloride sublimate method, 487 granules, 486 protoplasmic, 486 marginal, Held's method, 484 methods, 479 et seq. » stains, Aiiglade and Morel, 483 Da Fano, 484 Held, 484 Oppenheim, 486 Weigert, 480 Benda, 482 Mallory, 483 Neurokeratin, 439, 441 Neurosomes, 415 Neutral, balsam, 226 dye, 120 gentian, 396 rerl, 179 et seq. blood, 382 granules, 389 kidney, 394 mucin, 392 paucreas, 395 Neutralisation of carmine mass, 235 NeUVille, silver impregnation mass, 237 INDEX. 585 Nicolas, 32 embedding in gelatin, 93 reptiles, 275 NicoUe, 168 Nicotine, foi narcotisation, 12 Nigtosin, 170, 182, 408 Nikiiorow, 102 Nile blue, connective tissue, 354 fat, 362, 368 Nissl, 4:10, 411, 415, 450, 480 brain hardening, 403 bodies, 410 et seq. stains other than Nissl's, 412 granules, nature of, 130 methylen blue and erythrosin, 415 method, precautions against fading, 413 modifications, 413 Nitric acid, and acetic, for maceration, 247 bleacher, 257 and chlorate of potash, for macera- tion, 247 corrosion, 250 decalcification, 252, 253 for fixation, 38 fixative for neurofibrils, 417 formol, 64 maceration, 247 nerve tissue, 404 Noack, 83 Noll, corrosion, 250 Nordmann, mast cells, 355 Nosema, 546 Nowak, sublimate formol, 63 tactile corpuscles, 342 Nuclear stains, coal tar, 159 et seq. Nuclease, 308 Nucleoli, Gatenby on, 308, 310 malachite green for, 181 Nucleolini, 311 Nucleus, " vital " stains, 310 Nudibranchs, 502 Nuttall, Cooper and Robinson, 506 Obeisteiuer, brain, 403 Obregia, 469 method for paraffin and celloidin sections, 115, 119 Obst, nucleoli, 314 Octopus, 501 Odenius, maceration, 247 Odier, Golgi method, 465 Oestergren, 13, 515, 517 Ohlmacher, 167 alcoholic corrosive, 47 fluid, biain, 405 formaldehyde process, 165 myelin, 452 picric acid, 176 Oils, 65 et seq. See also under definitive name, e.g., Cedarwood. winter green, 271 Okajima, elective stain for reds, 388 fat, 368 Olmachec, 404 Olt, manipulation of frozen sections, 109 Opalina, 533 Ophiothrix, 519 Ophiuridea, 519 Oppel, lattice fibres, 393 Oppenheim, neuroglia granules, 486 Oppitz, 201 Orange G., 172, 173, 177 Orcein, 212 embryonic cartilage, 376 elastin, 353 method, Unna, 351 water blue, 340 Orchella, 211 Orcin, 212 Organic acids, fixation by, 51 Orientation methods, 273 et seq. for blastoderms, 273 • in embedding, 80 et seq. Origanum oil, 68 for celloidin sections, 102 Otr, 451 Marchi method, 450 Orth's fluid, 260 Orthoptera eggs, 286 Orton, 503, 508 Osmacet, 380 Osmium chloride, 50 Osmium tetroxide, reduction, 29, 32, 124 acetic acid maceration, 246 bleaching, 31 restoration of staining, 31 for blood, 380 gold method (Viallane), 205 ICopsch method, 327 nerve tissue, 404 and fat, 356 et seq. and picric, 56 pyrogallol method, 208 regeneration of, 30 fixation by vapour, 30 after-treatment, 30 sublimate, 48 vapour method, Cramer, 330 Ossifying cartilage, 254, 377 Osteoblastic areas, 378 Ostracoda, 505 Wo. Ostwald, 125 Ova, of Echinoderms, fixation, 260 fixation of mammalian, 265 frog's, 275 et seq. Inseota, Pisces, 259 treatment of uterine eggs of mam- mals, 266 Ovary as index to pregnancy, 263 Ovens, paraffin, 79 Oveihardened tissue, rejuvenation of, 462 Overton, 31, 34 iodine vapour, 51 Oviatt and Sargent, 233 586" INDEX. Oxalic acid, for maceration, 247 Oxidised hsematoxylin (Unna), 155 Oxidiseis as fixers, 21 Oxydase reaction, 390 Oxygen, place in tissue, 390 Pacini, 220 blood, 380 Pal, 472 Faladino, 453 Palladium chloride, 50 decalcification, 252 Palythoa, 522 Fansch, injection, 242 Pancreas, 395, 396 inclusions, 338 Pancreatin, digestion, 248 Paneth, cells, 392 Paper trays, embedding, 73 Papillae foliatse,-342 See also under Corpuscles. Pappenheim, 356 hemopoietic tissue, 386 mast cells, 355 panoptic stain, 383 pyronin and methyl green, 172 Paracarmine, 142 Paraffin, pure, 91 overheated, 92 sections, flattening, 90 clearing and mounting, 91, 111 et seg. embedding, 76 et seq. shaping block, 83 et seq, cooling, 82 bath, 77 Paramoecium, culture, 537 Paravicini, 418 Parietal cells, 393 Paris green, 160 Parker, 508 bleaching, 257 methylen blue, 194 and Floyd, sheep brain, 402 Parlodion, 95 Parmenter, Amblystoma, 306 Parolein, 228 Parthenogenesis, 259 Partington and Huntingford, 32, 364 Fartsch, cochineal alum carmine, 137 decalcification, 254 Passarge and Krosing, 354 Paton, 272 Bielschowsky modification, 430 Patten, 286, 502 moUusca, 504 orthopterous eggs, 286 orientation during embedding, 81 Patterson, chick, 273 Fauropoda, 505 Pavlov, myelin, 446 Peabody, corrosion, 249 Pearl, worms, 509 Fedicellina, 500 Pekelharing, 297 Pelagic fish ova, 281 Fenfield, Golgi apparatus, 438 Holmgren's trophospongium, 439 Fensa, reconstruction method, 261 Peppier, 549 Pepsin, digestion, 248 Peptic cells, 392 Peptonum siccum, 233 Perchloride of iron impregnation, 209 Perdrau, Bielschowsky method, 434 Perinyi's fluid, 39 P^rez, fly pups, 286 Periplaneta eggs, 285 Permanganate of potash, bleacher, 31, 256 Henneguy's mordant, 165 for osmic solution, 29 Perophora, 282 Peroxide, of benzol, blood, 382 of hydrogen^ bleaching, 256 for killing, 17 of magnesium, 256 of sodium, 256 Perrier, worms, 509 Perrin, 122 Pernsini, 453 Peter, 140 reconstruction method, 262 yolk stain, 260 Peters, R. A., culture of Paramceoium, 537 Petromyzon, nerves, 405 Fetronne, 387 Petrunkewitsch, Hymenoptera, 286 mercur6-nitric, 48 Pfltzner, 182 protozoa, 541 safranin, 166 Phalacrocera, 286 Phalangida ova, 287 Phenol, solution for fixation, 47 Phenylen brown, 161 Philippson, 339 FhloiQglucin decalcifying mixtures, 254 Phloxin, 180 Phoronis, 512 Phosphatides, 356 et seq. Phospho-molybdic acid haomatoxylin, Mallory, 157 Phospho-tungstic acid fixative, 38 Phosphoric acid decalcification, 252, 254 Photographic negative varnish mount, ing medium, 228 Photoxylin, 95 Physa eggs, 284 Fianese, 175 methylen blue eosin, 181 Ficraminic acid, 176 Ficcic acid, 54 decalcification, 252 ! plasma stain, 176 INDEX. 587 Picric acid — contd. and alcohol, 55 for maceration, 248 Picro-acetic, 55 Picio-carmine, 140 general nerve stain, 414 Picro-chromio, 56 Picro-hydroohloric, 56 decalcifier, 254 Picio-indigo-earmine, 212 Picro-nigrosin, 182, 213, 350, 408 Picro-nitric, 55 decalcifier, 254 Picro-osmic, 56 Picro-platinic, 56 with formol, 63 Picro-Saurefuchsin, 176, 350 Picro-sublimate, 48 Ficro-sulphuric, 55 Pictet's liquid, 219 Piiik salt, 418 Pintner, 29, 515 Pisces, embryology, 279 et seq. Pitfield, flagella, 549 Pittock, reconstruction method, 262 Pizon, Ascidia, 282 Placenta, glycogen, 295 Planaria, 44, 517 Plankton, general preservative, 525 Planorbis ova, 284 Plants, Golgi apparatus, 325 Plasma cel,s, 354 et seq. fibrils, 339 stains, 171 et seq. BoUes Lee, 314 Plasmiodium, 546 Plasmosome, 310 et seq. Plastic reconstruction, 261 Plastochondria, 315 Flatino-aceto-osmic, 38 Platinum chloride, 49 Platner, neurokeratin network, 441 Kemschwarz, 211 Pleschko, 194 Du Plessis, 515 Pleurobrachia, 524 Pluteus, 520 Podwyssozki, 167 fluid, 36 Polaillon, perohloride of iron impregna- tion, 209 Polariscope, 332 for fat, 361 et seq. in nervous system, 398 Policard, 547 Politzer, 496 Polumordwinow, 413 Polychoerus ova, 288 Polychrome methylen blue, mast cells, 365 nerve cells, 476 for nerve tissue, 414 Polychrome toluidin blue, 196 Polyclads ova, 289 Porifera, 525 et seq. decalcification, 251 Post-chroming, 326 Post-impregnation for gold, 207 et seq. Post-mortem changes, 25 Post-osmicating, 326 Potash method, for skeletons, 378 Potassium, 301 See under Bichromate, bichromate, maceration with, 245 chlorate and nitric acid, for macera- tion, 247 iodide, brain, 404 permanganate, for maceration, 246 sulphocyanide, for maceration, 245 Potter, 446 Fouchet, bleaching, 256 Pranter, 353 Precipitates, Golgi method, 466 Prenant, 181, 301, 497 Preservation media, 216 ■ by alcohol, ether, xylol, or toluol glycerine mixtures, acetic acid, cedarwood oil, 4 Preyer, 13 Priapulus, 512 Prickle cells, 339 Primerose soluble, 180 Fritchard, 498 Progressive stains, coal-tar, 159 Propylic alcohol balsam, 227 Protease, 308 Protozoa, 526 et seq. Borrel's blue, 534 Borrel method, 532 cell inclusions, see pages 316 — 338 collection, 529 culture; 527 et seq. of paramoecium, 537 current stains, 543 et seq. determination of life cycle, 530 Drew-Griffin live slide, 530 embedding, 542 examination in a coloured medium, 540 of faeces, 538 et seq. fixing and preserving, 541 et seq. flagellates, 533, 547 general fixation and staining, 531 et seq. general morphology, 531 hjematozoa, 546 killing by heat, 12 iodine, 541 Eomanowsky methods, 535 immobilisation, 539 staining Coccidia, 532 ciliates, 533 hsemamoebse, 533 intra vitam, 540 stains for flagella, 548 sporozoa, 546 sphserozoa, ^46 588 INDEX. Protozoa — contd. Taylor's culture method for amoebse, 536 trypanosomes, 533, 539, 547 Frowazek, 179 Prussian blue, aqueous mass, Ranvier, 240 acid cold, Beale, 239 cold, Beale, 239 glycerine mass, cold, Ranvier, 239 Ranvier, 236 reaction, 297 et seq. Prussic acid, 16 Przesmycky, nuole\»s staining, .310 Fulmonata, nervous system, 501 Purcell, Atta ova, 287 eyes of Phalangida, 508 Purpurin, 212 Fusateri, taohiol, 425 Puschkarew, 542 Pyridine, 59 with luoidol, 59 nitrate-pyridine fixative, 418 Pyroligneous acid decalcification, 252 Pyronin, 172 and methyl green, 172 pancreas ducts, 395 Pyrosin B., 180 Pyroxylin, 97 Pyrrhol cells, 388 Pyrrol blue, 389 De Quervain, 26, 398 Quinolein blue, 182, 367 Baadt, blood stain, 385 Babl, 341 chromoformio, 35 cochineal, 137 embryological fixation, 260 lens, 343 picro-sublimate, 48 platinum chloride, 49, 50 platinio sublimate, 260 superheated paraffin, 89 teleost eggs, 280 Rabl-Buckhard method for salmon eggs, 280 ^* Bacovitza, mollusca, 504 Baffaele, 281 Raja, electric organ, 346 Bana, embryology, 277 Randolph, 14 Banke, neuroglia method, 487 Bansom, 516 Banson, Cajal's method, 426 Eanvier, 182, 340, 356, 393 alcohol, 58 bladder, nerves, 349 chromic acid maceration, 246 carmine gelatin mass, 234 cornea, 343 Banvier — ctmtd. electric organs, 345 formic acid, gold, 204 -eleidin granules, 341 eye of Triton, 494 lemon juice, gold, 204 maceration with alcohol, 244 nerve endings, gold, 345 silver method, 345 pioro-carmine, 141 silver impregnation mass, 237 method, 198, 200 Vom Bath, pioro-sublimate, 48 Eawitz, 38, 184, 502 brain hardening, 404 carmine, 138 indulin, 409 mucin, 392 picro-nitric, 56 V. Recklinghausen, silver, 200 Beconstruction from sections, 261 Bed blood cells, elective stain, 388 gelatin mass, 234 Beddingius, 314 Bedenbaugh, IS Van Bees, 285 Befractive indices of media, 216 Begaud, formol-bichromate method, 323 myelin stain, 442 Begenerating nerve tissue, 421, 425 Regressive stains, 162 et seq. coal tar, 159 Behm, 227 Nissl method, 413 Beichenbach, deoapoda ova, 288 Beinke, 177, 341 maceration, 248 orange method, 171 Bejsek, corrosion, 249 Rejuvenation of tissue, Golgi, 462 by veronal or chloral, 421 Remak and Goette's fluid, for frog eggs, Renault, 214, 343, 390 Bengel, 38 Beptilia, embryology, 274. e< '.seq. brain, 405 Resins, 225 et seq. Besorcin-fuchsin, 353 for embryonic cartilage, 377 Betina, 493 et seq. of arthropods, maceration, 248 bleaching, 255 maceration, 247 Retterer, smooth muscle, 348 tendon, 347 uterine eggs of mammals, 267 and Lelievre, 377 and Zenker, injection, 242 Retzius, 191 Rhumbler, 75 Ribbons, paraffin, 86 and 89 et seq. Bichards, 14 Bieder, 367 INDEX. 589 Ringer's solution, 218 Bio-Hoitega, Bielschowsky, 431 neuroglia, 490 — 492 Bipart and Petit's fluid (copper), 53 Bitter, 282 Robertson, 449, 451 tsetse flies, 539 and Maodonald, 474 Robin, injection masses, 233 et seq. and Ranvier, 232 Robinski, silver, 200 Rochon-Duvigneand, 495 Roessle and Yoshida, 394 RoUett, 343 maceration, 246 Bomanowsky, stains, 384 et sej. Boncoroni, rodlet of, 421 Boosvelt, 210 Bosaniline, 120 Rose, bone, 370 Rose B, 180 Rose de naphthalin, 169 Rosein, 169 Rosenstadi, 340, 509 Rosenthal, fat, 367 Rosin, 181,413 Ross, blood, 383 thick film method, 534 Rossi, 449 blood, 380 flagella, 549 Rossolimo, 451 Rotatoria, 512 Jennings, 288 Rothig, 169 axis cylinder stain, 453 mammals, 263 methylen-azur, 408 Rouget, 191, 200 Rousseau, 497 decalcification, 251 desilicification of sponges, 255 sponge, 526 Rousselet, 15, 512 preparing aqueous mounts, 23 ) Rubaschkin, neuroglia, 482 Rubin, .169 method for neuroglia, 486 Rubin S, 171 Ruffini, 347 Russell, glycogen staining, 290 _ Russo, Ophiothrix, 519 Rnzi6ka, 314 Ryder, 105 Sabin, micro-injection, 271 Saefftigen, 513 SaSrosin, 180 Safranin, 165 et seq. elastic tissue, 352 and light green, 181 mucin, 391 myelin, 452 and wasserblau, 351 Saguohi, 396 pancreas cells, 338 Sahli, 226 brain hardening, 403 myelin, 452 Sainton, 400 Sala, 440 axis cylinder dendrite stain, 459 Salamandra, embryology, 277 Salicylic acid, fixation, 63 Saling, Tenebrio, 506 eggs, 287 Salivary glands, 392 Salkind, lead gum embedding, 106 Salmonidee, embryology, 280 Rab'.-Riickhard method, 280 Salts, for fixing, 40 solutions, 218 for maceration, 244 sublimate, 46 Samassa, 468, 525 Samter, 75, ^88 Sanchez, 425 Sand, 482 neurofibril method, 434 Sandal-wood oil, 68 Sandarac, 228 media, Gilson, 227 Sanders, 473 Sankey, 408 Sansom, Camoy modification, 53 Sanzo, fixing apparatus, 260 Sarcolemma, 344 Sata, fat, 367 Sattler, 201 Saurefuchsin. See under Acid Fuchsin. Saurerubin and Orange G, 393 Sauer, kidney, 394 S'aureviolett, 181 Savini, 177, 415 Sazepin, 506 Scala, 542 Scarlet B. See Scharlach. Scarpatetti, 408 Schafet, 232 Schaffer, 251, 261, 300, 350, 356 bone, 370 decalcification, 252, 254 thionin for bone and cartilage sec- tions, 376 Scbaper, 262 Scharlach R, 356 et seq. fat, 367 Herxheimer, 367, 368 Schaudinn, fluid, 546 Woodcock's modification, 541 Sctaaxel, 309 Schiefferdecker, 67, 68, 444 celloidin masses, 241 eye, 496 maceration mixture, 247 , and Kossel, 356 Schlemmer, Bielschowsky, 431 Scbmaus, 407 590 INDEX. Schmidt, gastropod embryology, 283 Sohmorl, thionin for bone and cartilage sections, 376 Schneider, 300 aceto-carmine, 138 Schonemann, 262 Schreiber, Golgi method, 464 Schridde, 356 blood, 381 mitochondrial method, 325 Schrotter, 408, 449 Schuherg, malaria, 547 Schulemann, 389 Schultze, 219, 496 chrome hsematoxylin, 157 frog embryology, 278 iodised serum, 219 muscle, 348 osmium hsematoxylin, 158 oxydase reaction, 390 potash method, 378 tendon, 347 Schumacher, 354 Schiirmayer, 539 Schiitz, 425 Bielsohowsky, 430 Schwaihe, 407 cochlea, 497 Schwarze, 517 Sclavo, 549 S. G. Scott, 119, 180, 498 blood, 381 Romanowsky stain, 386 neutral balsam, 226 standard hsematoxylin stain, 312 Scott and Osborn,triton embryology, 277 Scyphistoma, 524 Secretion granules, 315 Section-grinding, 108 Sections, crumpling, 88 flattening, 86, 90 mounting, Henneguy's method, 114 Seeliger, 520 Segregation granules, 389 Sehrwald, 468- Golgi methods and precipitates, 466 Seidenmann, 192 Seiler, alcoholic balsam, 226 carmine and indigo, 212 deoaloifier, 254 Selachia embryology, 280 el seq. Selenka, 269 Seligmann, 493 Sensory ganglia, 424 Serial section mounting, 111 Serum, for maceration, 243, 244 media, 219 Severeanu, injection, 242 Shearer, 511 Sheep, brain, 402 Sheldon, 446 Shell, mollusc, 503 Shellac, for brittle sections, 89 embedding, 108 Shipley, 271, 310 benzidine dyes, 389 and Macklin, trypan blue, 378 Shun Ichi Ono, glycogen, 338 Siebenmann, 498 Silver, carbonate, neuroglia method, 492 impregnation, double-staining, 202 for elastin, 354 fixation, 201 marine animals, 202 metallic stain, 198 et seq. for nerve. See under Golgi, Ramon y Oajal, and Bielsohowsky. nitrate, reduction, 201 picrate, lactate, acetate, for staining, 201 Simarro, neurofibrils, 419 Siphonophora, 524 Siphunculus, 511 Siredon, embryology, 276 Sjovall, 435 formol and osmic acid, 331 Skeletons, cartilage, 377 Skin nerves, 341 Slides, cleaning of, 112 Slow-worm eggs, 275 Small objects, embedding, 75 Smears, blood, 379 of gonads, 307 et seq. lucidol fixation, 59 Smirnow, 341 Golgi method, 465 Smith, eyes of gastropods, 502 and Mair, 366 Loriain Smith, Nile blue, 368 Smooth muscle, 348 Snails, asphyxiation, 16 Snake blastoderms, 274 Snessarew, Bielschowskj', 352 Soap masses, 92 Sobotta, 269, 280 Amphioxus, 281 mammals, 263 Soda carmine, 140 Sodium chloride and alcohol, for macera- tion, 244 nitrite, 233 sulphalizarinate, myelin, 449 Solferino, 169 Solger, bleaching, 256 muscle,_ 344 salivary glands, 392 Solvents, for paraflSn, 76 Soulier, maceration, 245 De Souza, pjT^idine, 59 Spalteholz, method of clearing, 270 Spee, ?2, 270 Sphserozoa, 546 Spicules, sponge, 526 Spielmeyer, 442 Spinal cord, 424 axis cylinder and dendrite, 459 hardening, 399 See under Nervous System. INDEX. 591 Spindles, fixation, 301 Spiral filaments, 439, 440 Spleen, 393 Sponges, 525 'desilicifioation, 255 embryos and larvae, 52(i sections, spicules, 526 Sporozoa, 546 Spuler, 140 sublimate formol, 63 Squire, 173 blueing sections, 151 glycerine jelly, 223 Staining, adjective, 131 in bulk, 6 with carmine, theory of, 136 effect of heat, 124 electrolytes in, 131 hindrances to, aids to, 25 nature of, 121 progressive, 132 regressive, 132 removal of dyes, 125 substantive, 131 two kinds distinguishable, 128 unsafe criterion of chemical consti- tution, 133, 134 vessels, 6 Stappers, 506 Starfishes, narcotisation, 13 StatoWasts, 282 Stauflacher, oyclas eggs, 284 Steensland, 460 Stein, 497 decalcification, 251 Stempell, 546 Stephens, 549 Stephenson's high ref active mounting medium, 224 Stirling, maceration, 245 Stoeltzner, 300, 442 Stohr, 214 Storch, 482 Strahnber, 477 Zur Strassen, 614 Ascaris ova, 290 Strasser, 262, 407 Stratum granulosum, 340, 341 Streaker, brain, 402 Streeter, myelin, 448 Van der Stricht, decalcification, 264 thysanozoon eggs, 289 Strieker, gum embedding, 108 Strong, 398, 449 brain of acanthias, 405 copper bichromate, 64 Golgi method, 463 iron alum fixation, 50 Stropeni, 355 Strychnin, for narcotisation, 16 Students, guide for, 556 Studnicka, 362 Stylaiia, asphyxiation, 16 Styrax, 370 Styrax and liquidambar, 228 Sublamin, 49 Sublimate, bichromate, 43 Golgi method, 470 See under Mercury and Corrosive. substitution stains, 164 Suchannek, 3 anilin oil, 69 bergamot oil, 68 mounting medium, 227 Suchanow, Golgi apparatus, 438 Sudan III., for blood, 383 Daddi, 367 fat, 356 et seq. Sulima, 14 Sulphonic acid, 121 Sulphuric acid, maceration, 247 Sulphurous acid, 43 bleacher, 256 decalcification, 253 for teeth, 372 Sumita, 300 Summers, method for celloidin sections, 116 Sumner, 279 Suschkin, chick embryos, 273 Suspensoids, 123 Sussdorf, 391 Sustaining apparatus, of medullary sheaths, 439 Sympathetic ganglia, 424 Synapta, 518 Syrup media, 219 Szecsi, lucidol, 59 for blood, 382 Szent-Gyorgi, eye, 494 Sziitz, 185, 208 Tachiol, 425 Tactile corpuscles, 341 et seq. hair, nerve endings, maceration, 247 Tadpoles, intra vitam staining, 179 Taenzer-Unna, orcein method, 353 Tafani, 498 Taguchi, Indian ink mass, 240 Tannin, for mounting, 220 Tannin-fuchsin, for fiagella, 548 Tap water substitute, 161, 313 Tartuferl, 343 eye, 496 Taylor, Sister Monica, amoeba culture method, 536—537 chromosome methods, 303 Teeth, Carter, 369 et seq. decalcification, 253 embedding through carbon bisul- phide, 372 lymphatics, 373 soft parts, 371 et seq. vessels, 373 Tegumentary organs, 339 et seq. Teleost eggs, 279, 280 embryology, 259 592 INDEX. Teljatnik, 451 Tellyesniczky, acetic bichromate, 41 Temnocephala ova, 289 Tendon, 344, 347 silver, 200 Tenebrio eggs, 287 Terpinol, 69 as mounting medium, 228 Test-eells of Afeoidia, 282 Theory of dyeing, electrical, 124 of fixation, 18 of staining, 120 Tberidlum, 287 Thiersch, 212 Thin, 496 Thionin, 162, 168 cartilage, 376 for Golgi apparatus, 438 for intra vitam staining of nerve, 196 mucin, 391 pancreas, 396 for skeletons of cartilage, 377 Thionine ph^niqn^e (NicoUe), 168 Thiophen green, 182 Thoma, decalcification, 253 indigo-carmine mass, 239 and Fromherz, corrosion, 249 Thome, 173 Ihompson, Arthur, reconstruction method, 262 Thomson, J. G., Giemsa stain, 545 Thread cells, 521 Thyme, oil of, 68 Thymus, 394 Thyroid, 394, 395 Thysanozoon ova, 289 Tigroid substance, 410 et seq. Timofejew, 196, 393, 405 Tirmann, 300 Tischaikin, 3 Tissue culture, 650 et seg. Toison, staining fluid for blood, 382 Tolu balsam cement, 231 Toluidin blue, 169 for cartilaginous skeletons, 377 and erythrosin, 415 for intra vitam staining of nerve, 196 myelin, 449 Niesl granules, 412 — 413 Toluol, 70 Tomaselli, neurofibrils, 418 Tonkoff, 183 Torpedo, electric organ, 346 Tortoise embryos, 275 Tower, Moniezia, 515, 616 Tozer, 513 Tracheae, 508 Tracheata, 605 Trachymedusee, 523 Trematodes, 516 et seg. ova, 289 Trenkmann, 549 Triacid mixture, 175 Trichina, 514 Trichlor-aceiic, 63 decaloifier, 254 Trimming blocks, Eternod, 83 Triple stain, Bonney, 178 Triton (Molge), embryology, 277 Trophospougium, 439 True fats, 356 Trypan blue, 389, 390 cartilaginous skeletons, 377 dental pulp, 373 violet, 390 Trypanosomes, 533, 539, 547 Trypsin, digestion, 248 Trzehinski, 406 Tschaskin, 389 Tschernyschew, 446 Tsetse flies, 539 TuUberg, 15 Tunicata, 499 kiUing, 12 ova, 281 et seg. Turbellaria, 517 et seg. embryology, 288 Turpentine, clearing, 69 for mounting, 227 dissolving fat, 322, 329 UexkiiU, 16 Underwood, 375 TJnio, methylen blue, 192 Unmasking iron, 299 Unna, 32, 34, 350, 388, 391 carbol-pjnroniu-methyl green, 172 collodion mass, 96 half -ripe haematoxylin stock, 153 on haematoxylin staining, 145 keratin, 341 mast cells, 355 methylen blue and Saurefuohsin, 351 orcein method, 351 oxidised haematoxylin, 155 polychrome methylen blue, 186 Rongalit white, oxygen test, 390 safranin and wasserblau, 361 sniooth muscle, 348 water-blue orcein, 339, 340 Upson, 407, 477 Uranium acetate, 54 nitrate silver method, for Golgi apparatus, 436 Urea and Bouin's fluid, 306 and chromosome fixation, 305 Urodele, embryology, 276 Ussow, cephalopoda, 282 Vanadium chloride process, 475 " haematoxylin, 157 Varnishes and cements, 229 et seg. Vaso-dilators, 232 et seg. Vassale, 460 Weigert method, 444 and Donnagio, Golgi method, 465 INDEX. 593 Vastarini-Cresi, embryonic cartilage, 377 silver, myelin, 451 Vejas, 408 Vejdovsky, 514 Venderovio, 407, 450 Venetian soap, 411 Venice turpentine, 227 for cementing, 230 Ventral cord, insect, 608 Veratti, 468 Veretillum, 523 killing, 12 VerhoeS, 354 Vermes, embryology, 288 et seq. general, 509 et seq. Vernon, 309 Vert d'Alcali, 160 Lumiere, 159 Verworn, 14 Vesuvin, 161 mast cells, 355 Viallane, 509 osmic gold method, 205 Vialleton, cephalopoda, 282 silver impregnation of chick, 274. Victoria blue, 169 mucin, 392 neuroglia stain, 483 green, 181 Violet B, 184 of Lauth, 168 Virchow, 33, 280 Vital stains, benzidine, 390 mitochondrial, 332 See under Intra Vitam. Seharlach VIH, myelin, 453 Vivante, bone, 375 Vivisection Acts, caution against, 265 Vogt and Tung, 511 Cestodes, 515 Cucumaria, 518 worms, 509 Volk, 17 Vosmaer, reconstruction method, 261 and Pekelharing, sponges, 526 Vosseler, wax feet, 243 Waddington, 13, 541 Waite, 288 Waldeyer, 498 decalcification, 252 Walsem. 449 Walton, tissue culture, 552 Ward, 16 Gephyrea, 511 Warnke, 407 Wasbburn, mollusc ova, 284 Washing out, after fixation in alcohol, formol, acetic acid, picric acid, nitric acid, corrosive, osmio, chrome, 26 liquids for, 26 Wasielewski, sporozoa, 546 Wasserblau, 183 blood-platelets, 386 and orcein, Unna, 340 and Safranin, 351 spleen, 394 Wassermann, 300 Watasi, cephalopoda, 282 Watch-glass, embedding in, 74 Water-baths, paraffin, 79 Water-blue. jSee under Wasserblau. Watery media, 217 Wax feet, 243 et seq. Webb, gum mass, 110 Weber, 512 Siphonophora, 524 Wedl, 211 Weed, 271 Weidenteich, 341 blood, 380 Weigett, 177, 450, 461 elastin stain, 353 fibrin stain, 388 hsematoxylin, 150 method for celloidin sections, 118 myelin, formol material, 444 stains, 442 et seq. neuroglia stain, 480 picro-Saurefuchsin, 215 Weigert-Pal, myelin method, 446 Weigl, Mann-Kopseh method, 328 Weil, 109 bone, 370 teeth sections, 373 Wellings, intra vitam staining of teeth, 373 Wermel, 381 Wernerj smooth muscle, 348 Wester, 507 Weysse, 269 Wharton's jelly, 391 Wheeler, eggs of Orthoptera, 286 White, bone sections, 370 Whitman, brains, 405 frog embryology, 276 pelagic fish ova, 281 Hirudinea, 511 Wickersheimer, 220 Widakowich, 270 Widmann, lens of Arachnida, 509 Van Wijhe, ammonia carmine, 141 cartilaginous skeletons, 377 Wilhelmi, 518 Will, reptile embryology, 275 Willebrand, blood stain, 383 Wilson, J. T., and Hill, J. P., 269 Wilson, Alcyonaria, 522 stain, 535 Wimmer, 482 Winiwarter, 177, 270 Wintergreen oil, for clearing, 271 Wislocki, 390 Von Wistinghausen, 155 De Witt, 3 4 Witte, pancreatin, 249 38 594 INDEX. Wittmaaok, 497 myelin, 451 WolfE, 388, 428 bladder, 349 WoUrum, elastin stain, 353 WoUschwaiz, 648 Wolter, 453 chloride of vanadium process, 475 Wolters, 376 myelin, 447 Woodcock, faeces, 538 and Wilson, Schaudinn fixation, 541 Woodger, 365 Woodland, 518, 521 Woodworth, reconstruction, 261 WooH, 168 Worcester's liquid, 280 Worms, 509 Wright, blood platelets, 386 Eomanowsky stain, 535 Wynn, 447 Xylol, 70 Yellow gelatin mass, 237 Yolk stain, Peter, 260 See under Fat and Cytoplasmic Inclusions, 316 et seq. Zacharias, 139, 505 acetic alcohol, 52 protozoa, 548 Zaleski, 300 Zander, 507 Zawaisin, cornea, 343 Zenker, fluid, 48 note on, 323 Zernecke, Ligula, 516 Zettnow, 549 Ziegler, decalcification, 253 teeth sections, 372 Zieglwallner, glycogen and fat stain, 296 Ziehen, gold sublimate, Golgi method, 475 Ziehl, carbolic fuchsin, 169 Zimmermann, 301, 352 Gfolgi preparations, 469 nucleoli, 314 Zinc, 300 chloride, 50 Zoantharia, skeletons, 522 Zograf, 542 Eotifers, 512 Zoja, 189 Ascaris ova, 290 Zosin, 453 Zschokke, 377 Zurn, 495 Zwaardemaker, safranin, 166 THE WHITEFRIARS PRE8B, LTD., LONDON, ^SD TONBEIDOK Sij-iSm-i