COLUMBIA LIBRARIES OFFSITE HEALTH SCIENCES STANDARD HX00077577 RECAP m ' i. .r> ■ , ,i ,..,iy.. , « t t«,. I. . . .', ), \ t' >■ ;' .' ..„ll" m iiwM, tntI)fCitpotBttogarli Irpartm^ttt of ^Ijgfiuilogg Digitized by the Internet Archive in 2010 with funding from Open Knowledge Commons http://www.archive.org/details/textbookofphysio01mcke A TEXT BOOK OF PHYSIOLOGY. in,iiinnrU!\TfO(.ocv A TEXT BOOK OF PHYSIOLOGY BY JOHN GEAY M'KENDEICK, M.D., LL.D., F.E.S. PROFESSOR OF THE INSTITUTES OF MEDICINE IN THE UNIVERSITY OF GLASGOW, FELLOW OF THE ROYAL COLLEGE OF PHYSICIANS OF EDINBURGH. INCLUDING HISTOLOGY BY PHILIPP STOHE, M.D., OF THE UNIVERSITY OF WiJRTZBURG. IN TWO VOLUMES. VOL. I.— OENEEAL PHYSIOLOGY. NEW YOEK: MACMILLAN AND CO. 1888. I/. I ^ B'C D Eh F G ' MoLc Muivn, deL acL Nax, Ifanfwrb UJJv. GENERAL PHYSIOLOGY: INCLUDING THE CHEMISTRY AND HISTOLOGY OF THE TISSUES AND THE PHYSIOLOGY OF MUSCLE. BY JOHN GEAY M'KENDPtICK, M.D., LL.D., F.R.S., PROFESSOR OF THE INSTITUTES OF MEDICINE IN TUE UNIVERSITY OF GLASGOW, FELLOW OF THE ROYAL COLLEGE OF PHYSICIANS OF EDINBURGH. NEW YORK: MACMILLAN AND CO. 1888. V. t PRINTED AT THE U^fIVERS^TY PRESS, QLASOOW, BV ROBERT MACLEHOSE. VIRO • ADMIRABILI ACERRIMO . IN . NATVRA • INVESTIGANDA IN . INTERPRETANDA • SAGACISSIMO lOANNI • BVRDON ■ SANDERSON QVI . LARGIS • INGENII ■ LVMINIBVS QVODCVNQVE • ATTIGIT ■ ILLVSTRAVIT SANO . IDEM . IVDICIO • ATQVE ■ AEQVO SOLIDA • SEMPER • VANIS • PRAETVLIT ■ VTILIBVS • HONESTA ATQVE . OMNIA . POSTHABVIT • VERITATI HOC • MVNVS • QVALECVNQVE D. D. lOANNES • G • M'KENDRICK PREFACE, Although this work has been modelled to some extent on my " Out- lines of Physiology," now out of print, it is essentially a new book, and as it aims at giving a more detailed account of the subject, the title of "Outlines of Physiology" has been abandoned for that of a "Text Book of Physiology." I have not endeavoured to give anything like an encyclopaedic account of the modern researches that have built up the physiological science of the present time, but rather to weave into a con- secutive narrative the main facts and principles of physiology, as these present themselves to my mind, after nearly eighteen years of experi- ence as a teacher of the science. Probably no science is advancing with greater rapidity, if one may form an opinion from the large number of original papers that are announced, month by month, and from the large number of investigators in the physiological laboratories of the Con- tinent, of America, and of our own country. Thus it is year by year becoming more difficult for a teacher to keep abreast of the wave of progress, and still more difficult to assimilate the new facts and to incorporate them with his previous knowledge. Each teacher will also view the subject more especially according to his predilections for the morphological, the chemical, or the physical side of the science, and it is not easy in the preparation of such a work as this to be im- partial and to present the science in all its completeness to the reader. An explanation may be expected as to my object in introducing another large work on physiology when in recent years several excellent and voluminous treatises have issued from the press and have met with X PREFACE. great acceptance. My chief reason is to have it in my power to place in the hands of my students a text book appropriate to the course of instruction in physiology, which it is my duty to give annually in the University of Glasgow. The present work contains most of the theoretical teaching given to the student, and I hope that with it in his hands, it will be possible for me to do more and more in the way of demonstration and of practical instruction. At the same time, I am not without the hope that the nature of the liook may be such as to com- mend it to a wider circle of readers, more especially to members of the medical profession who desire to become acquainted not only with the facts but also with the methods of physiology. The general plan of the book is to give an account both of methods and of results, and to introduce illustrations as far as possible |from the best sources. No expense has been spared to obtain good illustrations, not merely diagrams, but illustrations that will give the reader a fair idea of the appearance of the thing represented. The reader is recom- mended carefully to peruse the descriptions of the various figures, as, in these, information is often given which is not contained in the text. The work is primarily divided into two parts. First, that relating to the general physiology of the tissues, which forms the subject of the first volume ; and, second, that relating to the special physiology of organs, to be discussed in the second volume. This arrangement is simple and comprehensive. After an introductory section dealing with general notions as to living matter, more especially with reference to the great doctrines regarding energy, which form the basis of modern science, I proceed to discuss the nature and properties of the chemical .substances found in the body and the nature of the chemical reactions with which the phenomena of life are associated. In this section I have introduced a chapter explaining to the physiological student the views he should hold as to the true value of chemical formulae. This chapter, in the preparation of Avliich I received valuable assistance from Professor W. Dittmar, F.E..S., I introduced after much consideration, and because I know that in the minds of most students there is not a little confusion regarding this matter. No doubt such a chapter would be more appropriate in a strictly chemical work, but my experience as a teacher PREFACE. xi assures me that it will not be without its value even in a physiological text book, did it serve no other purpose than to shoAV how little we yet know of the molecular structure of organic chemical substances. In the preparation of this section I derived much assistance from Dr. Arthur G-amgee's Physiological Chemistry and from Beaunis' Physiologie Bumaine. I venture to direct the attention of the reader to the chapter on pig- ments in which these interesting substances are more fully discussed than has yet been attempted in any text book of physiology. The writings of Dr. C. A. MacMunn of Wolverhampton have largely assisted me in writing this chapter. Dr. MacMunn, as is well known, has made this subject his own special field of research, and from his stores of knowledge he gave me the valuable measurements of wave-lengths. He also kindly prepared the chart of spectra which forms the frontis- piece of this volume, and he added further to my indebtedness by read- ing the proof sheets of the chapter on pigments and by furnishing me with, many valuable suggestions. I consider myself fortunate in having secured Dr. MacMunn's co-operation, and I offer him my most cordial thanks. The next section deals with the physiology of the tissues. Here I was met by a great difiiculty. In my previous work scarcely any description of the microscopical structure of the tissues or organs, or Avhat is termed histology, was introduced, and the omission was regarded by critics as diminishing the value of the book. My own feeling was in favour of omitting the discussion of histology from a physiological text book, but after consulting several physiologists, in whose judgment I place great confidence, and after taking into account the wishes of my students, I found that the balance of opinion was in favour of introducing histology. At the same time, I am bound to admit that several physiologists gave good reasons for omitting histology. Having resolved to introduce this aspect of physiological science, the next difiiculty was whether I was prepared to face the trouble and cost of preparing a new set of histological woodcuts. After some consider- ation, I solved the problem by purchasing from Mr. Gustav Fischer, the well-known publisher in Jena, a set of electrotypes of the woodcuts in Professor Philipp Stohr's Lehrbuch der Histologie, published last year, and also the right of translating the work, and of ineori:)orating it with my 3fii PREFACE. book. This arrangement was effected Avith the cordial consent of the author, Dr. Stohr, whose name I therefore place on the title page. Dr. Stohr's work commended itself to me on account of the excellence of its descriptions, the fidelity of its illustrations, — which are not diagrams but drawings of real preparations, — and the value of its practical directions I have therefore made free iise of it, introducing special descriptions and illustrations, where these seemed to be needful, and I have done this the more readily as the directions given l)y Dr. Stcihr are substantially those followed from year to year in the practical classes held during the summer session in the University of Glasgow. Having Dr. Stohr's work at my command has also led me to introduce more in the way of practical directions as to histological methods than I might otherwise have done, so that the present volume will be a guide to the student during the practical work of summer, and at the same time serve as a systematic text book. One unique feature of Dr. Stohr's work is that he gives an account of the method by Avhich each preparation figured was prepared. These descriptions of methods I have relegated to an appendix and numbered consecutively so as to admit of ready reference. Before taking up the physiology of the tissues, I have discussed their origin in the light of recent investigations regarding the phenomena of fecundation and the minute structure of cells and of nuclei. This is a novel arrangement, so far as recent systematic works are concerned, but it has commended itself to me as one leading to a philosophical view of the whole subject. The cpestions as to the origin of the tissues and the mysterious Avay by which they are impressed with hereditary characters are of profound importance and have a bearing not only on physiological but on pathological theories. The portion dealing A^ath the theories of heredity has been read by Professor E. Eay Lankester, F.R.S., who, Avhile not agreeing with me in m}' general conclusion, has giA^en me A^aluable suggestions and criticisms. The Avhole of this section has also been read by Mr. J. H. Fiillarton, M.A., B.Sc. Section III. deals Avith the contractile tissues, the studj' of Avhich requires physical appliances. I still hold that AAdthout burdening a student by requiring of him a knoAA'ledge of complicated apparatus, it is possible to giA^e him, shortly, such information regarding methods as AAdll enable him to take an intelligent view of results. Instead, hoAvever, PREFACE. xiii of introducing a description of apparatus in the parts describing results, I have discussed apparatus and methods as far as possible in separate chapters, so that the reader may pass over these if he chooses. The importance of the uses of electricity in practical medicine and surgery appears to me to justify the account given of electrical apparatus, for assistance in the preparation of which I am indebted to Mr. Thomas Gray, of the Physical Laboratory of this University, and an accomplished electrician ; and the immense value of the graphic method in all sciences dealing with movements also warranted me in giving a brief account of its chief instruments. As to the introduction of physical questions into a text book of physiology, a good deal can be said on both sides. No doubt if students of medicine and practitioners read good text books on general physics, or the excellent works on physiological physics that have ajDpeared in recent years, there would be little or no necessity for introducing these matters into a text book of phj^siology, except in so far as all physiological questions ultimately resolve themselves into physical problems. But the fact is that the teacher has usually to deal with students who know little or nothing of physics. The examination in mechanics required for the registration of a medical student is of no use, but is just sufficient to worry him and exhaust his energies without con- ferring any real benefit in the shape of a knowledge of the principles of physical science. Had he a course of instruction in general physics before beginning the study of medicine, matters would be on a different footing, but that is not at present available. Consequently the teacher need not lecture on the appearance of a muscle as seen with polarized light without, in the first instance, stating the more elementary facts regarding polarized light, and without explaining the construction of a polarizing apparatus. Again, before beginning the discussion of muscle in which the physio- logical teacher must make use of electricity, he is obliged to give some explanation of the nature of voltaic cells, of induction coils, and of the general facts regarding resistance, and of the detection and measurement of currents. For these reasons I have introduced certain details as to physics just as I am obliged to introduce them in my lectures. At the same time, I admit that this is so far a provisional arrangement, and if the time arrives when the student, beginning the study of physiology, has already been well grounded in even elementary physics, it may be abandoned. I have in the meantime chosen the mode of teaching xiv PREFACE. which experience has shown mc to lie the most useful under the circumstances. The chapter on the electrical fishes is longer than might be exi)ectcd in a general treatise on jihysiology, but I have thought it right to enter into details regarding these remarkable animals, as their physiology has an important bearing on questions as to the functions of nerve and muscle. I am much indebted to Professor Burdon-Sanderson for aiding me with a description and with drawings of the electric organ of the skate, in the investigation of which he and Mr. Francis Gotch, M.A., have been recently engaged and the results of Avhich have not been published. I have also to thank Mr. Gotch for looking over the proof sheets of this chapter and for valuable suggestions. In the preparation of the entire volume, I have gratefully to acknow- ledge the valuable services of my assistants. Dr. J. M'Gregor-Robertson, M. A., and of Dr. William Snodgrass, M.A. Dr. Snodgrass has read all the proof sheets twice over, and Dr. M'Gregot'-Robertson has read the final revise. Both have given me great help and excellent suggestions. Mr. J. T. Bottomley, M.A., F.E.S., and Mr. Magnus Maclean, M.A., have also aided me in the discussion of various physical questions. I have also to thank Professor Rutherford, F.R.S., Professor Marey, the Cambridge Scientific Instrument Company, Messrs. Churchill, Blackie & Son, A. & C. Black, Cassell & Co., Collins T.' i''''r i,ii frnn: ?".rri-Trn i ri-°-n. ZI. CariiTTTiKg- di "rriTfr TxEna. T^y "DariillliESE ir Thttij^ T mryt. M. Sl 21. 22. 3{L i^boo^ JMc Ciirfa-jtHrr ^teioyfeiL "WKomm ^ laiS-rm -Mrrnr "WPttit— yr) I'r "C::^, _ - . . - SL Wffinnm, 38. Yrei 3&. C^sQB, ST. Fr^li, « * 17 41 4f !. DJagtamrfAlwMKp auB ef Lj^^ HirwMel i lrM i , Affict, .32. WapwatwHm, JR^q^ -M. H-^ciiii. - frey, . 13» .>5. Bilimbiii. FreKy . 1^* -■j*;. Spectmii: of C"_l;. ; /_ --r KSJini;. 1* -57- Spectrum c: T. ^ - iTSA*;. 1* .56l Speelnimc: : . - KSkau. 1-^ -5©. Spectr-- ; : J _ : - .ffBto.?. IS «). Pigme-: r. - i%8y, ^"^ 6L Cholesteniu ... - Firj, . -^ ^Q. Fat C^b, showing Crystals, . Fmj. 149 <>3. Si )tie4«n > oliirTiiwt ^ of Ton Flexjchl, .... - -^ ftl. laoEsfie. ^'Tf!'- -^~ €9u QsaOateafline^ ... .Frs;;/^ loi> 6& TjiaealFanKaf Sdmoarfeetei. M^inhjoll W,i2^d,'ifter Zivf. !■*!» 67. Types of Spate Fonatttat in Srfifmmjeetes, . JfarsM? ir««l, mf^erZa^, 130 eS. BadHas Antfaadsy JfawftrfT Wkfi ^&r J&k*. IM ^. bMnliator, ... ^^ 70. Thezm»zega]ataF, 1^ 7L DngnBt diownig liaiSa&ta3aaD& . . . 302. Du Bois-Reymond's Electromotive Molecules, . 303. Hermann's Fall Rheotome, .... 304. Diagram showing the Arrangement of the Fall Rheotome, ....... 30-5. Torpedo Galvani, ...... 306. Nerve-Tuft in Torpedo, 307. Terminations of Nerve on the Plate of Torpedo, 308. Diagram of Prism of Torpedo, .... 309. Electric Plates of Torpedo, .... 310. Section through Electric Plate of Gymnotus, . 311. Transverse Section of Spinal Cord of Gymnotus, 312. Electric Nerve Cell of Gymnotus, 313. Portion of Electric Organ of Malapteriirus, 314. Diagram of Electric Organ of common Skate, . 31.5. Semi-diagrammatic View of Disc from Electric Organ of common Skate, .... 316. Diagram showing the Current Curves in Electric Discharge of Torpedo, 317. Motorial End-plate in Muscle, .... 318. Mode of Nerve-endings in Glands, Marty, Gscheidlen, C>/on, . M'Gregor-Rohertson, Cyon, . Bur don- Sanderson, Bur don- Sanderson, Ewald, Eivald, Eioald, Ranvicr, Friisch, Fritscli, Fritsch, Fritscli, Burdon-Sanderson Bur don-Sander son, Burdon-Sanderson, PJiiiger, PAGE 444 445 445 446 447 449 449 450 451 453 453 454 456 457 . 458 . 462 . 463 . 463 . 463 . 464 . 466 . 467 . 468 . 469 . 470 . 471 . 481 . 484 . 484 ERRATA. Page 55, line 8 from bottom, for Amines read Amides. Page 225, line 1 from top, for expression read extrusion. Page 310, line 4 from bottom, before Marshall inseH Melland and. TEXT BOOK OF PHYSIOLOGY. SECTION I. GENERAL INTRODUCTION. Chap. I.— NATURE AND OBJECTS OF PHYSIOLOGY. Physiology is the science which treats of the phenomena normally occurring in living things. It is divided into Vegetable and Animal Physiology, according as the life-history of plant or of animal is made the subject of consideration. Human Physiology, a depart- ment of the science which forms the special subject of this treatise, concerns itself with the phenomena occurring in man. The physiology of man, or indeed of any living being, cannot be studied with success if we regard merely the changes happening in the body of an individual, or even if we attend only to the phenomena of the species as a whole; for much of our knowledge depends on the study of operations occurring in a great variety of living beings. When we contemplate any living being, we observe, first, a peculiar physical structure or conformation of the solid parts ; second, a determinate chemical composition of its several solid and fluid parts; and, third, the occurrence of certain chemical, physical, and vital changes. In the physical structure of the body, certain parts may be readily isolated, and these may each perform a special office in the economy. Such parts are termed organs, and the office which each organ performs is called its function. Life, so far as the individual is concerned, may be said to be "the sum of the several functions performed by the various organs, or the active state resulting from their concurrent exercise." (Allen Thomson.) The body may be studied both in the dead and in the living condition. In the dead state its structure and composition are the subjects of anatomical and of chemical research The living state I. A 2 INTRODUCTION. is more especially the })ro^^nce of the i)liysiologist, it being his duty to study the functions, or the various phenomena manifested by the organs incliA'idually, and as part of a system, during life. The economy of the human body is one of great complexity, and in its study the physiologist finds it necessary to extend his investigations over a wide field. His information is derived partly from observation and experiments made directly upon man himself, both in the healthy and in the diseased conditions, but very largely also from investigations into the functions performed by the organs of other animals. Many of the facts of phj'siology with which w(; are at present acquainted have been derived from observations and experiments made on the humbler animals, but the apparently identical nature of many vital actions, more especially as mani- fested by the elementaiy tissues and in the simpler organs, and the general plan of structure on which organs are formed throughout the range of the animal kingdom, warrant us in applying these facts generall}^ to the elucidation of the functions of the human body. Physiology derives its facts from the observation of the phenomena of li'vang beings, made ^nth scientific accurac}^ and aided by deduc- tions from facts belonging to other branches of science, more especially anatomy, chemistry, and physics. A general acquaintance, therefore, Avith these sciences is of paramount importance to anyone about to enter upon the study of physiology. The student ought, in the first instance, to be acquainted with the structure of the human body and of its various organs, and he should also be familiar Anth the general plan of structure met A\dth in the great general subdivisions of the animal kingdom. Such anatomical knowledge {Human and Comparative Anatomy) must be obtained by dissection and by a careful comparison of organs in different groups of animals. During the past forty years, much knowledge has been accjuii-ed regarding the minute structure of the various tissues forming the body. This constitutes the department of science called Histulofjij. As many functions are j^erformed by elements of the body which are of microscopic size, it is evident that we must be accurately ac- quainted \nih. the anatomical structure of these elements. Such knowledge, therefore, belongs equally to the anatomist and to the physiologist. The anatomist must describe the form, size, and general relations of these minute parts, while the physiologist directs his attention specially to the phenomena they manifest Avhile alive. Both are engaged in the study of the same textural elements, but from different points of view — the one morphological, the other physio- NATURE AND OBJECTS OF PHYSIOLOGY. 3 logiail. Here, as in the study of parts visible to the naked eye, knowledge of structure and of function are really, in the language of Goodsir, different aspects of the same truth. To acquire a wide knowledge of the functions of the body, it is not sufficient to study it, even as regards its structure, only in its full-grown or mature condition. It is necessary to examine it at various stages of its growth, and to trace minutely the first formation and early development of the embryo, the origin of the primary tissues, the formation of the more complicated organs and systems of organs, and the process of growth of the foetus and child. This research is designated by the name of Embryology, and in recent times it has furnished important information regard- ing the history and bearing of vital phenomena. Embryology may be conveniently divided into two sections: (1) The history of the development of any single organism from the ovum onwards {Ontogeny); and (2) the history of the evolution of the group or race from lower to higher forms (Phylogeny). Thus comparative embryology has both an anatomical and a physiological side ; on the anatomical it is the basis of morphology, or all that relates to organic form, and on the physiological it gives a solution of many of the problems of function. From the facts and laws of Chemistry the physiologist derives in- formation regarding the chemical composition of the solid textures, as well as of the various secreted and circulating fluids, and of the variations to which their composition is subject in diff"erent conditions of the body. Physiological chemistry has as its ultimate aim the correct statement and explanation of the chemical processes occurring in the body, and it is generally regarded as the most difficult department of the parent science. The laws of Physics are applied by the physiologist to the investiga- tion of all the obvious motions of solids or fluids occurring in the animal organism and also to the explanation of those molecular changes of which a physical explanation can be offered. A knowledge of physics is indispensable in examining the functions of such organs of special sense as the eye and ear, and in studying the influence of light, heat, and electricity, in modifying vital phenomena. It may further be remarked, that while the researches of the physiologist are directed strictly to establishing the knowledge of the functions of the living economy in a state of health, he not unfre- quently receives assistance from the pathologist (Pathology), who having his attention called to the organic and functional changes that occur under the influence of different forms of disease, takes advantage as it were of so many experiments made to his hand by nature, and 4 INTRODUCTION. is thus better ahle to discriminate the share which each particular condition has in the production of these complex results. The physiological study of the functions of the human body de- pends then on the follo^\^ng means of investigation, viz.: (1) The observation of the phenomena of the body in a healthy state ; (2) The occasional comparison of the healthy state with moi'bid conditions of the economy; (3) The experimental variation in man or animals of the conditions in which vital operations take place ; (4) The anatomi- cal examination, by dissection and microscopic research, of the structure of the organized parts of the body at various stages of its growth or in the fully-formed state, or as found in a simpler condition in the bodies of the lower animals or even in plants ; (5) The chemical analysis of the solids and fluids, and of the substances which are taken into or are ejected from the system, and the history of the chemical changes which occur in the living body ; and (6) the appli- cation of the facts and principles of physics where these are admissible. As the object of physiology may be said to be "to ascertain the conditions of bodj^ and mind which are necessary to life and health," so the most important practical end which the human physiologist has in view is to furnish data for guiding the physician in detecting diseases with readiness, in distinguishing their different kinds Math accuracy, in endeavouring to preserve health, and when disease shall have occurred to allay suffering and to restore the natural condition. A knowledge of the conditions of health must precede the study of disease, and a rational system of surgical and medical treatment has its foundation in an acquaintance Avith the principles of physiology. But, apart from its practical aspect, physiology considered as a pure science, presents an attractive field of study, taking a A\ade range over the domain of nature and bringing the mind into the contempla- tion of some of the most profound problems and of some of the most sublime truths. Chap. II.— MATTER AND ENERGY. The permanence of matter and the permanence of energy are two great generalizations which form the groundwork of physical science, and they must be recognized in dealing "\vith the phenomena of the living body just as clearly as in considering the facts of chemistry and physics. 1. Matter. — In its jDassage through the living body matter cannot be created or destroyed ; if it apparently disappears it is only trans- formed into another condition. It need hardly be said that chemistry MATTER AND ENERGY. 5 only became an exact science when the permanence of matter was recognized. No portion of matter, however minute, can either come into existence or go out of existence in any chemical operation. The study of the chemical transformations in or by a living body is an important branch of physiology, and it is becoming more and more the aim of physiologists to obtain quantitative as well as qualitative results, feeling assured that as they succeed in doing so, will they bring their science nearer to the platform of physics and of chemistry. Measuring and weighing in units of length, time, volume, and mass are operations as important in physiology as in physics. As the metric system is now in use over the greater part of Europe and is spreading in America, it should be uniformly adopted in Great Britain, at all events in scientific literature, and I would strongly re- commend the student to become familiar with it. To assist him, I give the following short resume for reference : — 1. Length. — One metre is equal to 39-371 inches, 1 foot is equal to 30 '48 centimetres, and 1 inch is equal to 2 5 '4 millimetres. It may be noted that in the metric system the Latin prefixes denote division and the Greek prefixes denote multiplication. Thus decimetre, centi- metre, millimetre, and micrometre denote the tenth, the hundredth, the thousandth, and the millionth of a metre respectively; and decametre, hectometre, and kilometre denote ten, a hundred, and a thousand metres. These measures may also be squared or cubed. In Fig. 1, 4 inches are placed alongside of 1 decimetre. 3 Z d Fig. 1. — Comparison of a decimetre with 4 inches : a, centimetres ; i, millimeti-es ; c, -|ths of an inch ; d, inches. The micron, /x, the loVotli of a millimetre = -001 mm. and is now used in the measurement of objects of microscopic size : thus, the average diameter of the red blood corpuscles in man is 77 /x or '077 mm. 2. Time. — The unit of time is the mean solar second. In many physiological observations, fractions of a second, hundredths, or even thousandths, must be accurately determined by means of specially designed chronographs. (See Graphic Method.) 3. Mass and Weight. — In the metric system the unit of mass is 1 gramme, which is the mass (or weight) of a cubic centimetre of water INTRODUCTION. Fig. 2.— Square Centimetre. Via. 3.— Cubic Centimetre. Fio. 4.— Balance. MATTER AND ENERGY. 7 at its temperature of maximum density, 4° C. (See Figs. 2 and 3.) One gramme is equal to 15-4321 grains avoirdupois. It is also con- venient to remember that if we compare imperial units (Avoirdupois) Avith the metric system, 1 grain = yoV-o pound = -0648 gramme ; 1 dram - ^\-q pound ; 1 ounce (16 drams) = y'gth pound = 28*35 grammes ; 1 pound (lb.) = -4536 kilog. In Troy weight, 1 ounce — 480 grains = 31*10 grammes, and in Apothecaries' weight, 1 scruple = 20 grains, 1 drachm = 3 scruples, and 1 ounce = 8 drachms. In weighing, a good balance is indispensable. (See Fig. 4.) It consists of a light rigid beam resting on a fixed horizontal axis at its centre. A scale-pan is hung at . each end of the beam, one to receive the standard weight and the other to receive the body to be weighed. When there are no weights in the scale-pans, or when these contain equal weights, the beam is horizontal. Both the beam and the scale-pans are sus- pended on agate knife-edges working on agate planes. A good balance, loaded with a kilogramme in each pan, will turn with -0007 gramme, or about i^oio-o"oth of the weight in either pan. The weights are kept in a suitable box, as seen in Fig. 5. FiQ. 5. — Box of Weights. 4. Volume.— The unit of volume in the metric system is 1 cubic centimetre. In this connection it may be noted that 1 pint = "568 litre (the litre being the capacity of 1 cubic decimetre) ; 1 quart = 1*136 litre; 1 gallon - 4*544 litres; 1 litre = -22 gallon; 1 fluid ounce (in sale of drugs) = 1*8 cubic inch, or *0284 litre; 1 cubic foot = *02831 cubic metre; and 1 cubic metre = 1*308 cubic yard. 8 INTRODUCTION. The A-olunu! of Huids is measured liy flasks having a capacity when filled up to a mark on the neck of from 100 to 2,000 cubic centi- metres (Fig. 6) ; by graduated cylindrical glasses, as shown in Fig. 7 ; or by graduated pipettes, as in Figs. 8 and 9. Fig. 6.— Litre flask, J nat. size. 25CC 10 CC Fig. S.— Pipette for 10 cb. cm., i nat. size. Fio. 7.— Flask, iliiiit. size, graduated for 100 ub. cm. Fio. 9.— Pi- pette, for 25 ob. cm., J nat. size. The knowledge of the physiologist regarding the molecular phenomena occurring in living things is stiU so imperfect that he has no occasion to follow the physicist in his speculations as to the ultimate nature of matter. The atomic theory of Democritus and Leucippus "glorified," as Professor Tait writes, "in the grand poem of Lucretius," Newtonls conception of hard, finite atoms, the substitution by Boscovitch for MATTER AND ENERGY. 9 the material atom of a mathematical point, "towards or from which certain forces tend," or the splendid vortex-atom hypothesis of Sir William Thomson, in which " matter, such as we perceive it, is merely the rotating parts of a fluid which fills all space," throws no light at present on vital phenomena, except in so far as these can be brought within the domain of physical science. It is possible, however, to conceive that certain vital phenomena may be due, in their essence, to special properties inherent in the constitution of matter, but the physiologist is still far from the discussion of such questions. He finds that living matter is composed of certain solid, liquid, or gaseous constituents, and that it is the assemblage of these in varying propor- tions that gives to living matter its peculiar properties. The pheno- mena of life are never manifested by solid, liquid, or gaseous matter individually, but only when matter is in a peculiar colloidal condition or mass which, when death occurs, will split up into solid, liquid, and gaseous substances. At the same time, it is becoming more and more evident that vital actions such as growth, secretion, and the contraction of living muscle, depend on molecular, it may be, atomic changes occurring in living matter, changes as much beyond direct inspection as the molecular movements in a conductor of electricity, or in the isomeric transformation of cyanate of ammonia into urea. 2. Energy. — Physiology, in common with the science of physics and of chemistry, has received a strong impetus towards exactitude and comprehensiveness from modern generalizations regarding energy. The term energy must be clearly distinguished from the term force, and before entering into a consideration of energy we must under- stand clearly what is meant by the terms force and work. The notion of force is suggested by the sensation of pressure or of resistance experienced when we move a body with the hand or foot, as in lifting a weight from the floor to the table, but it will be observed that this is merely a sensation or feeling due to what is termed the muscular sense and that we must distinguish between the sensation and the cause of the sensation. Suppose the muscular sense perverted by disease, the weight might still be lifted from the floor to the table, but our notions of the force would be quite different. There is no proof then of the objective reality of force. But the weight has been lifted from the floor to the table and there has been a transference of what is called energy from one portion of matter (the muscles of the arm and body) to another (the weight resting on the table). " When- ever such a transference takes place, there is a relative motion of the portions of matter concerned, and the so-called force in any direction is merely the rate of transference, or of transformation, of energy per 10 INTRODUCTION. imit of length for displacement in that direction." (Tait, Recent Advances in Physical Science, p. 16.) Force, then, is rate of transfor- mation of cnerg}' in time. The British unit of force is the "poundal," defined as that force which applied to one pound of matter for one second generates in it a velocity of one foot per second. In the centimetre-gramme-second system (c.G.s.), the imit of force is the "dyne," similarly defined as that force which, acting on one gramme for one second, generates in it a velocity of one centimetre per second. The next conception it is important clearly to grasp is that of IFc/rk. Work is done when resistance is overcome, and the quantity of work done is measured by the prodiict of the resisting force and the distance through Avhich it is overcome, in the direction in which the force acts. The British absolute unit of work is the "foot-poundal," which is the work done by a poundal acting through a foot. The al)solute unit of work on the metrical system is the "erg," which is the work done by a dyne acting through a centimetre. Again, work may be measured in gravitation-units — the British being the "foot-pound," which is the work required to overcome a force equal to the weight of a pound through the space of a foot ; the unit in the metrical system being the " centimetre-gramme," defined as that required to overcome a force equal to the weight of one gramme acting through the space of one centimetre. In the study of the Avork done by a contracting muscle, the unit usually employed is the "millimetre-gramme," similarly defined. We are now in a position to understand what is meant by saying that Energy is the power or capacity of doing work. When a weight is lifted from the floor to the table, work is done in overcoming the attraction between the earth and the weight, and the energy of the system — the earth and the weight — is increased. The weight on the table is now in a position to do work ; for example, it might, by proper arrangements, be allowed to descend slowly, as the weight of a clock, and do work in overcoming the friction of the wheels, the resistance of the air to the motion of the pendulum, and in giving rise to the waves of sound by which we might hear the ticking of the clock. Before it begins to descend, the weight possesses Potential Energy, or energy of position. A bent boAv, a coiled mainspring of a Avatch, a head of water, are familiar instances of this kind of energy. Another mode of energy is the energy of motion or Kinetic energy. Thus, suppose a certain amount of work is clone in lifting a weight, the weight acquires energy of position (potential) ; if it be now allowed to fall to the ground its energy of position is gradually transformed into energy of motion (kinetic), and it can be shoAvm that "the amount of potential energy lost MATTER AND ENERGY. 11 in every stage of the operation is precisely equal to the amount of kinetic energy gained." (Tait, op. cit. p. 19.) It follows from this that energy, like matter, can neither be created nor destroyed. It may dis appear, but there has only been a transference from one material thing, or system of material things, to another. It may suddenly appear, but it has not originated de novo : it has appeared at the cost of potential energy somewhere else. Further, it is known that when a transfor- mation of energy takes place " there is always a tendency to pass, at least in part, from a higher or more easily transformable to a lower or less easily transformable form." (Tait, op. cit. p. 20.) The doctrine of the Conservation of Energy asserts, in the language of Clerk Maxwell, "The total energy of any material system is a quantity Avhich can neither be increased nor diminished by any action between the parts of the system, though it may be transformed into any of the forms of which energy is susceptible." (Clerk Maxwell's Matter and Motion, p. 60 ; also proved by Von Helmholtz.) This great doctrine was enunciated after much speculation, calculation, and experiment, "with regard to the nature of heat. So long ago as the beginning of the 1 8th cen- tury Locke wrote the following passage : "Heat is a very brisk agitation of the insensible parts of the object, which produces in us that sensation from which we denominate the object hot ; so what in our sensation is heat, in the object is nothing but motion." (John Locke, Natural Philo- sophy, 1706.) Professor Tait shows {op. cit. Lecture II.) that Sir Isaac NeAvton was in possession of many of the facts of the conservation and transformation of energy. Count Rumford demonstrated "that the great quantity of heat excited by the boring of cannon could not be ascribed to a change taking place in the calorific capacity of the metal, and he therefore concluded that the motion of the borer was communicated to the particles of the metal, thus producing the phenomena of heat. ' It appears to me,' he remarks, ' extremely difficult, if not quite impossible, to form any distinct idea of anything capable of being excited and com- municated in the manner the heat was excited and communicated in these experiments, except it be motion.' " Sir Humphry Davy, sometime after the date 1799, showed that by rubbing two pieces of ice against one another in the vacuum of an air pump, part of the ice was melted, although the temperature of the receiver was kept below the freezing point, and he inferred that "the immediate cause of the phenomena of heat is motion, and the laws of its communication are precisely the same as the laws of the communication of motion." (Davy's Elements of Chemical Philosophy, p. 94.) In 1834, Faraday discovered important relations existing between magnetism, electricity, and light. In 1840, Joule showed, "1st, that the heat evolved by any voltaic pair is proportional, cceferis paribus, 12 INTRODUVriON. to its intensity or electro-motive force ; aiul, 2n(l, that the heat evolved hy the combustion of a hody is pro^jortioual to the intensity of its affinity for oxygen," thus establishing relations between heat and chemical affinity. Similarly, in 1843, Joule showed relations between magneto- electricity and heat; and in 1844, he proved "that the heat absorbed and evolved by the rarefaction and condensation of air is proportional to the force evolved and absorbed in those operations." In 1842, Mayer announced that he had by agitating water raised its temperature from 12° C. to 13° C, without however indicating the force employed; and, about the same date, and founding largely on physiological considerations regarding animal heat and the heat of the blood, Mayer attempted to calculate the mechanical equivalent of heat. This important determin- ation was finall}" experimentall)' made by Joule in 1845 and 1847, when he employed a paddle-wheel to produce fluid friction, and obtained the equivalent from the agitation of water, sperm oil, and mercury. (See Joule's Scientific Papers, vol. I., p. 298). Heat then being a kind of energy, it can be measured in terms of the unit of energy, as, for example, by the foot pound, the centimetre- gramme or erg, or it may be expressed in thermal units. The thermal unit is the quantity of heat required to raise the temperature of unit mass of water unit degree betAveen the limits of 0° C. and 40° C. Thus the British thermal unit is the "pound-degree," that is the heat required to raise a pound avoirdupois of water from 60° F. to 61° F., and the French thermal unit is the " calorie " or kilogramme-degree, the heat required to raise a kilogramme of Avater from 15° C. to 16° C, This statement enables one to understand what is meant by the dynamical equivalent of heat, or the value in terms of a unit of Avork of a unit of heat. Joule found by experiment, as already indicated, that 772"55 foot-pounds of work are required to raise 1 pound of Avater from 60° F. to 61° F., the intensity of gravity being that of the sea-level at GreenAvich, or 1389-26 foot-pounds are required to Avarm 1 pound of AA^ater from 0° C. to 1° C. Reduce 1389-26 feet to metres and Ave have 423-437 metres. The AA'ork done therefore is eqvuA^alent to (approximately) 424 kilogrammetres, if the unit of heat be the calorie aboA^e defined. To couA^ert kilogrammetres into calories diAade by 424, and to convert calories into kilogrammetres multiply by 424. The dynamical equiA'alent, or Joule, denoted by J, may be stated thus: The same energy AA'hich elevates 424 kilogrammes 1 metre high Avill, as heat, raise 1 kilogramme of Avater 1° C, or a Aveight of 424 kilogrammes, if alloAved to fall from a height of 1 metre, Avould, by its concussion, produce as much heat as Avould raise the temperatiu-e of 1 kilogramme of Avater 1" C Difficulty of experimentation has hitherto prevented the accurate determination of MATTER AND ENERGY. 13 the mecliamcal equivalent of light, electricity, or magnetism; but there is sufficient evidence that these also are modes of energy, and that they may be therefore transformed or transmuted, just as the energy of molar motion can become the energy of molecular motion or heat. Consult Von Helmholtz' Lectures on Scientific Subjects, No. 7 ; Prof. P. G. Tait's Properties of Matter; Prof. Tait's Recent Advances in Physical Science; Joule's Scientific Papers ; Grove — The Correlation of Physical Forces ; also, Address on Continuity. Chap. III.— GENERAL PRINCIPLES OF BIOLOGY. Before entering upon the study of the functions of a specific organism, it will be instructive to consider some of the general characteristics of living things, and to endeavour to grasp some of the conditions — chemical, 'physical, and vital — which govern the origin, development, life-history, and death of all living beings. This preliminary discus- sion of the General Principles of Biology prepares the way for the more detailed treatment of the Physiology of Man. The leading- characteristics of living beings or of living matter may be considered under the follo"vving heads : — 1. Physical Structure. — Solid and fluid matters invariably co-exist in living bodies : the solids contain fluids in their cavities or interspaces. No living matter ever assumes a crj^stalline form, but crystals may be embedded in it. The colloidal state, in which matter is soft, diffluent, and readily permeated by water, oxygen, and the crystalloids, is so characteristic of living stuff that Graham, its first investigator, termed it the dynamical state of matter. The colloidal condition, however, is not peculiar to living stuff, inasmuch as silicic acid and peroxide of iron may assume this state. All the solid parts of living matter are more or less moist; many have the character of softness, and they readily absorb water, becoming swollen by imbibition. When water is absorbed by starch there is an evolution of heat. This property of swelling up by absorption of water has led to the conception that the molecular structure of such substances as the walls of cells, and starch grains, may consist of very minute solid particles (micellce of Nsegeli), each particle being surrounded by a layer of fluid. Accord- ing to this view, in a perfectly dry organic substance the micellae would be in contact on all sides, and when it absorbed water the water would penetrate between the micellae, forcing them asunder against the opposing action of cohesion. Another view is that of Strasburger, which appears to explain the phenomena of imbibition more satisfactorily. Rejecting the micellar theory of ISTsegeli, he 14 INTRODUCTION. supposes that " the force which l)iucls together the molecules is of a chemical as opposed to a physical nature ; that they are held together not by cohesion, but by chemical affinity : he regards them as being linked together, probably by means of multivalent atoms, into mole- cular networks, the water present being retained in the meshes by intermolecular capillarity/' (Vines' Plujaiologij of Plants, p. 33.) This theory assumes that living colloidal matter consists of a dis- tensible molecular network, capable of absorbing water until the capillary attraction is greater than the chemical affinity by which the molecules are bound together, and when this limit is passed the molecular structure is desti'oyed. In the case of protoplasm, ^ it may be supposed that the molecules are in a state of incessant change, and that the absorption and the liberation of water i)lay an important part in vital phenomena. 2. Chemical Composition — Of the sixty-five or sixty-eight elements, not more than eighteen or twenty have been found in living things. Chief among these are oxygen, hydrogen, nitrogen, and cai'bon. It is interesting to note that the first three can only jDass into the solid state imder enormous pressures and at a low temperature, an indication of their great molecular mobility. Oxygen enters into combination with hydrogen, carbon, and even with nitrogen, an element remark- able for its chemical indifference. Hydrogen and carbon are chemically indifferent to other substances at ordinary temperatures. Carbon is allotropic, appearing in the unlike forms of charcoal, graphite, and the diamond. Along Avith these there are associated, of the non- metals— sidphur, phosphorus, and chlorine ; of the alkalies — sodium and potassium; of the alkaline earths — calcium and magnesium ; and of the metals — iron. In addition, there are minute quantities of silicon and fluorine, and there may be iodine, bromine, manganese, aluminium, copper, and lead in the tissues of particular plants and animals. It is interesting to notice that if we take the classification of the elements generally accepted by chemists we find that organic matter is built up of representatives of the hydrogen group — hydrogen (1), chlorine (35-37), and bromine (79"75); of the iodine group — iodine (126"53) and fluorine (19); of the oxygen group — oxygen (15'96), sulphur (31-98); of the nitrogen group — nitrogen (14-01), phosjihorus (30-96); of the carbon group — carbon (11-9.7), silicon (28); of the sodium group of metals — sodium (22-99), potassium (39*04); of the calcium group of metals, calcium (39-9); of the magnesium group of ^ Protoplasm, irp^ros, first, -rfKaap-a, anything formed. A term first used by Von Mohl to describe the mucilaginous granular contents of the vegetable cell ; now applied to the simplest form of matter constituting the physical basis of life. GENERAL PRINCIPLES OF BIOLOGY. 15 metals, magnesium (23 '94); and of the iron group of metals, iron (55 "9) — all the other groups being unrepresented. Excluding the metals of the alkaKes and alkaline earths, iron is the only metal essentia] to some of the higher forms of living animal matter. Of the fifteen non-metals, nine are represented, whilst of the fifty-three metals we find only five representatives, excluding those of rare and probably of accidental occurrence. Further, if we consider that the earth's solid crust consists chiefly of oxygen, silicon, aluminium, iron, calcium, magnesium, sodium, and potassium, and that water consists of oxygen and hydrogen, we see that carbon and nitrogen in particular are characteristic of living matter, and it may further be stated that no living matter can exist ^vithout the presence of the four, oxygen, hydrogen, carbon, and nitrogen. Their compounds form, from the chemical point of view, the basis of life. The elements are so combined in li^'ing matter as to constitute com- pound bodies which may be separated as such by chemical processes. Such compounds are termed i^roximate principles. For example, phos- phate of lime is a proximate constituent of bone, but phosphoric acid and oxide of calcium are not proximate constituents ; in bone they do not exist individually, but united to form the salt, phosphate of lime. Chief among proximate substances is water, which as a rule forms more than three-fourths of the weight of a living body. An organism may, however, contain almost no water and still have the potentiality of life, as has been shown in the case of dried seeds and of dried rotifers and infusoria. In the process of freezing, also, water may separate from the organic stufi" and become ice, and yet on careful thawing it may be taken up again, and the structure may return to its former condition. In these instances, the molecular changes on which life depends, and for the play of which the presence of water is necessary, have been arrested, and it is only on the return of water that life is renewed. Certain proximate principles are the same as those met "\\-ith in the crust of the earth or in the water of oceans and rivers, such as chlorides of sodium and potassium, sulphates of soda and potash, phosphates of soda, potash, lime, and magnesia, and fluoride of calcium. The proxi- mate substances more characteristic of the tissues of plants and of animals are compounds formed of carbon, hydrogen, and oxygen, such as starches, sugars, fats ; or formed of carbon, hydrogen, oxygen, nitrogen, and sulphur or phosphorus, such as albumin, glutin, or lecithin. Compounds formed of three elements have, especially in the dry con- dition, a considerable amount of chemical stability. Thus dry starch will not readily decompose. On the other hand, compounds con- 16 INTRODUCTION. taining four or more elements arc much less stable. They tend easily to disintegration. They exist only within a limited range of temperature and pressure, and under the action of bodies in a more active molecular state (ferments) the complex organic molecule falls to pieces, not usually resolving itself into its elements, but into simpler groups of elements. Thus a molecule of albumin may split up into simpler molecules of leucin, tyrosin, and other less complex bodies, such as Avater and urea. This molecular instability, probably, is one of the conditions of vitality, and as it is always most highly manifested by the compounds containing nitrogen, it has been conjectured that it may be due to the Aveak affinities of nitrogen-molecules for the other simpler molecules existing in organic matter. For example, two well- known explosives are gun cotton and nitroglycerine. Gun cotton is cellulose, CgH^oOg, in which 3 atoms of hydrogen are replaced by 3 atoms of a stable nitrogen compound, NOg — thus : CgH7(N02)305. Again, nitroglycerine is glycerine, CgHgOg, in which 3 atoms of hydrogen are replaced by 3 atoms of the same compound, NOg, thus : C3H5(N02)303. Another example is picric acid : first one atom of hydrogen in benzol, C^Hg, is replaced by an atom of hydroxyl, HO, thus : CgHj.HO, or phenol ; then 3 atoms of the hydrogen in phenol are replaced by 3 atoms of NOg, forming picric acid, thus : C6H2(N02)3HO. These compounds, especially the former two, are re- markable for chemical instability, and they offer an analogy to the constitution of living stuff. A sudden change of temperature, a vibra- tion, a concussion, may cause the molecule to fly to jDieces, and the fragments are of course simpler in structure. We shall see that all vital actions are characterized and accompanied by the appearance of chemical substances much simpler in composition than the substance which was the seat of these actions. A living body is continually undergoing a series of chemical changes of composition and decomposition, as a result of which there is an incessant renovation of the molecules of the organism. Chemical changes are a necessary condition of the action of living matter, or it may be said that the living state is always associated with chemical change; part of the living matter dies, is decomposed, or rather its decomposition is its death, and the dead matter is then thrown out of the organism. New matter is added from without, and thus there is a perpetual exchange between the organic and the inorganic worlds, which may be termed the ciradation of niatter. 3. Organic Form and Mode of Gro\vth. Li^dng bodies are organized, that is, they are composed of dissimilar or distinct parts arranged in a certain order, and each j^art performs a determinate office GENERAL PRINCIPLES OF BIOLOGY. 17 or function in connection with the maintenance of the life of the whole. There is reason to believe that this is true even of the apparently simple structure of an amoeba, as new modes of investigation reveal details of structure which no doubt have a special physiological significance. The external form of living beings is consistent with a certain morphological type. At the beginning of existence the typical form is nearly spherical ; afterwards, in the process of growth, a form is developed peculiar to the sjjecies. The spherical form is not only characteristic of the organism at the com- mencement of life — the egg or ovum, Fig. 10 — but is seen also in the primitive elements which compose the developed organism. (See cells.) Organized matter may be composed of molecules, granules, cells, fibres, membranes, and tubes. The mode in which new particles of matter enter a living organism furnishes also a distinctive character. A crystal grows by new moleciiles of similar composition to itself being applied directly to its surface, whilst a living thing absorbs the dead matter intO' its own substance and converts what was previously dead into living matter like itself. In other words, dead bodies increase in size by apposition, living bodies grow by metabolism^ and intussusception. ^ The manner in which the growth of crystalline forms may be modified by various physical conditions so as even to simulate organic forms has been the subject of various exhaustive inquiries, which have thrown light on the mode of origin of concretions, on the formation of shell, and on the deposition of earthy matter in soft tissues. In 1857, George Rainey showed that certain crystalline matters, when deposited in viscous or gummy solutions, assume globular and cell-like forms. ^ His method consisted in saturating a solution of gum arable with carbonate of potash (specific gravity 1-4068), placing the fluid in a small wide-mouthed l)ottle, so as to fill it half full, and introducing into the bottle two clean Fig. 10. Ovum from an Echiuoderm ^ Metabolism, from fiera^oXt], change ; the power living matter possesses of changing other matters brought under its influence. First used by Schwann. ^ Intiissusception. Intus, within; suscipio, I receive. The absorption of matters into the substance of living material. ^ On the Mode of Formation of Shells of Animals, of Bone, and of several other Structures by a Process of Molecular Coalescence. By Geo. Rainey, M.R.C.S. Lond., 1858. See also Quart. Jour, of Micros. Science, 1858, and On the Influence, of Colloids on Crystalline Form and Cohesion, by William Miller Ord, M.D., etc. Lond., 1879. I. B 18 INTRODUCTION. slides of glass, leaning against each other at the top. The upper part of the bottle was then filled by gently pouring in a solution of gum arabic in water, of a specific gravity of r0844:, and the mouth of the bottle was covered with a sheet of paper. The whole was laid aside for a period of three weeks or a month. The slides Avere then removed and were found to be coated with a deposit of carbonate of lime. The carbonate, however, instead of being deposited in a crystalline form was in the condition of fine molecules, and by examining such slides at various intervals it was shown that these molecules coalesce so as to form spheres, which in turn may coalesce to form larger ones. The perfect spheres exhibit both radial and concentric markings, and in polarized light show .a well marked cross. (See Figs. 11 and 12.) The presence of the Fig. 11.— Precipitation of Carbonate of Lime, showing the crystalline form passing into globular forms, (a) Crystalline form ; (6) angles of crystals rounded off ; (c) ovoid forms ; (d) coalescence of ovoid forms. ^.=.o=^ov=fe(7_p oo g, ^d "s m%mm j;y. -oce 4'cb "^ QMi Fig. 12.— Precipitation of Carbonate of Lime from a Viscous Solution on a slide of glass, showing (beginning on the left hand) (a) finely mole- cular forms, then globular forms (6) ; and these forming larger and larger bodies by molecular coalescence (c, rf.) On the right hand, large bodies showing concentric circles and radiating lines (/). colloidal gum, according to Eainey, " annuls the polarity of the crystal, and allows the molecules of the crystal to obey simple laws of common and mutual attraction." This aggregating of the molecules he terms " molecular coalescence." His words are : "When the molecules of pure carbonate of lime, that is carbonate of lime uncombined with a viscid substance, come into existence they immediately commence arranging themselves in straight lines, and thus when collected together form rectilineal figures or crystals ; but when the impure carbonate, that is car- bonate combined with a viscid substance, comes into existence, under similar cir- cumstances, its molecules assume a curvilinear disposition, and hence become collected into globules." (Eainey, op. cit. p. 31.) Further, if the spheres thus formed are plunged into solutions of gum of difierent specific gravity, the spheres break up along the radial lines and crumble into molecules. This is "molecular disintegration." Eainey showed that in the soft parts under the shell of the young GENERAL PRINCIPLES OF BIOLOGY. 19 lobster, crab, or shrimp, bodies exactly like thosg thus artificially pro- duced may be found. I possess a number of Rainey's original prepara- tions, and after the lapse of thirty years they show all the gradations of form depicted in Figs. 11 and 12. Professor Harting, of Utrecht, investigated this matter independently of, and even prior to, the appearance of Rainey's papers. He obtained similar results by allowing carbonate of lime to separate out of an albuminous or colloid fluid. " He states that the albumin contained in the calcosphcerites [globular bodies of Eainey] undergoes a chemical change bringing it near to chitin, and says that the same is effected in albumin by chloride of calcium. To this moditication he gives the name calcoglobulin. He finds in his experiments explanations of the structure of the shells of Lamellibranchiata and some Gasteropoda, of the calcareous plates of the bones of Sepia, of the shells of Foraminifera and loculi of Bryozoa, the spicules and sclerites of Alcyonaria ; and states that the last mentioned forms are produced when cartilage, previously impregnated with calcium chloride, is placed in a solution of potassium carbonate mixed with a little sodium phosphate. " (Ord, op. cit. p. 7.) Dr. Ord has modified Rainey's method with important results bearing on the formation of bone and especially on the crystalline forms assumed by various urinary sediments. Another striking example of the production of organic-like forms from dead matter is the formation of processes from myelin or protagon by the action of water. ^ Beat up the yolk of an egg in 30 cc. of absolute alcohol, boil for one or two minutes, and then filter on a flat plate. In the small amount of filtrate obtained a gelatinous yellow stuff appears. This is impure protagon or myelin. Allow the alcohol to evaporate. Place a small portion of protagon on a slide, lay a cover-glass gently over it, place it on the stage of the microscope, and focus so as to see the edge of the mass. This will be seen to be amorphous. Then allow a drop of water to pass below the cover-glass and immediately beauti- ful snake-like forms will shoot out, bend, curl, and assume grotesque . - . -rr t -FiG- 13.— Protagon or myelin forms, lOrmS such as are seen m Xlg. 13. curved and spiral. Processes shoot- -Kir , 1 1,1,1 , • in? out from a mass of protagon on Montgomery showed that by acting addition of water. on protagon with water, albumin, or glycerine, forms may be ob- tained simulating organic fibres, varicose nerve fibres, the broken down matter of the spinal cord or brain, and even cell-like bodies. ^ Montgomery, On the Formation of so-called Cells. Lond., 1867. 20 INTRODUCTION. All of these observations indicate that forms similar to those pro- duced in and liy living matter may be artificially produced in dead matter, and that molecular processes of coalescence, disintegration, and imbibition, may play an important part in the formation of shell, in the development of the curious spicules of sponges and synaptidae, in the deposition of calcareous matter in tendon, in the walls of vessels, and in fibrous membranes (phenomena familiar to the pathologist), in the formation of the hard tissues of teeth, and in the calcification of cartilage, one of the early stages of ossification. 4. Dynamical Characters. — We have already seen that the various forms of physical activit}-, heat, light, electricity, motion, chemical affinity, are all related and convertible so that they are now regarded as but various modes of one energy, potential or kinetic. But plants and animals exhibit activities peculiar to themselves, such as those of assimilation and growth, irritability, contractility, reproduction, and nervous actions, which are all manifestations of energy. Is this the same energy as is active in the world of dead matter, or have li^ang things an energj' of their own 1 This is a question of pro- found importance. The older physiologists thought that the energy of living things Avas an entity or principle derived from the germ, and quite different in kind from the various forces of the outer world. This energy was described under such terms as vital force, life, the vital principle, and it was supposed to exert power not onlj^ over the matter introduced into the body, but also over the outer forces acting on the body. Further, instead of this \dtal force being correlated to the outer forces, it was supposed to be in some way opposed to them, so that when the vital force ceased to act, the natural forces came into play and acted on the body as if it were composed of dead matter. Such views were generally held until 1845, Avhen Mayer first broke ground by setting forth that all change in the living organism, animal or vegetable, " lies in the forces acting on it from ^Wthout." There can be no cjuestion that Mayer first directed the attention of physiologists to this aspect of the subject, and that it was largely from physiological considerations that he was led to his speculations regarding the physical forces. In 1850, Dr. W. B. Carpenter applied to the explana- tion of physiological phenomena the doctrine of the correlation of the physical forces, and showed that the vital forces were generated in li^dng bodies by the transformation of light, heat, and chemical action, and that the energy thus derived from the outer world was given back to it again as heat and motion, and to a less degree in most animals as electricity and light. In 1859, these views were supported and extended in a modified form by Professor Joseph GENERAL PRINCIPLES OF BIOLOGY. 21 Le Conte, who argued that the chemical forces, set free in the plant and animal, were transformed into vital force, and that by this action matter was raised from the plane of dead matter to the higher plane of living matter. Both of these vsriters, however, laid special stress on the view that the medium of this transformation of physical into vital force was organized or living matter. This is clearly shown in the words of Dr. Carpenter : "It is the speciality of the material substratum thus furnishing the medium or instrument of the metamorphosis which establishes, and must ever maintain, a well-marked boundary line between physical and vital forces. Starting from the abstract notion of force as emanating at once from the Divine Will, we might say that this force, operating through inorganic matter, manifests itself as electricity, magnetism, light, heat, chemical aflinity, and mechanical motion ; but that when directed through organized structures it aflfects the operations of growth, develop- ment, and chemico-vital transformations and the like, and is further meta- morphosed, through the instrumentality of the structures thus generated, into nervous agency and muscular power." (Carpenter, Trans, of Royal Society, 1850, p. 752.) A careful study of the properties of protoplasm, more especially in the vegetable cell, and of the action on it of physical forces, such as those of heat and light, and the influence of the teaching of Herbert Spencer in his Principles of Biology, have led to the general adoption of these views; and the principle of the conservation of energy, as affecting the interactions of the forces manifested by dead and living- matter, is now, to the physiological inquirer, what it is, in the language of Clerk Maxwell, " to the physical inquirer, a principle on which he may hang every known law relating to physical actions, and by which he may be put in the way to discover the relations of such actions in new branches of science." (Clerk Maxwell, Matter a7ul Motion, p. 60.) Let us now discuss how these principles apply to the phenomena of plant and animal life. Plants containing chlorophyll require comparatively simple chemical substances as food. All plants absorb oxygen from the air, and all green plants, under the influence of sunlight, absorb carbonic acid. This carbonic acid is decomposed, the carbon being retained in the cell, whilst oxygen is liberated. Further, nitrogen exists in the juices of plants, but there is no evidence that it is directly used by the living matter. Plants may also absorb a small amount of ammonia from the air, and they obtain water chiefly from the soil. The roots obtain from the soil, or from the water in which the plant grows, ammonia and its salts (the nitrate, sulphate, and phosphate), nitrates of soda, potash, lime, and magnesia, chlorides of the alkalies and 22 INTRODUCTION. alkaline earths, sul])hates of potash and lime, phosi)hates of soda, potash, ammonia, lime, and magnesia, and iron in the form of a salt. From the substances thus derived from the air and the soil, the plant obtains the elements necessary for its existence, namely, carbon, hydrogen, oxygen, nitrogen, sulphur, phosphorus, })otassium, calcium, magnesium, and iron, and in some plants, chlorine. The first four — carbon, hydrogen, oxygen, and nitrogen — are absolutely necessary for those metabolic processes on which the life of the plant depends, whilst the others apparently assist in the performance of these processes. In addition, plants may contain silica. The carbon is obtained by plants containing chlorophyll by the decom- position of carbonic acid under the influence of light, but plants destitute of chlorophj'll (fungi) cannot thus decompose carbonic acid, and they obtain the requisite carbon from organic matter in which carbon is united to hydrogen. Hydrogen is obtained from water, oxygen from the air, and nitrogen from ammonia and its salts by plants of lower organization, and from nitrates by plants of a higher kind. The sulphur necessary for building up proteid matter is yielded by sulphates ; the phosphorus which exists in chlorophyll and protoplasm, by phosphates ; and the bases, potassium, calcium, and magnesium, by various salts. Although iron does not exist in chlorophyll, it appears to be necessary for its formation. From these simple materials, the protoplasm of the plant builds up more complex bodies, some non-nitrogenous and others nitrogenous. This process of building up may go along with the reverse process of pulling down; or, in other words, synthetic and analytic changes may go on, even simultaneously. The new matter supplied by the food may be, by these processes of constructive or destructive metabolism, divided into three portions — one part by constructive metabolism is used for building up the tissues of the plant (growth); a second portion, also by constructive metabolism, is built up into certain bodies which are stored \\\) in the tissues of the plant, constituting reserve material; whilst the third portion, by a process of destructive metabolism, may be decomposed in the tissues of the plant, never entering into the composition of these tissues, and the products of decomposition are excreted as useless. In plants, synthetic are carried on to a much greater extent than analytic processes ; synthesis indeed is characteristic of the lives of all plants containing chlorophyll. By such processes the plant builds up either non-nitrogenous or nitrogenous substances. The formation of non-nitrogenous organic substances, such as starch, is known to be directly connected with the action of chlorophyll, imder the stimulus of light. In green GENERAL PRINCIPLES OF BIOLOGY. 23 vegetable cells, the protoplasm contains small corpuscles, the chloro- phyll corpuscles, and it has been shown that these consist of a kind of matrix permeated by the chlorophyll, probably dissolved in an oil. The green matter of chlorophyll, called by Hoppe-Seyler chlorophyllan, has a formula of Cj9H22N203(P 1), and is believed to be a lecithin com- pound containing phosphorus. It is important to note that it is a highly complex body, formed originally from the protoplasm of the cell. Vegetable cells containing chlorophyll, under the influence of sunlight, as already said, decompose carbonic acid and liberate oxygen. This process is not to be confounded with the respiration of the pro- toplasm of the cell, which consists in the absorption of oxygen and the liberation of carbonic acid ; but is the first step in the constructive metabolism by which the chlorophyll builds up starch. Without light no starch is formed, nor, on the other hand, can it be formed without carbonic acid, even when the chlorophyll is exposed to light. The rapidity of the process in certain plants is very remarkable. Thus " Kraus found that starch grains made their appearance in the chlorophyll corpuscles of Spyrogyra within five minutes after exposure to bright sunlight, within two hours in diffuse daylight; in Funaria thej^ made their appearance after two hours' exposure to sunlight, and after six hours' exposure to diffuse daylight." (Vines, Physiology of Plants, p. 147.) It is probable that chlorophyll-corpuscles form starch by the union of carbonic acid and water to produce formic aldehyde, which, by polymerization,^ becomes starch, thus : COg + HgO = CHgO (formic alde- hyde) -fOgj then 6CH20 = CgHj20g; and CgH-^20g (grape sugar) - HgO = C(.Hjq05 (starch). But the starch is really formed by the dissocia- tion of the protoplasm under the action of the chlorophyll, so that the process may be, first, the formation of formic aldehyde, then the building up of the protoplasm, and finally a dissociation of the proto- plasm, one of the products being starch. In addition to starch, sugars, fats, cellulose, and organic non-nitrogenous acids may be formed. In a similar manner, nitrogenous bodies may be built up, although physiological chemists have not advanced so far in this direction. It has been supposed that nitrates and sulphates absorbed as food may be decomposed by organic acids (oxalic acid) and that the nitric and sulphuric acids thus set free may combine with a non- nitrogenous organic substance, such as formic aldehyde. Again, bodies of the nature of amides (that is ammonias in which hydro- gen is replaced by the radicle of an organic acid), such as asparagin, 1 Polymeric is a term given by Berzelius to organic compounds possessing the same composition but differing in molecular weight. 24 INTRODUCTIOX. leucin, etc., may be fonncd either as steps towards the Imikliug up of protoplasm or as products in its destructive metabolism. For our present piu'posc it is sufficient to note that nitrogenous matters may also be formed synthetically. Now arises the question as to the source of the enei-gy that enables the protoplasm of the i)lant-cell containing chli)ro})hyll to perform these remarkable synthetic processes. There can l)e no doubt that the energy is the radiant energy of light, more especially of the red and blue rays absorbed by chlorophyll. It has been ascertained that a solution of chlorophyll presents certain absorjition bands Avhen ex- amined with the spectroscope. It is the light of just those parts of the spectrum that is most active in the decomposition of carbonic acid by green plants, and it is remarkable that the maximum amount of absorption of light liy chlorophyll occurs at that part of the spec- trum where, according to Langley, of America, we have the maximum of energy. Fiuther, Engelmann has showTi that bacteria, placed along with a filament of Cladophora in the solar spectrum under the micro- scope, collect roimd the filament in those regions that coincide with the absorption bands of chlorophyll, and that this is an indication of oxygen being evolved from the chlorophyll coiijuscles in the Cladoi^hora, more especially where they are exposed to raj-s of the spectrum at the junction of the orange and the red and also at the blue, just beyond the line F. Light has been found to have an influence even on the absorption of food materials by the roots. The initiation of these changes is due to heat, a somewhat elevated temperature being essential to the active life of all plants; but in the later stages, the heat, as a soiu'ce of energy, is, relatively to the light, of less import- ance. The importance of light to the plant can hardly be overstated. Keep a green plant in darkness and, although it may appear to groAV, it loses weight by the exhalation of carbonic acid and watery vapour, and if kept too long in darkness it will die. Under the influence of light, assimilation will go on and the plant will gain in Aveight. The true respiration of the plant does not seem to be affected by light. Thus a plant containing chlorophyll lives on simple food elements and, iinder the influence of light, bmlds these up into more complex substances. In doing this, the kinetic energy of light is stored up and becomes potential. Such products as starch, gums, glutin, cellulose, may be regarded as matter containing stores of energ}\ They may be decomposed by various chemical operations, or they may be completely oxidized and converted into carbonic acid and water, but in these oxidations heat w-ould again become kinetic and could be employed to do work. Thiis the fiirnace of a steam-engine might be GENE HAL PRINCIPLES OF BIOLOGY. 25 fed with starch or vegetable oil and the energy liberated from these might to a certain extent be converted into motion. The green plant then, from the dynamical point of view, is storing up energy. On the ■other hand, plants destitute of chlorophyll cannot live on such simple materials as can green plants, nor can they obtain energy directly from the radiant energy of the sun. Like animals, they require food containing complex organic matters representing potential energy. Both classes of plants, however, expend energy, either as ^owth, as movement, as heat (more especially during germination ^nd at certain stages of the reproductive process), or, to a small extent, as light or electricity. The green plant stores more energy than it expends : it is, as already explained, more concerned in building up complex substances than in decomposing these into simpler ones, and hence the life and growth of such a plant is practically unlimited. Thus, immense accumulations of energy have been made and are being daily made by the vegetable world, and the energy of the sun's rays is being stored up, to be liberated again as heat in combustion, or as heat or motion in the bodies of living .animals. We have now to consider the question from the point of view of the animal kingdom. Some of the lowest forms of animals originate by a fission or division of the parent organism, but, above this low level of existence all originate in a cell, the o\Tim, or egg. After fecundation, this structure passes through numerous stages of de- velopment until the body of the animal is formed. During this process of growth the animal requires food, and, like the plant, it ■also requires a supply of energy to carry on the internal changes in its body, possibly to move its body from place to place and to supply energy for an expenditure going on in the form of heat, and to a •smaller degree, of electricity and of light. In the first instance, ■energy as heat is required to start the processes of assimilation and of growth. The development of all ova requires certain favourable conditions as regards temperature; but, like the plant, neither the ovum nor the adult animal form receives in the form of heat more than a small fraction of the energy it needs. Heat in both instances initiates or provides suitable conditions for the changes in the body peculiar to life, and it will presently be seen that the production of Jieat, and thus of the condition favourable to living actions, is one of the invariable accompaniments of such actions. Food is now supplied. With the exception of oxygen, no matter can be used by an animal in the form of an element, nor can an animal live on the comparatively simple compounds that, as we have seen, constitute the food of plants 26 INTRODUCTION. Animals obtain oxygen from the air, or, in the case of aquatic organisms^ from the oxygen dissolved in the Avater. The food-stuffs, -with the exception of a few Rim})le com})ounds such as water, common salt, etc., are derived either from the bodies of other animals or from plants, and a little consideration will show that all food-stuffs must ultimately have been derived from plants. Substances, then, formed in plants constitute the food of animals. These food-stuffs have, as already explained, been constructed by the plant and they represent energy in the potential condition. The substances essential to the formation of a diet for an animal derived directly from plants con-sist of (1) albuminous or proteid matters, as represented hj glutin or legumin ; (2) fatty matters, such as olive oil ; (3) carbo-hydrates, such as starch, sugar, gum; (4) mineral matters — phosphates, sulphates, chlorides of the alkalies and alkaline earths, and iron ; and (5) water. All of these, after undergoing various changes, chemical and physical, are ultimately assimilated by the living matter of the animal's body and pass through metabolism of the most complicated kind. Thus, part of the matter mav, by constructive metabolism, be built up into the tissues of the animal, bone and muscle and nerve and the tissues of the various organs ; another portion may also, after constructive metabolism, be stored up as reserve material in the form of fat and glycogen ; whilst a third portion ma}', by destrvictive metabolism, be split up into simpler substances which may be excreted without having entered into the composition of the body. Most, if not all, of these opera- tions are intimately connected with oxidations. Oxygen is absorbed in respiration and, directl}^ or indirectly, it combines Avith the matter in the food-stuffs and simpler products are formed. It is not im- probable that, in the first instance, constructive metabolism occurs by which the oxygen, proteids, fats, carbo-hydrates, salts, and water- are built up into the highly complex matter, living protoplasm, and that thus there is, for a short period, a still further conversion of energy into the potential condition even in the animal. This condition, how- ever, does not last. Animal protoplasm is characterized by molecular instability : its large and complex molecule can exist only in very limited conditions, and a sudden change in these conditions, or what is termed a stimulus, causes it to break up into simpler bodies. Thus energy is liberated and appears in the kinetic form as heat, and probably as motion. The decomposition, in the first instance, of li-vdng protoplasm is like a molecular explosion, shattering the complex molecule into fragments. These fragments, however, may still be complex, and the process may be repeated, simpler bodies being formed, and ultimately Ave find that the matter throAvn out of the body as useless is carbonic- GENERAL PRINCIPLES OF BIOLOGY. 27 acid, water, a nitrogenous substance called urea (two molecules of am- monia in wliich two atoms of hydrogen are replaced by CO), and saline matters. Tbese substances represent only a small amount of energy, and it will be observed that they are essentially the same as the simple substances constituting the food of the plant. Thus the complex bodies built up by the plant may become for a time part of the still more complex living matter of the animal, and then, by oxi- dations, this complex living matter is decomposed into simpler matters. In the latter operation, energy is set free and appears in the kinetic form as heat, motion, electricity, light, and sound. Thus the body of animals produces heat ; there are movements of the whole body, as in locomotion, or of portions of it, as in the respiratory movements or the beating of the heart ; and to a smaller extent, electricity (especially in electric fishes), light, and sound may be manifested. Energy becomes kinetic chiefly as heat and motion, and even motion (internal and external) is ultimately resolved into heat. Contrast now the plant and the animal — the plant transforms kinetic into potential energy, the animal transforms potential into kinetic energy. But neither the plant nor the animal is wholly concerned in the one operation. The j^lant also converts potential into kinetic energy, but relatively to a small extent; whilst the animal also converts kinetic into potential, but relatively to a small extent. It will be observed, finally, that whilst the plant furnishes potential energy to the animal, and the animal liberates this as kinetic energy, the kinetic energy of the animal is not re-transformed into the potential energy of the plant. The plant must therefore always receive new supplies of energy from the solar rays. The following illustration "wdll still further elucidate this interest- ing subject. It is well known that muscles and nerves may exhibit some of their vital properties after the death of the animal and that this is especially the case with the so- called cold-blooded animals such as the frog. Suppose a preparation is made consisting of the gastroc- nemius muscle of the frog "with sciatic nerve attached, as in Fig. 1 4. an instrument termed a myograph, one form of which is sho\^Ti in Fig. 15. The upper end of the femur is secured by the clamp C, sliding on the pillar B, and the tendo Achilles (./ in Fig. 14) is attached by a hook to Fig. 14. — Xerve - muscle Preparation. F, femur ; JV, nerve ; J, aperture for hook. This preparation is connected with 28 INTRODUCTION. the horizontal lc^•el• A7i, whicli carries a marker ,/.• this marker is brought into contact with G, a smoked ghiss plate that can be moved horizontally in sliding grooves. The nerve is stretched over wires coming from a battery or induction coil, so that it may be irritated by an electric current. When the nerve is irritated the muscle contracts, lifts up the lever EE, and the marker / draws a ^■ortical line on the plate G. The plate is Fig. 15. — Myograph, an instrument for recording the conti-action of a muscle. A, wooden stand; B, vertical brass pillar ; C, sliding forceps or clamp for holding upper end of femur of nerve-muscle preparation ; D, D, short vertical piUars on the top of which the lever EE works ; F, scale pan for weight ; /, marker ; G, glass plate ; K, counterpoise to keep / in contact with G ; H, counter- poise to lever EE. The nerve-muscle preparation is covered with a glass shade, to the walls of which pieces of wet blotting paper are attached, thus forming a moist chamber to preserve the nerve from drying. then pushed on, the nerve again stimulated, and thus a series of vertical lines is obtained, indicating the work done by the muscle in lifting a weight placed on the scale pan F. (See Fig. 16.) It can thus be shown that the muscle does work in lifting the Aveight, and by a refined method of inquiry it can also be demonstrated that with each muscular contraction heat is produced. Energy thus becomes kinetic as heat and motion. This energy is not derived from the battery used in irritating the nerve. GENERAL PRINCIPLES OF BIOLOGY. 29 The electricity is used merely as a stimulus to the nerve. When the nerve is irritated a molecular change is generated in the nerve ; this travels down the nerve with a certain velocity (nerve-current), and when Fig. 16.— Examples of tracings taken with myograph. Plate moving in direction of arrow. it reaches the muscle it sets up molecular changes in it Avhich result in a contraction, attended by the phenomena of heat and motion. Nor is there any quantitative relation between the strength of the electric cur- rent employed in irritating the nerve and the amount of work done by the contracting muscle. A very feeble current would be quite sufficient, just as the pull of a hair-trigger would be enough to set free the energy from a charge of gunpowder in a rifle. The energy then that has become Idnetic as heat and motion was stored up in the muscle. Further, it might be shown that the contraction of the muscle is associated Avith chemical changes in it, the splitting up of its complex protoplasm into simpler bodies. Thus energy is liberated in accordance with the principles already explained. This energy, potential in the muscle substance, must have been derived from the food of the frog, and if we trace back the history of this food we find it came originally from plants, and that the plants formed it from simple materials, in the processes transforming the energy of light into potential energy. Finally, at one end of the chain we have matter in the form of simple substances along Avith the kinetic energy of light, and at the other we have again matter in the comparatively simple conditions of carbonic acid, water, and nitrogenous substances, and kinetic energy as heat and motion. This experiment also shows that muscle substance may be regarded as a magazine or store of energy, which may be liberated by the molecular action of the nerve. We shall find many examples of this important relation between nerve and muscle and between the external modes of energy and the action of living matter. Just as nerve energy liberates the energy of the muscle, so light and sound liberate the energies of the sense organs on which they act, and these again liberate energies in the nerve centres, which in turn liberate the energy stored up in muscle or gland. 6. Evolutional History of Living Beings. — The evolution of a living being is determinate : it has a commencement, an existence, and an 30 INTRODUCTION. end; it passes through different phases, which succeed regularly and in a certain order. The same statements ma}/ be made, with a certain amount of truth, regarding a crystal, but its life history is distinguished from that of a living animal by the absence of -waste and of repair, and by its mode of gro\\'th, already referred to. Living beings have a certain individuality. Among the higher grades each member has a certain independence, although related to all ; but this characteristic may almost disappear in the lower classes of plants and of animals. All ^^dng organisms take their origin in a germ which was developed in a parent — that is a previously-existing being having essentially the same structure and properties. Every plant and animal, accordingly, have the power, at one stage of their existence, of producing a germ by which the species may be perjjetuated. This germ, which is known as the spore, seed, or egg of plants and animals, is a cell — the simplest form of structure. After its separation from the parent body, it is capable of independent existence, and, under favourable external influences, of groAving or developing into a new individual, in most respects similar to that from which it derived its origin. Living beings have formed a continuous series, from the first appear- ance of life upon the earth until now. Offspring usually possess more or less of the character of their parents ; and they may transmit pecu- liarities — either acquired by new conditions of existence, or received from their parents — to their descendants. This is known as heredity. The evolution of energy undergoes changes diuring the life of an organism. Usually the production of energy increases up to a certain maximum, and then slowly declines, so that in the life history of each individual there is a period of maximum vital activity. In certain species of animals, however, there are successive phases of rej^ose and of movement, as in the encysted conditions of infusoria, the meta- morjDhoses of insects, and the occrurence of hybernation. In other organisms also, vital phenomena may appear to be entirely suspended under certain conditions. For instance, some of the rotiferce may be kept in a state of dormant vitality for a considerable time by the simple process of drying. During its life an organism undergoes change of form, increase of ita mass, and development of its organization. This development is not, however, continued indefinitely. The organization becomes at last complete for each organism, reproductive functions are exercised, and then gradually the Avear and tear become greater than the power of upbuilding, and the organism breaks down. Death at last neces- sarily terminates its evolution. "NMien this occurs, the body is sub- GENERAL PRINCIPLES OF BIOLOGY. 31 mitted to the action of external agencies, botli physical and chemical, which ultimately reduce it to the simple elements of which it was at first composed. It is important to remember that an organism is affected during ■every instant of its life by the medium in which it lives. The medium furnishes the materials requisite for existence. Dead matter is supplied to take the place of Kving matter, and external modes of energy — such as heat and light — act upon it at the same time. There is thus an •action and reaction between the organism, and the conditions in which it lives. These conditions may be conveniently summarized by the term environment In this adaptation of relations every living organism has a power of suiting itself to modifications of conditions %vithin certain limits. This power is known as its variability, and it is a necessary condition of the existence of every living being. To recapitulate, the essential characters of a living being are the following : — 1. Molecular complexity; heterogeneity of parts; and chemical in- stability of the organic compounds forming it. 2. Waste and incessant repair of organic materials. 3. The conversion of kinetic into potential energy as the framework of the body is built up or stores of reserve material are formed. 4. Liberation of kinetic energy in various modes, and, in particular, as mechanical movement, heat, and electricity. 5. Organization, or the adaptation of certain parts of the body to particular functions. 6. A regular evolution from origin to death. 7. Origin from a parent, and the possibility of producing the elements of offspring. 8. A power of variability and of adaptation to external conditions. 6. Theories of Life. — Numerous efforts have been made to define life, of which the follomng are examples : — Aristotle says, " Life is the assemblage of the operations of nutrition, gTOwth, and destruction;" Lamarck states that " Life, in the parts of the body possessing it, is that state which permits organic movements, and the movements which constitute active life result from the application of a stimulus ; " Bichat says, "Life is the sum total of the functions which resist death;" Treviranus calls it "The constant uniformity of phenomena, with diversity of external influences;" Laurence says, "It consists in the assemblage of all the functions or purposes of organized bodies, and in the general result of their exercise ;" Duges calls it " The special activity of organized bodies ;" Beclard's definition is " Organization in 32 INTRODUCTION. action ;" and Herbert Spencer asserts that " Life is the continual adaptation of internal relations Avith external relations." It will be observed from the above definitions that authors have felt the necessity of presupposing some organized structure, the existence of which is taken for granted in their definitions. The dictum of B^clard, " Organization in action," is, on the Avhole, the best. In the history of physiology several distinct schools of thought have from time to time had the ascendency. Aristotle and Galen laid the foundations of anatomy, and in their physiological speculations they adopted the leading tenet of Hippocrates that there exists a principle Avhich he termed ^ o ^^ OS o Ph Blood. Serum. Blood Clot. Lymph. Urine. Milk. Bile. Excre- ments. Chloride of Sodium, - 58-81 72-88 17-36 74-48 67-26 10-73 27-70 4-33 Chloride of Potassium, 29-87 26-33 Soda, - - - . 4-15 12-93 3-55 10-35 1-33 36-73 5-07 Potash, - - - - 11-97 2-95 22-36 3-25 13-64 21-44 4-80 6-10 Lime, . - - . 1-76 2 -28 2-58 0-97 1-15 18-78 1-43 26-40 Magnesia, 1-12 0-27 0-53 0-26 1-34 0-87 0-53 10-54 Oxide of Iron, 8-37 0-26 10-43 005 ... 0-10 0-23 2-50 i Phosphoric Acid, - 10-23 1-73 10-64 1-09 11-21 19 00 10-45 36-03 Sulphuric Acid, - 1-67 2-10 0-09 2-64 6-39 Carbonic Acid, 1-19 4-40 2-17 8-20 ... 11-26 Silicic Acid, - 0-20 0-42 1-27 4-06 ■ 0-36 3-13 38 THE CHEMISTRY OF THE BODY. On considering these tables, we may note the folk) wing points : — (1) That about |- of the total mineral matter in the solids con- sist of phosphoric acid and of lime ; (2) the large amounts of potash and of phosphoric acid in muscle ; (3) the large amounts of potash and of phosphoric acid in the brain ; (4) the contrast as regards potash especially between the analyses of the liver and of the lungs ; (5) the large amounts of soda and oxide of iron in the spleen ; (6) the contrast of the analyses of blood, blood serum, and blood clot as regards chloride of sodium, potash, and phosphoric acid ; (7) the general resemblance of lymph and urine to serum, except as regards potash and soda; (8) the large amount of chloride of potassium in blood clot and its absence from serum ; (9) the large amounts of chloride of potassium, lime, and phosphoric acid in milk; and (10) the large amount of soda in bile. It will be observed that the analyses do not state precisely the amount of the various salts that are assumed to exist, but only the quantity of bases and of acids, and it would be impossible to allocate to the bases the requisite quantity of acids to form salts. Further, the phosphoric and sulphuric acids obtained in such analyses are derived partly from the decomposition of organic compounds, and they do not all exist in the tissues in com- bination with bases. It will therefore be seen that an analysis of the ash gives only an imperfect view of even the inorganic com- pounds. To get an approximate estimate of the amount of the more important salts another method is adopted in place of analyzing the ash, namely, that of estimating the quantity of the salt absorbed and eliminated daily. 1. Chloride of sodium and potassium} — The chief saltof sodium is chloride of sodium (NaCl) or common salt, which exists in all the tissues and in all the fluids. Probably 200 grammes may be taken as the average amount in the 1)ody of an adult. From 15 to 20 grammes are eliminated daily chiefly in the urine, whilst a smaller amount is separated in sweat and in the excrements. To make up for this daily loss, common salt is taken in food, either as an ingredient of the food-stuff or as an adjunct. In carnivora, the amount in the food is sufficient for the wants of the organism ; but herbivora and man appear to require an additional quantity. Bunge states that the food of herbi- vora is rich in carbonates, phosphates, and sulphates of j^otash, and that ^ The tests for the mineral substances are no doubt familiar to the student, and the chemical processes, volumetric or gravimetric, by which the amount of each constituent may be ascertained, will be described iu treating of the methods of analyzing the urine. The student of medicine, in particular, should make himself conversant with the more common reactions of the inorganic substances. THE INORGANIC CONSTITUENTS. 39 these salts, reacting on the chloride of sodium in the blood, are decom- posed, chloride of potassium (KCl) and phosphates, carbonates and sulphates of soda being formed. These salts, then, in excess in the blood, are eliminated by the urine, Avhilst the deficiency of chloride of sodium must be met by additional supplies in the food. On the other hand the food of carnivora is deficient in salts of potash, and thus the amount of chloride of sodium in the food is quite sufficient as less of it is used in the manner indicated. If chloride of potassium be substituted for chloride of sodium, disturbances arise by-and-bye from a deficient amount of the latter salt. The tissues, however, retain common salt very tenaciously, so that during a dietary devoid of salt, the salt disappears slowly from the urine. (Beaunis, op. cit. vol. i. p. 79.) A portion of the salt thus introduced may be supposed to pass through the body unchanged, but during its passage no doubt affecting nutri- tive processes. Thus it facilitates the absorption of albuminous matters, and it increases the metabolic changes in the blood or tissues, as shown by the increased amount of urea eliminated in the urine after the administration of salt. As already explained, a portion of the common salt taken in food is decomposed, giving its chlorine to produce chloride of potassium, a salt indispensable to muscular fibres and to the blood corpuscles. The relative amounts of chloride of sodium and of chloride of potassium in some of the principal fluids is shown, per 1000 parts, in the folloAving table — Blood, Blood Coi-puscles, Vl&sva&i, [Liquor Sanyuinis), - - - - Lymph, - Chyle, Gastric Juice, Pancreatic Juice (from permanent fistulse), Pancreatic Juice (from temporary fistula), Bile, ....-.--- Milk, - - Urine, NaCl. KCl. 2-70 2-05 3-67 5-54 0-35 5-67 5-84 1.45 0-55 2-50 0-93 7-35 0-02 5-53 0-28 0-S7 2-13 11-00 4-50 2. Salts of Soda and Potash. — This table shows that the blood cor- jiuscles are rich in KCl, and the plasma rich in NaCl, and it is a general fact that KCl exists largely in the solid parts such as blood corpuscles, musciilar fibre, and nervous tissues, while NaCl exists in the fluids of the body. At one time it was supposed that the salts of potash had a high nutritive value, but the experiments of Panum in- dicate that this is not strictly the case. No doubt, as a rule, animals 40 THE CHEMISTRY OF THE BODY thrive better on a diet containing salts of potash, hut in small doses these have a stimulant rather than a nutritive action, increasing especially the action of the heart, both as regards force and frequency of beat. In larger doses the activity of the heart is "weakened. It would appear that salts of soda are more abundant in embryonic and early life than in adult life. Thus, Bunge found that for each Idlogramme of weight there were the folloAving proportions of soda and potash — Rabbit's Embryo, - Rabbit, 14 days old, - Kitten, 1 day old, - Cat, 19 days old, - Cat, 29 days old. Dog, 4 days old. Adult Mouse, - XaoO. K.O. 2-183 1 -630 2-666 2-285 2 292 2-589 1-700 2-605 2-967 2-691 2-790 2-684 2-667 3-280 3. Salts of ammonia. — Ammonia may lie foimd in the urine, sweat, and gastric juice. About -7 gramme is eliminated daily by the urine, and the urine of herbivora contains less than that of carnivora. Thus, Salkowsky shows that in normal acid urine the ratio of ammonia to the total nitrogen is from 1 to 17 to 1 to 20-5, while in the alka- line urine of the rabbit it is from 1 to 54 to 1 to 57. It is well known that under the action of an organic ferment (see Urine), ammonia ax'ises from the decomposition of urea, and it has been suggested that possibly the small amount found in the urine, and in the sweat or expired air, may arise from the decomposition of urea in the intestine, the ammonia formed being absorbed by the blood- vessels and then eliminated by the kidney. (Beaunis.) In normal circumstances, there is no decomposition of urea in the blood leading to the formation of carbonate of ammonia, and Feltz and Hitter found that this did not occur even after injecting the ammoniacal ferment into the blood. 4. Salts of Lime. — Fluoride of calcium and phosphate, carbonate, sul- phate, urate, and oxalate of lime exist in the body. The fluoride is found in the enamel of tooth and in bone. Phosphate of lime is found everywhere, but more especially in the bones and teeth, which may contain from 60 to 70 or 80 per cent, of this salt. In these structures it exists as the tribasic phosphate (Ca32P04). The ash of all tissues, except elastic tissue, contains phosphate of lime, and urine contains phosphate of lime in sobition, whilst alkaline urine, as in herbivora, shows it in a state of suspension. (Beaiuiis.) Carbonate of lime exists THE INORGANIC CONSTITUENTS. 41 in the otoliths (Fig. 17) or concretions in the internal ear, in the urine, in saliva and salivary concretions, and along with phosphate of lime in the bones, teeth, and hairs. Sulphate of lime has been found in bone, in the blood, and in pancreatic juice, whilst urate and oxalate oi lime appear in urinary deposits. The salts of lime are derived from the food, more espe- cially from vegetable products and from water, in which lime exists as bicarbonate. It would appear that even the small amount present in ordinary water may be sufficient for the wants of the organism. The carbonate in the food or in the Fig. 17.— otoliths of carbonate of lime, con- T IT- T i- sisting (if small thick oolumnar crystals, water is changed durmg digestion combinations of rhombohedrons and hexagonal into phosphate, and this passes p"®™®' into the blood and tissues. Valentin supposed that this change of carbonate into phosphate might go on even in the tissues, as he found that young bones were rich in carbonate of lime, and that the carbonate ^ave place to the phosphate as the bones grew older. Another source ■of carbonate of lime might be found in the formation of carbonic acid from the decomposition of such organic acids as tartrates, malates, citrates during their passage through the body. Salts of lime are eliminated by the bowels and kidneys, in herbivora, chiefly by the former channel, and in carnivora by the latter. The special function ■of the lime salts is to give firmness and solidity to the tissues, more especially to the bones. If supplied in deficient amount, or if they are removed from the solid tissues to too great an extent, the bones in particular may be imperfectly developed, or, yielding to the superincumbent weight, they may become deformed. Further, it appears that when food or drink contains too little of the salts of lime for the wants of the body, these salts are taken from the bones and muscles, indicating that they may have some other part to play in the metabolism of the body with which we are unacquainted. 5. Salts of Magnesia. — Phosphate of magnesia (Mg32P04) is usually found in small amount along with phosphate of lime in all the solids and fluids. Gorup Besanez states that in muscles and in the thymus gland the amount may be even greater than the amount of phosphate of lime. It is eliminated by the kidneys from carnivorous animals as phosphate held in solution in the acid fluid, whilst in the urine of herbivora it exists as the ammoniaco-magnesian, or triple phosphate, 42 THE CHEMISTRY OF THE BODY. or as the i^hosphate of magnesia. Excrements iisiially contain the same compound salt, along with palmitates and stearates of magnesia. The function of magnesia salts is unknown. 6. Iron forms an essential constituent of haemoglobin, the colouring matter of the blood, and traces exist in the chyle, lymph, bile, milk, urine, gastric juice, pigment of the eye and hair. (Beaunis.) The blood of an adult contains about 3 grammes of pure iron, and in the spleen of the dog as much as "24 gramme per 100 volumes has been found hj Picard. The function of the iron has to do A\dth the resi^iratory properties of haemoglobin, the substance which conveys oxygen from the lungs to the tissues. It is daily eliminated in the faeces as the sulphide of iron. 7. Hydrocliloric Acid (HCl) exists in the free state in gastric juice. 8. Phosphoric Acid and Phosphates. — Phosi^horus, combined -with oxygen to form phosphoric acid (PO^), is derived from lecithin, nuclein, and glycero-phosphoric acids — three complex organic bodies, to be considered later — and it unites with soda, potash, lime, and magnesia to form the various phosphates already alluded to. There are three phosphates of soda : (a) Na,PO^, {b) Xa^HPO^, and (c) NaH.PO^ ; three i^hosphates of potash, («) K3PO4, {h) K2HPO4, and (c) KHgPO^; two phosphates of lime, (a) Ca32P04, and (&) CiiM.^2V0^, phosphate of magnesia, Mg32POi; and the ammoniaco-magnesian, or triple j^hos- phate, NH^Mg^PO^ + 6H2O. (Fig. 18.) An adult man eliminates by the kidneys from 2*5 to 3*5 grammes of phosphoric acid daily. Carnivora eliminate phosphates chiefly by the kid- neys, only -^-Q of the total being separated by the excrements as phos- phate of lime and magnesia ; whereas in herbivora the carbonates replace the Fig. is. — Phosphate of ammonia and . magnesia— three-sided prisms bevelled phosphateS in the Unue, and the phoS- .at both ends and on one of the edges. , ^ • n • i phates appear chiefl}^ in the excrements. Compounds of phosphorus are evidently of great importance in the body, as indicated by their presence in the blood corpuscles, the muscles, and nervous tissues. 9. Carbonates. — These are always found in the ash of animal matters, l)ut their presence may be due to the decomposition of organic acids. Still, the blood contains alkaline bicarbonates (NaHCOg and KHCO3), and they may also be found in the urine, lymph, and saliva. Carbonates are derived from the food, directly or indirectly, by the decomposition of organic vegetable acids, such as malic, tartaric, or citric acids. Again, as -will be seen hereafter, carbonic acid may l^e regarded, along Anth water, as the last of the series of bodies produced THE INORGANIC CONSTITUENTS. 43 by the decomposition of non-nitrogenous matters in the body, such as fats and carbo-hydrates. 10. Sulphates. — These are also always found in the ash of animal matter, but as sulphur is an essential constituent of albuminous matter, the sulphuric acid thus found arises from the oxidation of the sulphur during the process of incineration. The presence of sulphates in the ash is therefore no proof of the existence of such in the organic matter. Sulphates, hoAvever, are found in small quantity in the tissues, in the blood, and in the fluids, except milk, bile, and gastric juice. (Beaunis.) These are derived partly from the food and partly by oxidation of the sulphur in albuminous matter. That such oxidations do take place has been proved. Doses of sulphur are followed by an increase in the amount of sulphates eliminated. A diet rich in albuminous matter produces the same influ.ence, whilst a vegetable diet has the reverse effect. Possibly albuminous matters may be decomposed, and sulphates be thus formed. At all events, the increased elimination of urea fol- lowing a diet rich in albuminates is accompanied by an apparently correlative increase in the amount of sulphates. From 1'5 to 2 '5 grammes of sulphuric acid are excreted by the kidneys of an adult man daily. The urine may also eliminate phenol-sulphuric acid and con- jugated acids of a similar kind to the extent of "2787 gramme daily. {v. Velclen.) The urine of cats and dogs may contain alkaline hyposulphites. The excrements contain sulphide of iron, and, by the decomposition in the intestine of albuminous matters rich in sulphur, sulphuretted hydrogen may l^e produced. Chap. II.— THE CHEMICAL CONSTITUTION OF THE ORGANIC CONSTITUENTS. The organic compounds obtained from the body of a plant or of an animal were at one time supposed to be of a different constitution from those of inorganic nature. The chemist could decompose mineral matters into their elements and he could build them vqy again, but whilst he knew the amount of carbon, hydrogen, and oxygen in a given weight of starch, he could not build up this body from these elements. Hence it was assumed that the laws regulating the construction of the complex sub- stances present in living bodies were different from those which rule in- animate matter. The synthesis of urea — a crystalline body found in the urine in 1828 by Wohler — disproved this notion, and since then many organic bodies have been built up by the chemist. In the process, some insight has been obtained into their chemical constitution, the artificial distinction between inorganic and organic chemistry has been removed. 44 THE CHEMISTRY OF THE BODY. and Kekiile, one of the greatest authorities on such a matter, writes as follows — "The chemical compounds of the vegetable and animal kingdom contain the same elements as those of inanimate nature. "We know that in both cases the same laws of combination hold good, and hence no difterences exist between organic ami inorganic compounds, either in their component materials, or in the forces which hold these materials together, or in the number and the mode of grouping of their atoms. We notice continuous series of chemical compounds whose single members, when only those which lie close together are compared, exhibit strong analogy, so that between these no natural di\-ision is perceptible. If, however, for the sake of perspicuity a line of demarcation is to be drawn, we must remember that this boundary is purely arbitrary and is not a natural one, and may be drawn at any point which seems most desirable. If we wish to express by organic chemistry that which is usuallj- considered under the name, we shall do best to include all carbon compounds. We, therefore, define organic chemistiy as the chemistry of the carbon compounds, and we do not set up any ojiposition between inorganic and organic bodies. That to which the old name of organic chemistry has been given, and which we express by the more distinctive term of the chemistiy of the carbon compounds, is merely a special portion of pure chemistry, considered apart from the other portion only because the large number and the peculiar importance of the carbon compounds render their special consideration necessary." i Xotwithstanding the Aveight of Kekule's great authority, it must, how- ever, be said that in the above passage he overshoots the mark, and that there is a real difference in character between organic and inorganic carbon compounds. Contrast, for example, sugar with prussiate of potash, and it ^nll be seen that although both bodies are carbon compounds they are tj-jDCS of structure of an entirely different character. Organic carbon compounds have been happily defined by Schorlemmer as hydrocarbons and their more immediate derivatives, and organic chemistry is therefore that of the hydrocarbons. This definition is probably as good a one as can be given of organic substances. It is clear then that we have to do now "odth the chemistry of carbon compounds. The student is at once brought face to face with a vast series of bodies the chemical constitution of which is represented by complex formulae, and he soon finds also that the same substance may be represented by different formulae according to the notions of the com- position of the body held by difi'erent chemists. It is of interest there- fore to understand what such formulae mean and how the chemist is able to represent the constitution of an organic l^ody by formula?, because there is a great danger of attaching too much importance to formulae and of imagining that they really represent the grouping of the atoms. ^ Kekule, Lehrbuch d. Org. Chemie, i. 11. The above, on comparison with the original, was modified from the translation of the same passage given in Roscoe and Sohorlemmer's Treatise on Chemistry, vol. iii. part 1, p. 32. CHEMICAL CONSTITUTION OF ORGANIC CONSTITUENTS. 45 AVithout encroaching except to a very limited extent on the pro- vince of organic chemistry, it will be useful to give a brief account of the leading conceptions of the organic chemist in this most difl&cult department of the science. Carbon is the one element essential to organic compounds, and the peculiarities of the chemical substances found in the body depend to a large extent on the chemical properties of this all-important element. It is a tetrad element, and its atomic weight (12) is capable of uniting with at most four atomic weights of hydrogen (1) or of any other mon- atomic element. The simplest hydrocarbon compound, marsh gas or methane, CH^^, is a saturated compound, that is, methane cannot combine with chlorine, bromine, or other monatomic element, but one or more of the atoms of hydrogen may be exchanged for an equivalent amount of another element. Thus— Methane. Meth}-! Chloride. Methene Cliloride. Chloroform. CH4 CH3CI CH.Cl^ CHCI3 Carbon Tetra-chloride. Carbonic Acid. Hydrocyanic Acid. Cyanogen Chloride. CCI4 CO2 HCN CICN The above may all be termed substittition derivatives of the first hydro- carbon, CHj. But carbon may unite with itself, or, in other words there may be many atoms of carbon in a carbon compound, and these may unite with each other and thus add greatly to the complexity of the substance. As shown by Kekule, it is this fundamental property possessed by carbon, of its atoms uniting among them- selves, which distinguishes it from all other elements, and this is also the cause of the large number of bodies that may be derived from the different groupings possible with three or four or more carbon atoms. Thus, if two atoms of carbon com- bine, two atomicities of the group are satisfied whilst six remain free; if three atoms of carbon combine, there are eight free atomicities ; and if four atoms of carbon combine, there are ten free atomicities ; and so on. Each of these free atomicities may be satisfied by combining with one of a monatomic element — say hydrogen — or two with a diatomic body — say oxygen — and in this way the compound may become a saturated compound, which cannot combine further with elementary bodies, but from which new bodies may be formed by substitution. The addition of each atom of carbon to the end of the chain raises the combining power of the substance by two. Thus — H HH HHH HHHH I II III I I I I H— C— H H-C— C— H H--C— C-C— H H— C— U— C— C— H I II III Jill H HH HHH HHHH Four. Six. Eight. Ten. 46 THE CHEMIST liV OF THE BODY So that 7i-atoms of C Avill oombine with 2/( + 2 atoms of any monatomic substance. Each of these saturated compounds is the basis of a series of bodies, as shown in the following table — Hydrocarbons. Alcohols. Aldebyde. Acids. Ketones. CnHju + j (./uxljn + .jU C„H,uO C„H.,„0, 0„H,„0 ch; CH40 CH.,0., CaHg CoHgO C.H,0 C..H40, ... CsHg C^HsO c;hsO c.;Heo: C^HgO ^411 10 C.HjoO 04H80 C.HgO, C.HsO c'h;. C'sHisO C5H10O ^"5^100.2 C5H10O CgH|4 CeHj.O CgHjoO CgHioO., CrHis QHieO C.H^ C-HhO, CsHi8 CsHigO CsHieO CgHjgO, C<,H.,o CflH.joO ^9^,30.2 CjoHga CiqHo.jO C10H20O2 CuH.24 ^12^-26 Cv^-2,0, C13H28 Cl4H;jO ... C14H.28O2 Each of the individuals in this series of hydrocarbons ditfers from the one above it by CHg. Such a series is termed homologous, and it is well known that the boiling points of the substances in a homologous series rise by nearly equal increments. Since 1862, series of hydrocarbons represented by CnH.2,, + 2, etc., have been grouped under the name of paraffins. It was found that American petroleum contained a mixture of homologous bodies repre- sented by this formula, and their relation to the alcohols, aldehydes, acids, ethers, etc., already kno^vn to the organic chemist, was recognized. From each of these hydrocarbons numerous substitution products may be obtained. Thus, from any body having the general formula CnHou + o substances may be derived by the substitution of OH, XH^, CHo . OH, CO . OH for one atom of H, or double each of these group.« of OH, etc., for 2H, and so on. Further, from each hydrocarbon, CnHon + 2, by theory at least, compounds poorer in hydrogen are formed by the Avithdrawal of one or more couples of atoms of hydrogen, and from each of these again substitution compounds may be produced. Thus, we have the folloA^dng series of non-saturated hydrocarbons, each of which mav be the basis of a group of chemical substances' — CnHan CnsHsn - 'J C„H2n-4 CnH2u_6 CnB[2n - 8 C.H,„_i4 CuHon - IB CnHsn - 18 CiiH-in - 22 CuHo,, _ 04 C11H2D - 26 Etc. CHEMICAL CONSTITUTION OF ORGANIC CONSTITUENTS. 47 Thus, the series C,^R,n + o, as shown in the above table, contains the well-known monatomic alcohols, such as methyl, ethyl, and propyl alcohols, and the corresponding acids — formic, acetic, propionic, etc. The second series (CnHa,,), starting with olefiant gas, CgH^, contains the diatomic alcohols or glycols, GJi^n + ^^i, and the acids derived from these: (1) acids having the formula CnHanO^, such as giycollic acid, and (2) acids having the formula CnHo^.^Oj, of which oxalic acid is a representative. The third series (C^Hon-o) is illustrated by acetylene, C^Hg; the fourth series (C„Hon_4) by terebinthene, C^QH^gj the fifth series (Ci^Hon-e) hy benzol, C^Hg; the sixth series (Cj.Hsn-s) ^^J cinnamene, CgHg, etc. A good example of bodies related to such a series as any of the above we have in the fifth series, Ci^H2„_g which gives benzol, CgHg, and it in turn is related by substitution to numerous bodies, such as CgHj.OH, CgHg . COH, CgHg . CO . OH, CgHg . NH2, etc., just as CH4 yields the corresponding compounds, CH3.OH, CH3COH, CH3.CO.OH, CH3.NH2, etc. There is, how- ever, a far greater difference betAveen CH3 . ISTH.^ and CgH^ . NH., than between CH3 . NHg and any XH^ substitution product of, for instance, C0H4, C3Hg, etc. So marked are these differences that the benzol derivatives are classed as aromatic compounds to distinguish them from the more immediate derivatives of the paraffins which are termed fatty compounds. Eeference has already been made to the relation of the alcohols to the paraffins. Thus they may be represented by replacing the H of the hydrocarbon by OH. They are therefore substitution derivatives, and the analogues of metallic hydrates. For example, beginning with the hydrocarbon ethane, C^Hg, we have CgHg . OH, ethylic hydrate or ethyl alcohol, analogous to Na . OH, sodium hydrate. In like manner, there is at least one alcohol for each hydrocarbon. But it is well known that there are alcohols having the same percentage composition and yet differing in their products of oxidation and decomposition. The same general formula therefore might represent different substances. For example, CgHgO represents propyl alcohol, but it is well known that there are two propyl alcohols — the one yielding by oxidation first an aldehyde and then an acid, and the second a ketone. To explain this, the chemist devises two formulae. He supposes that in primary propyl alcohol there is a group of atoms, CH^ . OH, which is monatomic, and that in secondary propyl alcohol there is a diatomic groujD, CH . OH. CH, . OH, Primary propyl alcohol is then represented by the formula 1 C2H5 48 THE CHEMISTRY OF THE BODY. and secondary proj)}'! alcohol by CH . OH. In the oxidation of the 6H3 first, the aldehyde is formed l)y the removal of the H^, from the group C.OH CH., . OH, leaving 1 , or C.jHgO, propyl aldehj'de, and the acid ^2 5 (^Q OH by the substitution of for H., in the same group : 1 » or C2H5 CgHgOg, propionic acid. Again, in the case of the oxidation of the secondary propyl alcohol, a ketone is formed by the removal of CH, CH3 HH from the diatomic group, CH . OH— thiis : CH . OH, then CO , or I I CH, CH3 CgHgO, propyl ketone, or acetone. The ketone, when oxidized, cannot give any C, acid, because the group COH is wanting; it yields CO.,, H^O and acetic acid. In like manner there are primary, secondary, and ter- tiary amyl alcohol, and four butyl alcohols are theoretically possible. Compounds therefore having the same percentage elementary com- position and molecular weight, and which show a similar behaviour under certain reactions, whilst they differ to a greater or less extent in other reactions, or at least in physical properties as boiling point, specific gravity, etc., ai-e termed isomeric. Thus, as already pointed out, there are four isomeric butyl alcohols, all having the same formula, C4H9 (OH), and the products obtained by various reactions on these are also isomeric. Oxidation of the first butyl alcohol produces butyric acid (C^HgOg); of the second, isobutyric acid (C^HgO^); of the third, ethylniethyl ketone (C4HgO) ; and of the fourth, a mixture of acetic and formic acids. Finally, bodies having the same percentage composition, but different molecular weights are pobjmeric. Thus, aldehyde C2H4O and CgH^.^Og paraldehyde, are polymeric; and the same is the case mth the hydrocarbons of the general formula CnHon, and with formal alde- hyde, CH^O, acetic acid, C2H4O2, lactic acid, CyH^Og, and grape sugar, CgH^206- ^^ ^"^^^ ^^^ observed that the molecular weights of the polymeric bodies are integer multiples of the same empirical formula. The alcohols hitherto considered are monatomic, but there are also diatomic and triatomic alcohols, and also bodies of a similar kind referable to the higher series of hydrocarbons. Thus the diatomic alcohols or glycols are represented by the formula C„H2„(OH)2. It was pointed out that the primary monatomic propyl alcohol contains the group of atoms CH, . OH, and its relation to the primary propyl CHEMICAL CONSTITUTION OF ORGANIC CONSTITUENTS. 49 glycol may be represented by supposing two sucb groups existing in CHg.OH the latter. Thus 1 ^' ^ or CHg , is primary propyl alcohol, CH2 . OH ^2^5 CH3 and CH2 is primary propyl glycol. A diatomic alcohol is analo- CH2OH gous to the oxide of a dibasic metal, such as Ca(OH).,. Again, we find the triatomic alcohol haiang the general formula C^Hon+aOs, of which the best example is glycerine, of great importance in connection with the chemical constitution of the fats, as will be sho"\vn in another chapter. A triatomic alcohol is analogous to Bi(0H)3. Another group of bodies constitutes the ethers produced by treating the alcohols with dehydrating agents — 2G2H5(OH)-H20=(C2H5)20, or CoHg-0-C,H5. Ethyl alcohol. Ethyl ether. Ethyl ether. Observe also that ethylic ether (02115)20 is analogous to XagO, sodium oxide ; and ethylene ether, CgH^O, to CaO, calcium oxide ; and glycerine ether (03115)203, to 81503, bismuthic oxide. By the oxidation of the alcohols, another group named aldehydes is formed. They are bodies intermediate between alcohols and acids,, and they may be described as hydrocarbons in which the monatomic radicle group COH replaces H (Hydrocarbon, CH^) — CoH5(0H) + = CH3 . COH + H,0. Ethyl alcohol. Ethyl aldehyde. Further oxidation produces the corresponding acids, which may be re- garded as compounds of the same radicles with CO . OH — CH3 . COH + = CH3 . COOH. Ethyl aldehyde. Acetic acid. Thus each alcohol is related to a monobasic acid, and we have COH . OH, formic acid : CgOHg . OH, acetic acid ; C3OH5 . OH, propionic acid ; C^OHy.OH, butyric acid ; C5OH9.OH, valerianic acid ; and CgOHi^^.OH, caproic acid. These acids form metallic salts, com])ou7id ethers, amides^ and haloid derivatives. Thus, to form a salt — 2(CH3 . CO . OH) + K0CO3 = 2(CH3 . CO . OK) + CO. + H^O. Acetic acid. Acetate of potash. By acting on ethylic hydrate with acetic acid, acetic ether is formed, thus — CH3 . CO . OH + C2H5 . (OH) = CH3CO . OC2H5 + H2O. Acetic acid. Ethyl alcohol. Acetic ether. I. D 50 THE CHEMISTRY OF THE BOUV. The compound ethers in tvirn, ■when acted on l)y aninionia, yii'lf Carbon Compounds, p. 44). "Most carbon compounds are colourless when in the pure state; but there exist also a great number having characteristic colo\irs, and many of these are used as dye stuffs or for the preparation of pigments, such as indigo, the colours of madder-root, cochineal, aniline colours, etc. It appears that the colour of these bodies depends on their chemical constitution. Odour and Taste. The odours of volatile carbon compounds vary very much, as the following examples show : — Spirits of wine, ether, acetic ether, acetic acid, chloroform, camphor, oil of cloves, etc. Compounds having a similar constitution often possess a similar smell. Thus the marsh gas hj'di-ocarbons all possess a faint smell of flowers, which is more or less perceptible according to the volatility of the body. The compound ethers of the fatty acids smell like various kinds of fruits, and are on that account used bj- confectioners and perfumers. Most sulphur com- pounds, in which this element is not combined with oxygen, are characterized by their disagreeable odour, and many chlorides have a smell similar to that of chloro- form. Relations between odour and chemical constitution certainly exist ; but only a few such are known. Thus the amines or compound ammonias have an odour resembling that of ammonia, and many aldehydes, compounds which readily absorb oxygen from the air, have a pecialiar suffocating smell. The taste of carbon compounds is equally as varying as their odour ; we find here also that analogous constitution produces a similar taste, and the alkaloids, as quinine, strj'chnine, etc., have an intensely bitter taste, whilst the taste of the alcohols of polygenic radicals, as glycerine, mannite, and siigar, is pleasantlj' sweet. Solubility. A great number of carbon compounds are soluble in water, others only in alcohol, ether, acetic acid, benzine, etc. These different solvents are made use of in separating and purifying them. In homologous series the first members are generallj^ more soluble in water than the higher ones. Thus in the series of the alcohols and fatty acids, the lower members are miscible with water in all pro- portions, whilst those following next dissolve only in certain proportions, and the highest are insoluble in water. All hydrocarbons are either very sparingly' soluble or quite insoluble in water ; by replacing in them a part of the hydrogen by hydroxyl or oxygen, compounds are formed which are more soluble, generally in proportion to the more oxygen they contain. Thus butyl, C4H10, is almost in- soluble in water ; butyl alcohol, C4Hc,(0H)o, readily soluble ; and butylene alcohol, C4Hg(0H)j, mixes with water in all proportions. Succinic acid, C^HgOj, is more soluble than butyric acid, C^HgOn ; and malic acid, C^HgOg, is very deliquescent." It remains now to give in this chapter a classification of the organic compounds met Anth in the body. Two classifications might be given — the one based on chemical, the other on physiological considera- tions. Thus, one might classify the substances under the headings of the various series of hydrocarbons, but as not a few of the substances CHEMICAL CONSTITUTION OF ORGANIC CONSTITUENTS. 55 of interest to physiologists cannot yet have their place accurately fixed, I prefer to give a physiological classification — that is, one in which they can be grouped according to the r6le they perform in the general economy. I. THE NITROGENOUS include— I. THE PROTEIDS, OR ALBUMINOIDS. No formula can be given. A. Tkue Albumins : 1. Albumins— {I) Serum albumin (blood), and (2) egg albumin (white of egg). 2. Globulins— {!) Vitellin (yolk of egg), (2) myosin (muscle), (3) para- globulin (blood), and (4) fibrinogen (blood). .S. Fibrin (blood clot). 4. Proteim—(l) Casein (milk), (2) alkali albumin, and (3) syntonin (muscle). 5. Peptones— (I) Albumin peptones, (2) gelatin peptones (both digestive products). 6. Crystallizahle Alhuminoids—(\) Hfemoglobin (colouring matter of blood), (2) Vitellin plaques (?). 7. Soluble Ferments— {\) Ptyaliu (saliva), (2) Pepsin (gastric juice), (3) Pancreatin (pancreatic Juice), (4) Tripsin (pancreatic juice), (5) Inversive ferment (saliva ?), (6) Rennet (stomach of calf), (7) Lactic ferment (intestines), (8) Steatolytic ferment (pancreas), (9) Blood ferment (blood). B. Albuminous Derwatives : 1. Obtained by change of physical conditions or by chemical action from tissues— (1) Paralbumin (cysts), (2) Colloid matter (diseased liver, etc.), (3) Amyloid matter (diseased liver, kidney, etc.), (4) Mucin (mucus), (5) Nuclein (nuclei of cells), (6) Spermatin (semen). 2. Developed in tissues and obtained by boiling, etc. — (1) Collagen (yielding gelatin), (2) Chondrigen (yielding chondrin), (3) Elastin (from elastic tissue), (4) Keratin (from epidermis, etc. ). II. FATTY NITROGENOUS MATTERS- 1. Phosphoglyceric acid, CsHgPOg, nervous matter. 2. Cholin, or neurin, CgHigNO^, bile, etc. 3. Lecithin, C44HgoNPOg, in nervous tissues, blood corpuscles, yolk of egg, etc. 4. Cerebrin, CJ9H33NO3, in nervous tissues. III. AMINES— 1. Urea, CH4N2O, urine, etc. 2. Oxaluric acid, C3H4N2O4, urine. 3. Allantoin, C4H6N4O3, embryonic fluids. IV. AMIDES— 1. Glycocolle, or glycin, C0H5NO2, bile, etc. 2. Leucin, CgHjgNOg, pancreas, spleen, intestinal canal, etc. 3. Tyrosin, C9H11NO3, pancreas, intestinal canal, etc. 56 THE CHEMISTRY OF THE BODY. 4. Creatin, C4HgN302, muscles, etc. 5. Creatiniu, C4H7N3O, uiine, etc. 6. Tauiiii, C0H7NSO3, muscles, lungs, f;uces. 7. Oystin, C3H7NSO0, urine, etc. 8. Sarcosin, C3H7NO2, muscle. V. NITROGEXOUS ACIDS— 1. Sulphocyanic acid, CNHS, saliva. 2. Uric acid, C5H4lN'403 — and its derivatives, Parabanic acid, C3N2H.2O;;; Alloxan, C4H2iS'._,04 ; Guanin, CjHgXaO ; Sarcin, or hypoxan- thin, C5H4N4O ; Xanthin, C3H4N4O2 ; Carnin, CVH3N4O3— urine, etc. 3. Hippuric acid, CgHj,N03, urine. 4. Inosinic acid, CioH]4N40j], muscle. 5. Cryptophauic acid, C^oHjgNoOio. urine. 6. Bile Acids — (I) Glycocholic acid, C2CH43NO6. (2) Taurocholic acid, C.,6H45NSO;. (.3) Cholalic acid, C24H40O5, and its derivative, dyslysin, C.24H3s03. (4) Choloidic acid, C24H.J8O4. VI. SALTS formed by union of organic acids and inorganic bases. These will be discussed under the heads of their respective acids. 1. Hippurate of soda, C9H8NaN03, urine. 2. Hippurate of lime, CgH^CaNOs, urine. 3. Urate of soda, C5H3NaN403, urine. 4. Urate of potash, C5H3KN4O3, urine. 5. Oxalate of lime, C2HCa04, urine. 6. Glycocholate of soda, C26H42NaNOg, bile. 7. Taurocholate of soda, C26H44NaNS07, bile. 8. Sulphocyanide of potassium, CNKS, saliva. 9. Pheuolsulphate of potash, CgHjO. KSO3, urine. VII. NITROGENOUS BODIES CONTAINING NO OXYGEN— 1. Trimethylamine, CgHgN, urine. 2. Naphthylamine, CjoHgN, urine, fa?ces. 3. Indol, CgH-N, freces, etc. 4. Skatol, C9H9N, fceces. 5. Pyrrol, CH3N, faeces. VIII. PIGMENTS— 1. Blood Pigments: (1) Haemoglobin, no formula; (2) Haematin, C34H34N4Fe05 ; (3) Haematoidin. 2. Bile Pigments: (1) Bilirubin, C16H18N2O3 ; (2) Biliverdin, C16HJ8N2O4; (3) Choletelin, C16H18N2O6 ; (4) Bilifuscin, Ci6H2oN204; (5) Biliprasin, CisH.-,2N206 ; (6) HydrobUirubin , C32H44N4O7. 3. UEiXEPiGiLENTS : (1) Urobilin, C32H40N4O-; (2) Indican, CsgHgiNOi-. 4. Other Pigments: (1) Lutein, yolk of egg, etc.; and (2) Melanin, eye, etc. (no formula). CHEMICAL CONSTITUTION OF ORGANIC CONSTITUENTS. 57 II. THE NON-NITROGENOUS include— I. ALCOHOLS: (1) Ethylic alcohol, CgHglOH), urme, etc.; (2) Cholesterin, C2gH440.HoO, bile, nervous tissues, etc. ; (3) Glycerine, C3Hg(0H);j, intestine, etc.; (4) Phenol, CgHgO, faeces, urine, etc. IL FATS: (l)Tristearin,C3H5(O.Ci8H3gO)3; (2)Tripalmitin,C3Hg(O.Ci6H3iO)3; (3) Triolein, €3115(0.01811330)3; (4) Soaps, such as tristearates, palmit- ates, and oleates of potash and soda. III. CARBOHYDRATES— 1. Glucoses, CgHjoOg, : (1) Dextrose, (2) Chondroglucose, (3) Levulose, (4) Mannitose, (5) Galactose, and (6) Inosite (muscle sugar). 2. Sucroses, C^2H220ii : Sucrose (cane sugar), (2) Lactose (mUk sugar), (3) Maltose. 3. Ainyloses, (CeHioOg)o : (1) Starch, (2) Glycogen, (3) Dextrin, (4) InuLin, (5) Gums, and (6) Cellulose. IV. THE NON-NITROGENOUS ACIDS— 1. Acetic Acid Series, Cr^^sS^i '• (1) Formic acid, CHjOg ; (2) Acetic acid, C2H4O2 ; (3) Propionic acid, CgHgO^ ; (4) Butyric acid, C4H8O.2 ; (0) Valerianic acid, CgH^oOo ; (6) Caproic acid, CgHijOg ; (7) (Enanthylic acid, CjR-^fi^; (8) Caprylic acid, C^B-^^0„; (9) Pelargonic acid, CgHjgOo ; (10) Capric acid, CjoH^oOg ; (11) Lauro- stearic acid, C12H24O2 ; (12) Myristic acid, C14H08O2 ; (13) Palmitic acid, CigH3202 ; (14) Margaric acid, C17H34O2 ; (15) Stearic acid, CasiiseOa- 2. Glycollic Acid Series, CnHsnOa : (1) Carbonic acid, CH2O3 ; (2) Gly- coUic acid, C2H4O3 ; (3) Lactic acid, C3Hg03 ; (4) Oxybutyric acid, C4H8O3 ; (5) Oxy valerianic acid, C5H;lo03 ? (6) Oxycaproic acid, CSH12O3. 3. Oxalic Acid Series, CnH2n-04 : (1) Oxalic acid, C2H2O4 ; (2) Malonic acid, C3H4O4; (3) Succinic acid, C4Hg04; (4) Adipic acid, CgHj^o04; (5) Pimelic acid, C7H13O4 ; (6) Suberic acid, CgHi404 ; (7) Sebacic acid, C10H18O4. 4. Oleic Acid Series, Cn^^--20i: (1) Acrylic acid, C3H402 ; (2) Crotonic acid, CiHgOo ; (3) Angelic acid, CgHgOo ; (4) Oleic acid, C18H34O0. Chap. IIL— THE PROTEIDS OR ALBUMINOIDS. A. Chemical Chaeactees. These organic compounds of complex chemical constitution are the most important of all proximate principles, inasmuch as none of the phenomena characteristic of life can occur without their presence. They form the substratum of all tissues, and especially are they found in protoplasm, forming the chief mass of the nitrogenous matter of plants and animals. As a type of these substances we may take egg albumin, a viscous, non-crystalline, colourless, inodorous, taste- less substance, which analysis shows to consist of carbon, hydrogen, 58 THE CHEMISTRY OF THE BODY. oxygen, nitrogen, and sulphur. Tlic name proteid {jrpoiTeiov, })re- eminence) was first given b}^ Mulder to a substance he obtained by the action of potash on albuminous matter, and he considered it to be the basis or radicle of all albuminous substances, with the h3q3othetical formula CygHogN^Oig. It has been shown, however, that no such definite chemical compound exists, and that what Mulder oljtained was merely albuminous matter more or less modified. The composition of the pro- teids, according to Hoppe-Seyler, varies between the following numbers — C H N S From 51-5 6-9 15 "2 0-3 20-9 To 54-5 7-3 17-0 2-0 23-5 In addition, they always contain a small quantity of the chlorides and phosphates of the alkalies, however carefully they may have been purified. Considerable difference of opinion still exists among chemists as to the constitution of these bodies. The view of Mulder, above alluded to, that all the proteids contain the same radicle, protein, is now abandoned. Nasse has attempted to get an insight into their molecular construction by studying experimentally the mode in which the nitrogen exists in them. He treated various proteids in the dried and powdered state with caustic baryta for 40 or 50 hours and examined the products of decomposition. The conclusion arrived at is that part of the nitrogen is held fii'mly, Avhilst part can easily be displaced, shomng that it exists in different states of combination. Comparing the action of caustic baryta on albumin with its action on compounds, the molecular structure of which is fairly well known, such as amines, amides, or leucin, Nasse thinks that " a definite quantity of the loosely-combined nitrogen of proteids is combined as in amides ; another part as the niti^ogen in creatin or the more loosely-held portion of the nitrogen of uric acid ; and most of the remainder as in the acid amides and the difficultly expulsible nitrogen of creatin or sarcosin." ^ More extended researches in a similar direction have been made by Schiitzenberger who heated coagulated albumin or other proteids with caustic baryta for several hours. Ammonia and acetic acid were thus obtained, and a friable light yellow residue remained, to the extent of 96 per cent, of the albumin used. It would appear in the first instance that the action of the baryta caused a fixation of water and a decom- position into (1) the fixed residue in which a portion of the nitrogen is firmly combined ; (2) volatile products, such as pyrrol, indol, etc. ; (3) ammonia, the nitrogen of which is about ^ part of the total ^ Watt's Diet, of Chemistry, vol. vii. p. 1023. Nasse's Experiments — Siudien- ilber die Elweisshorper. Pfltiger's Archives, vi. p. 589. THE PROTEIDS OR ALBUMINOIDS, 59 nitrogen in the albumin ; (4) carbonic acid ; (5) fatty acids, more especi- ally acetic acid; and (6) oxalic acid. The following equation illustrates this decomposition — C24oH39oN6g075a3 + 57 HoO = C019H431N48O106 + I6NH3 + 3C.H,04 + SCO, + Albumin. Water. Fixed residue. Ammonia. Oxalic acid. Carbonic acid. 4C2H4O2 + C4H5N. Acetic acid. Pyrrol. The fixed residue, by the prolonged action of the baryta and a high temi^erature, yields (1) tyrosin, CgH^-^NOg; (2) amido-acids of the fatty series, such as leucin (amido-caproic acid), butalanin (amido-valerianic acid), alanin (amido-propionic acid) ; (3) glutamic and aspartic acids ; (4) giutimic acid; (5) tyroleucin; (6) leucei'ns, which are combina- tions of leucins and tyroleucins; (7) acids of the general formula C2iiH2„_iN06; and (8) gluco-proteins, bodies of a sweetish taste, having the formula CiHouNo04 (n = 10 or 12). The bodies most easily obtained were tyrosin and leucin, the former to the amount of from 2 "3 to 3*5 per cent. The leucins, having the general formula CnHoa+jNOo, and the leuceins, having the general formula CaH.,ii_i]Sr02, are derived from the splitting up of the gluco-proteins. The longer the action of the baryta is continued the greater is the proportion of leucins and leuceins formed, but at the beginning of the process, and whilst the solution is diluted, gluco-proteids form the chief part of the fixed residue. These in turn are split up into leucins and leuceins, and into intermediate compounds of gluco-proteins with leucins and leuceins. Thus, according to Schiitzenberger, albumin is an amido-derivative, a ureide, which by hydration splits up into a large number of amido-derivatives.^ The researches of the chemists have been conducted of course on dead albumin. But, as already pointed out, the dead state is very different from the living, and we must beAvare of drawing conclusions as to the chemical condition of living matter from an analysis of the substance after death. This was emphasized so long ago as 1837 by John Fletcher, a very suggestive writer, whose speculative opinions are still well worthy of study j^ but it was not until 1875 that the chemical difference between living and dead protoplasm was again pressed on the attention of physiologists by E. Pfliiger.-^ Dead albumin, more especially in the dry state, can be kept without change for a great length of time ; ^ Schiitzenberger, Becherches sur V Albiimhie et les Matltres Albuminoldes, Bulletin de la Soc. Chemique, v. 23 and 24. Also Watt's Bid. ofChem. v. viii. p. 1682. ~ Fletcher, Budiments 0/ Physiology. Edinburgh, 1837. ■' Pfltiger. See his Archives, vol. x. p. 251. 60 THE VHEMISTRY OF THE BODY. it is indifferent to oxygen, iiiid it has the character of chemical stability. But when it becomes living all those characters are reversed. It is now unstable, readily decomposing into simpler substances, and, as has been well said, " the molecule of albumin begins to live by breathing oxygen." ^ Pfliiger supposes that Avhen assimilated the nitrogen of the proteid matter passes from the state of more or less stable amides in the dead condition to the more unstable condition in which it exists in cyanogen and its com- pounds. Further, Pfliiger showed that no better proof could be given of the difference between the constitution of the dead and of the living molecule of albumin than the affinity of the living state for oxygen. He introduced li^^ng frogs into chambers containing no oxygen and kept at a low temperature, and observed that all the processes of life continued for many hours, that is to say there Avas constant self-oxidation. By a rearrangement of the atoms or groups of atoms of the molecule of dead proteid matter it becomes alive. It has a strong affinity for oxygen, and it may then split up into simpler bodies, some of Avhich do not exist among the decomposition products of dead proteid matter. Whether Pfliiger 's conjecture, that the constitution of living proteid matter is that it contains cyanogen radicles in which the nitrogen is loosely combined, be true or not, there can be no doubt his mode of vie^ving the subject has been of great value to science. Follo"\\ang in the same direction are the able researches of Loew and Bokorny.^ Approaching the subject from the point of view of chemists interested in the chemical changes in the plant, they made the remark- able discovery that liAdng protoplasm (or proteid matter) has the property of reducing silver from a weak alkaline solution of silver nitrate, whilst dead proteid matter has no such effect. The observa- tion was made ^wiih. the protoplasm of various alg^e, but the results were not so satisfactory with the protoplasm of the higher plants, and still less so with that of animal tissues. This is accounted for by LoeAV and Bokorny by the statement that animal protoplasm is extremely sensitive to even weak silver solutions, quickly dying, and that thus the usual reaction does not occur. By careful experiment and reasoning, they show that the efiect is not due to anything in the vegetable cell except protoplasm, and they then inquire what substance of a chemical nature in the protoplasm would have such a reducing effect. They come to the conclusion that in the protoplasm there is something of the nature of an aldehyde. They then direct attention to the formation of proteid matter in plants, shoA^ang that these absorb ^ Gamgee, Physiological Chemistry, vol. i. p. 22. - Oscar Loew and Thomas Bokorny, Die Chemische Kraftquelle im Lthendtii Protoplasma. Munich, 1882. THE PROTEIDS OR ALBUMINOIDS. 61 inorganic nitrogen-compounds, such as nitrates and sulphates of ammonia. These are probably decomposed in the plant by the action of the salts of organic acids, setting free nitric and sulphuric acids, and these in turn may be further split up, so as to supply the nitrogen and sulphur required in the formation of proteids. Further, Loew and Bokorny start with formic aldehyde, and by uniting it ■v^^.th ammonia they produce a body termed the aldehyde of aspartic acid.^ Aspartic acid, C^HylSTO^, is obtained by the decomposition, under the action of acids or alkalies, of asparagin, C^HgN^O,, a substance of the nature of an amide (the amide of succinamic acid, CHg . NH2 . CONH2 . COOH), frequently found in leaves and in young shoots kept in the dark. The accumulation of asparagin in young- shoots kept in the dark is explained by supposing that in these circum- stances some of the proteid already in the shoot decomposes, yielding among other substances asparagin, and that this asparagin does not meet Math the appropriate non-nitrogenous matter mth which it could com- bine to form proteid. Loew and Bokorny show the synthesis of a proteid in the following manner — NHo . CH . COH (1) 4CH0H + NH3 = ' I +-2E.fi. CH2 . COH Formic aldehyde. Ammonia. Aspartic aldehyde. Then by polymerization of the aspartic aldehyde, we have — (NHo.CH.COH \ (2) 3^ "I ^=CioHi,N304 + 2H.,0. / CHo.COHJ By further polymerization, in the presence of a sulphur compound, we have — (3) 6(Ci2Hi7N304) -h HoS + 6H2 = G7.H112N1SSO2. + 2HoO, a formula representing the composition of ordinary albimiin. The synthesis may also be illustrated in another manner. Kepresenting the aldehyde of aspartic acid thus — COH I CH— NH, CH.— COH, 1 The weak point in this theory is that the aldehyde of aspartic acid is unknown to chemists, but Loew and Bokorny say that it has not been diligently looked for, and that it is no doubt unstable and evanescent. 02 THE CHEMISTRY OF THE BODY. we obtain hy coiulciisation, as above, the product — COH I CH— NH2 C— COH il CH I CH— NH„ I C— COH II CH I CH— NHo I CH., I COH Then in a manner similar to the building up of complex liodies by successive duplications, along with condensation, we have six of the above grouped as follows to form proteid matter — CHOH — CHOH CHOH — CHOH CHOH — CHOH [a] I I I I I I CH— NH, CH— NH, CH— NH, CH-NH., CH— NH, CH— NH, [h] I "I 'I 'I "I "I C— COH C— COH C— COH C— COH C-COH C— COH (<■.) II II II II II !l CH CH CH CH CH CH (f^) I I I I I I CH-NHo CH— NHo CH— NH, CH— NH, CH— NH.. CH— NH., (e) I ^ i 'I "I "I "I C— COH C— COH C-COH C— COH C— COH C-COH ( /) II II II ' II II :i CH CH CH CH CH CH (.7) I I I I I I CH— NHo CH— NH., CH— NH, CH— NH., CH— NH., CH— NH. (A) I "1 "I 'I '1 "I CH, CH, CH, CH.. CH., CH., (i) I ' I ■ I " I ' I ' I ' CH— OH CH— OH - CH— OH CH— OH— CH— OH CH— OH (/.) — > • ^— The formula of this product is C72H^^4Nj8024, differing from the formula Lieberkiihn gave for albumin, C.^2Hii2Ni8022> S, by 2 atoms of H, 2 of 0, and 1 of S. This ingenious formula also shows how, by cleavage of the molecule of albumin, various bodies may be produced. Thus, a carbohydrate might arise from a and k ; from the rows 5, e, h, rich in N, by taking up 0, we obtain guanin, uric acid, kreatin : lines g and d constitute a benzol nucleus ; from i a fatty acid group, which by three-fold condensation leads to stearic acid. All these substances have been obtained from proteid matter, either by the chemist in the laboratory, or as the result of physiological experiment. THE P ROT BIDS OR ALBUMINOIDS. 63 Similarly, by cleavage in the vertical direction, and the absorption of water, leucin, tyrosin, and other bodies may be produced. Further, Loew and Bokorny attribute the remarkable energy of living proteid matter to the fact of the existence in it of an aldehyde group of atoms, in which molecular movements are constantly taking place. Thus, representing any monatomic radical by R, we have an aldehyde ^0 expressed by the formula, R C . Now, in such a compound two affinities are opposed— (1) the to the H, and (2) the C to each of and H. A struggle occurs between the antagonistic actions, causing much atomic movement, and one may conceive the follo'v\dng changes to occur in rapid succession — (1) (2) (3) (4) E. C , R C , R C , R C \H -^ \H \0H. If the affinities in 2 and 4 be satisfied by combining with another substance, there will then be a state of comparative rest ; but a neigh- bouring aldehyde group would prol^ably impair the stability, and again set up molecular action. Thus — CH— NH, CH— NH I'll C— C==0 = C — C— OH. !l \ II \ H H Group in active Group in passive albumin. albumin. A striking fact in support of Loew and Bokorny's theory that active proteid matter contains a chemical substance of the nature of an aldehyde is that a weak solution of hydroxylamine quickly kills pro- toplasm, a result that may be explained by assuming that a compound is at once formed by the action of hydroxylamine on the aldehyde. Thus— ^ H0\ .^X— 0— H R_C + hJn = R— C +H— 0— H. \H H/ \H Aldehyde. Hydroxylamine. Aldoxan. Water. Or, C,H30| ^ HO N =r CH3 . CNHOH, or C0H4NHO + H2O. Acetic aldehyde. Hydroxylamine. Acetic aldoxan. I have entered somewhat fully into these theoretical views regarding the composition of proteids because it is only by a consideration of such that we can understand how it is that when proteids are oxidized there is the simultaneous production of fatty acids, of aromatic compounds. G4 THE CHEMISTRY OF THE BODY. and of bodies analogous to urea. Further, the consideration of the complexit}' and the instability of the molecule leads the mind to the conception of certain forms of vital action being due to molecular move- ments of a chemical character. Let us next examine some of the more obvious chemical characters of the proteids. These may be demonstrated by making a filtered solution of egg albumin (white of egg). " Only certain of the proteids are soluble in water, all are soluble Avith the aid of heat, in concentrated acetic acid, and in solutions of caustic alkalies ; they are insoluble in cold absolute alcohol and in ether." (Gamgee, op. cit.) It can be readily sho^vn that the albumin is precipitated (1) by the strong mineral acids ; (2) by acetic acid and ferrocyanide of potassium ; (3) by acetic acid and a large amount of a concentrated solution of any neutral salt of the alkalies or alkaline earths ; (4) by basic acetate of lead ; (5) by mercuric chloride ; (6) by tannic acid ; and (7) by alcohol. In the pre- sence of free alkali they are slightly soluble in hot alcohol. The follow- ing special tests may then be applied — (1) Boil and then add a few drops of strong nitric acid ; the precipitate formed is insoluble in nitric acid. Serum and egg albumin solutions show opalescence at a tem- perature of from 60° to 65° C, and coagulation occurs from 72° to 73° C. (Hoppe-Seyler.) (2) Add acetic acid and a solution of ferrocyanide of potassium ; a white precipitate. (3) Acidulate "VAdth acetic acid, and add a concentrated solution of sodium sulphate ; a precipitate. (4) MiUon's reaction. Dissolve 1 part by weight of mercuiy in 2 parts of nitric acid of a .specific gra\dty of 1"42, and dilute each volume of liquid Avith tAvo A'olumes of Avater. Add a feAv drops of this solution to solution of proteid, and on heating, the fluid becomes of a i:)urple-red colovur. (5) Xantho-protein reaction. Add a fcAv drops of strong nitric acid ; boil ; cool ; and then add a few drops of solution of ammonia ; a yelloAv colour is produced if proteid matter is present. (6) PiotroioskVs reaction. Add a few drops of a solution of sulphate of copper and of a solution of caustic soda or potash; heat; a violet colour results. (7) Adamkieiuicz's reaction. Add excess of glacial acetic acid and then concentrated sulphuric acid; a violet colour AAdth feeble fluorescence is produced. B. Pha^sical Characters. If a solution of albumin, such as serum-albumin, in serum of blood, Avhich also contains saline matters and crystalline organic bodies, be placed in a dialyzer (see Figs. 19 and 20) in distilled water, it Avill be found that the crystalline matters pass through the parchment mem- brane into the Avater, AA^hilst the proteid matter remains in the dialyzer. THE PROTEIDS OR ALBUMINOIDS, 65 Solutions of other proteids behave in a similar way, unless they are combined with saline matters, when a portion may pass through. Fig. 10. — Hoop Dialyzer, consisting of a sheet of moist parchment paper stretched and tied over a ring of gutta- percha. Proteids belong to the group of colloids, thus described in the classical researches of Thomas Graham — " Although often largely soluble in water, they are held in solution by a most feeble force. They Fig. 20.— Another form of Dialyzer, in which the parchment is stretched and tied across the wide end of a bulb. appear singularly inert in the capacity of acids and bases, and 'm all the ordinary chemical relations. But, on the other hand, their peculiar physical aggregation with the chemical indifference referred to, appears I. E GG 'i'HJ^ CHEMISTRY OF THE BODY. to be required in substances that can interfere in the organic processes of life. The plastic elements of the animal l)ody are found in this class. As gelatin appears to be its type, it is proposed to designate substances of the class as colloids, and to speak of their peculiar form of aggregation as the colloidal condition of matter. Opposed to the colloidal is the crystalline condition. Substances affecting the latter form will be classed as crystalloids. The distinction is no doulit one of intimate molecular constitution." ^ Along -with other organic bodies, solutions of the proteids have the l^rojierty of rotating the plane of the rays of polarized light. A general knowledge of the principles on which the optical properties of various organic substances are examined is required of the physiological student, and as the use of the polariscope will be frequently referred to, I shall here give a brief account of the nature of polarized light, and of the structure of a Polarizing Apparatus. The nature of polarized light is thus happily illustrated by a distin- guished Avriter on Physics — ' ' Ordinary light consists of vibrations taking place always in planes at right angles to the direction of the ray, but ia all directions in those planes. That is, if the ray travels along the axle of a wheel, the vibrations composing it are all iu the plane of the wheel, but are executed along any or all of its spokes. The effect of reflecting light from certain substances, or of passing it through certain crystalline substances, is to cause all the vibrations to take place in the same direction — that is, along one spoke of the wheel and the spoke opposite to it. The light is then said to be polarized. Now, if the wheel, without being rotated, be slid along the axle, the spoke along which the vibrations take place will trace out a plane. When no rotative force is applied to the polarized light, the vibrations all take place in this plane, and the light is said to be plane polarized. If we twist the reflector or crystal, which we use as a polarizer, round the direction of the ray as an axis, we shall shift this plane in the same way as if we cause the wheel to turn on its axis and so shift the spoke along which the vibrations take place ; but when the wheel is slid along the axis, this spoke will still trace out a plane — only that plane will not be the same as before. That is, if we turn the polarizing mirror or crystal, we turn the plane of polarization, but the light still remains plane polar- ized. We cannot detect by the eye in what plane light is polarized, or, indeed, whether or not it is polarized at all. In order to do so, we have to take advan- tage of the following natural law : Transparent bodies which have the power of polarizing light in any given plane are opaque to light already polarized in a plane at right angles to that plane." ... " Thus to determine in what plane light is polarized, we have only to take a crystal which has the power of polarizing light in a certain plane fixed with regard to its axis, and to turn it round till the light ^ See Young's edition of Graham's Physical and Chemical Researches, p. 553. The paper from which quotation is made appeared in Phil. Trans. 1861. THE P ROTE IDS OR ALBUMINOIDS. 67 Fig. 21. -Diagram showing action of Nicol's Prism. is extinguished. We then know that the light is polarized in a plane at right angles to that plane in the crystal." ^ Light is readily polarized by passing it through an optical apparatus, called a JSHcoVs Prism. This consists (see Fig. 21) of two prisms of transparent calc-spar or Iceland spar A BCD, CDEF, cemented together with Canada balsam at the faces CD, and the faces AB, EF, cut so as to make an angle of 68° with theedges^ E, BF. Theincident ray m?iisdivided into two rays, both of which are polarized ; the one, no, termed the ordinary ray, follows the ordinary laws of refraction, remaining always in the plane of incidence and having a constant index of refraction ; the other, ne, called the extraordinary ray, being polarized perpendi- cularly to the principal section of the crystal, has its direction not confined to the plane of incidence, " unless that plane coincides with or is perpendicular to the principal section, and its index of refraction, excepting in the last- mentioned case, varying continually with the angle of incidence. " - By the Nicol's prism, the ordinary ray suffers total reflection in the direction oP, whilst the extraordinary ray passes in the direction ef, and thence issues as fo parallel to mn. An eye placed at g would see one image formed by the extraordinary ray nefg. Hence only one ray passes through a Nicol's prism, that is, a ray polarized in a plane perpendicular to the principal section, or the short diagonal of the rhomb ab. One Nicol's prism, suitably mounted, is termed a polarizer, because by itlight is brought into a polarized state ; if now another Nicol's prism be held between the first one and the eye, it will be found that no light passes through when this second prism is so rotated as to have its principal section at right angles to that of the first, that both are perfectly transparent when their principal sections are parallel, and that light of diminished intensity is transmitted in intermediate positions. This second Nicol's prism is termed the analyzer, because by it the state of the light polarized by the first Nicol is determined. If now a beam of light, polarized by a Nicol's prism in a horizontal plane, be passed through a tube containing a solution of albumin or of sugar, its plane of polarization on emergence will be rotated to one side or the other of the hori- zontal. To understand this, we must conceive the case of two rays of light of the same amplitude travelling along the same path, polarized so as to be at right angles to each other, and differing in phase by a quarter of a wave length, then the resultant wave would have its molecules rotating in circles transversely to the direction of the ray. This is termed circular polarization, and the effect is the same as "if the ray were polarized in one plane, and that plane were made to rotate round the direction of the ray as an axis, making a complete revolution during the time of one vibration."^ Mr. Gordon gives an ingenious mechanical ^ Gordon, Physical Treatise on Electricity and Magnetism, vol. ii. p. 206. 2 Watt's Diet, of Chem. vol. iii. p. 6o4. ^ Ihid. vol. iii. p. 674. 68 THE CHEMISTRY OF THE BODY. illustration of this state of matters : '' Let us, as before, represent the plane of polar- ization by the direction of the spokes of a wheel, and let us slide the wheel backward and forward on the axis, and cause it to rotate at the same time so that the spoke is always in the plane of polarization at the point where the wheel is on the axis. The case of natural rotation will then be illustrated by considering the tiirning of the wheel to be produced by the guiding action of a spiral or long screw thread cut on the axis. Thus, as the wheel moves along, the spoke traces out a twisted surface, while, when the wheel is slid back again, the spoke comes back along the same surface."^ Further, the direction of the movement of the ether molecules from the centi-e of the circle, or in other words, the direction of the plane of polar- ization, may be to the right, like the movement of the hands of a watch, or to the left, the revei'se direction, and substances rotating to the right are dextro-rotatory, whilst those to the left are hT?vo-rotatory. This effect on polarized light is due to the molecules of the body in solution, and it follows that the amount of rotation will depend on the length of the column of the solution and upon the strength of the solution. As the degree of rotation varies according to the wave lengths of the rays of the spectrum, the light chosen should be yellow rays, such as are emitted from a colourless gas flame in which common salt is volatilized. On these principles instruments are constructed termed Polarimefers, or when employed for solutions of sugar, Sarrharinteters, of which a convenient form, made by Soleil, is shown in Fig. 22. Fio. 22. — Saocharimetor of Soleil. Suppose then, to put the matter in a simple form, that the fluid to be examined is placed in a glass tube surrounded by brass, and closed at both ends with plate glass discs, to fit watertight, screw-caps pressing firmly on the discs. The length of the column of fluid is usually 1 decimetre. The tube may be placed on the support in the position of the dotted line (Fig. 22), between two Nicol's prisms at a and b. The one Nicol is the polarizer, the other the analyzer. Set the two Nicol's prisms so that their principal sections are at right angles and then, as above explained, on looking through the apparatus at a yellow light the field will be dark so long as the tube is not interposed. When the tube is put in position the field is at once lit up, and then the analyzing prism must be rotated until the light disappears. The angle through which the analyzer has been turned measures the rotation produced by the solution in the tube. With this arrange- ment the difficulty is in determining when the light disappears, and to obviate this an appeal is made to the sensitiveness of the eye to colour. It is well known that when a plate of the uniaxial crystal of quartz, cut at right angles to the optic axis, is placed between two Nicol's prisms with their axes crossed (that is with the field dark) a series of brilliant colours appears — red, yellow, green, blue, according to the thickness of the quartz plate. This is because quartz exhibits ^ Gordon, 073. cif. p. 208. THE PROTEIDS OR ALBUMINOIDS. 69 circular polarization in a striking Avay. It will also be found by experiment with different colours that to cause the colour to disappear the analyzer must be rotated through a different angle for each colour, an angle constant for a given thick- ness of plate for each colovir. Thus, with a quartz plate 3 "75 mm. thick the angles of rotation are as follows : — Medium red, 56° "25; orange, 71°'25 ; yellow, 90° ; green, 101°'25 ; blue, 120°; indigo, 142°"o ; and violet, 165°, as shown in Fig. 23. Observe, the angle is least for red and greatest for violet. Some varieties of quartz are dextro- whilst others are Isevo -rotatory, but for a given thickness of plate in both cases the angle of rotation is the same. Experiment has shown that with a quartz plate 3'75 mm. thick, when the principal sections of the polarizer and analyzer are parallel, the red and violet rays are transmitted with only slightly diminished intensity, the resultant colour being purple, which passes quickly to red or violet on rotating the analyzer. This special tint (sensitive- tint, transition-tint, couleur sensible, teinte de passage) is used in optical arrangements as a standard tint because it is easy for the eye to determine when this tint has been obtained and when it disappears. Soleil has taken advantage of these facts in the construction of the Sacchari- meter, a diagram of which is shown in Fig. 24. The light from a gas flame is Fig. 23.— Diagram showing the angular rotation of a beam of white light (polarized by a Nicol's prism whose principal axis is parallel to A A) caused by its pas- sage through a quartz plate 3'7j mm. in thickness, r-r' plane of vibration of red, o-o of orange, 2/-?/' of yellow, gr-gr' of green, h-h'oi blue,i-i'ofindigo,andr-i;' of violet. u. r Q e-f§- Ellld^]^^ r'l.linil^l Fig. 24. — Diagram showing the optical arrangements in Soleil's Saccharimeter. For further description, see text. polarized by a Nicol's prism, c {d m. Fig. 22) ; and it then passes through a double plate of quartz 3-75 mm. thick (at h in Fig. 22). One half of this plate is dextro- [d), whilst the other is lajvo-rotatory {g), as seen in diagram, C^oHg^N^PO^o, C.^H.^gK^POg, Cg^Hg^NPOg,^ and C39Hg2lSr,POg), and the lecithins — bodies of great instability, and hence of uncertain composition. ^ ^ For a full critical account of this difficult subject, see Gamgee's Physiological Chemistry, vol. i. pp. 425-447 ; also Dr. Thudichum's Research, Reports of Medical Officer of the Privy Council and Local Government Board, 1874, p. 113 et seq. 84 THE CHEMISTRY OF THE BODY. B. The Amides. 1. Urea. — The typical substance belonging to this group is urea, CH4N0O. It may be regarded as constructed on the ty})e of two mole- cules of ammonia, in which two hydrogens are replaced by the dyad radical CO, thus- N N NH., -CO = CO NH., CON.H^. It is thus a carbamide. This view shows how, by uniting ^^dth Avater, urea is readily changed into carbonate of ammonia — C0|NH:+g:0 = C0|g;^g^0r(NH,),C0, Urea. Carbonate of ammonia. Again, urea is related to cyanic acid, which may be looked on as a carbamide, that is, ammonia in which two atoms of hydrogen are replaced by CO— N N CO H ' or HCNO. Cyanic acid. Cyanic acid Avith water changes into carbonic acid and ammonia, and cyanate of ammonia is readihT- transformed into urea merely by mole- cular rearrangement — HCNO -f- HoO = CO. + NH.„ and NH4CNO' - CN0H4O. Cyanate of ammonia. Urea. Urea, found chiefly in the urine, is also met -with in the blood, lymph, chyle, bile, aqueous and vitreous humours of the eye, and in many of the liquids of the body. It is also found in nearly all the organs, and especially in the liver, spleen, and lungs. About .30 grammes on an average, Avith a mixed diet, are excreted by the kidneys of an adult man in 24 hours. It is always increased in amount by a diet rich in proteids ; but it does not disappear from the urine even during starvation, as the individual, in a sense, then lives on the tissues of the body, and practically the diet, if one can apply the term to food thus supplied, contains protcid Fig. 2."). — Urea. a. Four-sided prisms ; I). Indefinite crystals, such as are usually formed in alcoholic solutions. THE AMIDES, 85 matter. When obtained pure, it consists of elongated, prismatic, four-sided crystals, terminated by one or two oblique surfaces. (See Fig. 25.) Nitric acid forms nitrate of urea, a salt which takes the form of octahedra, or lozenge-shaped tablets, or hexagons (Fig. 26). Oxalic acid forms flat or prismatic crystals of oxalate of urea (Fig. 26). Urea is O.o Fig. 26. — Nitrate and Oxalate of Urea, a, a. Nitrate of urea ; &, b. Oxalate of urea. inodorous, and has a bitterish-saline taste. It is soluble in water and alcohol, but not in ether. It is not precipitated by acetate, nor by subacetate of lead, but it is thrown down by mercuric nitrate, forming a whitish precipitate, having the formula (CO]Sr2H4)2Hg(N03)2 + 3HgO, and containing urea and mercuric oxide in the proportion of 10 to 72. Mtrous acid, chlorine, hypochlorite and hypobromite of soda decompose it into nitrogen and carbonic acid — CONOH4 + N203 = CO2 + 2HoO + 2N2. CONgH^ + H0O + 3CI2 = COo + No -t- 6HC1. CON0H4 + SNaBrO = COo -K No + 2H20-(-3NaBr. On the reaction of mercuric nitrate with urea depends Liebig's well-known volumetric process for the estimation of the amount of urea in a fluid, and on its reaction with hypobromite of soda is founded the method of determining the amoiint of urea from the amount of nitrogen evolved, devised first by Knop and since modified and improved by many chemists. These methods will be described in treating of the analysis of the urine. By fermentation, under the action of a specific organism {Micrococcus Urece), it is transformed into carbonate of ammonia — CON2H4 -t- 2H2O = (NH4)2C03. Urea. Carbonate of ammonia. When dry chlorine is passed over urea, the following reaction occurs — 3(CON2H4) 4- SCla = C3H3O3N3 + N2 + 5HC1 + NH4CI. Urea. Cyanuric acid. 86 THE CHEMISTRY OF THE BODY. When heated with a mixture of caustic potash and })ermanganate of potash, it is decomposed into carbonic acid and ammonia. There is still considerable obscurity as to the origin of urea, but the evidence will be better appreciated after a study of the metabolic changes occur- ring in the tissues, and more especially after the study of the functions of the liver and kidneys. It is sufficient to state here that there is no evidence showing that it is derived from uric acid. No doubt it is a product of the splitting up of nitrogenous matters. We have seen that decomposition of the albumin-molecule may produce leucin, ty rosin, glycocolle, and amido-acids or amides ; and these substances, especiall}' the two first, are found in the alimentary canal, and probably are thence absorbed and carried to the liver. In cases of acute atrophy of the liver, urea is said to disappear from the urine, and it has been supposed that this occurs because the hepatic cells cannot effect such chemical changes on leucin and tyi'osin as normally produce urea. Further, the direct injection of leucin and glycocolle into the bowel increases the amount of urea, and " all the nitrogen of the leucin and of the glyco- colle appears in the urea." (Beaunis.) It will be seen that this is strong presumptive e'vddence in favour of the Aaew that urea may be a decomposition product of such bodies ; but one must admit that the effect maj' be due not to a direct transformation, but to the action of leucin and its allies on the general metabolism of proteid matter in the liver. It has also been sho^^ai that amides, such as glycocolle, sarcosin, and taurin, when introduced into the body so as to be carried to the liver, increase the urea (Salkowski), but they may appear in the urine as uramides, that is, bodies formed by a combination of NHCO (cyanic acid) "vnth the amide introduced. Thus glycocin and cyanic acid form hydantoinic acid, sarcosin (methyl-glycocolle) with cyanic acid forms methyl-hydantoinic acid (Schultzen), and taurin with cyanic acid forms tauro-carbamic acid (uramo-isthionic acid) (Salkowski) — NH2 . CH, . CO . OH + CO . NH = NH . CONH, . CH, . CO . OH. Glycocolle. Cyanic acid. Hydantoinic acid. NHo . CH„ . CHo . SO, . OH -f CO . NH = NH . CONH, . CH, . CH, . SO, . OH. Taurin. Cyanic acid. Tauro-carbamic acid. There is e\ddently then some relation between glycocolle, sarcosin, leucin, tyrosin, and urea, although the chemist has not succeeded in effecting a direct transformation of any of these bodies into urea. But if not directly derived, may not urea originate from certain bodies in their turn springing from the amides, glycocolle, sarcosin, etc., or at all events having a common origin "sWth these 1 For example, suppose by decomposition of albuminoids that cyanic acid were produced, then one can conceive urea originating thus — THE AMIDES. 87 NHCO + NHCO + HjO = CON^H^ + CO.,. Cyanic acid. Cyanic acid. Urea. Carbonic acid. Chemists have not yet found cyanic acid as a product of the decom- position of the amides, so that this origin of urea is doubtful. Another theory that has been put to the test of experiment with unsatisfac- tory results is, that urea may be produced by the formation from albuminoids of ammonia, and that the carbonate of ammonia produced by the union of ammonia with carbonic acid may then, by losing water, become urea. To test this view, salts of ammonia have been administered to animals, and the effect, if any, on the elimination of urea has been scrutinized. Kniriem and Salkowski state that the urea eliminated by the rabbit is increased by giving the animal chloride of ammonium or nitrate of ammonia; in the dog, however, Salkowski did not ob- serve this effect. The point in dispute is thus stated by Beaunis — " This fact, which led Voit and Feder to deny the results obtained by Kniriem and Salkowski, has been explained by Schmiedeberg and his pupils, by showing how differently the dog and the rabbit are influenced by acids. Schmiedeberg and Walter have shown that in the dog, the ingestion of hydrochloric acid increases notably the elimination of ammonia by the urine ; this ammonia, necessary for the elimination of hydrochloric acid, the latter takes from the organism ; but if, instead of the hydrochloric acid being introduced in a free condition, it is introduced in chloride of ammonium, there is no formation of urea, because the ammonia is re- tained by the hydrochloric acid, which, in place of borrowing the ammonia from the organism itself, employs that directly supplied. This is shown by the fact that if a dog receives carbonate of ammonia instead of the chloride of ammonium, a part of the carbonate appears in the urine as urea. If, however, following the example of Munk, the organism of the dog is placed in the same conditions as that of a rabbit by rendering the urine alkaline by a vegetable diet, then the chloride of ammonium only appears partially in the urine, a half, or less, of the salt intro- duced contributing to the formation of urea." ^ If then ammoniacal salts increase the amount of urea eliminated, we may suppose with Salkowski that cyanic acid arising from the decomposition of proteids unites with the ammonia to form cyanate of ammonia, which, by molecular transformation, then becomes urea. Normally then the reaction would be thus — CO.NH 4- CO.NH + H^O = CON2H4 + CO2. Cyanic acid. Cyanic acid. Urea. whilst the reaction after the administration of ammonia woidd be — 2C0NH + 2NH3 = 2CO]Sr2H4. Cyanic acid. Ammonia. Urea. Thus, whilst it is clear that urea results from decomposition of proteid matters, the exact steps by which this is accomplished are unknowai, ^ Beaunis, op. cit. vol. i. p. 147. 88 THE CHEMISTRY OF THE BODY. and the question whether nrea may arise from decomposition onl_y, or from the synthesis of products of decomposition, is still unsettled. 2. Oxalurk acid, C-jH^NoO^. — This substance is urea in which one atom of hydrogen is replaced l^y the residue of oxalic acid, that is oxalic acid CgHqOj, minus hydroxy], HO or C^O.jH — CO NH., NHo Urea. CO NH., NH'. C,0;)H. Oxaliiric acid. Heated in the presence of water, it is decomposed into oxalic acid and oxalate of urea — 2(C3H4N204) + 2(H,0) = Oxaluric acid. Water. (CH4N,0).C,H,04 Oxalate of urea. + C,H,0,. Oxalic acid. The nature of this acid Avill be further referred to in discussing uric acid and its derivatives, of which oxaluric acid is one. 3. Allantow, C^H^N^Og, is also a derivative of uric acid, found in the amniotic and allantoic fluids of the emliryo, in the urine of neAvly-borii children, and in the urine of certain animals, such as the clog and cat. It may be formed artificially by treating uric acid with water and per- oxide of lead, or by acting on uric acid Avith potassium ferrocyanide and caustic potash. It forms shining colourless prisms (Fig. 27), is taste- less, and is soluble in 160 parts of water at 20° C. and in 30 parts of boiling water. Heated with nitric or hydrochloric acids, it is changed into uric and allanturic acids ; sulphiuric acid splits it up into carbonic acid, carbonic oxide, and ammonia, Avhilst, when boiled with baryta water, it gives oft* ammonia and i:)recipitates oxalate of bariimi. When 1 part of gly- oxalic acid is heated with 2 parts of urea to 100° C. for eight or ten hours, allantoin is formed, and from this reaction it is regarded as the diureide of glyoxalic acid, thus — NH - CH - NH - CO - NH., CO I NH - CO Pelouze discovered an acid, allanturic acid, C-.HjoNp,Og(?), produced from allantoin by the action of dilute nitric acid, thus — H„0 = Fio. 27. — Allantoin. 2(C,H6N,03) Allantoin. C^HjoNfiOe Allanturic acid. CONgH^. Urea. THE A MIDO-A GIBS. 89 Chap. VL— NITROGENOUS PROXIMATE PEINCIPLES. —Continued. The Amides, or Amido-Acids. When we examine the chemical constitution of plants, we find they contain non-nitrogenous and nitrogenous bodies. As examples of the former, we have (1) carbohydrates, such as cellulose, starch, dextrin, gum, and cane and grape sugar ; (2) the varieties of the essential oils and resins ; and (3) vegetable acids. The nitrogenous compounds comprise in addition to proteids, the colouring matters of plants, the j)rincipal of which is chlorophyll, and organic bases termed alkaloids. These bases contain nitrogen, in addition to carbon, oxygen, and hydrogen. The following are examples — Aconitine, Asparagiae, Atropine, Biberine, - Br u cine, - Caflfein, - Cinchonine, Codeine, - Conine, - Morphine, Narceine, Narcotine, Nicotine, - Papaverine, Quinine, - Kinoline, Sinapine, - Strychnine, Thebaine, Theine, - Theobromine, C30H47NO7 C4H8N2O3 - CiyH^NOa C19H21NO3 C2sF,6N204 CsH.oN^O, C,oH24N,0 C18H21N2O3 C3H15N - C17H19NG3 C^H^gNOg C^^HasNOy ^loHi4N2 - C.,oH2iN04 C,oH24N,0, C9H7N - C16H23NO5 C^iH^^N^Oa Ci9H,iN,03 C8H10N4O2 C7H8N4O2 - Aconitum napellus. Asparagus officinalis. Atropa belladonna. Biberis vulgaris. Strychnos nux vomica. Tea, coffee, etc. Cinchona bark. Opium. Conium maculatum. Opium. Opium. Opium. Tobacco. Opium. Cinchona bark. Cinchona bark. White mustard. Strychnos nux vomica. Opium. Tea, coffee. Cacao beans. Chemically, these bodies are regarded as compound ammonias — that is, bodies in which one, two, or three equivalents of hydrogen are re- placed by radicles. Thus conine may be rejoresented as ammonia in which two atoms of hydrogen are replaced by the monatomic radicle, C4H7, and nicotine as a condensed ammonia in which all the atoms of hydrogen are replaced by the triatomic radicle CgHy. Example— (C4H,)^ H MN = aH,,N. Conine. C5H7 CgHy Nicotine. No = C,„a4N, In the animal economy certain bodies are found which apparently CgH^NO, Cystin, - - C,H-NSO., C,,H„N03 Sarcin, - - CgH.N.O^ CHgNO., Xanthiu,- - CsH^NA c;h7no;s Guaiiin, - - C5H5N5O C:HgN30, Alloxan, - - C,H,N,0, C4H7N3O Urea, - CH4N,,0 90 THE CHEMISTRY OF THE BODY. have a somewhat similar chemical structure. They differ in chemical composition from the vegetable alkaloids, however, by containing less nitrogen and oxygen in proportion to the amount of carbon. These substances are — Leucin, . - - Tyrosin, - Glycocin or glycocoUe, Taurin, Creatin, Creatinin, - It is probable that certain of these bodies are amides — ammonias in which one or more atoms of hydrogen are replaced by radicles of an acid, or amido-acids — that is, acids in which one or more hydrogen atoms of the radicle of the acid are replaced by NH^. Thus lU'ea, as already explained, may be regarded as carbamide, or ammonia containing the diatomic radicle of carbonic acid. Example — g^N, CO Ho ( N., = CH^K.O. ^^■f ' h: i " ■ Ammonia. Badicle of CO2. Carbamide. Urea. In like manner, on the type of one molecule of water, glycocin may be regarded as amido-acetic acid, leucin as amido-caproic acid, and sar- cosin, one of the derivatives of creatin, as amido-methyl-acetic acid. In the amido-acid group of the latter substance one atom of hydrogen is replaced by CHg (methyl), thus — CsHioONHolo H J ^ C.,H0(CH3)NH., ) , hT or C2H5NO, Glycocin or amido- acetic acid. or CeHigNO, Leucin or amido- caproic acid. or C3H-NO0 Sarcosin or methyl- glycocin, or amido- methyl-acetic acid. The amides are often conjugated bodies, and they may behave either as bases forming unstable compounds with acids, or they may play the part of feeble acids, which, A\"ith the loss of water, unite with alkaline bases. The chief point of physiological importance is their instability^ especially under the conditions prevailing in the living body. 1. GlycocoUe, or Glycm, or Glycocin, CoHjXOg, as above sho^vn, is amido-acetic acid. It forms a constituent of glycocholic acid, one of the acids of the bile. Combining Avith benzoic acid, it forms hippuric acid. "When uric acid is acted on with warm hydriodic acid, it splits up into glycocoUe, iodide of ammonium, and carbonic acid, a reaction which indicates that glycocoUe is related to uric acid — CgH4lir403 + SHI + 5H2O = CoHgNO. -f 3NH4I + SCOj. Uric acid. Hydriodic acid. GlycocoUe. Iodide of Carbonic acid. ammonium. THE AMIDO-ACIDS. 91 The decomposition of gelatin yields glycocolle among other substances, and it is possible that glycocolle may be produced in the liver from gelatin absorbed from the alimentary canal. Glycocolle, when thus formed, unites Avith cholalic acid to produce glycocholic acid, and it may also unite with any benzoic acid present to form hippuric acid. In the intestine, no doubt, glycocholic acid is decomposed, and the glycocolle liberated passes into the blood. It has been suggested that it may then be decomposed into methylamine and carbonic acid, thus — CH. . NH., 1 CO . OH . = N H H + CH3 CO Glycocolle. Methylamine. This methylamine (not found free in the body), may, in certain circumstances, form trimethylamine, which sometimes is present. Again, the oxidation of glycocolle produces carbonic acid, oxamic acid, oxalic acid, and water; and oxamic acid, ill turn, is readily decomposed into ammonia and oxalic acid. In a pure state, glycocolle appears in the form of rhombohedric or prismatic crystals (Fig. 28), having a sweetish taste. These are soluble in cold water, and they are insoluble in cold alcohol and ether. The solution is acid. When a solution of glycocolle is boiled with a solution of hydrated cupric oxide, a blue solution is formed, from which bluish needle-shaped crystals separate on cooling. 2. Leucin, C^-^^O.j, is amido-caproic acid, leucic acid, or oxy-caproic acid, that is to say, the hydroxyl of leucic acid is replaced in leucin by the radicle NH,. Thus — CH3 CH2 . OH CH2 . NH2 Fig. 2S.— Glycocin, or glyco- colie, or glycin. It is related to an acid. (CHs)^ CO. OH Caproic acid. (CHa)^ CO. HO Leucic acid or oxy- caproic acid. (CHa)^ I CO. HO Leucin. It has already been pointed out (p. 59) that leucin exists in consider- able amount among the decomposition products of proteids when the proteids are split up by the action of strong caustic alkalies or sulphuric acid. Leucin is also formed when proteids are decomposed by putre- faction and by pancreatic digestion. It is found in many of the tissues and organs, more especially in the pa creas, and in smaller quantities 92 THE CHEMISTRY OF THE BODY. ill the liver, kidneys, spleen, thymus, lymphatic glands, and the brain. In some pathological conditions, such as leidcremia and acute atrojjhy of the liver, it is found in the urine in the form of yellowish- hrown lialls (Fig. 29) or spheres, highly refractive, and consisting of masses of fine needle-shaped crystals. Leu- cin is inodorous, it is soluble in water, slightly in alcohol, l)ut not in ether. Its solu- tions are neutral in reaction and almost tasteless. Leucin originates in the body by the decomposition of proteids. In turn, it splits up into fatty acids and ammonia. Thus, by heat it is decomposed into carbonic acid and amyl- amine — ■^^^ Fig. 29. — Spheroidal crystalline masses of leucin. a, A very minute simple spherule ; b, hemispher- oidal masses ; c, c, a;,^gi-egates of small globules ; d, a larger globule supporting two halves ; e, /, large spheroids richly studded with minute seg- ments ; (I, g, (I, ri, laminated globules, some with smooth, some with rough surface, and of very various sizes. CH.,.NH., 1 CH., . NH., 1 :GB.^)i (CH2)3 CO . OH . CH3 Leucin. Amylamine. + CO., By hydriodic acid it is decomposed into caproic acid and ammonia, thus — CH.,.NHo CH, (CH.)4 CO . OH . Leucin. + 2H = (CH2)4 + NH,. CO . OH . Caproic acid. With concentrated sulphuric acid, leucin yields ammonia and valeric acid, and with permanganate of potash, oxalic acid, carbonic acid, valeric acid, and ammonia. Possibly, in the body, similar decomjoosi- tions may occur, which accounts for the traces of these substances found in some organs. Thus, valeric acid has been found in the sweat and in epidermic matters containing leucin. It has also been conjec- tured that leucin may be one of the steps towards the formation of urea, and this is supported experimentallj- by the fact that the admini- stration of leucin to dogs increases the amount of urea eliminated by the Iddneys. THE AMIDO-AGIDS. 98 3. Tyrosin, CgHj^^NOg, is the amide of oxyplienylpropionic acid, and is related on the one hand to a fatty acid, propionic acid, and on the other, to phenic acid, an aromatic substance, thus — CH, CH3 CH3 CH3 I I I I CH. CH . NH. CH . (CfiHj .OH) C . (CgHi . OH)NH, I ■ I ' I ■ ! CO .OH CO . OH CO .OH CO . OH . Propionic acid. Alaain. Oxyphenylpropionic acid. Tyrosin. Here observe that one atom of hydrogen in propionic acid is replaced by the radicle oxyphenyl (C^H^ . OH), and when the remaining hydrogen in oxyphenylpropionic acid is replaced by NH.,, tyrosin is produced ; or, as shown above, tyrosin may be looked on as alanin (the amide of propionic acid), in which one atom of hydrogen is replaced by oxy- phenyl. Tyrosin is usually found along "vvith leucin, and like it, results from the decomposition of proteids. Occasionally found in the urine, it is probably entirely decomposed in the body, and it has been sho^vn that the administration of tyrosin to a living animal is not followed by its apjjearance in the urine, but by the appearance of .phenol and phenol-sulphates, indicating its relation to the aromatic compounds. Tyrosin crystal- lizes in slender, colourless, mi- croscopic needles (Fig. 30). It is slightly soluble in cold water, but is insoluble in alcohol and ether. Whilst burning, it smells like burnt horn. Oxidation by bichromate of potash yields oil of bitter almonds, hydrocyanic, benzoic, formic, acetic, and car- bonic acids. Firia's test : Heat the substance with a few drops of concentrated sulphuric acid in a watch-glass ; when the solu- tion is cold, add a little water and a few morsels of chalk, when there will be effervescence ; filter ; evaporate to small bulk, and add two drops of a neutral solution of perchloride of iron ; a violet colour indicates tyrosin. in water ; add several drops of a neutral solution of mercuric nitrate ; boil, and a rose-coloured fluid with a reddish precipitate indicates tyrosin. Fig. 30. — Acioular crystals of tyrosin. a, Single crystals ; b, 6, smaller and larger groups of the same. Hoffmann's test: Dissolve substance 91 THE CHEMISTRY OF THE BODY 4. Creatin, C^HgNgOo + HoO, is related to another amide, sarcosin, (methyl-glycocin, or amido-methyl-acetic acid). This substance, sarco- sin, united to cyanamide, gives creatin, thus — CH..NH, CH.J CH,.N " " " CO. OH Glycocin or Methyl, .iinido-acetic acid. CO. OH Methyl glycocoUe or sarcosin. + C.N I I NH, Cyanamide. CH.,.N C.NH CO. OH NHo Creatin. Another view is that it is related to urea, in which one atom of hydrogen is replaced by sarcosin, less one molecule of hydroxyl — /CH3 /CH3 NHo I CO I NH., CHo . N I CO -H NH, I CO CH., . N N CO I H \H Urea. Sarcosin, less hydroxyl. Creatin. This vieAV is supported liy the fact that creatin boiled vnth. baryta water yields sarcosin and urea — C4H9N3O, + H,0 = C3H7NO. + CON0H4 Creatin. Sarcosin. Urea. Creatin is found in the muscles, nervous tissues, the blood, the liquor amnii, and the testicle, but not in glands. It appears in the form of colourless rhombohedric prisms (Fig. 31). It is soluble in water, almost in- soluble in alcohol; its solutions are neutral. By oxidation, creatin gives oxalic and carbonic acids and methyluramine, C^H^-Ng. Although found in the muscles, it has not been proved that it is increased in amount by causing the muscles to contract, and its presence appears to depend more on the natiire of the diet than on a breaking clown of the proteid matter of the muscles during activity. An animal diet increases, whilst a vegetable diet decreases its amount. With an animal diet, an allied substance, creatinin, into which creatin is readily transformed, appears in the urine, but it has been held that this excess of creatinin comes from the creatin in the animal food. 5. Creatinin, C^H-XgO, closely related to creatin, is also related to a body named methylhydantoin, being transformed into it and ammonia when heated T\ath baryta water to 100° C. — CH2 . N(CH3) CH. . N(CH3) Fig. 31.— Creatin. I CO I I CO . NH Methylhydantoin. C.NH CO . NH. Creatinin. THE AMIDO-ACIDS. 95 It is readily derived from creatin. Thus "vvitli hydrocMoric acid, or even when creatin is subjected to prolonged boil- ing, it parts witli water, and creatinin is formed, thus — when creatin is heated C4H9N3O, Creatin. CiH^NaO + HoO Creatinin. Fig. 32.— Creatinin. Creatinin forms large, brilliant, colour- less prisms, having an alkaline taste (Fig. 32). It is soluble in water and alcohol, scarcely soluble in ether. It has a strongly alkaline reaction. Oxid- ized, it yields methyluramine, C2H7N3. A concentrated solution of chloride of zinc (not acid) produces a finely granu- lar precipitate, or groups of fine needles or prisms, of a double chloride of zinc and creatinin ; this chloride, treated with sulphide of ammonium, reproduces creatin by taking an equivalent of water — CjHyKjO + H^O = C4H9N3O2 Creatinin. Creatin. Test. — A solution of creatinin, as in urine, acidulated by nitric acid, gives with phospho-molybdic acid a yellow crystalline precipitate, soluble in hot nitric acid. The transformation of creatin into creatinin does not occur in the blood (which contains no creatinin), and it would appear that it occurs in the kidneys, as there is always creatinin and not creatin in the urine. Ligature of the ureters, however, is not followed by the appear- ance in the kidney of creatinin, but of creatin, pointing to the transformation happening, not in the substance of the kidney, but in the urine. As to the possible transformation of creatin into urea, there is conflicting evidence. Munk found that the administra- tion of creatin to man and the dog was followed by an increase in the amount oi creatinin in the urine, and also by an increase in the amount of urea. The change of creatinin into lu-ea was supposed by Oppler, Zaleski, and others to occur in the Iddney, as extirpation of the kidney was found by them to cause not an increase of the urea in the blood, but of creatin in the muscles. On the other hand, if the ureters were ligatiured, urea accumulated in the blood and the amount of creatin in the muscles was normal. Similar experiments by Voit and Meissner, and by Voit and Gscheidlen have, however, given negative results. "V^Tien one considers the severe nature of the operations, and the impossibility of the animals surviving for more than a few hours, it 96 THE CHEMISTRY OF THE BODY. is not surprising tha tresults should be contradictory. The matter then stands thns — that whilst in the laboratory by various methods creatin can be decomposed into sarcosin and urea, there is no proof that this occui's in the l)ody. 6. Taurin, CoH^NSOy, is an organic body related to isthionic acid, or sulphurous acid in which an atom of hydrogen is replaced by a monatomic radicle oxyethylene ((CH2)o.0H), and in turn the replace- ment of the hydroxyl in isthionic acid by NHo produces taurin, thus — SO., . OH., CHo . OH CH., . OH CH., . NH, CHo CH, I SO., . OH Isthionic acid. CH., I SO, . OH. Tuurin. Sulphurous acid. Oxyethylene. "With cholalic acid, taurin forms one of the bile acids, taurocholic acid, just as glycocin, unites with the same acid to produce glyco- cholic acid. Another point of resemblance between glycocin and taurin is that cyanic acid combines with glycocin to form hydantoinic acid, whilst cyanic acid with taurin leads to tauro-carbamic acid, thus — CH., . NH, CH., . NH . CO CO. OH Glycocin. CH, . NH., I CH2 . SO. , Taurin. OH CO . OH NH, Hydantoinic acid. CH2 . HN . CO I I CH, . SO, . OH . NH, Tauro-carbamic acid. Tauro-carbamic acid exists in the urine and may originate by the union of taurin with cyanic acid. Taiirin, arising from the decomposition of taurocholic acid, is found in small amount in the intestinal canal and faeces, and also in muscle, in the lungs, in the urine of the ox, and in the liver and spleen of certain fishes. Its origin is unknown. From the small amount in the faeces, and the relatively large amount found in the liver, it is probable that jDart of the taurin is decomposed into simpler substances. As potash splits it U2) into carbonate of ammonia and the sulphite and acetate of potash, it has been conjectured that the alkalies of the bile may so transform it, and that the sulphates of the mine may Fig. 33. — Taurin. a. Six-sided prisms ; b. Irregular sheaf-like masses from an impure solution. THE NITROGENOUS ACIDS. 97 be partly derived from this source. Taurin crystallizes in the form of colourless six-sided prisms (Fig. 33). It is soluble in water, and is insoluble in alcohol and in ether. 7. Cystin, CgH-NSO^, is amido-lactic acid, thus — CH„ . OH CH3 "HS CH. I CO. OH Lactic acid. c ■■ I CO ^NHj OH Cystin. Fig. 34.— Cystin. It is found as a constituent of a rare form of urinary calculus, and occasionally in the urine, more especially of the dog. Its origin is unknown. It forms rhombohedric or hexagonal colourless plates (Fig. 34). It is insoluble in water, alcohol, and ether, but is soluble, like uric acid, in ammonia, in the mineral acids, and in a solution of oxalic acid. If heated on a silver surface with a little caustic soda, it gives a brownish-black spot of sulphide of silver. 8. Sarcosin, CgH^NO^, has been already referred to in treating of ci-eatin, and we have seen that it is methyl-glycoUe, C2H4(CH3)N'02. It has not been found in the body, but it may be obtained by heating creatin with baryta water. It crystallizes in rhombohedric colourless columns. It is very soluble in water, less so in alcohol, and not at all in ether. Chap. VII. -THE NITROGENOUS ACIDS. 1. Sulphocyanic Add, CNHS, united to potassium or sodium to form a sulphocyanide, is almost invariably found in the saliva, and it has occasionally been found in the urine, in milk, and in the blood. It may be detected by giving a red colour (sulphocyanide of iron) with a solu- tion of perchloride of iron. Some have supposed that it may originate from the decomposition of carious teeth, but it has been found where this condition did not exist. 2. Uric Acid, CgH^N^Og, is one of the most important bodies of this group, inasmuch as it is related to a number of nitrogenized substances, often met with in the solids and fluids. Much diversity of view still exists as to its chemical nature. Baeyer regards it as formed by the union of a radicle cyanamide with tartronic or oxymalonic acid — CO . OH fNH., CO . NH . CN I 2J I " I CH.OH + [CN = CH.OH + 2HoO CO .OH CO . NH . CN Tartronic acid. Cyanamide. Uric acid. I. G 98 THE CHEMISTRY OF THE BODY or, tAvo molecules of bydroxyl in tartronic acid are replaced by two cyanamides, which each lose an atom of hydrogen, and these, Avith the molecules of hydroxyl, form Avater. Another vieAv put forAA^ard by Medicus is to consider uric acid as containing a nucleus of 3 atoms of carbon, and that this Avith tAA^o residues of urea forms uric acid or a, di-iu^eide, thus — —CO I C— II — c— Nucleus. CO NH Residue of urea. NH— CO I I CO C— NH\ I II / NH— C— NH / Uric acid. CO. This suggests the relationshij:) of a number of the bodies allied to uric acid, such as alloxan, urea, parabanic acid, allantoin, oxaluric acid, xanthin, sarcin, and guanin, thus — NH— CO NH, NH— CO NH-C . OH I I ■ I CO— CO CO CO NH— CO Alloxan. NH., I CO NH. Urea. CO. OH NH— CO Parabanic acid. NH- CO NH- -C— NH- Allantoin. CO— NH, NH-CO NH— CO Oxaluric acid. CO C— NH ! II NH— C — N Xanthin. CH NH— CO CH 11 II N— C - Sarcin NH. -N- NH- I NH = C CH I^H— C - Guanin CO I C— NH II N- CH Most oxidizing substances coiiA^ert uric acid into alloxan or parabanic acid : and AA^hen boiled AAdth water and peroxide of lead, it yields allantoiTi. AVhen oxidized in the presence of Avater it giA^es up tAA'o of its hydrogen atoms to the oxidizing agent, while the dehydrogenized residue (dehy- duric acid) reacts A\dth AA'ater to form mesoxalic acid and urea — CsH^N^Oa Dehyduric acid. 4HoO CsH.Os Mesoxalic acid. 2CN2H4O Urea. Dilute nitric acid acts upon uric acid and produces alloxan, and this when heated AA-ith baryta AA^ater is resoU'ed into mesoxalic acid and urea — \- 2H„0 C^HoN^Og Dehydm-ic acid. C^NoH^Oi Alloxan. HoO CiNoH,04 Alloxan. Slesoxalic acid. CNoH.O Urea. CNjH^O Urea. Alloxan may be regarded as a mon-ureide of mesoxalic acid formed by THE NITROGENOUS ACIDS. 99 the union of one molecule of the acid with one molecule of urea, less two of water, and formed thus — C3H0O5 + CNoHiO - 2H2O r= C4N2H2O, Mesoxalic acid. Ureiu Alloxan. Again, the hypothetical dehyduric acid is a di-urekle of mesoxalic acid, that is, it is formed by the union of one molecule of mesoxalic acid with two molecules of urea, less four molecules of water, thus — C3H2OS + 2(CH4N20) - 4HoO = C3H2N4O3 Mesoxalic acid. Urea. Dehyduric acid. By hydrogenizing mesoxalic acid, tartronic acid is produced; and by hydrogenizing alloxan, we obtain another comjDOund known as dialuric acid. These bodies, tartronic acid and diakmc acid, bear the same rela- tion to uric acid as mesoxalic acid and lurea bear to dehyduric acid, thus — C3H2O3 C4N,H,04 CgN4H203 Mesoxalic acid. Alloxan. Dehyduric acid. C3H40g C,N,H,0, CsN.H.Oj Tartronic acid. Dialuric acid. Uric acid. Thus we have three groups of bodies related to uric acid : (a) an-ureides, non-nitrogenous acids, such as tartronic and mesoxalic acids; (h) mon- ureides, consisting of a residue of the acid added to one residue of urea, such as dialuric acid and alloxan; and (c) di-ureidcs, consisting of a residue of the acid added to two residues of urea, such as uric acid itself. It is important also to observe the origin of other bodies belonging to these groups. Thus mesoxalic acid, the most complex non-nitrogenous product that can be obtained by the oxidation of uric acid, is one of a series of an-ureides, each of which contains one CO group more than the preceding, thus — CH2O3 Carbonic acid. C2H2O4 Oxalic acid. CoHoOj Mesoxalic acid. Acted on by nascent oxygen, mesoxalic acid loses its excess of CO and becomes oxalic acid, thus : CgHgOg + = CO2 + C2H2O4. Hence when uric acid is oxidized beyond the point of producing mesoxalic acid, oxalic acid may be formed, and this may conjugate with one mol. of urea to form a mon-ureide, parabanic acid, or with two mol. of urea to form a di- ureide, mycomelic acid. If the oxidation of uric acid proceeds further than the oxalic acid stage, carbonic acid is formed, and this is also theo- retically capable of forming ureides. Thus allophanic acid is the mon- ureide of carbonic acid, but no di-ureide exists. Alloxan, the mon-ureide of mesoxalic acid, is formed from mesoxalate of urea by the elimination of two mol. of water, but if only one mol. of water is removed, a 100 THE CHEMISTRY OF THE BODY. related body, alluxanic acid, is produced. Similarly, oxalic acid yields two mon-ureides, parabanic acid and oxahiric acid. The relation of these bodies is seen in the following table, modified from that given by Odling. {Lectures on Animal Cheinistri/, p. 133.) Di-uroides. An-ureidos. CHoO. Carbouic. CMjSi Glyo.xalic. CM.fii Oxalic. C.H4O4 :Malonic. C^HjOg Tartronic. C3BUO5 Mesoxalic. Mon-ureides. C0N0H4O3 Allophanic. CgN^.HjO^ Lantanuric. C3N.JH4O4 Oxaluric. CgNoHoO^ Parabanic. C4N2H4O3 Barbituric. C4N0H4O4 Dialuric. C4N',Ho04 Alloxan. C4N4H6O:, AUautoin. €5X41140 Hypoxanthin. GgN4H402 Xanthin. C5N4H4O3 Uric acid. The substance termed alloxantin is foi'med by the union of two consecu- tive mon-ureides, dialuric acid and alloxan, ^Wth the elimination of Avater, thus — C4N0H4O4 + C4N0H0O4 - H,,0= C8N4H4O,. Dialuric acid. Alloxan. Alloxantin. It -will also be seen that xanthin is the di-ureide of barbituric acid, which again is the mon-ureide of malonic acid, and this in turn can be obtained from tartronic acid by deoxidation. Closely related to xanthin is hypoxanthin. Uric acid deoxidized by sodium amalgam yields a mixture of xanthin and hypoxanthin, and hypoxanthin may be con- A-erted into xanthin by oxidation with nitric acid. Uric acid is found in the urine, either in the free condition to a very ^,,-— :^ small extent or in the form of the urates of ^^^^^^ss;^^ . '^^ potash and soda, perhaps the most common forms of urinary deposit. The daily amount eliminated by an adult man Avith a mixed diet is from '5 to 1 gram.; -with a vegetable diet it may fall to '3 gram., and with a rich animal diet it may amount to as much as 1 '5 gram. It also is met Avith in urinary calculi and in the chalky deposits in the joints of gouty persons. Traces also are found in the blood, kidneys, spleen, lungs, brain, and muscular tissue. It forms a large part of the excrement of birds and of reptiles. Pure uric acid crystallizes in colourless rhombic rectangular or hexagonal plates, or in rectangular prisms. "WTien obtained from urine, it is coloured more or less by the urinary pigment, and forms rhombs, lozenges, or rectangular prisms, and these are grouped so as to Fig. 35. — Uric acid, a, a, Rhombs and rectangular prisms ; b, forms from human urine ; c, dumb-bell crystals. In right hand corner, rosette. THE NITROGENOUS ACIDS. 101 form rosettes, barrels, or dumb-bell like forms. (See Fig. 35.) Uric acid is tasteless and inodorous. Its solubility is small as it requires for its solution 1,900 parts of cold water and 15,000 parts of hot water; it is insoluble in alcohol and ether. Its solutions are feebly acid. It is dibasic. There are several well known salts of uric acid. (a) Urates of soda. These form the chief part of the deposit of urates commonly found in urine and are readily recognized by their disappearance on heating. There are two urates of soda — the neutral salt, CgHgN^OgNa^, which forms nodular masses, and the acid urate, CgHgN^OgNa, which is usually amorphous and rarely crystallizes in urine. The latter is soluble in 1,200 parts of cold water and in 25 parts of boiling water. The chalky deposits in the joints of gouty persons are almost entirely composed of this salt, and from these it may be obtained in forms represented in Fig. 36. ■^ ^ . Fig. 36.— Acid urate of (o) Urates of potash correspondina; to those of soda soda, a, Needles, usually T ,•: .^ *. , . aggregated ; 6, 6, spher- may be made by saturating a solution of potassium oidai masses. hydrate with uric acid, when they may be crystallized as fine needles, but they do not often occur in urinary sediments. i^ V ^ (c) Acid urate of ammonia, Q>^.^^.JiJ^^^, is found '^ ^ in alkaline urine, either as a pinkish amorphous powder or in the form of globular masses having little spicules proceeding; from them (Fia;. 37). The ^ . . -, \ & / Pjg_ 37._xjrate of neutral salt is unknown. ammonia. {d) Acid urate of lime, (G^.^^O^.^G^i, occurs in urinary sediments and calculi, and appears in the form of fine needles. (e) Acid urate oflithia, CgHgN^OgLi, is the most soluble salt of uric acid. "It may be artificially prepared by dissolving uric acid in a warm solution of lithium carbonate. It crystallizes in needles, which dissolve in 60 parts of water at 50° C, and do not separate when the solution is cooled." (Witthaus, Manual of Chemistry.) Tests for Uric Acid. — (1) Murexide test: Put a little of the substance to be examined on a white porcelain plate or lid, add two drops of nitric acid, heat and evaporate till dry ; if the substance is uric acid, it dissolves in nitric acid, and gives on evaporation a yellow or reddish- yellow residue, which becomes reddish-purple by adding a drop of caustic ammonia, and bluish-violet with soda or potash. The purple colour is readily brought out by bringing a glass rod dipped in ammonia solution near the residue after evaporation. (2) Dissolve the substance to be examined in a few drops of solution of caustic soda, and filter ; add 102 THE CHEMISTRY OF THE BODY. to the liquid chloride of ammonium in excess, and there will be a precip- itate of urate of ammonia which, by the addition of hydrochloric acid, yields crystals of uric acid. (3) The microscopic examination of the crystals so as to determine their form. (4) Garrod's test : To detect uric acid in serum, place about 2 c.c. of the serum in a flat watch-glass and acidulate with acetic acid ; a fine fibril of linen thread is placed in the liquid, which is set aside and allowed to evaporate for two or three hours ; the fibril is then examined microscopically when it Avill be found to have crystals of uric acid adherent to it. The ori[jin of u.ric acid is still douljtful. Its relationship to guanin, sarcin, and xanthin has already been pointed out. Guanin and sar- cin have been changed into xanthin, but the latter has not been con- verted into uric acid. Uric acid, however, reduced by sodium amalgam, has yielded sarcin and xanthin, and the ultimate products of the oxidation of all four bodies are the same, namely, parabanic acid, oxaluric acid, and urea. These four bodies — guanin, sarcin, xanthin, and uric acid — therefore, probably represent four stages of oxidation, of which uric acid is the last. Uric acid forms the principal product of the decomposition of nitro- genous matters in animals so different as birds and reptiles. In the bird there is rapid oxidation and a high temperature, while the reverse conditions exist in the reptile, and still the chief nitrogenous substance in the excrements of both classes is uric acid, a fact for which no satis- factory explanation can be offered. Even urea in the bird appears to be changed into uric acid, as it has been found that the ingestion of urea by fowls is followed by an increased elimination of uric acid. On the other hand, the ingestion of sarcosin by fowls diminishes the amount of uric acid, and in other animals, inhalations of oxygen or of protoxide of nitro- gen, the administration of sulphate of quinine, and a vegetable diet, also diminish it. Quinic acid and benzoic acid and an animal diet increase the amount of uric acid eliminated. Further, it is supposed that a portion of the uric acid formed is eliminated as such, while another portion is transformed by oxidation into other substances. Thus it may readily be changed, first into alloxan, then into parabanic acid, then into oxaluric acid, then into oxalic acid and urea, and ultimately into carbonic acid and water, thus — (1) C5H4N4O. + H,0 + = C4H2N,04 + N0H4CO. Uric acid. Alloxan. Urea. (2) C4H2N,04 + = C3H2N0O3 + CO.. Alloxan. Parabanic acid. (3) CsH^N^Os + H,0 = C3H4N2O4. Parabanic acid. Oxaluric acid. THE NITROGENOUS ACIDS. 103 (4) C3H4N2O4 + HoO = CN^H^O + C2H2O4. Oxaluric acid. Urea. Oxalic aaid. (5) C2H2O4 + = 2C0. + H2O. Oxalic acid. Another view is that uric acid by oxidation is changed into allantoin and that this substance is resolved by successive stages, in which new compounds appear, into urea and parabanic acid, the latter being then split up into urea, carbonic acid, and water, as has already been shown, thus — (1) C5H4N4O3 + H^O + = C4H6N4O3 + CO2. Uric acid. Allantoin. (2) C4HSN4O3 + HoO = CN2H4O + C3H4N0O3. Allantoin. Urea. Allanturic acid. (3) 2{C3H4N203) = C3H2N2O3 + CsHgN^Oa. Allanturic acid. Parabauic acid. Hydaoto'inic acid. Allanturic, parabanic, and hydantoinic acids have not been found in the body, but allantoin is met with in the urine of the newly born. Fur- ther, Salkowski has found that the ingestion of uric acid by dogs is followed by the appearance of allantoin in the urine. The evidence both from chemical considerations and the observation of some pheno- mena of disease is in favoiu" of the view that a portion of uric acid may be resolved into urea, and that this transformation is aided by active oxidations. The substances specially related to uric acid are the following — (1) Guanin, C5H5N5O, has been found in the liver and pancreas. It is a white or yellowish, amorphous, odourless, tasteless substance, almost insoluble in water, alcohol, and ether, but easily soluble in acids and alkalies. Evaporated on a bit of platinum foil with a drop of fuming nitric acid, it gives a yellowish residue which becomes red on the addi- tion of a drop of caustic soda, and gives a purple colour on heating. Derived from the splitting up of albuminous matters, it probably gives origin to xanthin and ultimately to urea. (2) Sarcin or Hypoxanthin, C^H^N^O, has been found in the spleen, pancreas, supra-renal capsules, muscles, liver, marrow of bone, the urine, the liver in acute yellow atrophy, and the blood and urine of leucocythaemia. It occurs in the state of nodular masses formed of fine colourless needles, soluble in 300 parts of cold and 78 parts of boiling water. (Witthaus.) Oxidized by nitric acid it gives xanthin, thus — C5H4N4O + = C5H4N4O0. Hypoxanthin. Xanthin. (3) Xanthin, C^^HJ^^O^, sometimes termed xanthic oxide, is found in a rare form of urinary calculus, in human urine after the use of sulphur 104. THE CHEMISTRY OF THE BODY. baths and inunctions, and in the pancreas, spleen, liver, thymus, and brain. It is a pale yelloAv amorphous powder, slightly soluble in cold water, insoluble in alcohol and ether, and soluble in ammonia. Dissolved in a drop or two of nitric acid and evaporated, a yellowish residue is left which turns reddish-yellow on the addition of a drop of solution of caustic potash, and violet-red on being heated. Calculi of xanthin vary in size from that of a pea to that of a jngcon's egg. They are hard, brownish-yellow, smooth, shining, and show well-defined concentric layers. Their broken surface assumes a waxy polish when rubbed. (4) AUanioin and oxaluric acid have been described. (See p. 88.) (5) Caniin, C-HgN^O., -t- HgO, occurs in the form of chalky crystals, readily soluble in warm water but insoluble in alcohol and ether. It is intimately related to sarcin and it may be considered as a compound of that body and acetic acid, thus — CgfljN^O + C2H4O2 = C,H8N403 Sarcin. Acetic acid. Carnin. It may be obtained from extract of meat. Doses of from -05 to '2 gramme exert a slightly depressing influence on the heart. 3. Hippuric acid, C9H9NO3, or benzyl-glycocol, is glycocolle in which one atom of hydrogen has been replaced by the radicle benzyl, C^.Hg.CO, thus — CH, - NH„ CH, . NlCfiHg . CO)H I ' ' I " CO .OH CO . OH Glycocolle. Hippuric acid. It is isomeric with acetamidobenzoic acid, C.^Hg(C\,H30)NOo. AVhen heated with the mineral acids, or the alkalies, or when influenced by certain ferments, such as that of decomposing urine, hippuric acid is converted into glycocolle and benzoic acid — C9H9NO3 + H2O = C2H5NO, + C^HgOo Hippuric acid. Glycocolle. Benzoic acid. Hippuric acid is a constant constituent of the urine of herbivora and of human urine in the form of hippurate of soda (CgHgNaNOg . H2O) or of lime Ca(C9HgN03)2 • SH^O. Only a very small amount exists in urine in the free state. It crystallizes in transparent, colourless prisms (Fig. 38). Bj^ slow evaporation from dilute solutions, crystals arc obtained similar to those of the triple phosphate of ammonia and magnesia. (Frey.) It is soluble sparingly in water, and more readily in hot alcohol or ether. It dissolves unchanged in hydrochloric acid, and on boiling the solution it is converted into benzoic acid and gly- cocolle. The folloAving are the tests by which hippuric acid may be THE NITROGENOUS ACIDS. 105 Fig. 3S. — Hippuric acid, a, a, Prisms ; 6, crystals like those of the triple phosphate. readily distinguislied : (1) Lucke's test: Evaporate with excess of nitric acid ; heat residue strongly, and an odour of hydrocyanic acid may be per- ceived. (2) It gives a brown precip- itate with ferric chloride. (3) Heated with lime, it gives off benzin and am- monia. Hippuric acid may be formed from matters supplied in the food or from the decomposition of albuminous matters in the body. It is well known that it appears in the urine after the administration of benzoic acid, or of benzoates, or of articles of food con- taining benzoic acid, and this can be explained by assuming that it unites with glycocin, with the loss of a molecule of water, thus — CyHgOa + C3H5NO2 - H2O = C3H9NO3 Benzoic acid. Glycocin. Hippuric acid. Marchand, after taking 30 grains of benzoic acid, found 39'2 grains of hippuric acid in the urine. It is also found in the urine after the ingestion of cinnamic and mandelic acids. It would appear also that this reaction is the type of others. Thus, such aromatic acids as toluilic, anisic, and salicylic acids unite in the body with glycocin to form toluric, anisuric, and salicyluric acids. Quinic acid is changed into hippuric acid in the organism, probably first into benzoic acid. The large amount of hippuric acid in the urine of herbivora has not received a satisfactory explanation. In this case it is not derived from benzoic acid or quinic acid, as both of these substances are usually absent from the food of herbivora. Shepard and Meissner have main- tained that it is derived from the cuticular matter of plants, and from cellulose, and they state that the amount of hippuric acid is increased by a diet rich in such matter, such as the straw of cereals, whilst it is diminished by a diet of potatoes, carrots, or beet-root, substances rich in starch but containing little of the cuticular matter. These state- ments have not been unquestioned, and it has been pointed out that much may depend on the kind of animal, as the straw of oats increases hippuric acid in the urine of the ox, but not in that of the rabbit. (See Hippuric acid. Watt's Diet, of Chem., 2nd supplement, p. 647.) As hippuric acid is always found in small quantity in the urine even with an animal diet, it has been supposed that it may originate from the decomposition of proteid matter. It is conceivable that such a decomposition may give rise to benzoic acid and glycocoUe ; and, in 106 THE CHEMISTRY OF THE BODY. that case, hippuric acid Avould probably be formed. This union may occur in the liver (Kiihne and Hallwachs), or in the tissues (Bunge and Schmiedeberg), or in the kidneys (Shepard and Meissner) — most pro- bably in the latter. Thus, it has been found that after ligature of the renal vessels in the dog, no hippuric acid appears in the liver or in the tissues after the injection of benzoic acid. Again, Bunge and Schmiede- berg have found that when blood containing glycocolle and benzoic acid is passed through a kidney by an artificial circulation, hippuric acid appears both in the blood returning from the kidney and in the fluid that in these circumstances may exude from the ureter. No definite information exists as to the substance in the blood that thus yields hippuric acid. Coloured blood corpuscles and oxygen are neces- sary. Some have supposed that it may arise from the oxidation of tyrosin ; and others, that a substance exists which may, in certain conditions, become urea, and, in other conditions, hippuric acid. (Beaunis.) 4. Inosinic or Inosic acid, CjoHj^lST^Oj^^, is a substance found in the mother liquor of the preparation of creatin from muscle juice. It is uncrystallizable ; it is readily soluble in water, and alcohol precipitates it from its aqueous solution. The nature and properties of this sub- stance have not been fully investigated. 5. Cryptophanic acid, C-^qH-^^ .fi^^, is said by Thudichum to exist in human urine. It is an amorphous, gummy mass, transparent and nearly colourless, readily soluble in water, and less soluble in alcohol. It is said to have feeble acid properties. (See Journal of Chemical Society [2] viii. 132 ; see also Beaunis, Physiologie, vol. i. p. 138.) 6. Bile acids. The bile of most animals contains the sodium salts of amido-acids of complex constitution. Of these acids, two are found in the bile of man, namely, glycocholic acid and taurocholic acid, and these in turn are related to two others, cholalic acid and choloidic acid. (a) Glycocholic add, C^^^H^gNOg, the cholic acid of Gmelin, exists in human bile as glycocholate of soda in amounts varying according to diet. An animal diet diminishes the quantity, while a vegetable diet has the contrary effect. It is therefore readily found in ox bile and in the bile of herbivora generally, and the amount is smaller in that of carnivora. When obtained from ox bile, it forms brilliant, colourless, transparent needles, sparingly soluble in cold water, readily soluble in hot water and in alcohol, glycerine, and acetic acid, whilst it is almost insoluble in ether. The taste is at first sweet, and afterwards intensely bitter. The alcoholic solution is dextro-rotatory when examined with the polariscope, [«]d= +29°. By the action of potash, baryta, dilute sulphuric acid, or dilute hydrochloric acid, it takes up a TEE NITROGENOUS ACIDS. 107 molecule of water, and th^ri is decomposed into glycocin and cholalic acid, thus — Glycocholic acid. Cholalic acid. Glycocin. Sodium glycocholate, CggH^gNaNOg, crystallizes in stellate needles (Fig. 39), is readily soluble in water, less so in alcohol, and is insoluble in ether. With polarized light [a]D= + 25°-7. Hamersten states that the glycocholic acid of man differs slightly from that of the ox. The bile of sea fishes con- tains the glycocholates and taurocholates of potash, not of soda, and chiefly in the form of the soda salt. (h) Taurocholic acid, C26H4r,N07S, exists in human bile and in the bile of carnivora ; in small amount in the bile of herbivora. It is easily obtained from dog's bile, in the form of silky needles, deliquescent, and readily changed into an amorphous resinous mass. It is soluble in water and alcohol, forming intenselj^ bitter solutions. The alcoholic solution gives with polarized light [a]D= + 24°"5. Barium hydrate and acids give the following decomposition — C^gH^^NO^S + HoO = C24H40O5 + C^H^NOgS Taurocholic acid. Cholalic acid. Taurin. The same decomposition occurs in the presence of putrefying material, Fig. 39.— Glycocholate of soda. and in the intestine. (Witthaus.) TcmrocJwlate of soda, C2(; is soluble in alcohol and in water, and is very difficult to crystallize. When the glycocholates and taurocholates exist together in an aqueous solution, they may be separated by dilute sulphuric acid and a small quantity of ether, which pre- cipitates glycocholic acid alone. (c) Cholalic acid, C24H4(jO;;, the result of the de- composition of the conjugate bile acids, as already explained, is found in the intestines and faeces in the form of large, shining, deliquescent crystals (Fig. 40), slightly soluble in water, and readily soluble in alcohol and ether. With polarized light, [a] jj = -f 35°.^ By boiling with acids or heated to H^^NaNO^S, Fig. 40. — Cholalic acid. 200° C, cholalic ^ Hoppe-Seyler gives for cholic acid +aq., 31°"2 ; for cholic acid alone, 50° "2 5 for sodium chelate dissolved in alcohol, 31°'4 ; potassium chelate, 30° '8; for sodium chelate in water, 26°; and for potassium chelate, 25°. (Jl. for Cliem. Ixxxix. 257 ; Bull. Soc. Chem. v. 622, quoted in Watt's Diet, of Ghem. vol. vi. p. 342. 108 THE CHEMISTRY OF THE BODY. acid may lose one molecule of water, and become {d) choloidic acid, or (e) dyslysin. Thus — C,4H,oO,, - H,0 = C,,4H3804 ChoUilic acid. Choloidic acid. C,,H,oOg - 2H.,0 - C,4H3803 Cholalic acid. Dyslysin. Hoppe-Seyler regards choloidic acid as merely a mixture of cholalic acid and dyslysin, the latter being a neutral resinous body, insoluble in water and alcohol, and only sparingly soluble in ether. Tests fm' the bile acids. (a) Pettenkofer's test. — Add to the liquid a few drops of a strong solution of cane sugar and a few drops of con- centrated sulphuric acid, and keep at a temperature of about 70° C, and a cherry-red or dark reddish-purple colour appears. The presence of nitrates and chlorates delays the reaction. Albuminoids, lecithin, oleic acid, cerebrin, phenol, turpentine, benzin, tannic acid, salicjdic acid, morphia, codeia, amyl-alcohol, cod-liver oil, and camphor give a similar reaction. The red liquid produced by the reaction is dichroic, and gives two absorption bands between F and E. When examined by the spectroscope, the red liquid formed by the action of strong sul- phuric acid on albuminoids is not dichroic, and those Avith oleic acid and amyl-alcohol show no absorption bands. Still, as stated by Witthaus, the spectroscopic appearances with, different fluids are not sufficiently definite to serve as a check to the Pettenkofer reaction. (/3) Bogoinoloff's test. — Evaporate an alcoholic solution to dryness; spread out the residue on a white plate, and wet with one or two drops of concentrated sulphuric acid and one drop of alcohol. Shades of colour will be shown from centre to circumference — yellow, orange, red, violet, and indigo. (y) Strassburg's test. — Dip a morsel of filter pajDer in fluid, then into a solution of cane sugar, allow it to dry, and let a drop of concentrated sulphuric acid trickle down a glass rod to the centre of the paper; in a quarter of a minute the sjDOt Avill become translucent and transmit light of a violet colom\ Physiological characters of the bile acids. — From their constitution, one would infer that the bile acids are formed in the liver by the union of cholalic acid and taurin or glycocin. Glycocin and taurin, as already explained, may originate from the decomposition of albuminoids, and it is probable that cholalic acid has the same origin. This view receives some support from the similar behaviour of albuminoids, the bile acids, and cholalic acids towards sugar and concentrated sulphuric acid. Some have, on the contrary, suj^posed that the non-nitrogenous cholalic acid may be derived from fats. After their formation, the acids pass into THE NITROQENO US A CTDS. 1 09 the intestine, and there they are partially decomposed into cholalic acid, choloidic acid, dyslysin, glycocin, and taurin, and some of these products are reabsorbed into the blood. The remainder is voided in the faeces. In the blood, or in the tissues, the bile acids and their derivatives are no doubt decomposed still further. At all events, Avhen injected into the blood — in cases of biliary fistulse in rabbits, by which it was possible to collect the bile — it was found that only a third or a fourth part of the acid injected appeared in the bile, the remainder disappearing. Injections of the bile acids into the blood cause de- struction of red blood corpuscles, slowing of the pulse and of the respiratory movements, fall of arterial pressure, and lowering of the bodily temperature. Doses of from 2 to 4 grammes given to a dog, caused epileptiform convulsions, the passing of very dark- coloured urine, and death. The biliary acids may be detected in the blood and urine in jaundice and acute atrophy of the liver. Several important physiological questions relating to the action of the bile acids will be discussed in treating of the bile. Chap. VIII. —NITROGENOUS BODIES CONTAINING NO OXYGEN. 1. Trimetliijlamine, (CH3)3N", occurs normally in human urine, and it has been found in the blood of the calf, in cod-liver oil, ergot, cheno- podium, yeast, guano, herring-brine, and in many flowers. It is an oily fluid, having an odour of fish, boils at 9° C , is soluble in water, alcohol, and ether, and has an alkaline reaction. It is also found among the products of the decomposition of albuminous bodies. 2. Naphthylamine, C-^^^^, has been found among the oxidation pro- ducts of albuminous matter (Schiitzenberger), and naphthalene (C^^oHg) in minute quantity has been detected in the urine. (Hoppe-Seyler.) 3. Lidol, CgH^N, belongs to the group of aromatic bodies related to indigo ; indeed, it may be regarded as the nucleus of the indigo group. Thus various plants yield indigo, or indigo-blue, indigotin, CgHgNO. This, by the action of reducing agents, becomes indigo-white, or indi- gogen, CgHgNO. Again, a third body, isatin, CgIl5N02, is produced by the action of nitric or chromic acid on indigo, — CgHgNO -f = Cj^HgNOg. The parent of these three bodies is indican, CggHg^NO^^^, which is a colourless substance existing in the plants that yield indigo. Inclican, when boiled Avith acids, splits up into indigo-blue and a substance named indiglucin, CgHjoOg. Thus — - C06H31NO17 + 2H„0 = CgHgNO + SlCgHioOg). Indican. Indigo-blue. Indiglucin. Now, isatin, CgHpNOa, plus a molecule of water, HoO, yields isatic 110 THE CHEMISTRY OF THE BODY. acid, CgH-NO.j, wliich is a hydroxyl derivative of iiidol, or trioxindol. Thus, trioxindol may be expressed CjjH^N(H0)3. Trioxindol by re- duction yields dioxindol CgH^N(HO)o, and oxindol CsHgN(HO). Finally, Baeyer and Emmerling represent all these bodies by the fol- lowing formulne, in which the benzin nucleus, i^^i, pla}'s an important part — / COH /C— OH /C— OH / C— O CbH4\ C CeH4< C— OH CcHjx C-OH CgHj / C-0 \NH \NH \N— OH Oxindol. Dioxiudol. Trioxindol. C6H4< /'CH \NH \ I Thus indol may be obtained from indigo by converting the indigo into isatin, dioxindol, and oxindol, and then reducing the oxindol by zinc dust. The relationship of these bodies may also be illustrated by putting their formulae in series, thus — Indol, - - - CgH^N. Isatin, - - CgHsNO, Oxindol, - CgH^NO. Indigo-white, - CgH^NO Dioxindol, - CgHyNOo. Indigo-blue, - - CsHgNO. Isatyde, - - CsHeNOo. We pass from indol to indigo-blue by successive oxidations, and from indigo-blue to indol by successive reductions. It is of more importance, however, to observe that indol is also obtained by the decomposition of albumin through the agency of a ferment in the pancreatic juice. (Nencki and Kiihne.) It appears as an oily fluid, which, when mixed with water, solidifies to a crystalline mass, and on recrystallization from pm-e water, crystals of pure indol are obtained, which melt at 52° C. Indol has a peculiar fsecal odour, like that of naphthylamine ; it is readily soluble in alcohol and ether, and it acts as a very weak base. One of its characteristic properties is to give a reddish precipitate with dilute fuming nitric acid : this is soluble in alcohol, "and the alcoholic solution mixed with hydrochloric acid colours fir wood cherry-red, changing after a while to dirty brown-red." (Baeyer.) The history of indol, as a decomposition-jDroduct of albuminoids, prepares us for finding it in the intestines and in the faeces, as is the case, and it also exists in all putrefying albuminoids. Part of it thus formed in the intestine is evacuated and a portion may be changed into indican which is absorbed and finally excreted in the lurine by the kidneys. NITROGENOUS BODIES CONTAINING NO OXYGEN. \\\ 4. Skatol, G^^, or methyl-indol, C3Hg(CHg)X, may be represented l)y the formula — ^--N= CH C6H4j interpolation curves — " A piece of paper ruled into square inches and tenths has a scale of wave-lengths ruled off along one edge, and the edge at right angles to this has a scale corre- sponding to the scale of the instrument marked on it. The vah;e of the Fraun- hofer lines on the scale of the spectroscope is observed, and by a reference to the above numbers, their value in wave-lengths, they are then marked in their proper place on the scale with + +. A curve is then drawn through these marks as uniformly as possible. When a band or bright line has to be mapped out, all that is necessary is to take its reading on the scale ; then, knowing between what lines it is placed, we find its position on the curve, opposite which its wave-length is printed on the right hand edge." The measurements for oxy-hsemogiobin in one millionths of mm. are as follows — "In a concentrated solution the red rays only are transmitted, light is cut off completely at the red end at X706, a shading extends to X647 ; light is again completely absorbed at X591, and a shading extends to X610. In a more dilute solution one broad band embracing the two oxy-heemoglobin bands becomes detached from about X589 to X520. Still more diluted, the first band extends from X589 to X564:, with its darkest part from X585 to X569, and the second from X555 to X517, with its darkest part from X552 to X526. In a greater degree of dilution the first band reads X587 to X566, the darkest part being from X583 to X571, and the second from X550'5 to X523, the darkest part ranging fromX549toX529." The character of the absorption spectrum of oxy-hsemoglobin, with different strengths of solution in a layer one centimetre in thickness, as measured by the hsematoscope, is illus- trated graphically by Rollet (op. cit.) in Fig. 50. Here the line o x has on it at the proper distances the chief Fraunhofer lines, whilst the figures along the right hand border are percentages of the amount of oxy- haemoglobin present. Thus, Avith -1 per cent, the two bands are narrow and pale, they gradually become broader until they fuse at nearly "7 per cent., and at -9 per cent, no light passes 0.5 fL aC B Fig. 50. — Graphic representation of the amount of absorption of light by solutions of oxy-hfemoglobin of different strengths. The shading indicates the amount of absorption of the spectrum. 122 THE CHEMISTRY OF THE BODY. except in the red. It will be observed also that from a little OA-er -^ per cent, to the top there is a gradually increasing, though slight, absorption of the low red. Preyer has devised a spectro-colorometric method of estimating the amount of oxy-haemoglobin by preparing a standard solution, having the- absorptive power indicated in the diagram as on the level of the lines PP. This is the point at which the green is extinguished and only red rays are transmitted. The specimen of blood is diluted with water until it gives a similar spectrum (that is, to find the exact point at which the absorption of the green takes place), and then the percentage amount is estimated by the formula — x — K (b + w) b ' in which K- the percentage of the standard solution, b = the amount of blood, and ic = the amount of Avatcr, and x = the percentage required. Thus, suppose 2 c.c. of blood required 25 c.c. of water to give an absorp- tion spectrum similar to that of the standard spectrum, the percentage of which is -85 ; then (2 + 25) X " -85^ — - — ^= 11-4 per cent, of oxy-haemoglobin. By various methods the oxygen may be removed from oxy-haemo- globin. The following are reducing fluids : (1) Stokes' Fluid. Thus, to a solution of sulphate of iron is added a small amount of citric or of tartaric acid, and then ammonia until the reaction is alkaline. Ammonia does not precipitate ferrous hydrate in the presence of the vegetable acid, and a clear light green solution is obtained, which becomes darker by the absorption of oxygen on exposure to the air ; (2) a solution of stannous chloride, with a little tartaric acid and ammonia added until neutralization ; (3) a solution of ammonium sulphide. When any of these fluids is added to a solution of oxy -haemoglobin, and the solution is examined by the spectroscope, the two bands of oxy-haemoglobin disappear and in their stead there is one band, seen in A, Fig. 49 and in Plate I. 3. The spectrum is thus described by Dr. Gamgee — *' The spectrum of oxy-liEemoglobin had been described by Hoppe-Seyler when Professor Stokes made the remarkable discovery that when diluted blood is treated with certain reducing agents the colour of the liquid and its spectrum undergo remarkable changes ; the former loses its bright red and acquires a, brown colour, while the green interspace which had existed betM^een the absorp- tion bands a and /3 of oxy-hsemoglobin disappears, and instead of the two bands there appears a single one, less deeply shaded, and with less finely defined edges, extending between D and E. The band we may distinguish as absorption band 7." TEE PIGMENTS. 123 Dr. MacMunn states that "the broad hazy band extends from A59T to A536, and that the darkest part is from A573 to A542." In Fig. 51 there is represented the amount of absorption mth dif- ferent percentages of reduced haemoglobin, as in Fig. 50, showing oxy-haemoglobin. It will be observed that the absorption band diminishes in breadth as the solution becomes weaker, the blue rays becoming more visible. With very weak solutions, the single broad band never breaks into two narrow ones, but gradu- ally fades from view. With two weak solutions of equal strength, that of oxy-hsemoglobin may dis- tinctly show the two bands, whilst that of re- duced haemoglobin shows no band. Further, the general appearance of the spectrum of reduced hae- moglobin differs from that of oxy-haemoglobin. In the former less of the blue end is absorbed, and even in strong solutions some of the bluish-green rays pass through. This accounts for the difference of colour — the blood containing oxy- haemoglobin being scarlet, w^hilst that containing reduced haemoglobin is purple. The first kind of blood allows the red and orange-yellow rays to pass, and the second kind the red and blue-green rays. The difference is thus described by Professor Michael Foster — ^ " In dilute solutions, or in a thin layer, the reduced hEemoglobin lets through so much of the green rays that they preponderate over the red, and the resulting impression is one of green. In the unreduced hfemoglobin or oxy-hsemoglobin, the potent yellow which is blocked out in the reduced hsemoglobin makes itself felt, so that a very thin layer of haemoglobin, as in a single corpuscle seen under the microscope, appears yellow rather than red." Haemoglobin is widely distributed in the animal kingdom. Thus it has been found in the red blood corpuscles of all vertebrates except amphioxus (in which it is found in the plasma only) ; in the striated ^ Handbook Jor Physiological Laloratory, by Klein, Burdon-Sanderson, Foster, and Lauder Brunton. aCB D Eb Fig. 51. — Graphic representation of the amount of absorption of light by solutions of reduced haemoglobin of different strengths. The figures on the right border express percentages. 124 THE CHEMISTRY OF THE BODY. muscles of mammals and birds ; in the cardiac muscles and very active muscles of other vertebrates ; in Ophiadis lirens (an echinoderm.) ; in the blood of some of the annelidse ; in the fluid in the perivisceral cavity of the leech ; in the larva of Chironoimis (midge), although absent in other insects, and in mj-riapods and arachnids ; in the l^lood plasma of certain crustaceans ; in the blood of certain molluscs (Planorhis) ; in the mus- cular fibres of the pharynx of certain gastropods {Lymnceus, Paludina), and in the ventral ganglia of Aphrodite. Haemoglobin forms definite compounds Avith at least other two gases — {a) Carbonic-oxide Hcemoglohin. — When blood is shaken up with carbonic oxide it assumes a red arterial colour, and if examined spectroscopically two absorption bands are seen similar to those of oxy-haemoglobin. Careful measurement, however, shows that both bands are slightly nearer the violet end of the spectrum. The measure- ments given by Dr. MacMunn, calculated from Preyer's Map of Spectra, are : " First band, A583 to A564 ; second band, A547 to A529; or, in more concentrated solutions, A583 to A561 and A547 to A521."^ This com- pound, which forms crystals like those of oxy-haemoglobin, is remark- able for its stability ; it is not acted on by those agents which reduce oxy-hsemoglobin, but the CO may be driven ofi" by passing for a long time a stream of air or of a neutral gas. It also resists putrefaction for a long time, so that the two bands may be visible for months or years, whilst, when normal blood putrefies, the reduction of oxy-h^emoglobin occurs at once. (Gamgee, quoting Hoppe-Seyler.) Blood containing carbonic-oxide-haemoglobin gives a cinnabar red precipitate with caustic soda, due to the formation of a compound of carbonic oxide with haematin. The gas CO can displace the from oxy-haemogiobin. It is well known that carbonic oxide, given off during the imperfect combustion of carbon, as occurs in charcoal stoves, is a powerftd. poison, soon destroying life. This it does by combining with the haemoglobin of the blood, and thus interfering with the respiratory functions of that important body. (b) Nitric-oxide Hcemoglobin. — AVhen ammonia is added to blood, and then a stream of nitric oxide is passed through it, this compound is formed (Hermann), and it may be obtained in a crystalline form (iso- morphous) "with those of oxy- and carbonic-oxide-haemoglobin, whilst it also has a similar sj^ectrum. A stream of nitric oxide (NO) can dis- place carbonic oxide (CO) from the carbonic-oxide-haemoglobin in blood, thus sho"\Wng, as is well pointed out by Dr. Gamgee, that we have three compounds of htemoglobin with gases, increasing in stability in ^ Dr. Gamgee gives his own and the measurements of other observers at p. 105 of Physiological Chemistry, vol. i. THE PIGMENTS. 125 the following order : Oxy-ligemoglobin, carbonic-oxide-hseinoglobin, and nitric-oxide-hsemoglobin. 2. HcBmochromogen. — Wben bsemoglobin is acted on hj acids or alkalies it decomposes into hsematin, a substance containing the iron, and one or more albuminous bodies, to which the name globulin may be given. This, however, is not a direct decomposition, but, in the presence of oxygen, there is in the first instance oxidation, with the result of the production of an intermediate body, haemochromogen. The oxidation occurs so quickly that the new body and its spectrum can only be recognized with special arrangements. If, however, haemo- globin be decomposed in an apparatus ^ from which oxygen is excluded, by the action of alcohol containing sulphiuric acid or caustic potash, a purple-red fluid (in alkaline solutions) is obtained. This is haemo- chromogen, which, by oxidation, yields hsematin. Hoppe-Seyler supposes it to have the composition Cg^HggN^FeOs, ^^^1 gives the equation — 2(C34H36N,Fe05) -f- 0, = C,^^,,-^,Ye.fi^, + 2H2O. Haimochromogen. Hsematin. It is really a product of the decomposition of haemoglobin, although it is erroneously termed reduced hsematin by some authors. The sj)ectrum of this substance, seen in Plate I. 8, is thus described by Dr. MacMunn— "Obtained from blood treated by rectified spirit and ammonia by adding sulphide of ammonium. The second band varies much in breadth, the iirst being much better marked. In concentrated solutions, the first band reads A569 to X542, and the second band X535 to A.504. More diluted, first band, X569 to X542 — darkest part, X566 to X549 ; second baud, from edge to edge, \536'5 to A506. In a still more dilute solution, they read : First band, X566 to A.547, and second band, \535 to \514. If we distinguish the readings of the extreme edge of each band from the darker parts, we get : First band, A567'5 to A547, and the darker part from A.564 to X550'5 ; the second band, A.535 to A514. " 3. MethcemogloUn. — This is a substance intermediate between haemo- globin and oxy-haemoglobin, and it would appear that the oxygen it contains is more firmly united to haemoglobin than in the case of oxy- haemogiobin. It is produced when a solution of haemoglobin has been exposed to the air for some time, when it becomes acid and loses its blood-red colour, acquiring a brownish tinge, and the spectrum, as given in Plate I. 4, shows two absorption bands like those of oxy- haemoglobin and a new band in the red near C. This is the spectrum of an acid solution. If this be now rendered alkaline by the addition of ammonia, the band near C disappears, and another fainter band is ^ Apparatus figured La Gamgee's Physiological Ghemistrij, p. 119. 1-2Q THE CHEMISTRY OF THE BODY. seen before D. (See Plate I. 5.) On comparing the spectra 4 and 5 in Plate I., it will be seen also that the position of the band corres])onding to the a-band of oxy-hsemoglobin, that is the one immediately to the right of D, is moved a little farther to the right in the spectrum of alkaline methaemoglobin. Dr. MacMunn thus descriljes these spectra — Methemocjlobin. — Prepared by acting on an aqueous solution of sheep's blood with a solution of permanganate of potash : First band, \647 to X6'22 — often another feebler band may be observed from AGIO to \597 ; second band, '\587 to X571 ; third band, /\o52 to /\532 ; and a fourth band (?), X514 to X490. Alkaline methmnoijlohin. — Prepared by adding a little ammonia to the previous solution : First band, X610 to X597 ; second baud, XoS7 to X5l}7"5 ; third band, X552 to X523." The chief interest in methsemoglobin is connected with the facts that if the alkaline solution be shaken up with sulphide of ammonium, a solution is obtained giving the spectrum of reduced hajmoglobin, and if this latter solution be now shaken up with air, oxy-hsemoglobin is detected by its bands a and jB. These facts were first observed by Dr. Arthur Gamgee in his well-kno"vvn research on the action of nitrites on the blood, in Avhich he showed that blood mixed with nitx'ites contained oxygen that Avas not removed by a stream of carbonic oxide, nor was given up to a vacuum, and he believed that this was owing to some kind of combination having taken place between the haemoglobin and the nitrites. Later researches have supported the view that this nitrite-hsemoglobin is really metheemoglobin. This substance is not a hyperoxide of haemoglobin, nor a per-oxyhsemoglobin, because, as shoAATi by Hoppe-Seyler, it may be formed in circumstances in Avhich oxidation cannot occur. 4. Hcematin, C^^^^^eO-^ (or tAvice that formula), is formed along with globulin Avhen haemoglobin is decomposed. A solution may be readily made by adding acetic acid to blood. The blood be- comes of a brownish colour, and if it now be shaken up with ether, the latter floats to the top and an ethereal solution of acid haematin is obtained. This may be used for examination Avith the spectroscope, or the ether may be evaporated, and the residue, haematin, washed AA'ith ether, alcohol, and Avater. AVhen so obtained, it is a reddish-broAvn amorphous poAvder, having a metallic lustre, in- soluble in water, alcohol, and chloroform, but soluble in acidulated alcohol and alkalies. The spectroscopic appearances of acid haematin and of alkaline haematin are given in Plate I. 6 and 7. With acid haematin four bands are seen, the 1st in the red betAveen C and D ; the 2nd, a narroAV faint band close to D ; the 3rd, a broad band betAveen D and E ; and the 4th, broad and faint, between h and F. If the fluid is made alkaline Avith ammonia, then we see the spectrum of alkaline THE PIGMENTS. 127 hsematin, one broad absorption band near D, and a shaded dark part of the spectrum from a little to the left of E to midway between E and F, and complete absorption beyond this boundary. The exact details as to the position of the bands are thus given by Dr. MacMunn — "(1) Acid HcEmatin. — A rectified spirit and sulphuric acid filtered extract from sheep's blood. The 1st band from X647 to X605, dark part X641 to A622; 2nd band, very feeble from \593 to X571 ; 3rd band, from X550-5 to X529 ; 4th band, from X517 to X488 or X485. Prepared in the usual way by acting on blood with acetic acid, and shaking with ether, the ethereal solution gives bands with these measurements : 1st band, from X656 to X615 — darker part X647 to X630 ; 2nd band, from X597 to X577 ; 3rd band, from X557 to X529 ; 4th band, from X517 to A488. (2) J IhxUne Hcematin got by adding rectified spirit and caustic soda to blood and filtering. The whole band extends from X630 to X562 — the darker part from X619 to X581. The purer form of alkaline hasmatin prepared from pure hsematin gives a band, when examined in rectified spirit, from X627'5 to X562 — darker part from X619 toX581." A substance similar to hsematin (entero-ha^matin) occurs in the bile of snails {Sorby), and in that of the limpet and cray fish (MacMunn). 5. Hmmatopm-phyrin or cruentin, Cg<5H^2^8^i2> ^^ ^ body formed by the ■decomposition of haematin. When the latter is mixed with strong sulphuric acid, it is dissolved, and after filtering through asbestos, a clear purple-red solution is obtained. (Gamgee, op. cit.) If water be now added largely to this solution the greater part of the dissolved coloured substance is precipitated as a brown flocculent mass, and the amount of this is increased if alkalies be added to neutralize the acid. It is impor- tant to note that in this decomposition the whole of the iron of the hsematin separates and is found in the solution as a ferrous salt. Thus — C68H68N8Fe.,Oio + 2(ES0d + 0,= CggH^^NsOi^ + 2(FeS0J. Hasmatin. Sulphuric Hsematoporphyrin. Sulphate of acid. iron. Hoppe-Seyler has obtained hsematoporphyrin by the action of nascent hydrogen on hsematin, and MacMunn has found the same substance by the action of sodium amalgam on hsematin. The precipitate is hsema- toporphyrin. Like hsematin, it may exist in either an acid or an alka- line solution, and the two solutions give different and characteristic spectra. Acid hsematoporphyrin gives a broad dark band a little to the right of D (Plate I. 9), and a thin faint band to the left of B.^ Alkaline hsematoporphyrin (Plate I. 10) gives four bands : 1st, thin and narrow between C and D ; 2nd, a broader band near to D ; 3rd, another broad 2 Gamgee states, " The first (solution in strong sulphuric acid) exhibits a pretty dark band immediately below D and a sharply-defined band nearly intermediate between D and E." (Gamgee, Physiol. Chem. p. 117.) 128 THE CHEMISTRY OF THE BODY. band near E ; and 4tli, a "well marked dark hand between h and /'. The exact position of the bands is thus stated by Dr. MacMunn — " (1) Acid Hamatoporphyrin. — Id concentrated solution in sulphuric acid the bands read : 1st band, X607'5 to X593 ; 2nd band, X5S5 to X536'5. In a weaker solution : 1st band, X605 to X593 ; 2nd band or shading, from X5S5 to the next band which extends from \o67'5 to Xo-lO. If the fluid be diluted by adding a little recti- fied spirit to the sulphuric acid instead of adding acid, the bands read : 1st band, X610 to X591— darker part from X605 to X593 ; 2nd band or shading, X585 to X567*5 ; and dark band, X567 '5 to X540. For the bands of jmre acid hsematoporphyrin in rectified spirit and sulphuric acid the following are the measurements : 1st band, X605 to X591 ; 2nd, a shading from X5S3 to X564 ; 3rd band, X564 to X542. (2) Alkaline Hcematoporphyrin. — In solutions of this pigment, the edges of bands vary much according to concentration. The folloM'ing may be taken to apply to a solution of mean concentration : 1st band, X633 to X612"5, and dark from X630 to X615 ; 2nd band, X5S9 to X.564, and a shading before this which is most difficult to measure, and may be from X605 to X589 ; 3rd band, X549 to X529, which is the darker part ; 4th band, X518'5 to X4S8." MacMunn has found hsematojDorphyrin in the intcgximent of Uraster rubens (star fish),i in the integument of Limao: flavus, Limax variegatus, and in Atrion ater (slugs), in common earth Avorm, Lnmhricus terresfris, in Sole- curtus strigillatus, and he shows that polyperythrin, a pigment discovered by Moseley in various actiniae and deep-sea polypes, is probably identical ■with hsematoporphyrin. It is also found in the eggshells of some birds. A land of haematoporphyrin also appears in the urine of cases of Addi- son's disease, of acute rheumatism, and other diseases. Dr. MacMunn - has found in A-'arious common species of actinia, A . mesemhryanthemum, Bunodes crasskomis, Sagartia bellis, etc., a colouring matter related to haemoglobin, very similar to htemochromogen, and convertible into hfematoporphyrin. 6. Hcematolin, Cg^H^gNgO;, is another derivative of haematin described by Hoppe-Seyler. It is insoluble in sulphuric acid and in caustic lyes, thus differing from hsematoporphyrin. 7. Hccmatoldin. — In old blood clots such as occur in the brain, in the corpora lutea of the ovary, and in the coagiila of aneurisms, crystals of this substance are found in the form of regular or oblique rhombs or rhomboids (Fig. 52). The crystals are strongly refractive and trans- lucent, of a brick-red or yelloAvish-red colour. They are insoluble in water, alcohol, ether, acetic acid, and the dilute acids and alkalies. Caustic potash gives a bright red at first and then the crystals dissolve. 1 MacMunn, Quart. Jour, of Micros. Science, \811. Mso Journal of Physiology, vol. vii. See also MacMunn on the haematoporphyrin of Solecurtus strigillatus. Jour, of Physiology, vol. viii. No. 6. -MaclMunn, Pliil. Trans, of Royal Society, 1885. TEE PIGMENTS. 129 Concentrated acids and fuming nitric acid give a play of colours Anth. crystals of hsematoidin like the reaction of ♦ ^ these acids -with the colouring matter of the bile, the bodies gradually becoming brownish- red, green, blue, rose-coloured, and finally yellow. Haematoidin is also said to appear in a granular form. (Eobin.) There has been much discussion as to the true nature of haematoidin, some asserting that it is identical with bilirubin, one of the pigments of the bile to be hereafter described, while many others— Holm and Staedeler— ^^°- s^.-Crystais of H«matoidin on the ground of differences in chemical reactions, and Preyer, from differences in the spectra, have denied the identity.^ Fig. 53. — 1. Hcemin crystals from man — whetstone form; 2. Crystals of common salt ; 3. Hasmatoidin crystals from man, 500 diameters. 8. Hcemin, C^gilj^^Ye^O^^, is a derivative of hsematin, the appearance of Avhich is an important test for the presence of this substance, or in other words, of blood, in medico-legal inc^uiries. Sometimes termed Teichmann's crystals, they are rhombohedral plates or six-sided plates, of a brownish colour by transmitted light (Fig. 54). They are insoluble in water, soluble in hot alcohol, in hot ether, and in caustic potash. To obtain them, add to a drop of blood on a glass slide a few particles of common salt, and then a drop of glacial acetic acid ; place the slide in a warm place, or heat cautiously over the flame of a spirit lamp, and then examine with the microscope. The crystals will then be found as represented in Fig. 53, probably mixed with a few crystals of common salt. Fig. 54. — Crystals of Hasmin. The vie'w in Fig. 63 is more like what one may readily obtain from a drop of blood. ^ See on this point Gamgee's Physiolocj. Chem. p. Patlwlogy, p. 313. 120, and Wagner's General 130 THE CHEMISTRY OF THE BODY B. — The Pigments of the Bilk. The bile of mammals contains two chief pigments, bilirubin and biliverdin. From these, certain other pigmentary bodies may be derived. The general relation of these pigments may be sho^\^l in the first instance by contrasting their empirical formula? as follows — Derivatives Bilirubin, - Biliverdin, - Choletelin, - Bilifuscin, - Bilipvasiu, - Hydrobilinibin, - CeHisKA ^"leHisNaOg Cj6H,oN204 C32H40N4O7 1. JBilirvbin, CigHigX^Og, or Cg^H^gN^Og, may be obtained from fresh bile by acidulating and then shaking up with chloroform. The latter dissolves the bilirubin, and on draAving off the chloroform solution and evaporating it the coloiu-ing matter remains. It may be purified by being treated ^Yiih. alcohol. Bilirubin is either a reddish-orange amorphous powder or it may be found in the form of dark red needle-shaped or rhombohedral crystals (Fig. 5.5). These are insoluble in water, slightly solul)le in alcohol or ether, and readily soluble in chloroform, benzin, bisulphide of carbon, sulphuric acid, and the alkalies. Bilirubin, bj" oxidation, becomes changed into biliverdin and choletelin, and during the oxidation other transitory bodies are formed, some of Avhich On this fact depends Gmelin's reaction or test, used for the detection of the bile in the lu-ine. A few drops of bile are spread out on a white porcelain plate, and a drop of fuming nitric acid (nitric acid containing nitrous acid) is allowed to fall into the middle of the fluid film. At the point of contact of the two fluids, beautiful rings of colour appear in the follo^^-ing order : green, blue, violet, red, and yellow. If biliverdin alone is present, the play of colours begins "with the blue. The spectrum of the chloroform solution of bilirubin shows no absorption bands ; only an absorption of the ^dolet end of the spectrum to b. (See plate I. 13.) A solution that shows Gmelin's reaction, when examined spectroscopically, gives " a broad shading (probably composed of two distinct bands) in orange and yellow, and a black band extending from near b to beyond i^. ... In a very short time the shading in orange begins to fade, and at the time the oxidation process is completed and the colour of the solution has become Fig. 5u. — Crystals of bilirubin. have not been isolated. THE PIGMENTS, 131 yellow, nothing hut the band at F is left." (MacMunn, op. cit. p. 160.) Whilst bilirubin has never been obtained artificially by the chemist from the decomposition of haemoglobin, there are strong grounds for holding that in the body, and probably in the cells of the liver, this transformation occurs. The injection into the blood of substances that destroy the red blood corpuscles, such as large quantities of water, the bile acids, or ammonia, is followed by the excretion of bilirubin in the urine ; therefore it is fair to infer that the increased amount of bilirubin has come from the breaking up of haemoglobin. As, however, the direct injection of a solution of hsemoglobin into the blood does not cause an increase of urinary pigment, it is evident that the chemical transformations are not yet understood.^ 2. Biliverdin, C-^j-H-^giSr^O^, the chief pigment in the bile of herbivora, may be made by exposing greenish bile for some time to the air. The addition of hydrochloric acid throws down green flaky masses, amor- phous, insoluble in water, but soluble in alcohol. The alcoholic solu- tion has a deep green colour, and on evaporation an amorphous residue is obtained, soluble in glacial acetic acid, and the evaporation of the acetic acid solution yields biliverdin. (Beaunis.) It is then found to l^e an amorphous powder, insoluble in water, ether, and chloroform, but soluble in alcohol and sulphuric acid. Biliverdin is undoubtedly formed by the oxidation of bilirubin. A substance like biliverdin has been fovind. by MacMunn in various actiniae, and by Krukenberg in the shells of certain molluscs. MacMunn believes it may be looked on as excretory, being probably derived from other pigments like htemo- globin, or from those he terms histohfematins, pigments scattered through the tissues. 3. Choletelin, C^gH-i^glSr^Oc,, is an oxidation product of biliverdin, or it may be regarded as the substance formed at the end of the oxidation process of bilirubin. It is prepared by passing nitrous vapours into an alcoholic solution of bilirubin. When the alcoholic solution is poured into water, flakes of choletelin " of the colour of ferric oxide " separate, and these, by evaporation, may be obtained as a dry powder. " Chole- telin cannot be made to crystallize from any solvent. It dissolves very easily in the fixed alkalies, and their carbonates, also in ammonia, forming brown solutions, from which it is precipitated in flocks by acids. It is soluble in alcohol, ether, and chloroform. The alcoholic solution ^It is significant that urobilin can be obtained from haemoglobin and haematin, and from bilirubin by the action of reducing agents. (See below.) Moreover, after blood has been extravasated in large quantity into the tissues, the urobilin of the urine is much increased. 132 THE CHEMISTRY OF THE BODY. is precipitated by -water. With silver nitrate, it gives a precipitate only on the addition of ammonia. No pcrceptililc reaction is produced by hydrogen sulphide, or b}- zinc and hydrochloric acid." ^ It does not give Gmelin's reaction, as it has been oxidized beyond that stage. The spectrum of a similar pigment, to which the bands in ox and sheep bile are due, is shown in Plate I. 12, and is thus described hy Dr. MacMunn — " Cholohcematin, from bile of sheep. The bile is, after addition of absolute alcohol and acetic acid and filtering, agitated in a tap funnel Avith chloroform, which, after separation, shows this spectrum. On letting the chloroform stand for a day or so, a urobilin band also generally becomes visible. (1) The lile itself — 1st band, centre at A649 (not shown in plate); 2nd band, A613 to /\585 ; 3rd band, A.577"5 to A561-5; 4th band, A537 to A521-5 (?). Bands 2, 3, and 4 shown in plate. "(2) Chlcyroform sohition — 1st band, A654 to A636 ; 2nd band, A607 to A580-5 ; 3rd band, A.572 to A.560 ; 4th band, A536 to A516. Bands 2, 3, and 4 shown in plate." ^ 4. Bilifuscin, C^oH.^oN.fi^, may be obtained from brown gall stones from the human being. By means of ether, cholesterin is removed from the powdered gall stone, and the powder is then treated with Avater and a little hydrochloric acid. It is then thoroughly washed with water to remove all the acid, next extracted with boiling ether to remove fatty acids, and finally boiled Avith absolute alcohol. On evapor- ating the alcoholic solution, bilifuscin appears as a dark brown powder, or a black brittle mass.'^ By a somewhat similar process. Simony has extracted it from human bile.* It does not give Gmelin's reaction. It is readily soluble in alcohol, glacial acetic acid, and alkalies ; sparingly soluble in chloroform, but insoluble in water, ether, and dilute acids. The alcoholic solution absorbs the violet and indigo-blue parts of the spectrum. 5. BUiprasin, C-^^.-,^^0^^, is the name given by Staedeler to a green pigment obtained from gall stones. By exhausting the gall stones with ether, hot water, chloroform, and dilute hydrochloric acid, he obtained a brownish-green residue, from which chloroform dissolved a broAvn pig- ment (bilifuscin) and some bilirubin, whilst the undissolved residue gave to alcohol biliprasin. Its spectrum has not been studied. 1 Watt's D/c^. of Chem., Bile Pigments, vol. vii. p. 189. Quoted from Maly and Heynsius and Campbell. 2 See Jour. Physiol, vol. vi. Nos. 1 and 2, p. 24. ' Dr. Thudichum's Chemical Physiology, 1872. ■» Watt's Diet, of Chem. vol. viii. p. 325. THE PIGMENTS, 133 6. Bilicyanin, a blue pigment, may be obtained by adding an alcobolic solution of bromine to bilirubin suspended in chloroform. On evapora- tion the liquid assumes a blue colour, and on complete evaporation "there remains a shining dark mass, which appears green when spread in a thin layer upon porcelain, but dissolves with a splendid blue colour in alcohol. It is less soluble in ether, but more freely in ether-alcohol." ^ It gives two or perhaps three absorption bands in the yellow and in the green. 7. Another blue colouring matter has been separated from the bile of man and of the ox, sheep, pig, dog, and cat by E. Ritter, to which no name has been given. Bile is shaken with chloroform till a yellow solution is obtained. This solution is treated with carbonate of soda till the yellow colour disappears. On neutralization with hydrochloric acid, two layers are formed, one the yellow chloroform solution and the other the blue pigment. This pigment is insoluble in acids and chloroform ■ it dissolves in alkalies, and when the alkaline solution is neutralized with acids and exposed to air, a brown precipitate is formed, which becomes blue in a few days or only after a month, thus differing from an alkaline solution of reduced indigo which instantly turns blue on exposure to the air. 8. Bililiumin is a name given by Staedeler to a humus-like residue left after exhausting gall stones with water, alcohol, ether, chloroform, and dilute acid successively. It is probably impure biliverdin and mucus.^ 9. Reducible Product of the Oxidation of the Bile Pigment. — The following account of this substance from the paper on the subject by Stokvis is given in Watt's Dictionary of Chemistry, vol. vii. p. 189 — " The substance is formed as a secondary product in most cases of the oxidation of biliary colouring matter, whereby Gmelin's reaction is produced. It is colour- less, or of a light yellow tint, and is soluble in water, alcohol, and dilute acids. It becomes of a beautiful rose-red colour when boiled with reducing agents in alkaline solutions. The red solution gives in the spectrum a tolerably broad absorption band in the green. In concentrated solutions (thick strata) the band begins close on the line D and extends to h. In dilute solutions (thin strata) it occupies only two thirds of the space between D and E, ending short of E. Shaking with air causes both the rose colour and the absorption band to disappear. This bye-product differs from the bile colouring matter and other oxidation pro- ducts of the same, in being insoluble in chloroform and ether and in not forming ^ Watt's Diet, of Ghem. vol. vii. p. 188, quoting from Heynsius and Campbell. See also Dr. MacMunn, op. cit. p. 152. - For an account of Dr. Thudichum's views as to the pigments of the bile, see his Tenth Report of the Medical Officer of the Privy Council) 1867 ; also his Chemical Physiology ; also MacMunn's Spectroscope in Medicine, p. 154. Dr. MacMuun also gives an account of the spectra of the bile of various animals. 134. THE CHEMISTRY OF THE BODV. iasoluble compounds with neutral or basic lead acetate. It is precipitated how- ever by ammonia and basic lead acetate. This substance exists as such in the gall stones both of man and of the ox. It can be obtained from them by simply boiling with distilled water and extraction by dilute acids. It does not exist in fresh bile. It occurs in the urine of animals which have been starved for some days previously, in icteric urine, and in the urine of febrile diseases, such as small- pox, typhus, etc. It is not found in healtliy urine. It seems to be present in the alimentary canal, although in direct experiments with different kinds of food little or none could be found. In alkaline solutions it soon loses its characteristic pro- perties. Its occurrence in any liquid of neutral or acid reaction affords an indica- tion of the previous existence of bile pigment therein. In applying the test, the liquid is to be precipitated with lead acetate, excess of lead removed by oxalic acid, and the filtrate concentrated and boiled with alkalies and a reducing agent. If no reduction takes place, and if the other tests for biliary colouring matter have given a negative result, their absence may be safely inferred." C. — The Pigments of the Urixe. The urine contains at least two pigments, urobilin and indigo-blue, derived from indican. Thudiclium describes a third, named urochrome, which by oxidation becomes uroerythrin.^ 1. Urobilin, CogH^oN^O^, is believed to be closely related to bilirubin, and it may be termed hyclrobiliruUn. When bilirubin is dissolved in dilute potash lye, and sodium amalgam added, air being excluded, no hydrogen is evolved, the dark brown fluid becomes lighter in colour, in the course of a few days becoming yellow or brownish-yellow, and then hydrogen is given off". Hydrochloric acid added to this solution sepa- rates a pigment of weak acid reaction, which is soluble in alcohol, slightly in water, readily in ammonia and fixed alkalies, and in ether, glacial acetic acid and chloroform. This substance contains about 1 -5 per cent, less of carbon and about 1 -5 per cent, more of hydrogen, and therefore it may be called hijdrohiliruhin. Thus — 2(Ci6Hi3No03) + H, + H,0 := C32H,oN40, Bilirubin. Hydro-bilirubiii or urobilin. Biliverdin Avith sodium amalgam behaves in a corresponding way. There is every reason to believe that in the intestines a portion of these liile pigments is changed into hydrobilirubin, Avhich is then absorbed and excreted by the kidneys as urobilin. Urobilin is a dark reddish- Ijrown powder, soluble in water and alcohol, less soluble in ether. It forms a reddish yellow solution in chloroform. With chloride of zinc and ammonia it gives a rose-coloured solution, -svith green fluorescence. This substance gives a characteristic spectrum, seen in Plate I. 1 4, which is thus described by Dr. MacMunn — ^ This statement has however been disproved. THE PIGMENTS. 135 ' ' Spectrum of urobilin from normal urine thus prepared : The urine is pre- cipitated with neutral and basic acetate of lead, the precipitate separated by filtering, decomposed by rectified spirit containing sulphuric acid ; this extract filtered, diluted with water, and agitated in a tap-funnel with chloroform ; the chloroform separated off, filtered, evaporated, and the brownish residue redissolved in rectified spirit. Febrile urobilin differs from this in the greater shading and breadth of band and in other slight particulars. Stercobilin, found in fasces, also differs from it." It will be seen that there is a distinct broad band a little to the left of F. In Plate I. 15 the spectrum is shown of the same solution with the addition of ammonia and chloride of zinc and filtered. Here the band is narrower and fainter, and less of the blue is visible. Dr. MacMunn gives the position of the absorption band, thus — "(1) Chloroform solu- tion, A 504 to A 481 ; (2) Rectified spirit solution, A 504 to A 481 ; (3) with zinc, chloride and ammonia, A512"5 to A 496." The band is specially well marked in febrile urines, but Dr. MacMunn asserts that urobilin is invariably present in normal human urine, although the quantity may be so small that the absorption band cannot be detected unless the urine has been subjected to the treatment with acetate of lead above described, or oxidized by means of permanganate of potash or bromine water. According to Disqu6, there is a colourless form of urobilin in urine which may be called reduced urobilin, i.e. a ehromogen, which gives no absorption spectrum. This, he says, may be readily changed into urobilin by oxidation,^ either in the intestine or during the passage of the ehromogen from the intestine to the kidneys. There is no strong evidence for the existence of this substance. 2. Derivatives of Indican. — By decomposition indican may give rise to leucin, volatile fatty acids, and a reddish colouring matter, indigo -red, the indigruhin of Schunck, the urrhodin of Heller. Sometimes, also, indican inthe ^xrine m-Sij gire rise to indigo-Uue. (Seep. 109.) The uroxanthin of Heller appears to be indican. If the urine of cases of cancer of the liver, or of obstruction of the great intestine, be allowed to stand until putrefaction occiurs, an irridescent pellicle forms, which yields small crystals of indigotin, and the fluid may become blue. Indigo-blue also may be obtained from most specimens of normal urine, and quite easily from the urine of the horse, by mixing the urine with an equal volume of strong hydrochloric acid, then adding a solution of hypochlorite of calcium till a blue colour appears, and, lastly, shaking up with chloro- form. A blue chloroform solution of indigo-blue is thus obtained 1 Ludwig Disque. Ueber Urobilin. Zeitschrift fiir Physiol. CJhemie, Band ii. 1878. Hoppe-Seyler has obtained urobilin by the action of tin and hydrochloric acid on haemoglobin or hsematin. — Handhuch der Phydol. vnd Pathol. C'hejn. Analyse, s. 214. 136 THE CHEMISTRY OF THE BODY. The spectrum of this sohition is shown in Plate I. 16, and is thus described by Dr. ]Mac]\Iunn — " Spectrum of indirjo-hlue from normal urine. The urine is boiled with about a third of its bulk of hydrochloric acid, cooled, iind agitated with chloroform, which shows this spectrum, and also — although not here shown — a band at F, due to urobilin. The band before D is due pi'obably only to the blue constituent, indiijo-hlue, whilst that after D is probably due to the rechlish constituent, for this reason that the bluer the solution the deeper is the band before D, while the more it approaches violet the darker is the band after D. This test for the pre- sence of indican will detect it when Jaffe's hypochlorite of calcium test fails. Normal urine can generally be made to show the presence of indican by its means." 3. Urohcematoporphi/rin, or urohcematin, is a pigment found Ijy Dr. MacMium in the urine of cases of rheumatic fever and of Addison's disease. He believes it to be nearly related to the body prepared by Nencki and Sieber by the action of tin and hydrochloric acid on hsematin. The alcoholic solution gives the follo■\^^ng spectrum — " 1st band, A595 to A587; 2nd band, A.576 to A.566 ; 3rd band, A557 to A.541-.5; and 4th band, A.503 to 482-5."' ' 4. Urochroine, a substance described by Dr. Thudichum is said to be an amorphous substance of a yellow colour, easily soluble in water, less so in ether, very dilute acids, and alkalies, and least of all in alcohol.- 5. Vromdanin is another dark pigment separated by Dr. Thudichum from urine by the action of strong sulphuric acid. Its existence as an independent pigment is more than doubtful.'' D. — Pigment of the F^ces. In addition to the ordinary pigments of the bile, some of which are voided in the faeces, a variety of urobilin, to which the name stercohilin has been given, has been examined by Dr. MacMunn. He states that there is scarcely any difference between the urobilin fomid in the lurine of febrile patients and the stercobilin of human faeces ; but that there is a distinct difference in the spectrum of ordinar}' lu'obilin and sterco- bilin.'^ By covering faeces with absolute alcohol, chloroform, ether, rectified spirit, and sulphuric acid (1 in 15), and Avater, solutions Avere obtained Avhich gave the folloAving results Avith the spectroscope — [1] Alcohol solution, 1 broad band from A.506 to A482-5 ; (2) Avith caustic 1(7/. C. le Xobel, Archlrf. d. ges. Phys. B. xl. 1SS7. - Thudichum, Brit. Med. Journal, 186-4. 3 For an account of it see Watt's Diet, of Chem., 1st supplement, p. 1120; and cf. Hoppe-Seyler, Handhuchf. Physiol, und Path. Chem. Analyse, Vierte Aufiage. ^ MacMunn, Journil of Physiology, \o\. vi. Nos. 1 and 2. THE PIGMENTS. 137 soda, A521"5 to A505; (3) vvdth zinc chloride alone, 1 band A517"5 to A501. [2] Ether solution, 1 band which, A\dth acetic acid, extends from A504 to A482-5. [3] Chloroform solution, A507 to A486-5. (Compare this with the description of the spectrum of normal urobilin, given at p. 135.) On evaporating the chloroform solution in the water bath, Dr. MacMunn obtained a brown amorphous residue, which gave practically almost all the characters of febrile urobilin both as to solubility and spectrum. In none of the solutions did he detect un- changed bile pigments. If febrile urobilin and stercobilin were the same, then the amount of the former found in the urine woidd be the measure of the amount of the latter absorbed from the alimentary canal, a fact of considerable clinical importance. Dr. MacMunn thus sums up our knowledge of the relation of the urinary fascal and bile pigments- — • " It would appear that the stercobilin resulting from the putrefactive processes in the intestine, and accompanied by imperfectly changed biliary pigments, is taken up by the branches of the portal vein and carried into the liver, where it is probably again changed by the action of a ferment into a chromogen. A portion of this chromogen gets into the blood and is excreted by the urine as a chromo- gen. A portion may escape in the condition of biliary urobilin as such, and appear in the urine in a further oxidized condition or, owing to disturbance of circulation in the liver, a large portion of unchanged biliary urobilin may appear in the urine. Besides this, the urine under certain conditions may contain a pigment that has no biliary origin, and may be derived entirely from htematin ; while, in certain diseased states a reduction product of hsematin, having no con- nection with bilirubin or biliverdin, and closely related to hEematoporphyrin, may appear in the urine and, to a great extent, if not altogether, may replace urobilin. "1 E. — Pigments of the Tissctes. 1. The Histohcematins are a class of pigments probably first detected by Dr. Sorby, but elaborately examined and discussed by Dr. MacMunn, ^ found in the tissues of many animals, both invertebrate and vertebrate. Thus they have been found in the echinodermata, mollusca, arthro- poda, and vermes, where they may be detected in the absence of haemoglobin and its derivatives, which apparently they replace. With regard to the vertebrata, Dr. MacMunn says — "In vertebrata the search for histohsematins in the organs and tissues is attended with difficulty, OAving to the presence of haemoglobin, but fortunately the bands of the histohsematins can be recognized in the tissues or organs ^ MacMunn, Jour, of Physiology, vol. vi. p. 39. 2 MacMunn on Myohaematin and the Histohsematins. Phil. Trans, of Eoyal Society, part i. 1886. 138 THE CHEMIST IIY OF THE BODY. when squeezed out to a degree of thinness which no longer allows the bands of hiemoglobin to be seen, and in most cases the blood-vessels can be injected "vvith salt solution sufficienth" to eliminate the influence of the circulating hjemoglobin." Dr. MacMunn has detected them in the stomach wall, liver, kidneys, and intestinal Avail of various fishes; in the testes, liver, si:)leen, and stomach wall of the frog and other amphibians; in the liver, spleen, Iddney, and intestinal wall of various reptiles; in the spleen, pancreas, liver, kidney, and gizzard of the common rock pigeon and other birds ; in the liver, spleen, kidney, and stomach wall of the hedgehog, guinea-pig, rat, rabbit, dog, cat, pig, ox, and sheep. He also discovered a histohsematin in the thymus gland of a child of ten months old, and in the spleen, liver, and kidnej' of man. A haemo- chromogen was detected in the medullary portion of the human supra- renal body, and a histohfematin in the cortex. No trace was discovered in nervous tissues in invertebrates or vertebrates. No histohfematins have yet been isolated,^ as they are probably united to a proteid, the compound being changed by the action of the various solvents used. Dr. MacMunn, however, has made the important discovery that they can be reduced in the tissues, and that the appearance of an absorption spectrum of bands indicates a reduced state of the histohsematins, the " bandless " specti'um characterizing them in the oxidized condition. The histohfematins are allied to the hgemochromogens, the spectra being changeable into one like that of hsemochromogen ; and, as " their bands are intensified by alkalies and enfeebled by acids, intensified by reducing agents and enfeebled by oxidizing agents," they are capable of oxidation and reduction, and are therefore respiratory. They are concerned in the internal respiration of the tissues. The position of the bands is thus indicated by Dr. MacMunn — (1) Stomach tcall of cat — blood free: '"Ist band, A613 to A593 ; 2nd band, A569 to A563; 3rd band, A556 to A551. (2) Kidney of cat: 1st band, A613 to A596-5; 2nd band, A569 to A563 ; 3rd band, A556 to A550."2 2. Myohcematm. — This is a pigment, of a yellowish-red colour, also discovered by Dr. MacMunn in the muscles of certain beetles (Hydro- jphilus, Dytiscus), the common fly (Musca vomitoria), and other insects ; in spiders, crustaceans, molluscs, fishes, amphibians, rei^tiles, birds and mammals.^ He has also found it in the heart and voluntary muscles of man. "Its bands are seen Avith great distinctness in the muscidi ^ Although they can be got into sohitioii by means of ether, see below, Myohjematin. -MacMunn, "Fui'ther Observations on Myoha?matiu and the Histohsmatins, " Jour. ofPhi/sio'. vol. viiii Ko. 2. ^ MacMiinn, Phil. Trans, op. cit. part i. 18S6. THE PIGMENTS. 139 papillares of the human heart." By a process of freezing, combined \vith pressure, which apparently prevents the decomposition of the proteid compound in which myohaematin exists, Dr. MacMunn^ has obtained "a few drops of a reddish-yellow licjuid," which gives the characteristic spectrum, mixed Avith the spectrum of oxy-hsemoglobin. On reducing the latter with ammonium sulphide, " through the thin hazy band of reduced haemoglobin," the bands of myohaematin were apparent. The spectrum of myohaematin from the alar muscle of a meat fly is shown in Plate I. 11, and Dr. MacMunn remarks— "The spectrum is practically the same throughout the whole animal kingdom." He gives the position of the bands as follows — "(1) Heart of hare: 1st band, A613 to A600; 2nd band, A569 to A.563; 3rd band, A556 to A550. (2) Heart of rat: 1st band, A613 to A.596-5 ; 2nd band, A.569 to A.563 3rd band, A556 to A550." F. — Luteins or Lipockromes. In 1849, Dr. Thudichum- described a pigment which he obtained from the corpora lutea, from eggs, from butter, and from blood serum, to which he gave the name lutein. Considerable doubt has been thrown on the existence of all of these bodies, but a colouring matter gi\dng absorption bands does exist in the corpora luteit and in the yolk of egg.^ A yellow colouring stuff has been extracted by ether from the eyes of frogs after removal of the retinae. It has two absoqDtion bands between F and G, and it bleaches by sunlight. It has been called lipochrin, and is no doubt a lutein. The lutein pigment is held in solution in fatty matter. The name Upochromes has been given bj- Krukenberg to all animal pigments soluble in certain fat solvents and that give bands in the blue and violet. These are all luteins. They give in the solid state a blue or green colour with strong nitric and sulphioric acids, also generally with iodine dissolved in iodide of potassium. An immense number of lipochromes have been found not only among animals but also among plants. Carotin, C^gHg^O, may be taken as a typical Hpochrome. None so far has been found to contain nitrogen. ^ MacMunn has obtained myohiematin in solution from the pectoral muscles of pigeons by covering the blood-free muscles (finely diAided) with ether for some days, when a red juice exudes, which shows the bands of modified myohccmatin. J(mr. of Physiol, vol. viii. No. 2, 18S7. - Thudichum, Proc. Roy. Soc. xvii. 253. The yellow colouring matter was first separated from ovary of cow by Piccolo and Lieben and called huemolutein. An ethereal extract of corpora lutea boiled with potash has excess of water added, when small shinmg dichroic crystals are deposited. ^MacMunn on "Animal Chromatology, " Proceed, of Birmingham Philosoph. Society, vol. iii. ISS3. 140 THE CHEMISTRY OF THE BODY. G. — Chromophanes. These are colouring matters that have been separated hy Kuhne from the cones of the retina. Three such bodies have been isolated and their spectra examined.' "A large number of eyes (50 to .300) of doves or hens are bisected so as to cut off the anterior segments ; the vitreous humour being removed, the posterior segments of the eyes are placed at once in absolute alcohol : as soon as possilile the alcohol is poured aAvay and the eyes are thoroughly exhausted with ether. On evaporating the ether, a fier3'-red fat is obtained wliieh is dissolved in hot alcohol and saponified by the action of caustic soda, Avatcr being used to replace the alcohol as it evaporates. The hard soap -which separates from the mother liquor is well dried and treated successively with petroleum ether, then \n.t\i ether, and lastly Avith benzol, which dissolve in order — clilorophane, xanUiophane, and rhodophane." The probable function of these colouring matters in connection with the sense of vision \n[\ be dis- cussed fully in treating of that sense. All that is here necessary is to describe shortly the spectra of these pigments. a. Clilorophane is a greenish-yellow j^igment and gives the absorption bands seen in Fisc. 56. Spectrum. C D .^ ,4« Fic. 56. — Spectnim of Chlorophane — A, dissolved in ether oi- in petroleum ether ; B, dissolved in bi- srjphide of carbon. 1). Xantliophane is slightly soluble in petroleum ether, readily soluble in alcohol, ether, and carbon disulphide, and gives the spectrum seen in Fig. 57. Spectrum. a 5 C Z> E& E .^^ Fig. 57. — Spectrum of Xautliophnne — A, dissolved in ether ; B, dissolved in bisulphide of carbon. ' Gamgee's Physiological Chemistry, vol. i. p. 460. THE PIGMENTS. 141 c. Ehodophane is insoluble in petroleum ether or carbon disulphide, is soluble in oil of turpentine, in alcobol acidified with acetic acid, and in benzol, and it shows the spectrum given in Fig. 58. Spectrum. « IB m Fig. 58. — Spectrum of Rhodophane — A, dissolved in benzol ; B, dissolved in oil of turpentine. d. Ehodopsin or visual purple is a colouring matter associated with the rods of the retina, and sensitive to light. Kiihne discovered that it is soluble in " crystallized bile " thus prepared — " Colourless crystallized bile is obtained by evaporating ox-bile to dryness in the water bath, after mixing it thoroughly with much animal charcoal. The perfectly dry residue is treated with absolute alcohol, and a large excess of ether is added to the filtered solution ; by this means the salts of the bile acids are precipitated and ultimately acquire a crystalline structure. The precipitate, which consists of sodium glycocholate and taurocholate is termed ' crystallized bile.' " ^ As this substance is destroyed by the action of actinic light, care must be taken to open the eyes of frogs in a chamber lit by a sodium flame, and the retinae are moistened with about 1 c.c. of a 2 per cent, solution of the bile salts, and gently shaken for about one hour. A reddish-purple fluid is thus obtained which is a solution of rhodopsin. Light causes it to pass from purple, " through red and orange, to yellow, before becoming colourless." Thus "visual purple" is converted into "visual yellow" and the latter then fades away. The spectra of the two conditions are shown in Fia;. 59. A. a. : K Spectrum. 3 C D f h F ^, B. ■ 1 Fig. 59. — Spectriim of Rhodopsin— A, unaltered by- light, " visual purple "; B, altered by light, "visual yellow." ^ Gamgee's Physiological Chemistry, vol. i. p. 464. 142 THE CHEMISTRY OF THE BODY, H. — Black Pigment. A black |)it;iiioiit named Melanin occurs in the hexagonal epithelial colls forming the external layer of the retina (Fig. 60) in the connective tissue cells of the choroid itself, in the deeper layer of the epidermis of the skin, constituting the rete Malpighi, and it is often I.— PigmTntcfiis. fouud in various kinds of timaours. It also occiu's in the skin of fishes, amphibians, and reptiles, and in the feathers of birds. In amphibians, such as the frog, it is abundant even in the deep tissues and organs of the body. The ink of the cuttle-fish is of the same nature; but there is no doubt that quite different colouring matters have been included under the name melanin, Avhich only possess one attribute in common, \\z., blackness. Some of these pigments are non-nitrogenous, e.g. from birds and Seioia (cuttle-fish). It is insoluble in water, acids, alcohol, and ether. It is imperfectly dissolved in boil- ing caustic potash, a brown fluid being formed, thus distinguishing it from the particles of carbon sometimes found in lung tissue, which are quite insoluble in boiling caustic potash. The percentage composition varies from C, 51-7 to 58-3; H, 4-02 to 5-09; N, 7-1 to, 13-8; and 0, 22-03 to 35-44.1 The pigment of the choroid is said to contain a small amount of iron. I.— Other Animal Pigments. To attempt to give the characters of the numerous pigments existing in animals, more especially among the invertebrata, that have been described by Moseley, Ray Lankester, Krukenberg, MacMunn, and others in recent years, is quite beyond the aim of this work. Only a few are alluded to, specially \n.\h. the view of pointing out the wide range of many of these pigments, and the remarkable resemblance they bear to some of the pigments already described as relating to man and the higher animals. This branch of inquiry is still in its infancy, but enough has been done to show the resemblance between plant and animal pigments and the fact that not a few of them are concerned in respiratory processes. The follo-\^4ng are a few of the more imjDortant.^ 1. Chlai-ophyU occurs in many of the lower invertebrates (Spongilla, Hydra, Paramecium), and Professor Ray Lankester has sho^\^l that the ^Gamgee, Physiological Chemistry, vol. i. p. 304. -The inquirer wishing to study these pigments may refer to MacMunn's papers already frequently referred to, which are not only critical and full of the results of original inquiry, but also give many references to the literature of the subject. THE PIGMENTS. I43 corpuscles are part of the animal itself and not unicellular algae leading an independent existence, as Brandt supposed.^ It has also been found in the blood of caterpillars ^ and in the elytrse of cantharides beetles.-^ Dr. MacMunn has found in many animals (more especially in alcoholic extracts of the " liver and other appendages of the intestine answering to it"), a substance having a spectrum similar to that of the chlorophyll of plants, to which he gives the name entero-cMaivphyllA He has also recently found chlorophyll in several salt water sponges.-^ 2. Hcemocyanin is a blue isigment found in the blood of many inverte- brates. The blood of many molluscs and arthropods becomes bluish in colour after exposure to the air {Helix, Unio, Limulus, Odojms, Sepia., Scorpio, Cancer, Homarus, Portunus, Maja, Sqidlla, etc.). MacMunn found no absorption bands in that of Helix pomatia. Helix aspera, Paludimt vivipara, Lymnmis stagnalis, Homarus vulgaris, Cancer pagiirus, Carcimis mcenas, and Astacus flimatcdis.^ 3. Chlorocruorin is a green pigment discovered by Professor Ray Lankester in the blood of Sahella ventilabrum and Siplionostoina,~ which is capable of being oxidized and reduced by ammonium sulphide like haemoglobin. MacMunn has figured the spectrum of the blood of Sahella and of Serpida contortuplkata in the paper referred to,^ and shown its resemblance in some respects to that of hsemochromogen. 4. Echinoclirome is a brown pigment found in the perivisceral cavities of Echinus (Schafer, P. Geddes, and MacMunn), and also in Strongylocentrotus livichis by MacMunn, who first described its spectrum. When exposed to the air it becomes deeper in colour. Dr. MacMunn remarks with regard to the spectra figured in the paper — " A compari- son of these spectra shows hoAv remarkably unstable echinochrome is. It is on this instability that its usefulness as a respiratory substance depends." ^ Eay Lankester on the Chlorophyll Corpuscles and Amyloid Deposits of Spongilla and Hydra — Quart. Jour, of Micros. Science, vol. xxii. KS. See also MacMunn on the Spectroscope in Biology — Proceed, of Birmiwjham PhiloaoyMcal Society, vol. V. part ], and Quart. Jour. Micros. Science, vol. xxvii. p. 573 et seq. 2 Poulton, The essential nature of the colouring of Phytophagoiis Larvse — Proc. Boy. Soc. 1885. 3 Pocklington, Phar. Jour. Trans, vol. iii. ; MacMunn, By^it. Ass. Report, 1883. * MacMunn, Further observations on Enterochlorophyll and Allied Pigments — Phil. Trans. Royal Society, part 1, 1886. See also MacMunn's Notes on the Chromatology oi Anthea cerevs — Quart. Jour, of Micros. Science, March, 1887. 5 MacMunn, Proc. Physiol. Soc. March, 1887. ^MacMunn, Chromatology of Blood of some Invertebrates — Quart. Jour, of Micros. Science, 1885. '^ Raj Lankester, Jour, of A7iat. and Physiol. 1868. 144 THE CHEMISTRY OF THE BODY. 5. Fentacrinin, a pigment discovered by Professor H. N. Moseleyi in species of Pentacnnus dredged 1)}^ H.M.S. " Challenger." It exists either as a purple or a red pigment, and gives a spectrum of three bands, " one intensely black covers D, the second is between D and E, and the third, a broad dim one, stretches from h to F." Its spectra have been mapped and measured by MacMunn {Froc. Fhil. Soc. Birming. op. cit.), and he remarks — " It may be concluded, however, that Mose- ley's purple pentacrinin does bear a most remarkable resemblance to plant pigments. It also bears a resemblance to honelle'm, the colouring matter of Bonella liridis, which had long been taken to be a chlorophyll, and which Professor Lankester thinks may also be present in Ch(etopterus." 6. Adiniohcematin is a colouring matter found in Actinia mesembryan- themum by ]\IacMunn, which can be changed into hsemochromogen and haematoporphyrin. It is not actiniochrome, a pigment A^dely distributed in actinia?, chiefly in the tentacles. In Sagartia parasitim, a special pigment is found not identical Avith either actiniohaematin or actinio- chrome, and in Anthea cereus, JBimodes halii, and Sagartia hellis, a mix- ture of plant-like pigments is met with containing chlorofucin (an algal pigment), and derived from the "yellow cells" in the tentacles.- 7. Turacin is a pigment obtained from the feathers of the Cape lory, which is said to give a spectrvim like that of blood. (Church.) 8. Tetronerijthrin is a pigment found by Hoppe-Seyler in the rose- coloured rings round the eyes of certain birds. Merejkowski^ has found it in 104 species of invertebrates and fishes. This body is of great interest inasmuch as it is said to perform the fimction of haemo- globin as a carrier of oxygen to the tissues. In many invertebrates it takes the place of haemoglobin, and this no doubt accounts for its wide distribution. 9. Crustaceoruhin^ is the name given by Moseley to a pigment he found in some deep-sea decapod Crustacea. 10. Cochineal, from the insect Kermes cacti, and Lac-chje, from Coccus laccce, give no characteristic absorption spectrum. Carminic acid, from carmine, gives a spectrum somewhat resembling that of haemo- globin. 11. Aphidein is a pig-ment obtained by Sorby from Aphides. It gives an absorption spectrum, and it appears to be a true respiratory pig- 1 Moseley, Quart. Jmir. of Micros. Science, 1877. - MacMunn, Xotes on the Chromatology of Anthea cereus — Quar. Jour, of Micros. Science, vol. xxvii. IST.S., p. 573-590. ^It would appear that under the name " tetronerythrin " several lipochromes of a reddish colour have been included. ■* Probably identical with tetronerythrin. EXPLANATION OF THE PLATE OF SPECTRA. (FRONTISPIECE.) 3ctruin of diffused daylight with some of the principal lines of Fraunhofer, sctrum of oxy-hcemoglohin in a moderately dilute solution, p. 121. 3ctrum of reduced hcemoglohin, p. 122. sctrum of methcemoglobin, got by adding a solution of potassium permanganate to )n of sheep's blood in water, p. 125. ectrum of alkaline methcemoglobin, got by treating the last solution with a little a, p. 126. ectrum of acid hcematin, got by treating blood with acetic acid and agitating with ;he acid hsematin got by treating blood with rectified spirit containing sulphuric embles this but wants the full band at D, j). 127. ectrum of alkaline hcematin, got by treating blood with rectified spirit and caustic Chis differs from the alkaline hsematin produced by the action of rectified spirit ;ionia, as the latter does not give the band at D, but a dark hazy band in the green, ectrum of reduced hcematin or hcemochromogen, got by adding sulphide of am- . to the solution of alkaline ha;matin. The latter should be prepared by the action onia and rectified spirit, as in this case the hsemochromogen bands are better p. 125. ectrum of acid hcematoporphprin, prepared as follows :— Defibrinated blood is with rectified spirit acidulated with sulphuric acid, filtered, diluted with water, . in a tap-funnel with chloroform, the chloroform separated off and evaporated to , the brown residue divided in strong sulphuric acid and filtered through asbestos, pg a little rectified spirit this spectrum can be seen. By treating blood or hsemo- lirectly with strong sulphuric acid and filtering through asbestos a similar spec- j,y be observed, but it is better to proceed as above, as then the influence of the constituent is avoided, p. 127. Dectrum of cdkaline hcematoporpltyrin, got by poiuing the sulphuric acid solution ned under 9) into water, adding ammonia, removing by filtering brown flocks, which b, dissolving the precipitate in rectified spirit and ammonia, p. 127. ^ectrum of myoluematin, from alar muscle of a meat fly. The spectrum is practi- ie same throughout the whole animal kingdom, p. 139. jpectrum of cholohcematin from sheep's bile. The bile is, after addition of absolute land acetic acid and filtering, agitated in a tap -funnel with chloroform, which, jparation, shows this spectrum. On letting the chloroform stand for a day or \bilin band also generally becomes visible, p. 132. pectrum of a moderately dilute chloroform solution of bilirubin, which shows Sorption of the violet end of the spectrum, no bands, p. 130. pectrum of urobilin from normal urine thus prepared : — The urine is precipitated ^utral and basic lead acetate, the precipitate separated by filtering, decomposed ;fied spirit containing sulphuric acid, this extract filtered, diluted with water and I in a tap-funnel with chloroform, the chloroform separated off, filtered, ,ted, and the brownish residue redissolved in rectified spirit. Febrile urobilin fom this in the greater shading and breadth of band and in other slight parti- so does stercobilin, p. 134. 'he same solution with ammonia and zinc chloride (filtered), p. 135. pectrum of indigo-blue, etc., from normal urine. The urine is boiled with about af its bulk of hydrochloric acid, cooled and agitated with chloroform. It then shows sctrum, and also, although not here shown, a band at F due to urobilin. The band D is due probably only to the blue constituent, indigo-blue, while that after D is y due to the reddish constituent, for this reason that the bluer the solution the is the band before D, while the more it approaches violet the darker is the band This test for the presence of indican will detect it, when Jaffa's hypochlorite um test fails ; and normal urine can generally be made to show the presence of by its means, p. 136. e spectra were mapped by means of a Sorby's microspectroscope. In the chemical scope the bands are more hazy and some of the faint ones difficult to see. The of light was an Argand gas burner. EXPLANATION OF THE PLATE OF SPECTRA. (FRONTISPIECE.) 1. Spectrum of diffused daylight -with some of tlie principal lines of Fraunhofer^ p. 112. 2. Spectrum of oxy-hmmogloMn in a moderately dilute solution, i>. 121. 'd. Spectrum of reduced hcemoglobin, p. 122. 4. Spectrum of methcemoplohint got by adding a solution of potassium permanganate to a solution of sheep's blood in water, p. 125. 5. Spectrum of alkaline meth(emogJohin, got by treating the last solution with a little ammonia, p. 126. 6. Spectrum of acid hmmatin, got by treating blood with acetic acid and agitating with ether ; the acid heematin got by treating blood with rectified spirit containing sulphuric acid resembles this but wants the full band at D, j). 127. 7. Spectrum of alkaline hcematin, got by treating blood with rectified spirit and caustic soda. This differs from the alkaline hsematin produced by the action of rectified spirit and ammonia, as the latter does not give the band at D, but a dark hazy band in the green, p. 127. 8. Spectrum of reduced hcematin or hmmochromogen, got by adding sulphide of am- monium to the solution of alkaline hiematin. The latter should be prepared by the action of ammonia and rectified spirit, as in this case the hremochromogen bands are better marked, p. 125. 9. Spectrum of acid hcematoporphyrin, prepared as follows : — Defibrinated blood is treated with rectified spirit acidulated with sulphuric acid, filtered, diluted with water, agitated in a tap-funnel with chloroform, the chloroform separated off and evaporated to dryness, the brown residue divided in strong sulphuric acid and filtered through asbestos. On adding a little rectified spirit this spectrum can be seen. By treating blood or h£erao- globin directly with strong sulphuric acid and filtering through asbestos a similar spec- trum may be observed, but it is better to proceed as above, as then the influence of the proteid constituent is avoided, p. 127. 10. Spectrum of alkaline ha^matoporphi/rin, got by poiu-ing the sulphuric acid solution (mentioned under 9) into water, adding ammonia, removing by filtering brown flocks, which separate, dissolving the precipitate in rectified spirit and ammonia, p. 127. 11. Spectrum of myohcematin , from alar muscle of a meat fly. The spectrum is practi- cally the same throughout the whole animal kingdom, p. 139. 12. Spectrum of choloJuematin from sheep's bile. The bile is, after addition of absolute alcohol and acetic acid and filtering, agitated in a tap-funnel with chloroform, which, after separation, shows this spectrum. On letting the chloroform stand for a day or so a urobilin band also generally becomes visible, p. 132. 13. Spectrum of a moderately dilute chloroform solution of bilirubin, which shows only absorption of the violet end of the spectrum, no bauds, p. 130. 14. Spectrum of urobilin from normal urine thus prepared : — The urine is precipitated with neutral and basic lead acetate, the preciintate separated by filtering, decomposed by rectified spirit containing sulphuric acid, this extract filtered, diluted with water and agitated in a tap-funnel with chloroform, the chloroform separated off, filtered, evaporated, and the brownish residue redissolved in rectified spirit. Febrile urobilin differs from this in the greater shading and breadth of band and in other slight parti- culars; BO does stercobilin, p. 134. 15. The same solution with ammonia and zinc chloride (filtered), p. 135. 16. Spectrum of indigo-blue, etc., from normal urine. The urine is boiled with about athird of its bulk of hydrochloric acid, cooled and agitated with chloroform. It then shows this spectrum, and also, although not here shown, a band at f due to urobilin. The band before d is due probably only to the blue constituent, indigo-blue, while that after D is probably due to the reddish constituent, for this reason that the bluer the solution the deeper is the band before d, while the more it approaches violet the darker is the band after d. This test for the presence of indican will detect it, when Jaffa's hypochlorite of calcium test fails ; and normal urine can generally be made to show the presence of indican by its means, p. 136. These spectra were mapped by means of a Sorby's microspectroscope. In the chemical spectroscope the bands are more hazy and some of the faint ones difficult to see. The source of light was an Argand gas burner. THE FIGMENTS. I45 ment. Sorby shows that aphidein is related to cochineal on the one hand and to haemoglobin on the other. 12. Aplysiopurpurin is a purple colouring matter obtained by Moseley from the Doris (nudibranch). It also exists in Aplysia, the sea-hare. 13. Tyrian jpmjjU is the dye obtained from species of Purpura and Murex, and is a secretion of a layer of epithelium, " which is placed between the gills and the hind gut in the mantle cavity." Conclusions Eegarding the Pigments. The previous discussion of the nature and properties of the pigments enables us to appreciate their physiological significance and to understand in some measure the part they play in the animal body. In most, if not in all, animals there is one chief pigment such as the haemoglobin in the blood of the higher groups, and the entero-chlorophyll, haemocyanin, chloro-cruorin, echinochrome, or tetronerythrin^ of many of the lower. This chief pigment is mainly if not wholly concerned in respiration, by its property of combining readily with a certain amount of oxygen and of giving this up to a reducing agent. Thus it receives oxygen from the air or from the air dissolved in the water in which the creature lives, and it conveys this oxygen to the living tissues. These tissues practi- cally reduce it, and it returns to its non-oxygenated condition. In this process, however, it is not a stable substance. It undergoes decomposi- tion, becoming modified into histohaematins that permeate the tissues,, probably entering into their very substance and still exercising a respir- atory function. May it not be possible that these histohaematins act intermediately as regards the haemoglobin (or its representative) of the blood and the oxygen-seeking living tissue? Again, the haemoglobin may be resolved in the liver cells, or in the intestinal canal, or in the kidneys, and probably in other organs, into simpler bodies which then appear as the pigments of the various excretions, waste-products, in short, formed in the chemical operations of the body. Such are probably the pigments of the bile, of the urine, and of the faeces, bodies of no physio- logical value, and voided as useless, at all events in the higher animals. In some of the lower animals, however, such waste-pigments may not be useless, but if innocuous they may be stored up in the epidermal tissues, thus staining the covering of the body so as to suit the habits of the animal, or perhaps adorning it with beautiful colours that serve some useful purpose in the circumstances of its life. Lastly, certain pigments have a more specialized function. Such are the pigments in the eye-spots of many of the humbler forms, and the pigments of the rods and cones of ^ With regard to the latter, it has never yet been shown that it is affected by reducing agents. I. K 146 THE CHEMIST R Y OF THE BODY, the retina. These arc sensitive to light, and by the chemical changes occiu'- ring in them under the influence of light the ends of the optic nerve fibres may possibly be stimulated. The recent researches into the nattu-e of pig- ments are of profomid interest as giving us an insight into some obscm^e processes, and there is little doubt that after the comparative study of pigments has progressed much farther than it has yet done even more important generalizations may be possible than those I have ventured to make. Nor is the subject devoid of practical importance. The physician by studying the amount and nature of the iu"inary pigments, for instance, may obtain valual^le information as to the metabolism occurring in the liver and the changes happening in the bowel, and it is likely that a. more careful study of the pigments in some pathological conditions, such as Addison's disease, "oith the peculiar bronzing of the skin, may throw light on the nature of these obscure affections.^ Chap. X.— THE NON-NITROGENOUS MATTERS. These include the alcohols, the fats, the carbo-hydrates, and certain acids belonging to the acetic acid, the giy collie acid, the oxalic acid, and the oleic acid series of non-nitrogenous organic acids. I. The Alcohols. 1. Ethylic Alcohol, C^Hj . HO, common alcohol, is said by Bechanqj and Eajewski to be formed in small quantities in the body, so that it may be detected in the urine even in the absence from the food and drink of all fermented liquors. In these circumstances it must be formed by an alcoholic fermentation in the body, either in the intestinal canal, or in the tissues, most probably in the former. 2. Cholesterin, or Cholesteric Alcohol, C.,^.H^O . HO, a crystalline sub- stance in the bile, and forming the chief constituent of gall stones, is regarded by chemists as an alcohol belonging to the series C„Ho^_80. It was first obtained by Conradi, so long ago as 1775, from gall stones. It exists in the bile, and it has been obtained from blood, urine, nervous matter, yolk of egg, seminal fluid, red blood corpuscles, white blood corpuscles, milk, the spleen, the contents of the intestines, the meconium, the faeces, tubercular and cancerous deposits, cataracts of the eye, and atheromatous blood vessels. It has also been foiuid in peas, fixed oils, fat of Avheat, glutin, fat of rye, barley, maize seeds, young shoots of roses, and in yeast. To obtain it pure, pulverize biliary calculi, extract \nth ^ On this subject see Dr. ilacMunn's suggestive paper in the British Medical Journal, Feb. 4th, 1888, entitled, " On Addison's Disease and the Function of the Suprarenal Bodies. " THE NON-NITROGENOUS MATTERS. 147 iDoiling ether, distil off the ether, dissolve the residue in alcohol, and allow the solution to cool. The crystalline mass in the solution is heated for some time with alcohol containing a little caustic potash, and on cooling, a crop of crystals is obtained. These are washed with alcohol, and if it is desired to get them prue, they may be re- dissolved in and recrystallized from ether. The crystals are odourless and tasteless. They then appear as rhombic plates, often with one obtuse angle awanting (Fig. 61). Cholesterin is insoluble in water, in alkalies and dilute acids ; slightly soluble in cold, but very soluble in hot alcohol, and in ^ig. (3i.-crystaisofchoiesterin. ether, acetic acid, glycerine, benzol, petroleum, chloroform, and solutions of the bile acids. Anhydrous cholesterin fuses at 145° C, and solidifies at 137° C. Its specific gravity is 1-046. Solutions of cholesterin with polarized light give [aju = - 31°'6. Hot nitric acid oxidizes it so as to form cholesteric acid, a, CgH^QOg, a substance also produced by the oxid- ation of the bile acids. (Witthaus.) By oxidation with potassium permanganate, three acids may be obtained : cholesteric, /3, C26H42O4 ; oxy cholesteric, ^i^io^-^', and dioxy cholesteric, C.^gH^gOg. (Latschinofi".) There are two good tests for cholesterin : {a) If the substance is bruised with a few drops of sulphuric acid, and a drop or two of chloroform then added, a blue-red or violet colour is produced, which changes to green on exposure to air ; and ih) when a mixture of two volumes of sulphuric acid Avith one volume of ferric chloride is evaporated upon cholesterin a violet colour appears. When sulphuric acid is added to a solution of cholesterin in chloroform the upper fluid becomes blood-red or purple red and the under liquid has a green fluorescence. (Salkowski.)'^ A substance, isomeric with cholesterin (isocholesterin), has been obtained by E. Schulze from sheep's wool. The mode of origin of cholesterin in the body has not been clearly made out. Whether it is formed in the tissues generally, or in the blood, or in the liver, is not known, nor has it been determined conclusively that it is derived from the decomposition of albuminous or even of nervous matter. It is also doubtful if we can regard it as a waste- substance of no use in the body, as its presence in the blood corpuscles, in nervous matter, in the egg, and in vegetable grains of various kinds, points to a possible function of a histogenetic or tissue-forming character. 3. Glycerine, C3II5 . (0H)3, is a triatomic alcohol existing in the fats 1 See "Watt's Diet of Chem. vol. vii. p.' 329. 148 TEE CHEMISTRY OF THE BODY. of the body, which, as will presently be seen, are its ethers. It also exists in palm and other vegetable oils, and it is one of the by-products of the alcoholic fennentation. The chemist has been able to build it up synthetically " by heating for some time a mixture of ally! tribro- mide, silver acetate and acetic acid, and saponifying the triacetin so obtained." ("Witthaus.) The properties of glycerine are so well known as scarceh' to require description. It is soluble in water and alcohol, but is insoluble in ether and chloroform. It is a good solvent for many organic matters, as, for instance, the ferments of the salivary, gastric, and pancreatic juices. As wiU be seen in describing the digestive process, glycerine is pro- duced during pancreatic digestion. The fat of the food is decomposed by the action of the pancreatic ferment, and glycerine is set free. Some have held that this free glycerine is instantly absorbed into the blood, and that in the blood it is quickly decomposed into simpler substances. That the process is rapid is evident from the fact that no free glycerine can be detected either in the bowel or in the blood, and even after the injection of glycerine into the blood, there are none of the products of its oxidation (formic acid, acetic acid, propionic acid, etc.). The amount of carbonic acid, hoAvever, is increased, and consequently it has been supposed that it is decomposed into carbonic acid and water. Others have contended that glycerine may contribute to the forma- tion of glycogen, the carbo-hydrate of the liver. A diet rich in glycerine undoubtedly increases the amount of glycogen in the Kver, but it is not easy to follow the steps of the process by which this may be accom- plished. Thus "Ploesz has found in the mine of animals to which glycerine has been given a reducing body analogous to glucose, but differing in having no action on polarized light." (Beaunis.) This body is not glucose, but has been supposed to be an aldehyde of glycerine, CgHgOg, and by the combination of two molecules of this substance, with the eKmination of water, glycogen may be formed. Thus — 2C3H,03 - H,0 = CeHioOj Aldehyde of glyceriue. Glycogen. Again, it has been supposed that the glycerine may not be absorbed, but may be decomposed in the intestine into various acids, and in particular propionic acid, which may then form propionates voided in the faeces. It is not likely that the whole of the glycerine is thus disposed of. A more probable theory is that of Beneke, who suggests that as by the action of the gastric juice phosphoric acid is set free from the phosphates of the food, this free acid may imite Avith the free glycerine to form the important substance, j)hospho-glyceric acid, which again is concerned in THE NON-NITROGENOUS MATTERS. 149 the formation of lecithin. (Beannis.) On the whole, I am of opinion that the evidence is in favour of the view that glycerine contributes to the formation of glycogen. 4. Phenol, CgHg . OH, phenic acid, carboKc acid, has been found in small quantities in the urine and faeces, and is probably the result of pancreatic digestion. Staedeler has discovered it in the urine of the cow. When found in lurine, it is not in the free state, but in the condition of phenol-sulphuric acid or j)henol-sulphate of potash, CgHgO.SOg.OK. The simultaneous ingestion of phenol and of sulphuric acid, or of a sulphate, is said to increase the amount of the phenol-sulphates, and to cause a disappearance of the ordinary sulphates. The phenol of the urine may have originally been formed in the intestinal canal, but it is not imjDrobable that some substances introduced into the body may h& transformed directly into phenol. This peculiar combination of phenol and sulphuric acid is the type of a number of bodies to which Baumann has given the name of sulpho-conjugated acids. Phenol-sulphate of potash may be decomposed by acids and by heat into phenol and sulphuric acid. Many other compounds of a similar kind exist, such as thymol, pyrocatechin, pyrogallic acid, nitro-phenol, amido-phenol, sulpho-pyrocatechic acid, and chresol-sulphuric acid. (Beaunis, j). 115.) 2. The Fats. Animal fats exist diu"ing life in a liquid form, contained in small cells lying in the meshes of connective tissue, from which the fluid may be expelled by pressure (Fig. 62). The oils thus obtained have been termed palmitin, stearin, and olein. Stearin is the most consistent of the three, while olein is fluid at ordinary temperatures. Human fat is formed chiefly of a mixture of palmitin and olein, and contains only a very small quantity of stearin. Fats consist chemically of a combination of the triatomic alcohol known as glycerine, CgHgOg, with a fatty acid. Their struc- ture may be best comprehended by sup- posing that they are built on the type of ^^om'tiilm^^^^'' '^'^^^^'^^ quite free three molecules of water, in which three atoms of hydrogen are replaced by three equivalents of the monatomic Fig. 62.— On the cooling of fat, say after death, the fatty matters may assume the forms of groups of needle-shaped crystals, a, Single needles ; b, larger groups of the same ; c, groups of needles 150 THE CHEMISTRY OF THE BODY. radicle of a fatty acid and the remaining three hy the triatoniic radicle glyceryl. Thus — (H,0)3 Water. (H,0)3 Water. Radicle. C18H37 Radicle. CisHgiO Radicle of palmitic acid. Radicle of stearic acid. H Ho + - CifiH^iO Radicle of palmitic acid. C18H.55O Radicle of stearic acid. C3H5 Glyceryl. C3H5 Glyceryl. [ (Ci6H3iO)3G,H, |0, 1 Palmitin. (Cl8H;,,0),Cft |0, stearin. Of a,HooOfi C„H„„0„ Olein. 0, . 3H.,0 or CWH„„0„ + 3H.,0 A fat may thus also be considered as a compound ether, or, in other words, as consisting of palmitic, oleic, and stearic gl}^cerides, variously mixed together. Moleschott found in the body of a man of thirty years of age, weigh- ing 63-65 kilogr., about 1566 gi-ammes, that is, 2*5 per cent, of the body weight. Burdach gave an estimate of 5 per cent., but it is evident the percentage of fat will vary much in different individuals. The follomng table from Gorup-Besanez^ gives the percentage of fat in the chief organs and flmds of the human body — - Fluids. Percentage of Fat. Tissue or Oeoan. Percentage of Fat. Sweat, •001 Vitreous humour, •002 Saliva, •02 Cartilage, - 1-3 Lymph, •05 Bone, - 1^4 Synovia, •06 Crystalline lens, - 2^0 Liquor amnii, •06 Liver, 2^4 Chyle, •2 Muscles, 3 3 Mucus, •3 Hair, - 4^2 Blood, •4 Brain, 8^0 Bile, - r4 Egg, - - 11 •G Milk, - 4-3 Nerves, 22-1 Adipose tissue, - 82-7 Marrow of bone, 96-0 1. Tripcdmitin, C3H5(0 . C-^^(^Il.^-fi).^, exists in most animal and vege- table fats, and especially in palm oil. It occurs in the form of crystalline plates, very soluble in ether, and sparingly soluble in hot alcohol. It melts at 50° C, and solidifies at 46° C. 2. Tridearin, C3H.(0 . C^gH350)3, is the chief constituent of ordinar}^ ^ Gorup-Besanez, Physiologucken Chemie, 1867, p. 149. THE NON-NITROGENO US MA TTERS. 151 animal fats. It is, in tlie pure state, a hard crystalline solid, readily soluble in ether, and soluble in boiling, but scarcely soluble in cold, alcohol. 3. Triolein, 03115(0 . 0;^gH330)3 . SHgO, is the principal constituent of all fats that remain fluid at ordinary temperatures, and in the pure con- dition it is a colourless, tasteless, odourless oil, soluble both in alcohol and in ether. 4. Trimargarin, 03115(0 . 0^711330)3, has been obtained artificially as a crystalline solid. By this name is sometimes meant a mixture of tripalmitin and tristearin. 5. Trihutijrin, 03115(0.041170)3, is found in butter. It is a pungent liquid, and Avhen it decomposes, butyric acid is set free. 6. Trivalerin, 03115(0 . 05HgO)3, exists in seal oil, and in the fat of some marine mammalia. It is identical with the phoceninc of OheAoreul. (Witthaus.) 7. Tricaproin, 03H5(0 . 0(iHi,0)3 ; Trkaprylin, O.^JiO . 08H^50)3 ; and Tricaprin, 03115(0 . O^qH^qO).,, are found in milk, butter, and cocoa-butter. Fats are either derived from the fat in food or formed in the body. Their origin, function, and ultimate destination will be fully discussed in treating of nutrition. It is sufiicient here to say that they contribute largely by oxidation to the production of heat, and that they also are concerned in histogenetic processes. 3. The Carbo-Hydrates. The name of this important group indicates that they may be re- garded as composed of carbon with hydrogen and oxygen in the proportions that form Avater. Thus, glucose is OgH^gOg, or 0(H20)g. This is, however, a very imperfect view of their chemical constitution, as there are chemical considerations which point to some of the carbo- hydrates being aldehydes, alcohols, or ethers. They are classified as follows, the -H or - sign indicating that they are dextro- or Isevo- rotatory as regards polarized light — • 1. Glucoses. 2. Sacc:haroses. 3. Amyloses. nlCsHisOg) "(Ci-^H^Ai) nlCgHioOg) -1- Glucose. + Saccharose. + Starch. (Dextrose). + Lactose. + Glycogen. C hondroglucose . + Maltose. + Dextrin. - Lasvulose. Cellulose. Mannitose. Gums. ■f Galactose. Inosite. CHj, OH (CH. 1 OH), COH Glucose. 152 THE CHEMISTRY OF THE BODY. (1) The Glucoses. These may be regarded as the aldehydes of mannite, and they con- tain in their rational formula the group of atoms, COH, characteristic of aldehydes. Thus — CHjj . OH I (CH . 0H)4 and CH. . OH JIannite. 1. Glucose, glycose, grape sugar, diabetic sugar, dextrose, CgHjgO^,, exists in fruits, in honey, in the liver, thymus, heart, lungs, blood, and muscles, and in the saliva, sweat, faeces, blood, and urine of persons suffering from diabetes mellitus. Human blood contains on an average about "09 per cent. (Bernard.) It has also been found in the fluid of the amniotic and allantoic sacs of herbivora, and in the lurine of the foetal calf and foetal sheep. Glucose may be crystallized from an aqueous solution in white spheroidal masses containing 1 molecule of water, and from alcohol iu transparent anhydrous prisms. It is soluble both in hot and in cold Avater, and in alcohol. With polarized light, the result is [a]D= +52°-85. A solution of glucose heated with an alkali gives a brown or yellow colour, from the formation of glucic and melassic acids. In alkaline solutions, glucose reduces salts of silver, bismuth, mercury, and copper, in the case of the first three, the metal being precipitated, whilst cupric are reduced to cuprous compounds, with the separation of cuprous oxide. The folloA\dng are the chief reactions of glucose by which its presence may be detected, or its amount in any fluid determined — (1) Mooi-e's test. Heat "with an .equal quantity of solution of caustic potash, and a yeUow colour is produced, and, if a large amount of sugar is present, the colour is brown. In the latter case, there may be a molasses-like smell. (2) Trojnmer's test. Add to the solution a few drojDS of a Aveak solution of sulphate of copper, then a little of a solution of caustic potash ; the latter precipitates hydrated cupric oxide, which is soluble in excess of caustic potash, if sugar be present. Then heat to near boiling, and a deposit appears of cuprous oxide, which may be yellow, orange, or red, according to the amount of cuprous oxide reduced. If a large amount of sugar be present along with only a small quantity of cupric oxide, on heating, the blue fluid may become yellow, without precipitation of cuprous oxide, the latter being held in solution by the excess of glucose. (Witthaus.) (3) Fehling's test. A solution known as " Fehling's solution" is thus prepared — I. Dissolve 51-98 grammes of pure THE NON-NITROGENOUS MATTERS. 153 crystals of sulpliate of copper in 500 c.c. of water ■ II. Dissolve 259-9 grammes of pure crystals of EocheUe salts (tartrate of soda and potash) in 1000 c.c. of a solution of caustic soda ha-vang a specific gravity of 1"12. ^\^len required for use, 1 volume of I. is mixed with 2 volumes of II. Keep in the dark in a stoppered bottle, having the stopper parafiined. To apply the test, boil 4 or 5 c.c. of FehUng's solution in a test tube ; if it remain unaltered, add solution containing glucose drop by drop, and the fluid will become green, and a red or yellow precipitate of cuprous oxide is obtained. The reaction is precisely the same as in Trommer's test. In both we have an alkaline solution of cupric oxide along with glucic and melassic acids, formed by the action of the caustic alkali on the sugar, and, as these acids have a strong af&nity for oxygen, they rob the cupric oxide of oxygen, reducing it to cuprous oxide, which, being insoluble in an alkaline fluid, is precipitated. (4) B'ottgers test. A few cubic centimetres of the solution of glucose are mixed in a test tube with an equal volume of a solution of sodium carbonate (1 part of crystallized carbonate of soda and 3 parts of water), a few grains of subnitrate of bismuth are added, and the mixture boiled. In the presence of sugar, bismuth is thrown down by reduction as a brown or black powder. (5) Picric acid. To a solution of glucose add a few drops of a solution of picric acid, heat, then add a drop or two of caustic potash, and a brownish-red colour is obtained. Other substances give a similar reaction, such as creatinin. (6) Fermentation. Mix. a solution of glucose with yeast in a large test tube, and invert the test tube so as to have the mouth immersed in a solution of glucose ; keep at a tem- perature of 25° C. for ten or twelve hoiurs, and a quantity of gas, carbonic acid, wiU collect in the test tube, and alcohol ^xi\\ be formed in the fluid. (7) MiLlder-Neuhauer test. A solution of glucose is rendered faintly blue with indigo solution, and "faintly alkaline with sodium carbonate, and heated to boiling Avithout agitation." (Witthaus' Chemistry, p. 219.) It turns violet and then yellow without agitation, but if it is shaken the blue colour is restored. (8) Saccharimeter. A solution of glucose rotates the plane of polarization to the right.' It is often important to determine quantitatively the amount of glucose or diabetic sugar present in such a fluid as the lu-ine. The fol- lowing are the methods to be relied on. (1) Saccharimeter or Folarimeter. — Add to the fluid containing glucose a solution of acetate of lead, and filter. Place the filtered fluid in the ^ The precautions to be observed in applying any of the above reactions for the detection of sugar in the urine are not here detailed, as these are the special applications of general methods which can be best understood and appreciated in the clinical work of the hospital. 154 THE CHEMISTLIY OF THE BODY. tube of the saccharimeter, and take half a dozen readings of the angle of deviation. The mean will represent the angle of rotation, and the percentage of sngar is determined by p = ^^.^^ x I, in which p = the weight in grammes of ghicose in 1 c.c. of fluid, a = the angle of deviation, and / = the length of the tube in decimetres. The numbers 52-85 indicate the specific rotatory power of glucose = [a] c. A very ingenious spectropolarimetei' has been invented by E. v. Fleischl for the determination of the amount of ghicose in a sohition. It is specially adapted for the estimation of diabetic sugar in urine. One of its chief advantages is, that it avoids the difficulty of forming a judgment as to the identity of two coloured surfaces, as has to be done with one of the usual instruments. (.See p. 69, Fig. 2i.) Fio. 63. — Speotropolarimetur ot E. v. Fleischl. A lamp or gas flame is placed opposite the end of the instrument shown to the right of the figure. The light passes first through a Nicol's prism h, the Polarizer, then through two small quartz plates cc, so placed that the bisecting plane in which they meet lies horizontally. The one quartz plate is dextro-, whilst the other is Itevo-rotatory, and the thickness of each plate, 7 "75 to 8 mm., is such that the green rays between the Fraunhofer liiaes E and b of the spectrum are circularly polarized through an angle of 90°, the one set passing through the upper quartz to the left, and the other through the lower qiiartz to the right. The light then continues onwards through a tubej^, 177 "2 mm. in length, containing 15 c.c. of the solution, and having flat glass plates to close it at each end. It then passes through a second Nicol d, the Analyzer, with its principal axis horizontal, like that of the first Nicol b, and it will be evident that the light between E and b, extinguished by circular polarization through an angle of 90° by the quartz plates, will not pass through this second Nicol. Finally, the light passes through a direct vision spectroscope e. To give sharpness of definition to the spectrum thus obtained, there is a movable vertical slit at A-, immediately in front of the quartz THE NON-NITROGENO US MA TTERS. ] 55 plates c and c. Suppose then that the tube ff contained water or was empty, or suppose it not to be in its position, and a black cloth to be thrown over the part of the instrument at^so as to exclude light, on looking through a small telescope carrying the prisms e, two beautifully distinct horizontal spectra are seen, one over the other, and separated by a very thin dark line. Each spectrum, however, shows a dark band ia the green, owing to the extinction of that part of the spectrum by the right and left-handed rotation of the green rays by the quartz plates through 90°, and the two bands are exactly over each other, so that the lower edge of the one touches the upper edge of the other. The Analyzer is fixed in a tube carrying an arm bearing a vernier g, so that it oan be rotated round the surface and near the border of a circular metal disc seen in section at h, and this disc is graduated into degrees. The two bands in the spectra exactly coincide when the zeros of the vernier and of the scale correspond. If now the tube ff containing the solution of sugar is interposed, the two bands are seen to be shifted — the one to the right or the other to the left, according to the direction of rotation by the substance under examination. If the substance be glucose, which is dextro-rotatory, then the band corresponding to the dextro-rotatory quartz plate will be pushed to the right, and the extent of the movement is measured by rotating the Analyzer through the number of degrees required to cause the zero points again to coincide, and the two bands are made also exactly to coincide as to vertical position. Suppose then, that to produce this effect, a rotation of 6° is necessary, then the fluid in the tube contains 6 per cent, of sugar, that is 6 grammes in 100 cubic centimetres. The scale of degrees is such that each degree corresponds to 1 per cent, of sugar in the given length of column of fluid in the tube//. The green rays are chosen for exclusion, because they are the most suitable in the case of urine, which absorbs the violet rays (those having the greatest amount of rotatory dispersion), and, as already explained, the exclu- sion of these rays is effected by the determinate thickness of the quartz plates. It would have been equally easy, by altering the thickness of the quartz, to exclude the red rays ; but, as their rotatory dispersion is small, the instrument would not then have been so sensitive, or the blue rays, but in that case the bands would not have been so distinct. This instrument gives results to within ^V per cent, of sugar, and as it can be quickly applied without the use of monochro- matic light, and as it gives the percentage amount without any calculation, it is specially adapted for the wants of medical men. (2) Sir William FiobeH's method. — Determine the specific gravity of the fluid at 25° C, add yeast, maintain at 25° C. till fermentation stops ; again take the sjoecific gravity, and each degree of diminution of specific gravity represents •2196 gramme of sugar in 100 c.c. (or 1 grain to the ounce). This method is well suited for the determination of sugar in urine. (3) Volumetric process hy Fehlingh solution. — The strength of Fehling's solution is such that to reduce the cupric to cuprous oxide in 20 c.c. •1 gramme of glucose is required. The solution of glucose is diluted with nine times its bulk of water and placed in a burette. In a porcelain dish 20 c.c. of Fehling's solution are mixed with 40 c.c. of water and 2 c.c. aq. ammonia and heated to boiling under the burette. The fluid 156 THE CHEMISTRY OF THE BODY. in the burette is alloAved to mix with the bhic fluid in the porcelain dish, and as the cupric oxide is reduced to cuprous oxide, the blue colour gradually disappears. When the blue colour has entirely gone, the number of c.c. of the fluid in the burette is read off. This represents the number of c.c. containing -1 gramme of glucose, as 'l gramme is required to precipitate all the cuprous oxide in 20 c.c. of Fehling's solution. To control the observation, filter the fluid in the porcelain dish and divide into three portions. To the first add a few drops of acetic acid and treat with a solution of ferrocyanide of potassium. If the reduction has not been complete, and any free cupric oxide still exists, a reddish-brown colour is obtained. In that case, enough of the solution in the burette has not been added. This may be corroboi'ated by acidulating the second portion with a drop of hydrochloric acid and a few drops of ammonium sulphide, which mil give a black colour ^^dth any free cupric oxide. To the third portion, in the event of the first two giving no reaction, add a few drops of Fehling's solution and boil ; the absence of any yellow colour Avill show that there is no excess of glucose, that is, that we have not added too much of the fliiid from the burette. Suppose 40 c.c. of fluid in burette has been used and that it was diluted with 9 times its bulk of water. This would represent 4 c.c. of fluid containing -1 gramme of glucose, that is 2-5 per cent. (4) Pavijs Gravimetric method. — In a gravimetric process the cuproiis oxide is carefully collected, dried, and weighed, but this is a matter of great difficulty, and even in the hands of skilful manipulators is wanting in precision. It occurred to Dr. Pavy to dissolve the precipitated cuprous oxide and then to throw the copper down in the metallic form by galvanic action upon a platinum surface, and lastly to weigh the copper thus deposited. The process is fully described in Dr. Pavy's Croonian Lectures on Points connected vith Diabetes, pp. 41-49 (1878), and he sums up its theory as follows — " When sugar is boiled ■with the copper solution the change occurring stands in the relation of one atom of the former to five atoms of cupric oxide. One atom of sugar is oxidized by, or reduces, five atoms of cupric oxide. This is the founda- tion of the action involved in the operation of the test, and the calculation of the amount of sugar present is made accordingly. Taking 63 '4 as the atomic weight of copper, and ISO as that of glucose (CgHjaOg), 317 parts of copper will stand equivalent to 180 parts of glucose. Thus one part of copper corresponds to "5678 of glucose, and in calculating the amoi;nt of sugar in the blood analysis, the weight of copper deposited has only to be multiplied by "5678 to give its equivalent in glucose. The quantity of sugar in the amount of blood taken for analysis being thus determined, the required information is supplied for expressing the proportion for 1000 parts." The glucose found in the alimentary canal is derived from the THE NON-NITROGENOUS MATTERS. 157 conversion of starch into glucose by the action of the salivary and pancreatic juices. This glucose is absorbed and carried to the liver, where it is changed into glycogen, the peculiar amylose found in that organ and in many of the tissues. Part of the glucose in the bowel may, by fermentations, be changed into lactic or butyric acids — CgHi^Og = 2(C3Hg03), lactic acid, or 2{G^B.^^0^) = 3{C^B.f)^), butyric acid. The physiological importance of glucose will be shown in discussing the glycogenic function of the liver in relation to nutrition. By its oxidation into carbonic acid and water, heat will be produced. There is also evidence to show that the consumption of glucose is one of the chemical phenomena connected with muscular action, and with the activity of protoplasm generally. 2. Chonclro-glucose is a substance formed by the action of nitric acid upon cartilage. It reduces cupric oxide to cuprous oxide and is said to be fermentable, producing melitose, a kind of sugar obtained from manna. 3. Lcevulose, uncrystallizable sugar, is found in the intestine as the result of the inversion of cane sugar by the action of a ferment, when cane sugar is changed into equal parts of dextrose (glucose) and Isevulose. It has been discovered in the blood, urine, and muscles. Lsevulose is uncrystallizable, very soluble in water, insoluble in alcohol, and it reduces copper salts. With polarized light, it has a powerful laevo-rotatory action, - [a]D= - 106°. 4. Mannifose, or Mannite, is a yellow uncrystallizable sugar, like glucose in its general character, but having no action on polarized light. 5. Galactose is formed by the action of dilute acids on lactose, or milk sugar. With polarized light, [«]d= +83-33. Nitric acid converts it into mucic acid. 6. Inosite, Muscle sugar, is a sugar found in muscles (specially the muscle of the heart), in the kidneys, liver, lungs, pancreas, spleen, suj^ra-renal bodies, brain, spinal cord, testes, blood, and in urine. (Beaunis.) Professor Kiilz asserts that it exists even in nor- mal urine ; it certainly is found in the urine of diabetes, and sometimes in that of uraemia. It forms long clear shining crystals (Fig. 64) with two molecules of water of crystallization, and "usu- Fig. 64. -crystals of inosite. ally arranged in groups having a cauliflower-like appearance." (Wit- thaus.) Inosite is readily soluble in water and with difficulty in alcohol, 158 THE CHEMISTRY OF THE BODY. and is insoluble in absolute alcohol and ether. It has no action on polarized light. It is not fermentable. In the presence of inosite, hydrated cupric oxide is dissolved in excess of caustic potash, but on boiling the blue fluid no reduction occurs, as with glucose. The follow- ing reaction has been said to distinguish inosite, but it is difficult of application and does not give good results with inosite in a fluid con- taining other organic matters. Srhprer's test. Evaporate the fluid A\nth a drop or two of nitric acid on a platinum plate nearly to dryness : moisten the residue with ammonium hydrate; add a drop or two of a solution of chloride of calcium and again evaporate to dryness — a rose- pink colour may appear. The origin of inosite, in addition to Avhat may be introduced in the food, and its physiological uses are unkno^vn. (2) The Saccharoses. These are regarded as condensed glucoses, inasmuch as they arc formed by the comliination of two molecides of a glucose with the loss of one molecule of water. Thus — QHioOe + C6Hj.,06 - H,0 = Ci,H.,,Oii. 1. Cane sugar, beet sugar, does not occur in the body, but in relation to nutrition and to other carbo-hydrates it is important to notice some of its properties. Only those not familiar to everyone are here alluded to. It does not reduce cupri-potassic solutions in the cold, but it may do so with prolonged heating, with excess of alkali. Aqueous solutions ■with polarized light give [ft]D= + 73°-8. When boiled with water, it is converted into dextrose and IffiA^dose. Thus — C10H22O11 + H.O = CgHioOg + CfiHi.Os. Saccharose. Dextrose. Lajvulose. The solution now shows left-handed rotation ^rith polarized light as the left-handed rotation of Isevulose, [ajo = 106°, is only partially antagonized by the right-handed rotation of dextrose, [a]o = 52°-85. "With active yeast saccharose is first inverted by a special soluble ferment produced by the yeast cell, and then there is fermentation of the glucose thus formed. 2. Lactose, milk sugar, as the name implies, occurs in milk. It exists in the form of prismatic crystals, soluble in hot or cold water, and in acetic acid, but insoluble in alcohol and ether. "With polarized light the action is la]o= +59-3. Dilute mineral acids change it into galactose, and nitric acid oxidizes it to mucic and oxalic acids. It reduces Fehlino's solution. When a solution of lactose is mixed ^ith yeast the alcoholic fermentation goes on slowly, and during putrefaction lactose produces lactic acid. TEE NON-NITROGENOUS MATTERS. 159 When introduced into the alimentary canal, lactose is changed into galactose (a glucose) which is then absorbed. "With regard to the origin of lactose in the cells of the mammary gland, there are diverse opinions as to whether it may be formed from glucose taken in the food or produced from starch. The injection of glucose into animals during lactation has given unreliable results, and it cannot be said that the amount of lactose in the milk of such animals was sensibly increased. On the suppression of lactation, sugar may appear in the urine but it has not been satisfactorily determined whether it is glucose or lactose. 3. Maltose is a sugar formed during the glucose-forming action of the salivary and pancreatic ferments and of diastase on starch. It crys- tallizes like glucose, but differs from that sugar in being less soluble in alcohol and in exerting a dextrogyratory power three times as great. (3) The Amtloses. These may be regarded as the anhydrides of the glucoses. CfiHiaOg - HoO = CgH^oOg. Glucose. Starch. They are readily changed by the action of ferments and by weak sulphuric acid into glucose. Chemists hold that the formulae of the amy- loses should be expressed as a multiple of CgH^oOg, that is ??(CgH^()05). 1. Starch does not exist in the human body, but forms an important element of food. A solution gives with polarized light [a] ^ = -t- 216°. Heat causes starch grains to swell and burst, and at 200° C. it is changed into dextrin. It is also changed into dextrin by heating with water at 160° C, and prolonged heating changes it into glucose. Starch is also changed into glucose by hydrochloric and oxalic acids. It is insoluble in alcohol, ether, and cold water. Cold water causes the starch grains to swell enormously and to become glutinous, forming starch paste or hydrated starch. If then boiled ^vith water a solution of starch is obtained. A dilute solution of iodine gives a violet-blue ■\\ith starch, and if to a solution of starch made blue ^vith iodine, a solution of a neutral salt is added there separates a blue flocculent deposit of the so-called iodide of starch. As will be seen in the description of digestion, starch is converted into sugar by the action of the sali'\'ary and pancreatic jmces. 2. Glycogen is a form of animal starch found in the liver, placenta, cartilage cells, pus cells, colourless blood corpuscles, muscular tissue, and in embryonic tissues generally. It may be prepared from the liver by either of the two following methods : (1) Claude Bernard's method. The liver is cut into small pieces and thrown into boiling water ; the frag- ments are then bruised in a mortar for a (quarter of an hour in a very 160 THE CHEMISTRY OF THE BODY. little -water. This matter is then submitted to pressure and the fluid obtained is mixed with animal charcoal and filtered. The opalescent filtrate is mixed ^nth five times its volume of alcohol of 38° to 40° strength, and the glycogen is precipitated. The precipitate is collected and washed -\nth alcohol, and to obtain the glycogen pure it is boiled in a concentrated solution of caustic potash and is then reprecipitated with alcohol. The alcoholic precipitate is freed from the caustic potash by being washed with distilled water. (2) BrilcJ:e's method. The liver is thrown into boiling water. "\Mien hard it is bruised in a mortar -with a little water, and the bruised matter is heated gently for half an hour in a little more water. The milky fluid is then poured off" and is replaced by water. This is again boiled and the fluid again poured off" repeatedl)^ until the water has an opalescent tint. These washings are collected, cooled, and filtered, and acetic acid and potassio-iodide of mercurj^ are added so as to form a precipitate. This is filtered, and the filtered liquid is heated "vWth alcohol which precipitates the glycogen. This is then collected, washed with alcohol, and purified by the caustic potash method above described. The C[uantity may also be estimated by Briicke's method, by weighing the amount of the precipitate, or the glycogen may be converted into glucose by the action of weak acids, and the glucose thus formed maj^ be fermented. Briicke's method gives the best results both qualitatively and quantitatively. When so obtained, glycogen is a snow white, odourless, tasteless powder, soluble in water, and insoluble in alcohol and ether. Its solu- tions with polarized light are dextrogyrous to about three times the extent of those of glucose. In the presence of glycogen, hydrated cupric oxide is soluble in caustic potash, but no reduction occurs on boiling. Iodine gives ^^^th glycogen a wine-red colour. When boiled with nitric acid, oxalic acid is formed. The ferments of the salivary glands, and of the pancreas, glycerine-extracts of the liver, pancreas, muscle, lung, and brain, and dilute mineral acids change it into glucose. The iDhj'siological importance of gljxogen will be discussed in treating of the glycogenic function of the liver in its relation to nutrition. 3. Dextrin is a substance obtained by subjecting starch to a dry heat of 175° C, or by heating starch ^vith dilute sulphuric acid to 90° C, or by the action of the diastase in infusion of malt upon hydrated starch. It is a slightly yello^^'ish powder, soluble in water, forming mucilage, and its solutions ^vith. polarized light give [a]D == -i- 138° -88. Dextrin is said to occur in the blood and in muscle, and when a solution is injected into the blood it is changed into glucose. 4, Cellulose is the basis of vegetable tissues. (See Vines' Physiology of Plants, Lect. i.) A variety of cellulose has been found by Schafer in THE NON -NITROGENOUS MATTERS. 161 the mantles of Fi/rosomidce, Salpidce, and Fhallusia mamillaris. The reactions of this substance leave no doubt as to the identity of animal and vegetable cellulose.^ A similar substance is said to exist in the skin of the silkworm. - 4. The Non-Nitrogenous Organic Acids. These are acids formed in the body or introduced in the food, and usually united "with bases. Representatives of four groups of these organic substances occur in the body, belonging to the acetic acid series, the giycollic acid series, the oxalic acid series, and the oleic acid series. ( 1 ) Acids related to the Moxato jiic Alcohols ok Acetic Acid Series. These are a series of acids, all monobasic, and related to a series of radicles, CHo, CgH^, etc., each member of the series differing from the one before it by an additional increment of CH.,. They may be regarded as formed on the type of Avater in which the radicle of the acid is sub- stituted for an atom of hydrogen, or there is a substitution of for H., in the radicle, and this altered radicle is then united to hydroxyl — C.,H,0 H CsHg - H2 + = C.,H,0 : then : H H Ethyl. Radicle of acetic acid. Acetic acid. Another view is that they are derived from the primary monatomic alcohols by the substitution of for H^ in the gTouj) CH^ . OH — CH3 - CH, - CH. - CHo . OH, Normal butyl alcohol. CH3 - CHo - CHo - CO . OH, Normal butyric acid. The annexed series (see page 162) is sometimes termed the fatty acid series, because the higher substances, such as palmitic, margaric, and stearic acids, exist in fats. (See Fats, p. 150.) The follo^ving are the characters of the chief members of the group.-^ Formic acid, CH^O^, is a colourless liquid of strong and pic[uant odour ; volatile at 100° C. without residue ; is not precipitated by the nitrate of mercury ; heated Avith concentrated sulphuric acid, it decomposes into water and carbonic oxide, CH^Oo = CO + HgO. Acetic acid, C^H^O^, transparent foliated crystals ; changes at 17° C. to a colourless fluid, of a piquant characteristic odour, and of a very acid taste ; volatile Avithout residue. It is not precipitated by perchloride of iron, but if we saturate the acid vnth. ammonia, the licjuid becomes dark- ^ Schafer, Annates Chem. Pharm. clx. 312. ^ De Lucca, Comptes Bendus, lii. 102, Ivii. 43. ^ From the Appendix to Beauuis' Physiologie Humaine, vol. ii. p, 1441. I. L 162 THE CHEMISTRY OF THE BODY. >5 ^ M t: £: Si a ^ ^ g £ c -2 CO "-I s -3 2 _» o 5^ i< aj ^ cs ^ _2 O r; > . o » o • r3 >> o iT3 s o o - o ^"o.So ^5 'o s .'I' ^"^ ^ .^ I o fl Q w p: 1^2, -5 05 O O LO CO CI O o ■* O l-^ C5 — H CC O — ( ^ ^ — < CI Ol c^ O ffi M . • O 2 tiT o" d" o' o' 2 9 2 2 ^" o" P o" o o o o o |i! hd W ^P^ (6 i:^ ^ > 00000000 ■S -S -^ °c a 3 ^ ••? odop-iOi-il^p^ ^ - . ^' --r ^•- x~ n '^ O ^ "^ ooo;;; 2222ffiKKK o < ■fe o 1 ^- W_ tC^ i32^ W o o o o ^ 2 a 5 H PM m <5 M W M B Qi n ~T a O O O O H P^ M ^ oooooo oo o ^ m ~ '"d >. o O rt ^ ttl 4S ^ :j n) J» s m' "g 4^ o to rt ;i » ci 4J ^ 3 ^ _2 O X' . H CO "* CM < Cl5 o -V '"' 00 ^ W b C5 CO b f3 t^ r- ^ in ^ -* ^ 1 — 1 CO 1 fe a C) ^H CO ^ -+ CO CM ; o M >o lO G5 C-1 "J m r-~ CO - g§ 00 s ■■ 30 S" CO 1 ■P CO CM CO i T 1— H >H Tj* CO »-o £ 00 ^•2 5 <) "S CO 00 IN CO 00 CO CO CO P 00 (N 10 1 1—5 1— 1 10 CO '- • • • • 'C ■ ■ ■ p a c" .. s ., a" ' •^ (1> S &D W Tl 3. Hydrogen has been found in small quantity in the air of expiration and also in the intestinal canal ; it is said to increase in the great ^ Beaiinis, Physiologie Humahie, vol. i. p. 71. 170 THE CHEMISTRY OF THE BODY. intestine during a milk diet and to reach a minimum during a meat diet. Its origin in the intestine is probably due to fermentation. 4. Carbonic acid also exists in the lungs and intestinal canal. In the lungs it is derived from the blood, and in the intestinal canal it may come partly also from this source, but it is no doubt chiefly due to chemi- cal decomposition. The gas may be simply dissolved or it may be loosely united to the carbonates and alkaline phosphates. 5. Carhurettecl liijdrogeri has been found in the great intestine, where it is said to exist to the amount of from 5 to 10 per cent. It is increased by a leguminous, and falls to a minimum during a milk, diet. It is the result of decomposition. 6. Sidjihiiretted hydrogen is occasionally found in the intestinal canal and results from the decomposition of the albuminous constituents of the food or from biliary matters, both of Avhich contain sulphur. Chap. XII. -THE CHEMICAL REACTIONS IN THE LIVING ORGANISM. Hitherto we have been describing the substances obtained by chemical processes from dead animal matter, or from the secretions or excretions of the body. It is manifestly a much more difficult task to attempt to follow the chemical reactions which occur in the living body, and it may be at once admitted that much of our knowledge of such phenomena is based upon inferences collected from the general facts observed by the physiological chemist when he analyzes the substances obtained from the body, and studies hoAv they are affected by oxidizing and deoxidiz- ing agents, into what simpler substances they may be decomposed, or what more complex substances may be formed by their combination. "VYe are still ignorant of the exact phenomena and conditions of those obscure chemical processes which occur in living tissue, and which appear to be absolutely necessary for the manifestation of vital action, inasmuch as it is impossible to submit living tissues to the ordinary processes of chemical analysis, even on a microscopic scale, without de- stroying their vitality. The principal chemical reactions, however, may probably be classed into the great divisions of oxidation, decomposition, reduction, syn- thesis, and fermentation. A. Oxidation. Oxidation is the most common chemical reaction in the living organism. The albuminates, fats, and carbohydrates, by union vdth. oxygen, form a series of compounds, somewhat simpler in chemical composition, and lower in molecular weight than themselves. From CHEMICAL REACTIONS IN THE LIVING ORGANISM. VJ\ these, by a continued process of oxidation, bodies still less complex in cbemical composition are formed, and so on in successive stages until the elements which at first existed in albuminates noAV appear under the form of urea, carbonic acid, and water, and those of the carbohydrates and fats as carbonic acid and water. At present it is impossible to demonstrate the successive phases through which albumin may have to pass, but there is little doubt that many of these have been observed by chemists in the laboratory. Thus, by oxidation, albumin has been re- solved into leucin, tyrosin, glycocin, and fatty acids ; uric acid into urea, allantoin, oxalic acid, and carbonic acid ; guanin into xanthin, oxaluric acid, and urea ; creatin into sarcosin and urea, and fats into the Avhole series of fatty acids. It has also been found that the intro- duction into the living body of one or other of these substances has l)een followed by the appearance in the secretions of an increase of the materials into which this body may be resolved. Thus the introduction of uric acid increases the amount of urea and of oxalate of lime excreted. The oxygen of the air introduced by respiration is the agent which effects the oxidations ; but it is remarkable that, while in the laboratory these oxidations only take place at a high temperature and with very powerful oxidizing agents, such as permanganate of potash or nitric acid, in the living condition they occur at the temperature of the body and with much greater rapidity. To explain this, it has been assumed that the oxygen of the tissues is in the condition of ozone, which is known to have strong oxidizing powers even at comparatively low temperatures. Thus, at the temperature of the blood, and more especially in the presence of alkalies, or of alkaline carbonates, fats, glucose, and organic acids may be oxidized by ozone, but not by ordinary oxygen. (Gorup-Besanez.) No proof, hoAvever, has been offered that ozone, O3, exists in the blood ; indeed, its presence has been expressly denied by Schonbein. But oxygen in the nascent state, 0, that is at the instant it has been set free from ozone, O3, or from ordinary oxygen, O2, or from any chemical substance containing it, is well knoAATi to have powerful oxidizing properties, and it has been suggested that it is thus liberated in some of the chemical processes occurring in the body. It has also been pointed out by Hoppe-Seyler, Pfliiger, and others that the heat formed by molecular oxidation is quicldy carried away, and that the mean temperature of the tissues is no direct measure of the heat produced in them. Thus, a high temperature Avill be associated Avith the oxidation of an atom of carbon or an atom of hydrogen, in the body as out of it, but in the body the heat so pro- duced is quickly diffused. Much discussion has also taken place as to Avhether oxidations occur 172 THE CHEMISTRY OF THE BODY. in the tissues or in the blood. There can he no doubt that oxidations occur chiefly, if not wholly, in the tissues. As first shoAvn by Spal- lanzani, and corroborated by many modern physiologists (Valentin, Liebig, Claude Bernard, Hermann, and Paul Bert), living tissues con- sume oxygen and produce carbonic acid. On the other hand, blood left to itself in a vessel produces carbonic acid and consumes oxygen, but this is the beginning of putrefactive changes. A stream of oxygen passed through fresh blood does not increase the amount of carbonic acid given ofi' which would be the case if oxidation occurred in that fluid. Pyrogallic acid, ^\liich is readily oxidized, if injected in con- siderable qviantity into the blood, quickly appears in the urine, showing that it has passed through unchanged. But living tissues immersed in blood use up its oxygen and produce carbonic acid. The fact that nitrites are converted into nitrates, sulphites and hyposulphites into sul- phates, and that organic acids, such as malates and tartrates, are changed into carbonates when passed through the body, would at first sight indicate that oxidations may occur in the blood; but even these changes would appear to depend on the living tissues, as lactate of soda in con- tact with blood remains unaltered, but if injected into a vein in a living animal, it is quickly decomposed, "nath the formation of carbonates, which then appear in the urine. One must avoid entertaining too mechanical a view of the so-called oxidations in living matter. There is no such phenomenon as the direct union of oxygen with carbon or with hydrogen, as in burning these in the laboratory. If this were the case, there would be oxida- tions strictly so called, and we Avould find a constant relation between the amount of oxygen used and the amount of the combustion products. No such parallelism can be traced, nor, in many cases, can even the products of combustion be estimated quantitatively. Thus, one is led to infer that oxidations are one stage of complicated processes, possibly analogous to those of fermentation or putrefaction. On the oxidation processes occurring in the body, animal heat, mechanical work, and innervation largely depend. B. Reduction. The phenomena of reduction, which are so important in the life of a plant, do not occiu" so frequently in the animal body. The formation of fat from carbohydrates is an example : the carbohydrates lose oxygen, and become transformed into fats. The formation of indol, of tri- methylamine, and the conversion of benzoic acid into quinic acid, of iodates and bromates into iodides and bromides, and of indigo-blue into indigo-white, during the passage of these substances through the body, CHEMICAL REACTIONS IN THE LIVING ORGANISM. 173 are no doubt examples of reduction. It is likely also that reductions and oxidations may form part of the same process, or the one process may be carried through before the other. Thus, malic acid by reduction becomes succinic acid, which is then oxidized into carbonic acid and water. C. Decomposition. By decomposition we mean the splitting up of an organic substance into two or more chemical compounds, the combined molecular weight of which is exactly equal to the molecular weight of the first substance. Thus, taurocholic acid may divide into choloidic acid and taurin — CosH4gNS07 = C2H7NSO3 + C.,4H3s04 Taurocholic acid. Taurin. Choloidic acid. Occasionally, by dehydration or the removal of water, a simpler body may be formed. Thus, with the aid of heat, cholalic acid may be con- verted into dyslysin and water, and creatin into creatinin and water — C24H40O5 =. C.^HggO. + 2H,0 Cholalic acid. Dyslysin. C4H9N3O2 = C4H7H3O + H2O Creatin. Creatinin. Sometimes a molecule of water may be removed, and the residue may then split up into simpler compounds. Thus, oxalic acid may be divided into carbonic acid, carbonic oxide, and water — C2H2O4 - H2O = CO. + CO Oxalic acid. It would appear in some instances that a compound must combine with water before splitting up. This occurs in the saponification of fats, and in the decomposition of glycocholic acid into cholalic acid and glycocin — C,6H43N06 + H^O = C24H40O5 + C2H5NO2 Glycocholic acid. Cholalic acid. Glycocin. In like manner, creatin may be resolved into urea and sarcosin, and urea into carbonic acid and ammonia — CH4N2O + H2O = CO2 + 2NH3 Urea. Ammon. Carbonate. A special form of decomposition is dissociation where a compound resolves itself into two or more simpler bodies under altered physical conditions. Thus, oxy-hsemoglobin in the diminished pressure of a partial vacuum, and with the aid of heat, gives up its oxygen, and resumes it when the conditions return to those of ordinary atmospheric pressure and a temperature of 15° C. Bonders has suggested that a dissociation process may occur in respiratory exchanges. (See Eespiration.) 174 THE CHEMISTRY OF THE BODY. D. Synthesis. The formation of organic substances by synthesis in the li\ang animal body is still very imperfectly understood, but it is interesting to observe that many nitrogenous organic compoimds have been formed synthetically by the chemist in the laboratory. Thus urea, hippuric acid, glycocin, tavu-in, sarcosin, creatin, glucose, and oxalic, lactic, succinic, benzoic, propionic, acetic, and formic acids have been formed artificially ; but as yet it has been impossible to prepare the higher members of the series. It ^"s probable that in the living body more of the nitrogenous compounds are formed by analytical than by synthe- tical processes. One well-kno'wn example of a synthetical process is the formation of hippuric acid after the introduction of benzoic acid \vith food or medicine. In these circumstances, benzoic acid unites with glycocin to fomi hippuric acid, which makes its appearance in the urine — OyHfiO., + aHsNO., - CgHgNOs + H.O Benzoic acid. Glycocin. Hippuric acid. In some cases there may be a simple union of this kind, but in most the process is probably much more complicated. Thus, a complex substance may be decomposed or oxidized, and the simpler products are then used to build up another complex body. For example, the ingestion of toluol is followed by the appearance of hippuric acid in the urine. Here toluol by oxidation becomes benzoic acid, and this com- bining with glycocin produces hippuric acid. Many organic acids must thus be built up, by the union of various substances with glycocin. Again, when taui'in is given to a living animal, tauro-carbamic acid appears in the urine, by the imion of the taurin ^^•ith cyanic acid. Again, aromatic bodies unite with sulphuric acid to produce conjugated sulpho-acids. Thus, phenol is followed b}^ the appearance of phenol- sulphate of potash or soda, cresol by cresolsulphate, etc. There may also be in the first instance oxidations, such as benzol into phenol, anilin into amidophenol, and then, as explained, phenol gives rise to phenolsulphate. Syntheses pla}- an important part in building up the complex bodies existing in living matter, and we may consider that substances so all- important as fats, lecithin, and other compounds existing in nervous matter, haemoglobin itself, and albuminous bodies, are thus formed. How such processes are accomplished is not kno'siTi, nor must we sup- pose that there is only one way by which a complex chemical substance may be formed. It has been conjectured that the elimination of water plays an important part in synthetic operations, and that the bodie.=! CHEMICAL REACTIONS IN THE LIVING ORGANISM. 175 thus formed may be regarded as anhydrides of substances produced by the combination of the simpler bodies. Much of our knowledge on these points is still obscure, but it is remarkable that the triumphs of chemical science are in the synthetic production of complex organic bodies, and it is not unlikely that each successive step in this direction will lead to a better understanding of the similar processes occurring in the body in the upbuilding of its tissues. E. Fermentation. Certain substances have been long known to possess the property of exciting chemical changes in matters with which they come into con- tact. Such substances have been named by chemists ferments, and the process is known as fermentation. As fermentation plays a very important part in nature, and as in recent times it has been supposed to be the explanation of many processes occurring in the body, and to account for the origin of not a few forms of disease, namely, those included under the generic name of zymotic, it is necessary to refer to it here somewhat in detail. Chap. XIII. —FERMENTATION. The processes included under the name fermentation are so important as to demand careful study, more especially in these days when they are known to be of wide application to many phenomena of the living body, and also to some of the jDhenomena of disease. Not a few trans- formations occurring in the body, more especially during the process of digestion, are of the nature of fermentations ; and it is highly probable that changes of a similar kind are involved in nutrition. It is now also generally acknowledged that fevers and epidemic diseases owe their origin to the action of fermentive agents, and that as we gather knowledge of the natural history of these agents, their conditions of vitality, their mode of growth, their reproductive j)rocesses, and the substances they produce, we may hope either to suppress them or to confine their action within narrow limits. Further, the study of fer- mentation throws a flood of light on many of the general phenomena of biology in the lower plants and animals, and lays a sound foundation or the appreciation of physiological processes, as these occur in the higher animals and in man. When a fluid ferments, it becomes clearer or more muddy, as the case may be, and it usually froths and gives ofi" a gas. This is well seen if the sweet juice of a plant, such as grape juice, is exposed to the fair. The clear fluid becomes turbid, a froth gathers on the surface 176 THE CHEMISTRY OF THE BODY. owing to the evolution of gas, the temperature of tlie fluid rises, and a slimy deposit gathers at the bottom of the vessel. These physical changes are also known to be associated with chemical changes. Alcohol and various other substances are now found in the fluid, and the gas given off is carbonic acid, whilst the sugar has been used up, so that at the end of the j^rocess it has entirely disappeared. Further, there is a change of a biological character, for if the slimy deposit at the bottom of the vessel be examined with the microscope, it will be found to contain numerous minute unicellular organisms, various species probably of Toridce or Saccharomijcctes, Avhich were very few in number at the beginning of the process. Some of these phenomena must have been known from the most distant times, indeed since men began to drink the fermenting juice of the grape, or ynne, but the one that specially caught their attention was the effervescence or frothing, and as it resembled the bubbling of the surface of a boiling fluid, such a pro- cess Avas called a fermentation (from fervere, to boil). The practice of distillation, by which the alcohol was separated from the rest of the fluid, was knoAvn as early as the 8th century, and in the 13th century the alchemists succeeded in removing the water from the " spirits of wine," thus obtaining absolute alcohol. It was not, however, until about 1600 that Van Helmont showed that the gas given off in fermen- tation was the same gas as Avas obtained by the combustion of charcoal, or Avhat Ave noAv term carbonic acid. Then, it Avas ascertained that the production of the alcohol and carbonic acid AA^as at the expense of the sugar in the fluid ; and Lavoisier attempted to strike a balance betAveen the amount of sugar at the beginning of the process and the amount of alcohol and carbonic acid at its close. As AA'as to be expected, LaA^oisier always found that he could not account for the Avhole of the sugar, because he did not knoAV all the substances formed in the alcoholic fermentation, although he erroneously suj^posed acetic acid to be one of them. Gay-Lussac repeated these quantitative experiments and came nearer to the truth, but still there AA'as a discrepancy. In 1847, Schmidt, of Dorpat, shoAved that succinic acid and glycerine Avere formed ; and, at last, Pasteur established that, from 100 parts of cane sugar, equal to 105-4 of grape sugar, there Avere formed 51*1 of alcohol, 49-4 of car- bonic acid, "7 of succinic acid, 3*2 of glycerine — in all, 104'4 — and he supposed that about 1 per cent. AA^as taken up by the yeast itself. Even in this case the balance is not correct; but, making alloAvance for errors of experiment, it is sufficiently near to shoAv that in the process of fer- mentation the sugar is resoH'ed into a number of chemical substances. Such, then, is Avhat Ave may term the chemical side of the problem. Even the early observers could not fail in observing that, if pure FERMENT A TION. \ 77 grape jiiice is boiled and kept in a well-corked bottle to exclude the air, fermentation does not take place. Pure solutions of cane sugar or of grape sugar do not ferment under these circumstances, whilst impure, unfiltered, unboiled solutions readily undergo fermentation. This disposes of the conjecture one might make, that the atoms forming the sugar are simply rearranged so as to produce alcohol, carbonic acid, glycerine, succinic acid, and other bodies, just as the atoms of cyanate of ammonium, NH^CNO, may be rearranged so as to form urea, CH^NoO. Something evidently must be added to the grape juice to cause fermentation. As well put by Professor W. Dittmar,i an analysis of yeast does not reveal the existence of any substance capable of effect- ing this remarkable transformation. Take the most recent analysis of yeast by Schiitzenberger. An extract of yeast in boiling water showed — (1) a considerable quantity of phosphates ; (2) a large quantity of gum (arabin) convertible by nitric acid into mucic acid ; (3) leucin and tyrosin, to the former of which a sulphiuretted compound obstinately adhered ; (4) carnin, xanthin, guanin, hypoxanthin, and sarcin. It is remarkable how similar this analysis is to that of an animal substance, l)ut none of these chemical compounds can be supposed to effect the transformation of sugar into those formed in the alcoholic fermentation. By slow stages, however, the part played by the yeast came to be recognized. So long ago as 1680, Leeuwenhoek, with his simple microscope, saw that in fermenting fluids there were minute globular or ovoid j^articles. This was the first obserA^ation of the yeast-cells, and for many years they received little or no attention. In 1838, however, Schwann and Cagniarcl de la Tour demonstrated the vegetable nature of these yeast cells, and showed that they grew and multiplied in saccharine solutions, and for the first time it was asserted that fer- mentation in some way depended on the action of living things. Previous to 1838, Berzelius put forward the theory that the action of the yeast is what is called catalytic (Kara, Averts, separation), or a mere action of presence, similar to that of platinum-black on peroxide of hydrogen, causing the latter to give up an atom of oxygen. This theory Aras no explanation. Another view, first proposed by Liebig in 1848, is that there is no necessary connection between the fermentive process and the development of living organisms, and that the organisms may simply produce a substance, the molecular vibration of which may cause a rearrangement of the atoms of the substance undergoing fermentation. This is a modification of the mechanical theory of Berzelius above described. The spHtting up of sugar into carbonic acid and alcohol, by the action of the yeast plant, he places side by side with the decompos- ^W. Dittmar, article "Fermentation" in the Encydoi). Br'dami. 9tli edit. I. BI ]78 THE CHEMISTRY OF THE BODY. itiou of anhydrous acetic acid into acetone and carbon dioxide (a change brought about by heat), and with the change of an aqueous solution of cyanogen gas into oxaniide, which is brought about by the action of the merest trace of aldehyde. Just as the vibration produced by the alde- hyde determines the rearrangement of the atoms of cyanogen and water so as to constitute oxamide, so Liebig regards the rearrangement of the atoms of sugar as the result of a vibration produced by the chemical changes which take place in some unstable substance produced by the yeast-plant. The growth of the yeast-plant is, according to this idea, indirectly connected with the process of fermentation. " It is possible," he says, "that the physiological process stands in no other relation to the process of fermentation than that, by means of it, a substance is formed in the living cell, Avhich, by an action peculiar to it — resembling that of emulsin on salicin or amygdalin — determines the decomposition of sugar and other organic molecules. In such a case, the physiological action would be necessary for the production of this substance, but would be other'snse unconnected with the fermentation properly so called." ^ For many years this theory held its ground owing to the prestige of its illustrious author, and fermentation was held to be largely a chemical process. But facts came to light that again concentrated the attention of men of science on its biological aspect. It had been shown by Gay-Lussac that clean grapes or boiled grajje juice introduced into the Torricellian vacuum of a barometer tube kept free from fermentation for any length of time, but that if a single bubble of air Avere admitted fermentation soon appeared. Years afterwards, in 1838, in his celebrated experimental inquiry, SchAvann repeated Gay- Lussac's experiment, and shoAved that if the air bubble Avere admitted to the vacuum through a red-hot tube then fermentation did not occur. Clearly then it AA^as something in the air that caused fermentation, and this something Avas destroyed by heat. Further, it was noticed from time to time by many observers that active fermentation Avas ahvays accompanied by an apparent groAA'th of the yeast, and also that the pro- cess could be influenced by various physical and chemical conditions. Thus a temperature of from 20" to 24" C. Avas most favourable to it ; it Avas arrested at 60° C, and boiling destroyed the poAver of fermentation. On the other hand, it Avas arrested by freezing, but on careful thaAving, the process Avas resumed. Chemical substances, also, such as alcohol, bichloride of mercury, sulphuric acid, and sulphurous acid, impeded or arrested it. The next great step was made Avhen SchAvann endeavoured to identify ^ Article " Fermentation," in Supplement to Watt's Dictionary of Chemistry, Tol. vi. p. 612. FERMENT A TIO N. 179 the processes of fermentation and putrefaction. In the 17th century, Stahl, with that curious insight that gives his -writings almost a pro- phetic character, said that fermentation and putrefaction were essentially the same, and he explained both by disturbances in the molecules of the fermentive or putrefactive body. Schwann, however, full of views about the origin of the tissues from cells, and of speculations as to spontaneous generation, investigated putrefactive fluids with a knowledge of the existence in these fluids of vibrios and other living organisms. He found that if a putrescible fluid is boiled and excluded from the air no putre- faction occurs, but that if air is subsequently admitted, putrefactive changes soon ensue. Obviously then the air has to do with putrefaction. But if the air were previously passed through red-hot tubes there was then no putrefaction, and this warranted the conclusion that it was not the air itself but something in the air that caused putrefaction. He also made the important observation that the chemical substances affecting fermentation and putrefaction varied in their mode or degree of action. Thus white arsenic and corrosive sublimate, which poison both plants and animals, stop both putrefaction and fermentation, whilst nux vomica, which poisons animals but not plants, prevents putrefaction, whilst it does not arrest the -vdnous fermentation. These statements require modification in the light of more modern inc|uiries, but they merit attention here as being an important contribution to knoAvledge. Schwann's conclusions were supported by many important experi- mental inquiries. Von Helmholtz showed that the oxygen produced by electrolysis in a sealed up fermentable fluid did not cause fermentation. He also performed a remarkable experiment in which he placed a bladder full of boiled grape juice in a vat of fermenting juice and found that the fluid in the bladder did not ferment. Thus the cause of fermentation could not pass through the wall of the bladder. Mitscherlich showed that when in a tube a layer of fermentable fluid was separated from a layer of yeast by a septum of filter paper no fermentation occurred. Hoffmann proved that a layer of cotton wool had the same effect, and Schroeder and Dusch made the important demonstration that, if a putrescible fluid be boiled in a flask and the neck of the flask be plugged with cotton wool at the moment the flame is removed from underneath the flask, putrefaction will not occur in the fluid. It might be objected that putrescible fluid will keep fresh in a hermetically sealed flask because no air has been admitted, but this objection will not hold good when applied to the experiment -with the plug of cotton wool. Here evidently air, but filtered air, has been admitted, and still putrefaction does not occur. The cotton wool has sifted from the air the bodies that cause putrefaction, just as the wall of the bladder, the septum of filter paper, 180 THE CHEMISTRY OF THE BODY. or the layer of cotton wool, prevents the }):issage of the yeast cells from a fermenting to a fermentable fluid. Even if the neck of the flask be long drawn out and lient here and there in a zig-zag fashion Avithout sealing up the tube, a j)utrescil')le fluid will remain free from putrefaction in the flask. In this case the organisms that cause putrefaction are caught at the bends of the neck of the flask. The conclusion then is irresistible that the living organisms in the air and in the yeast are the cause of putrefaction and of fermentation. Such was the state of the question when, in 1857, Pasteur began those important researches Avhich, continued in various forms up to the present time (1888), have revolutionized this department of science. Pasteur, in particular, shoAved that each kind of fermentation was connected with the growth and development of a special organism, and that as the yeast cell was specially the cause of the alcoholic fermentation, so there Avas another organism for the lactic fermentation by Avhich the sugar of milk was changed into lactic acid, and a third which efifected the transformation of lactic into butyric acid. Further, he introduced the method of culti- A^ating certain organisms in fluids specially adapted to their wants and inimical or unsuitable toother organisms, and thus by repeated cultivations he demonstrated that it A\^as possible to obtain a fluid containing one organism, the physiological properties of Avhich might then be examined. Thus by soAving a mixture of organisms, a, h, c, and d, in a sterilized fluid suitable specially for the groAvth of a, and hj repeating this process many times AAdth the fluid of successiA^e cultivations, a fluid is obtained in Avhich only the organism a is found, the others having perished in the struggle for existence in exceptional conditions. This method has been of the greatest importance to science, and like maiij^ neAv methods has been the key to many problems. It AA^as in 1864 that Pasteur made the important observation that the ferment causing the butyric fermentation is capable of liAang and multi- plying in a fluid containing certain mineral matters, along Avith sugar or lactate of lime, and that it does this in the absence of free oxygen or of free air. In this case the presence of oxygen or of air arrests the fermentation, for if the air be again excluded, the fermentation Avill be resumed. This led to the investigation of the behaviour of various fer- mentive organisms, Avith the result of shoAving that the same rule pre- vailed AAdth many (as in the but3'ric fermentation), namely, that the fermen- tation is carried on most efficiently in the absence of free air or free oxygen. Further, it Avas ascertained that most fermentiA^e organisms may assume tAA^o conditions, one aerobic, that is, liAdng in the presence of oxygen or of air, and the other anaerobic, that is, liAang in the absence of the oxygen ; and it is Avhen it is in the latter condition, the anaerobic. FERMENT A TION. 1 81 that ail organism carries on the work of fermentation. If the organism receives a free supply of oxygen from the air, it consumes it, assimilates nutritious matters, and takes on a certain habit of growth, like the coloured moulds found on the surface of preserved fruits or on milk exposed to the air. On the other hand, if there is a limited supply of oxygen, or none at all, the organism removes oxygen from the fermentable matter and thus causes fermentation. The curious fact that these lowly organisms apparently may live without free oxygen is no exception to the general law that all living things require this gas, but they have a special method under certain circumstances of obtaining it, namely, by the decomposition of the fermentable matter. Thus Pasteur is justified in the aphorism, "La fermentation est la vie sans air" and this remark- able property points to the important role of fermentations in many of the phenomena of the living body. It is c|uite true that the actual removal of oxygen from sugar by the action of the yeast has not been demonstrated, and that the explanation is thus far theoretical ; but without this theory, it is difficult to account for the fact that some organisms do live and multiply without the access of either oxygen or air. A few flourish best with less oxygen than in ordinary atmospheric air. Thus Engelmann showed that some species gather close to a bubble of air, whilst others keep away from it and only come near when the bubble has lost a part of its oxygen. On the other hand, some, such as Bacterium aceti, require oxygen, and are truly aerobiotic. It would appear that at the beginning of fermentation oxygen is required, but that after it has begun, free oxygen may or may not be requisite according to the nature of the organism carrying it on. Thus, after many vicissitudes, the vitalistic theory of fermentation has been put on a secure footing. True ferments in the shape of living organisms have been found for the lactic fermentation, resulting in the jiroduction of butyric acid ; for the viscous, by which sugar may be changed into mannite ; for the acetous, by which sugar may pass through alcohol into acetic acid ; for the conversion of urea into carbo- nate of ammonia in decomposing urine ; and for the phenomena of putrefaction, in which, by the action of iaderiumr-foTms, albuminous and other matters are changed into simpler bodies. It is to be noted, how- ever, that while each fermentation is accompanied by the development of a special organism, the same substance, such as alcohol, may be one of the products of different fermentations. It has been urged by some that the multiplication of organisms in a fermenting fluid or in a putrefactive fluid is the consequence and not the cause of the fermentation or putrefaction. In the case of the alcoholic fermentation, this is disproved by the experiments of Von 182 THE CHEMISTRY OF THE BODY. Helmholtz, INIitsflierlicb, and others, -\vhicla showed that if the yeast cells were prevented from passing into a fermcntahle fluid by the interposition of an organic membrane, fermentation did not ensue. Further, as shoArn by Chauveau, the properties of vaccine lymph are due to the small oi'ganic particles in the fluid, and not to anything in the fluid itself; and it has been found possible to sterilize various fluids, that is, remove from them organisms cajjable of causing fermentation, by passing the fluids through thick septa of finely porous substances. A striking corroboration of the view that the organisms themselves are absohitely necessary we have in the fact that if the fluid containing a cultivation of a bacillus believed to be the cause of splenic fever be inoculated into an animal, it Avill die of the disease, but this result does not follow if the inoculated fluid has been pre^^ously elaborately filtered so as to remove the bacillus. If then the organisms are the cause of the fermentations, how do they act 1 Do they live on the fermentable matter and oxygen, and are the substances Ave call the products of the fermentation to be regarded in the light of excreted or Avaste matters 1 Or, do the organisms, as held by Berthelot, Fremy, Hoppe-Seyler, and others, produce a kind of ferment Avhich in turn acts on the fermentable matter? There are reasons for holding that the latter AdeAv is not far from the truth. It is quite true that no substance has yet been separated from yeast capable of causing the alcoholic fermentation other than the j^east cell ; but Berthelot has obtained from yeast a sohxble ferment capable of changing starch and cane sugar into glucose. Further, as Avill presently be seen, there are many soluble ferments kuoAvn to chemists, but these are all formed in the first instance in animal or vegetable cells that may be considered to be the analogues of the organized cells capable of causing fermentation. Hoppe-Seyler has AAdth great force pointed out that the action of the fermenting matter may not be a direct oxidation, but rather a reduction. Thus, in the lactic and alcoholic fermentations and in putre- faction there is the liberation of hydrogen, and this nascent hydrogen may seize hold of an atom of oxygen from ordinary oxygen, Og, to form water, Ho + Og = H^O + 0. The nascent oxygen thus liberated at once attacks any oxidizable matters present, or it may unite Avith more free hydrogen to form AA^ater, or it may unite Avith a molecule of ordinary oxygen, Og, to form a molecule of ozone, — - ^ . Thus the liberation of hydrogen may be the cause of active oxidations. On the other hand, suppose no free oxygen is present, then the hydrogen liberated attacks organic substances, reducing them. In this case the phenomena are not FERMENTATION, 188 those of oxidation, but of reduction, and we see how in fermentations and putrefactions there may be either oxidations or reductions according to the presence or absence of oxygen. Thus in putrefying fluids oxida- tions may be going on in the upper layers where there is abundance of oxygen, and reductions in the lower layers where the oxygen is deficient or absent. This explanation throAvs some light on the phenomena of so-called oxidations occurring in the tissues. It may be supposed that some of the molecular changes concerned in the nutrition of living tissues are similar to those of fermentations in the absence of an abundant supply of oxygen. In these circumstances, the hydrogen set free in turn liber- ates nascent oxygen, which then attacks oxidizable matter. Thus, according to Hopj^e-Seyler, supposing the oxidizable matter to be repre- sented by n, the changes would be represented by the equation — HH + O2 + % = H2O -(- On. It will be observed also that this is equivalent to a dehydration. Ferments may be classified under three divisions — (1) the soluble ferments ; (2) the organized ferments ; and (3) according to the nature of the chemical changes they are capable of exciting. (1) The Soluble Ferments. These are substances produced in the interior of animal or vegetable cells, and when obtained as free from extraneous organic matter as possible, they are solid, amorphous, colourless, tasteless substances, soluble in Avater, and precipitated from their aqueous solutions by alcohol, and the acetate of lead. Chemically, they resemble the deriva- tives of albuminates, but they contain no sulphur. Their chemical constitution is unknown. The following are a few examples of fermen- tive processes — 1. The conversion of starch into dextrin and glucose, produced by the action of the diastase of malt, of ptyalin in saliva, of a ferment in the pancreatic juice, and of all albuminous matters in a state of decom- position — 4(06HioO,) + 3H,0 = CgHioOg + ZC,B.^^O,. Starch. Dextrin. Dextrose. or, '(a) 3(C6HioOg) + H2O = CiaH^oOn + CgHioOg. Starch. Maltose. Dextrin. (h) 2(C6Hio05) + H.3O = Ci^K^On. Dextrin. Inverted siigar. 2. The transformation of cane sugar into inverted suoar, which is a 184 THE CHEMISTRY OF THE BODY. mixture of dextrose and Isevulose, and into glucose, accomplished by a ferment in the intestinal juice, and by a ferment in yeast — Ci2H.«0ii + H.O = CoHiaOfi + CgHiaOfi. Cane sugar. Dextrose. Lajvulose. 3. The conversion of glucosides, such as amygdalin, into glucose and various compounds, accomplished by synaptase or emulsin — CooH,7NO,i + 2H2O ^ 2C6H,.A + QHgO.NHC. Amygdalin. Glucose. Oil of bitter almonds. CisHigO, + H.O = CoHi.Og + CVHgOo. Salicin. Glucose. Saligenin. 4-. The conversion of glucose into glycerine and mannite — CgHj.iOg + H^ = CeH,40e. Glucose. Mannite. 5. The conversion of glycerine and of mannite into alcohol T)y the action of nitrogenous organic matter in a state of decom})Osition. (Berthelot.) 6. The splitting up of fats into fatty acids and glycerine by the action of a ferment in the pancreatic juice — C.VH104O6 + m.p = 3(Ci8H,,0,) + C3H8O3. Olein. Oleic acid. Glycei-ine. 7. The transformation of albuminates into peptones by the action of pepsin, the ferment of the gastric juice, and by a ferment in the pan- creatic and intestinal juices. The following are examples of the chemical changes effected in these processes of fermentation — 1. Simple isomeric transformations, as in the conversion of starch into dextrin and sugar. 2. Hydrations, as in the conversion of cane sugar into glucose. 3. Synthetic processes, as in the fermentation of glucosides. 4. The separation of water, and a change in the molecular condition of the remainder, as in the conversion of albuminates into peptones {hydrohjt ic ferments). It is to be noted that, in many instances, chemical changes, similai- to those produced by soluble ferments, may be effected by the action of heat and of the mineral acids. Thus, sulphuric and hydrochloric acids effect a hydration and change cane sugar into dextrose and Itevulose, milk sugar into galactose, and starch into dextrin and dextrose accord- ing to the above equations. Many of these ferments may be arranged under the following- physiological classification, which is also practical — I. Proteolytic- — those changing albuminous matter into peptones (acid FERMENTATION. 185 digestion by pepsin of gastric juice) or into tryptones and then into leucin, tyrosin, etc. (alkaline digestion l:'y tripsin of pancreatic juice). II. Aniylolytic — those changing starch into glucose with absorption of water, such as the ptyalin of saliva, a ferment in the pancreatic juice, and a ferment in the liver and in many tissues. III. Steatolytic — those decomposing fats with water, as a ferment in the pancreatic juice. IV. Inversive — converting cane sugar into inverted sugar. Such a ferment exists in the intestinal juice, possibly to a small extent in saliva and in hepatic cells. V. Blood ferment — causing union of the elements that form the fibrin of blood clot. Ferments of this class are the principal agents in the chemical transformation of food in the process of digestion, and in all probability processes of a fermentive character also occur in certain organs, as in the liver. (2) The Organized Ferments. These are living organisms, the type of which is the unicellular plant found in yeast, known as Saccharomycetes cerevisice, and including such organisms as many forms of fungi, vibrios, and bacteria. 1. Soluble ferments usually produce, as the result of their action, not more than one or two substances, but the organized ferments, as a rule, produce several substances. Thus glucose, in the presence of yeast not only may yield carbonic acid and alcohol but also glycerine,' succinic acid, acetic acid, fatty matter, a nitrogenous matter, and other products. 2. Organized ferments, as already explained, do not absolutely re- quire the presence of the oxygen of the air, as they have the power of obtaining oxygen from fermentescible matter itself In certain cases it would even appear that excess of oxygen arrests fermentation. Pasteur has shown that vibrios are killed by a strong current of oxygen passed through the fluid containing them, and Paul Bert has found fermentation to go on much more slowly under a pressure of five atmospheres of pure ■oxygen. 3. As examples of the fermentations excited by such organisms, we may consider the following — a. Alcoholic — - CgHi-A + H2O = 2C0o + 2(C2H60) + H2O. Grape sugar. Alcohol. b. Lactic acid — C12H2A1 + H,0 = 4(C3H603). Milk sugar. Lactic acid. 186 THE CHEMISTRY OF THE BODY. c. Butyric acid — 2(C3H603) - C^HgO, + 2C0, + 2H,. Lactic ficid. Butyric acid. d. Acetous — CgHeO + = C2H4O + HoO; then C0H4O + = aH402. -^cohol. Aldehyde. Aldehyde. Acetic acid. e. Putrefaction — This cannot be represented by an equation but albuminous and fatty matters are changed into leucin, tyrosin, fatt}^ acids, glycerine, ammonia, sulphuretted hydrogen, Avater, hydrogen, and possibly nitrogen. (3) Classification of Ferments according to tue Nature of the Chemical Changes they Produce. Hoppe-Seyler^ has given an important classification of ferments which is in the main chemical in its character, but it merits special attention because it illustrates the probable action of many fermentive substances whether soluble ferments or organized ferments. I. The conversion of anhydrides into hydrates — A. Ferments acting like weak mineral acids at the temperature of boiling water (100° C). 1. Conversion of starch or glycogen into dextrin and glucose — 4(CeHio05) + SHoO = CeHjoOg + SlCgHi-A)- starch. Dextrin. Glucose. 2. Conversion of cane sugar into glucose and Isevulose (fruit sugar) — CjoH2,0ii + HoO = CgHiA + CfiHiaOfi. Cane sugar. Glucose. Lasvulosc. 3. Conversion of a benzol-giucoside into sugar and a benzol deriva- tive by the action of emulsin — CisHjgO, + H,0 = CeHiA + C«H,{g|2'^^ Salicin. Sugar. Saligenin. Ci3H,eO, + H,0 = CfiHi^Oe + CeH.jgg^ Hulicin. Sugar. Palicylaldehyde. C,3Hi,03 + H,0 = CgHi^O^ + QH,{gg- 0^ Glucoside of sail- Sugar. Salicylic acid. cylic acid. C25H34O14 + 2(3^0) = '2[C,n,^,) + CeH,{g| + CeH.jggHa ArbutiD.2 Sugar. Hydrochinon. llethylhydrochinoii. C20H07NO11 + 2(H„0) = 2(C6HiA) + CgHg.COH.CHN Amygdalin. Sugar. Oil of bitter almonds. ^ Physiologisclie Chemie, 1877, p. 116 et seq. ^ Arbutin, a crystalline suljstance in leaves of red bearberry (Arctostaphylos uva ursi). FERMENTATION. 187 CisHjoOg + H,0 = CeHioOg + CioHuOs . OH Coniferin.i- Sugar. Conifer-alcohol. C14H18O9 + HoO = CfiHisOe + CgHgO^ Gluooside of vanillic Sugar. Vanillic acid, acid. 2 CgiHg^Oig + 2(H.,0) = 2(C6Hi208) + CigHi.Og Dapliinin.3 Sugar. C3iH5oOi,*+ 5HoO = SlCfiHiA) + Ci3H.3,03 Cg^HggOifi + 5H.0 = 3(C6Hi.,06) + C16H30O3 Jalapin.i Sugar. ■4. Decomposition of a sulphur compound into sugar, sulphuric acid and oil of mustard through myrosin — CioHigNSoOioK = CeHi.,06 + KHSO4 + C4H5NS Myronate of potassium. 5 Sugar. Sulphate of Oil of mustard. potassium. CgoH^NoSoOn = CeHiaOy + CieHo.N . SO^H + CgH.ONS White mustard. Sugar. B. Ferments acting like caustic alkalies at a higher temperature. 1. Decomposition of ethers and of the fats into an alcohol and fatty • acid. 2. Decomj)osition of amides with absorption of water — CH^N.O + HoO = (NHJ.COs Urea. Carbonate of ammonia. C9H9NO3 + H.,0 = C.,HgN02 + C^HgO., Hippuric acid. Glycocoll. Benzoic acid. CagH^gNSO, + HoO = C0H7NSO3 + CojH^oOg Tauro-cholic acid. Taurin. Cholalic acid. II. Ferments causing the transference of the oxygen from the hydro- gen atom to the carbon atom. 1. The lactic fermentation. Here one molecule of milk sugar, with the addition of a molecule of water, is changed into two mole- cules of sugar (dextrose and Isevulose), which in turn become four molecules of lactic acid. ■ 2. The alcoholic fermentation. The transference of from H to C is thus shoAvn — CH., .OH CO . (0H)o CH . OH CHoOH ) , , , , CH . OH + 2HoO = CH3 /-'^^co^o^- CH . OH ' CH3 \ A icohol CH . OH CHoOH S Alcohol. COH CO(OH)o Glucose. ' Coniferin, a glucoside in the cambium of coniferous woods, Ahks exceUa, Larix Europoea, etc. 2 Vanillin, neutral odoriferous principle of vanilla, fruit of Vanilla planifolia. 3 Dapliinin, a crystallizable glucoside in Daphne alpina and other species of Daphne. * Convolvulin, a resin from Convolvulus schiedeanus. * Jalapin, a resin obtained from scammony resin, yielded by Ipomcea orizabensis. s A salt in the seed of black mustard. 188 THE CHEMISTRY OF THE BODY. 3. Putrefaction. The following are examples — (CHO.,)oCa + Formate of lime. H.,0 n: CaCOg + CO., + -211, Carbonate of lime. (C-HsOaJoCa + Acetate of lime. H.O = CaCOg + CO.^ + 2CH4 Carbonate Jlarsh gas. of lime. "{CgHioOg) + Cellulose. nCK-fi) = 3»(C0,) + 3«(CHJ Marsh gas. Lactate of lime. CaCO,, Carbonate of lime. + 3C0., + 4H, + Ca(C4HA)2 Butyrate of lime (4) The Nature of the Organized ^'ebments. These organisms belong to the group of bodies to which the name of Schizomycetes was given by Naegeli in 1857, and they include such as cl Fig. 66.— Typical forms of Schizomycetes (after Zopf). a, micrococcus; 6, macrococcus, or " monas" ; c, bac- terium ; d, bacillus ; e, chlostridium ; /, monas Okenii ; (?, leptothrix ; h, i, vibrio ; le, spirillum ; I, spirulina (a form of Beggiatoa alba); m, spiromonas ; «, spirochaBte ; o, cladothrix. The gi-anules in h, f, and e are particles of sulphur. are often termed bacteria, bacilli, micrococci, microphytes, microbes, etc. They are minute unicellular bodies of the nature of saprophytic FERMENT A TION. 189 or parasitic schizophyta, devoid of chlorophyll, and multiplying usually by fission, and in some by a process of spore-formation. They vary in size from -001 to '005 mm. in length. The general appearance of the typical forms of these organisms is thus given by Professor Marshall AVardi (Fig. 66). The form may be one of the foUowdng — (1) Globular or spheroidal, usually termed micrococcus, or, if larger, macrococcus or monas. (2) Ilocl-like, cylindrical cells, three or four times as long as broad, hacterkmi, when the rods are short, or with rounded ends, or bacillus, when the rods are longer and usually sharply cut off at the end. (3) Filaments, consisting of single elongated filaments, or of several much elongated cells that have remained attached after di\dsion ; such are seen in leptothrix. (4) Spiral forms— 'li the sinuosity be slight, the body is a vibrio, and if more cork-screw like, a spirillum. (5) Plates or tablets, irregular branching groups of cells, which, by successive divisions both longitudinal and transverse, give rise to little groups of square-shaped cells lying in groups of four, six, or eight, as in sarcina. Some schizomycetes shoAv pleomorphism, that is to say the same organ- ism at one stage of its life, may appear in the form of a micrococcus, at a later, of a bacterium, and afterwards, as a filament or leptothrix. An example of such an organism is Cladothrix dichotoina,^ and the special forms have been regarded erroneously as different species of schizomy- cetes. On the other hand, many of these organisms maintain the same form, generation after generation. Schizomycetes are found practically almost everywhere, and if any putrescible fluid is exposed to the air for some hours, at ordinary tem- peratures, it soon swarms with micrococci and bacteria. They also abound in many animal fluids in the living body under certain conditions of disease, and some representatives may be found in small numbers in the tissues even in a state of health. In the alimentary canal, except where the secretions are highly acid, they are also plentiful. Each schizomycete consists primarily of a minute cell, never nucleated, formed of a mass of " homogeneous or slightly granular protoplasm with a pearl-like lustre, and without vacuoles ; this is enveloped by a membranous envelope Avhich is so delicate as to be scarcely per- 1 Marshall Ward, article "Schizomycetes," in Ennjdop. Britannka, vol. xxi. p. 399. - Figured in Marshall Ward's article. (Fig. 16, op. cit. ) 190 THE CHEMISTRY OF THE BODY. ceptible."^ This envelope in some cases consists of cellulose. Coloured pigments (red, yellow, green, or blue) have been fo\ind in some organisms. The protoplasmic matter consists of a colourless substance named mi/co-protein. Starch has been found in some bacteria, giving a blue colour with solutions of iodine. Minute crystals of sulphur have been found in filamentous schizomycetes (see Fig. QQ, f), and oily and fatty matters have been detected. Sometimes, after these bodies have multiplied by transverse fission until a long chain-like structure has been formed, the chain may break into fragments and the particles thus set free — bacteria, bacilli, or micrococci — form dense swarms which " become fixed in a matrix of their own swollen contiguous cell walls, and pass into a resting state as a so-called zoogloea." . They become entangled in this jelly-like matrix, and the zoogioeain itself may take on forms, spherical, ovoid, or filamentous, peculiar to the species. By-and- bye, the organisms in the zoogioea may become active, and, scattering in the surrounding medium, begin to grow and multiply. The multiplication of schizomycetes by spores is now a well-ascer- tained fact, and the various tyjjes of this mode of development are shown in Fia:. 67. K. \ ^ Oc '00°o I A A ^ a c 6 o a o ^ L i .^ H Fig. 67. — Types of spore-formation in schizo- mycetes. (After Zopf.) A, various stages in the development of the endogenous spores in a Clostridium {haciUus), the small letters indi- cating the order. B, endogenous spores of the hay bacillus. C, a chain of cocci of Icu- <;onostoc)(i,esenfc/-ioic7t's, with two resting spores, i.e. arthrospores. (After Van Tieghem.) D, a mobile rodlet with one ciliuni, and with a spore forming inside. E, spore-formation in (^t&ro-like(e),and SpiriUum-W^e (o, h, d) schizo- mycetes. F, long rod-like form containing a spore (these are the so-called " Kopfchcn- bacterien " of German authors). Gr, Yibrio- form with spore. (After Prazmowski.) H, Clostridium — one cell contains two spores. (Prazmowski.) I, Spirillum containing many spores — a, which are liberated at b by the breaking up of the parent cells. K, (Termina- tion of the spore of the hay bacillus (Bacillus subtilis) — the axis of growth of the germinal rodlet is at right angles to the long axis of the spore. L, germination of the spore of Clostri- dium butyricum — the axis of gi'owth coincides with the long axis of the spore. It is now well known that schizomycetes are found in the blood, tissues, and organs of animals and of man suffei-ing from certain specific diseases. One of the most striking instances of this occurs in ^ Marshall Ward, op. cil. FERMENT A TION. 191 splenic fever, where the blood abounds in Bacillus anfhracis, shown in Fia 68. Fig. 68. — Bacillus aDthriicis. (After Koch.) A, Bacilli mingled with ■blood corp\iscles from the blood of a guinea-pig, some of the bacilli dividing. B, the roiilets after three hours' culture in a drop of aqueous humour. They grow out into long leptotlmx-li^e filaments, which be- come septate later, and spores are developed in the segments, (x 650.) Much discussion has taken place as to whether the i^resence of such an organism is a casual, or even a concomitant phenomenon, or whether it is the real cause of the disease. It is beyond the province of this work to discuss a question that is chiefly pathological in its natui^e, and it is sufficient here to say that, from the purely physiological stand- point, the presence of such an organism in vast numbers cannot fail in being inimical to the well-being of its host. Even considering the affinity such organisms have for oxygen, it is clear that a struggle will ensue between them and the living tissues for this gas ; and, if the organisms secure the predominance, the living tissues must suffer. In addition, it is not improbable (rather highly probable) that the organisms may secrete alkaloidal substances of a poisonous nature which, in their turn, may exert powerful physiological effects on the tissues and organs. The growth and development of schizomycetes are influenced by various physical and chemical agents. (1) Temperature. — A temperature of about 35° C. is the most favour- able for the development of the majority of these organisms. Cohn ^ states that he has subjected bacteria to low temperatures without destroying their activity. He gives the temperatures as follows — 1 Cohn, Beitrclge zur Biologic der Pflanzen, 1870 ; Zweites Heft, s. 221. 192 THE CHEMISTRY OF THE BODY. Exposure for 12 hours 30 minutes to a temi)craturc of 0° C. ; for 1 h. 30 m., to - 16° C. ; for 1 h. 45 m., to - 17° C. ; for 3 h. 30 m., to - 18° C. ; for 4 h. 30 m., to - 18° C. ; for 5 h., to - 17°-5 C. ; for G h., to - 14° C. ; and for 7 h. 30 m., to - 9° C. He produced the cold by freezing mix- tures, and the lowest temperature he obtained was - 18° C. = 0° F. In 1870-71, Melsens^ exposed yeast and vaccine lymph to very low temperatiu-es ( - 78° C.) obtained by means of solid carbonic acid, without destroying the power of fermentation or of inociilation. Klein ^ states that "freezing destroys likewise most bacteria, excei)t the spores of bacilli, which siu-vive exposure to as low a temperature as - 15° C, even when exposed for an hour or more." Again, in another place, he says : "Exposing the spores of anthrax bacillus to a temperatm-e of 0" to - 15° C. for one hour did not kill them." In 1884, a r-emarkable series of experiments was described to the French Academy by MM. R. Pictet and E. Yung.-' These observers sealed up in small glass tubes fluids containing various kinds of micro- phytes, and placed them in a wooden box. The box was in the first place submitted for 20 hours to a cold of — 70° C, produced by the evaporation of liquid sulphurous acid in vacuo. The box was then sur- rounded by solid carbonic acid for 89 hours, and a cold of from - 70° to - 76° C Avas thus obtained. Finally the box Avas subjected for a third period of 20 hours to a cold produced by the evaporation of solid carbonic acid in vacuo — the temperature being estimated at from —76° to - 130° C, that is, a minimum temperature of 202° below zero F. They sum up by stating that the organisms were acted on by a cold of - 70° C. for 109 hours, followed by a temperature of - 130° C. for 20 hours. The organisms tested were Bacillus anthrads. Bacillus subtilis, Bacillus ulna, Micrococcus luteus, and a micrococcus not determined. Bacillus aiithracis retained its virulence when injected into a living- animal. The vitality of the others was not affected. Experiment showed that whilst cold seemed to kill some of the micrococci, a great number resisted it. Yeast showed no alteration under the microscope, but it had lost its powers of fermentation. Vaccine lymph exj^osed to the low temperatures did not produce a pustule on the left arm of an infant, whilst another sample of the same lymph introduced into the right arm of the same child produced a pustule. Pictet and Yung con- clude from their experiments that, in the conditions of cold indicated, many of the lower organisms were not destroyed. ^ Melsens, Compte>i Rendu^, tome Ixx. 1870, p. G29 ; also Comptes Rendus, tome xxi. p. 3"2o. - Klein, Micro-organisms, p. 35. " Pictet and Yung, Comptvs Rendits, tome xcviii. No. 12, p. 747. FERMENTATION. I93 In 1885, J. J. Coleman and the author' made numerous experiments in which very low temperatures were reached and maintained for many hours by the use of a special modification of the cold-air engines invented by Mr. Coleman, now largely used in the industry of importing meat into Great Britain. Although putrescible infusions and fresh meat con- tained in hermetically sealed bottles were exposed to a temperature of —83° C. for 100 consecutive hours, putrefaction soon began when the bottles were removed to a warm room. It was found impossible to pro- duce sterilization, that is, to kill the schizomycetes or their spores, by cold. On the other hand, high temperatures are more fatal. The spores of bacilli, hoAvever, may germinate after the fluid has been boiled (100° C.) for an hour, and it is said that they have mthstood a temperature of even 110° C. Dry spores resist a higher temperature than sj)ores in a moist state, and it would appear that ripe spores are more resistant than spores in the germinating state. Spores are killed also at a lower temperature in acid media than in alkaline media. Probably prolonged boiling Avould ultimately kill all spores. Tyndall^ ascertained that discontinuous heating, that is rej^eatedly boiling the solutions contain- ing spores for half an hour daily, sterilized the fluid in two or three days, although boiling the same kind of fluid (100° C.) for half an hour once did not produce this eff'ect. The explanation offered is that spores not killed by the first boiling may germinate before the second boiling, and if caught in the germinating state they are killed, whilst some not germinating may again escape. (2) TFater. — Schizomycetes germinate only in a fluid, but spores may remain alive in a dormant dry state for months or years. Thus Bacillus suhtilis has been kept alive in a dry state for years. More extended observations are required on this point with regard to many species, but it is obvious that they may live in the dry state and be wafted about by air currents. (3) Light. — Brilliant sunshine appears to be inimical to the spores of certain species, and flasks may be partially, if not wholly, steril- ized by being exposed to sunlight. On the other hand, it is not unlikely that light may encourage the development of other species, more especially in fluids and in the. vicinity of chlorophyll-bearing- organisms, as shown by Engelmann's experiment described at p. 24. Aerobiotic forms collect where the oxygen is being evolved from chloro- phyll, in the yellow-red part of the spectrum. ^ Coleman and M'Kendrick, Proceedings of Royal Institution of Great Britain, Lecture on "The Mechanical Production of Cold and Effects of Cold on Micro- phytes," 29th May, 1885. 2 Tyndall, Floating Matter in the Air, 1881. I. N 194 THE CHEMISTRY OF THE BODY. (4) Ozone. — The author has attempted to sterilize organic fluids and solids by means of ozone, but without success, although it was evident that ozonization of the air or of an atmosphere of oxygen delayed con- siderably the advent of putrefaction. (5) Pressure. — Paul Bert ascertained that putrefaction was prevented in flesh, moist bread, fruits, etc., by pure oxygen under a pressure of from 10 to 27 atmospheres, but that air under the same pressure had no effect in delaying putrefaction. (6) Chemical substances. — Schizomycetes resist the action of certain substances that might be supposed capable of destroying them. Thus Bacillus anthracis survived several weeks' immersion in absolute alcohol. On the other hand, they are killed by carbolic acid, salicylic acid, quinine, hydroxylamine, sulphurous acid, corrosive sublimate, chlorine, and the mineral acids. (5) Modes 'of Cultivating Schizomtcetes. Bacteriology, or the department of biology relating to the growth and development of the schizomycetes, has, during the past four or five years, become so important in connection with the germ theory of disease, as to have led to a remarkable develojDment of experimental methods by which these organisms may be studied under scientific conditions. To describe these methods in detail is beyond the scope of this work, and all that is attempted is to give the student a short sketch of the general princii^les on which bacteriologists proceed. Bacteria may be artificially cultivated in or upon media so prepared as to meet the requirements of microscopical investigation. As a rule, they grow well upon any organic substance containing nitrogen and carbon, of a slightly alkaline reaction, and at a suitable temperature. This substance may be either liquid or solid. Pasteur's fluid, e.g. con- sists of a solution of 1 part of tartrate of ammonia, 10 parts of candy sugar, and the ash of 1 part of yeast in 100 parts of water. Cohn's liquid consists of "5 grm. phosphate of potash, -5 grm. sulphate of magnesia, •05 grm. tribasic phosphate of lime, and 1 grm. tartrate of ammonia, in 100 grms. of water. Infusions or decoctions of fruit, vegetables, or flesh are also employed. A very serviceable bouillon (or beef-tea) may be made by boiling 500 grms. of finely minced beef in a litre of water for three quarters of an hour. It is rendered alkaline by adding a few drops of a saturated solution of carbonate of soda, and filtered to obtain a per- fectly clear fluid. This is placed in glasses with stoppers of cotton-wool and sterilized by exjjosure to the temperature of boiling water for about three quarters of an hour on three successive days — "discontinuous sterilization." FERMENT A TION. 195 A great objection to the cultivation of bacteria in liquids is that it is -exceedingly difl&cult to get a "pure cultivation" that is to say, a special variety of micro-organism growing without admixture of other kinds. Accordingly various solid or semi-solid substances are used, upon whose surface a single variety may be isolated and examined. Thus, the cut surface of a boiled potato affords a very suitable nidus for many varieties of bacteria. The potato is sterilized by being washed with a 1 to 1000 solution of corrosive sublimate. It is then boiled for about three quarters of an hour, cut into two |)ieces by a knife which has been :sterilized by being heated in a flame, and placed under a glass cover for protection. Bread rubbed to a fine powder, moistened with distilled water, and sterilized by discontinuous heating is frequently employed for the study of moulds and fungi. An exceedingly useful, firm, clear, transparent jelly is made in the foUo'v^ang way : 500 grms. of finely minced lean beef are soaked for 24 hours in a litre of water and strained through a muslin cloth so as to give a litre of meat-juice. To this are added 10 grms. of peptone powder, 5 grms. of common salt, and 100 ^rms. of the best (French) gelatin. The mixture is well shaken and heated till the gelatin is completely liquefied. It is then made slightly alkaline with a saturated solution of carbonate of soda, filtered while warm into glass vessels or test tubes, and sterilized by discontinuous heating. Sometimes 1 or 2 per cent, of grape sugar is added to the .above preparation. As this jelly melts at a temperature of 25° C, it is unsuitable for the cultivation of such organisms as grow only at temperatures more nearly approaching that of the human body (37°-38° C). To meet this objection, from 10 to 20 grms. of Agar-agar, a gelatinous substance •obtained from various kinds of seaweed, may be substituted for the gelatin, the other constituents remaining as above. The jelly so •obtained is not quite so clear as when gelatin is employed, and the process of filtration is rendered very slow by the tendency of the agar- agar to gelatinize when the temperature falls below the boiling point. For the growth of certain micro-organisms, such as the bacillus of tuber- culosis, blood-serum is employed, made firm by heating to 68° C for .about an hour. If heated to a higher temperature, it becomes grey, opaque, and unsuitable for microscopical research. It is sterilized by being heated to a temperature of from 56° C to 58° C, tAvo hours daily for about eight days, or if the blood of a healthy animal is carefully drawn off into cold sterilized vessels, the serum may be obtained at once free from micro-organisms and requires no further sterilization. Before any of the above-mentioned substances can be used for the cidti- vation of bacteria, all instruments and utensils that may come in contact 196 THE C HEM IS TR Y OF THE BOD T. with them must be completel}- sterilized. All metallic instruments re- quired, such as knives, scissors, forceps, needles, and wires may be sterilized by being heated in the flame of a Bunsen burner for about half a minute. Glass utensils, such as covers, slides, and test-tubes are placed in an oven at a temperature of 160° C, and after the lapse of about half an hoiu" are found to be sterilized. For the sterilization of nutrient media, a long cylindrical vessel "vnth a jacket of felt and a perforated cover is- used in which water is kept boiling and the substances to be sterilized are suspended in the rising steam for about three cpiarters of an hour for three successive days. But, if the substance contains much albumin, as, e.g. blood-serum, it is then warmed for about three or four hours- daily to a temperature of from 56°-58° C. for a week, during which time all germs which have been present at the beginning of the process have developed into mature forms and, as such, been destroyed. Suppose, now, we wish to make a pure cvdtivation of a special micro- organism, we may proceed by various methods. If the potato is to be used, a small ci[uantity of the material supposed to contain the desired variety is taken upon the point of a sterilized knife and rubbed evenly over the cut surface of the potato, care being taken not to touch its- circumference at any part. From the centre of this surface, a minute portion is now taken and rubbed over the surface of a second half; a little of the second is conveyed to a third, and so on, until six or seven halves have been successively inoculated one from the other. It will- be readily understood that only an exceedinglj- minute portion of the original material can be present ujDon the surface of the sixth or seventh potato. All the pieces are placed under a glass cover, and they are kept from drj'ing by placing moist blotting paper beside them. In the course of a few days, a vari-coloiu-ed film will probably be seen covering the surface of the potato first inoculated, composed of colonies of various species of micro-organisms. On the second potato, this layer ■vsoll be slightly broken up as the germs which have given rise to the 3'oung colonies have been more separated from one another than in the first case. On the sixth or seventh there Avill probably be only one or two small colonies which have sprung from single germs, and in these we have a pure cultivation of a special variety ready for examination or further separate cultivation. If the gelatin or agar-agar preparations are to be used, three test-tubes about half full of the jelly are placed in a water bath at a temperature high enough to liquefy the jelly. A small quan- tity of the material to be investigated is introduced into one of the test-tubes and stirred about in it by means of a sterilized platinum wire, the end of which has been bent round so as to form a. FERMEXTATIOX. 197 small loop. The "wire is no'w sterilized in the Bimsen flame, cooled, and by its means a small drop of the liquid in the first tube is trans- ferred to that of the second, and intimately mixed with it. Similarly, from the second a small drop is transferred to the third. As "with the cultivation on the potato, so here there can be very little of the original material in the third test-tube. In order, no"w, to obtain a some"what large surface upon "which the micro-organisms may gro"w separate from one another, the mouths of the test-tubes are sterilized by gentle heating in the flame, and the licjuid gelatin or agar-agar is poured on to cold sterilized glass plates, "where it quickly forms a thin layer of firm jellv. The plates are covered "with a glass shade, the air being kept moist as described above, and the gro"wth of the cultivation observed. The special variety of micro-organism, if present, may be recognized either by the naked eye appearance of the colony, or by microscopical examination of a small portion of it, stained or unstained as may be desired. Let us suppose that the variety "we "wish to examine has been isolated and recognized, "we may -wish to cultivate it further by itself. In this case a minute portion of a colony is Lifted on the point of a sterilized platinum "wire, the cotton-"wool stopper of a gelatin or agar- agar tube is removed, and the "wire is thrust do"wn through the jelly and cj[uickly "withdra^m, lea"\"ing, as it passes out, the micro-organisms adherent to the jelly, or, if the jeUy has become firm in an inclined tube, so that it has a long sloping surface, a line may be dra"wn upon this surface "with the end of the platinum "wire, lea^ang the micro-organisms exposed to the air in the tube. Here the micro-organisms gro"w, and their appearance may be noted from time to time. Gro"wth may go on at the ordinary atmospheric temperature, or it may require the tempera- ture of the human body (ST'-SS" C). In this case, tubes containing agar-agar or blood-serum are used, and are placed in an incubator, one form of "which is represented in Fig. 69. In this the temperature is maintained constantly at the required height, any changes being pre- vented by the use of a thermo-regulator, one form of "which is sho"wn in Fig. 70. It consists of a three-"way tube (/), the lo"wer end of "which passes down through a caoutchouc stopper to near the bottom of a test tube {u) containing mercury. In very sensitive instnunents, a diaphragm through "which the tube passes is placed at the centre of the test-tube, and immediately belo"w this diaphragm is a small quantity of a mixture of alcohol and ether, "which, "when heated, expands much more rapidly than mercury. To the upper end of the tube {t) is fixed an india-rubber tube bringing gas. In this end of the tube, ho"wever, there is a caoutchouc stopper (not seen in the diagram), through -which passes a smaller tube (/), the lo"wer end of "which is cut in a slanting direction 198 thl: chemistry of the body towards the mcrcur3\ Gas can only pass into the three-way tube through the smaller tube (t), and thence it is led by the horizontal arm Fig. to. — Thermo-regulator. u^ test-tube with mercury ; I, three- way tube ; 2, smaller tube through which gas passes into I ; a, small aperture in i ; c, tube to burner. Fig. 69. — Inoubator. (c) and an india-rubber tube to a burner placed below the incu.- bator. There is a minute opening in the tube (^) at («), the object of which will soon become evident. When the instrument is to be used, the test-tube is passed into the incubator through an aperture in its cover, and the gas is lit. As the temperature inside the incubator rises, the mercury expands, and is forced up into the tube (/) until it reaches the tube (i). Continuing to rise, it gradually shuts off the gas, and the flame becomes smaller ; but if the mercruy still rose, the stream of gas would be cut ofi" altogether, and, the flame being extinguished, the mer- cury would fall and the gas escape were it not for the minute aperture (a), which always permits a very small cpantity of gas to find its way to the burner, and prevents the extinguishing of the flame. But the flame is now so small that the temperature of the incubator falls, the column of mercury descends, the tube (i) is opened, the gas streams. FERMENT A TION. 199 into (/), and the flame enlarges. The lower end of the tube (i) is placed at such a height above the surface of the mercury that the flame will be lowered when the temperature reaches any desired point. The flame is guarded by a screen, and when once heated, the incubator remains at the same temperature for any length of time. By means of the incubator we can study the life history of many varieties of micro-organisms which would either perish or whose growth would be much retarded by exposure to lower temperatures. This is the more important because many such micro-organisms can, when introduced into the tissues of living animals, occasion serious disturb- ances in the vital processes of the part inoculated or even of the whole organism, and consequently the study of such forms is of the profoundest interest and importance alike to the physiologist and the physician. Much as has been done in this field of research, very much more still remains to be done. Much has still to be discovered about those varieties which have been isolated and cultivated artificially, and we know that there are still many forms which cannot be so cultivated partly because the different nutritive media in use are not suited to their requirements, and partly because we cannot supply the proper environment without which they must perish. Certain varieties can only, so far as we know, develop and be propagated in the bodies of living animals, some, indeed, seem to be limited to a single species of animal. Having once found a lodgment, however, they give rise to a series of symptoms, so definite in their order, so characteristic in their appearance, and so grave in their result, that we are frequently warranted in inferring their presence from the manifestation of the symptoms. We must not, however, regard any micro-organism as the specific cause of a definite disease unless it satisfies the following conditions-^ Firstly, it must be present in the tissues of every animal suffering from the disease, and not in others. Secondly, we must be able to isolate and cultivate it outside of the organism of the diseased animal, so that it is entirely free from contact with diseased tissue of any kind. Thirdly, after inoculation with the pure cultivation, of the tissues of healthy animals, it must produce in them the same disease. Consult on matters relating to physiological chemistry, in addition to the works referred to in the text, Hoppe-Seyler — Physiologische Chemie, 1877- 1881; Arthce, Gamgee — Physiological Chemistry of the Animal Body; Beaunis — Physiologic Humaine, vol. I.; the articles on the subject in Hermann's Handbvich der Physiologic ; Witthaus — Medical Student's Manual . of Chemistry, 1884. See also Prof. P. W, Latham's Croonian Lecture, 1886. 200 THE CHEMISTRY OF THE BODY. In discussing the pathology of Gout and Diabetes, he has advanced a theory regarding the formation of uric acid, which may be briefly stated as follows — " The glycocin, instead of undergoing in the human subject the normal change into urea, combines in the liver with two molecules of urea, derived from the other amido-acids, leucin, etc., and forms the compound CO ■{ NH— CHo— COOH ; that is a compound containing one more molecule of CONH than hydantoic acid, and allied to biuret and alloplianic ether. This substance dehydrated forms p^iNH— CO CO\NH— CHa, which, like hydantoin, is soluble in the blood, passes on to the kidneys, and is there conjugated with another molecule of urea, and forms ammonium urate — COJNH I + CO- JJil^ = H,0 + CgHaNA-NH^, COINH— CH. '^'■^^2 Urea. Ammonium urate. which is excreted ; but, a portion overflowing from the kidney into the general circulation and meeting with soda in the blood, is converted into sodium urate." ^See Lancet, 1884, vol. L, p. 485; and Lancet, 1885, vol. I., p. 1120.) 201 SECTION III. THE PHYSIOLOGY OF THE TISSUES. Chap. I.— HISTORICAL INTRODUCTION. The morphology and physiology of the tissues are included under the common term Histology. Before entering on the consideration of the individual tissues we shall take a historical survey of the steps by which our present knowledge has been attained. This survey will give an in- telligent comprehension of the more modern views held regarding the structure of living matter and the origin of the various tissues. It is evident that knowledge of the structure of the tissues must depend chiefly on the degree of excellence of the microscope, and on the skilful adaptation of methods of preparing these for examination by that instru- ment. ]S[o doubt simple lenses enabled Eobert Hooke, Malpighi, Grew, and Leeuwenhoek, in the 17th century, to discover some of the cellular elements, and by such simple instruments Fontana, in 1780, saw and described the nucleus of the cell, adipose tissue, striated muscular fibres, and the elements of nervous tissue. Little progress, however, was made until early in the present century, when, by the application of the prin- ciple of achromatism by Fraunhofer, and by its practical development more recently by Lister and others, the compound microscope became a trustworthy instrument. Since then each improvement in the micro- scope, as to quality of lenses, modes of illumination, and mechanical adjustments, has been accompanied by a contemporaneous advance in histology. The application of the instrument has also been facilitated by the gradual development of methods of prejmring the tissues for examination. Fontana, so long ago as before 1781, appears to have been the first to apply reagents to substances under the microscope, and he used alkalies and acids and even syrup of violets as a coloiu-ing matter. ^ Little, however, was done in this way until about the middle of the present century. Since then scarcely a year has elapsed without the invention of an improved method. ^ Camoy, La Biologic Gellulaire. 202 THE PHYSIOLOGY OF THE TISSUES. Thus Purkinje and Bauschel and Burdach used acetic acid as a reagent to clear up the tissues ; Gerlach showed how to fill the minute blood-vessels with trans- pai'eut injection ; H. Midler devised the method of hardening tissues by steeping them for long periods in solutions of chromic acid or of its salts ; the plan of stain- ing certain elements was followed by Lord Sydney Godolphin Osborne, Gerlach, and Lionel Beale, with carmine, and by Waldeyer, with logwood or hematoxylin ; Rainey showed how to render tissues transparent by glycerine; Loukhart Clarke made it possible to render nervous tissues transparent liy passing them through alcohol, oil of turpentine, oil of cloves, and finally mounting them in Canada balsam ; Rollet and Schwartz invented the method of double staining, so that one element of tissue might be tinted red by carmine, whilst other elements were tinted yellow by picric acid ; Eanvier, Jtirgens, Di-eschfield, and others called to our aid the coal tar colours ; and Krause, His, and Von Recklinghausen introduced the use of salts of silver. Max Schultze, the use of osmic acid, Cohnheim, the use of chloride of gold, and F. E. Schulze, the use cf the salts of palladium- -these observers ha^■ing ascertained that certain elements of tissue reduce these salts, precipitating the metal in such a condition that by lines or marks the eye can follow details in structures that otherw ise would show no optical indication of their presence. By such operations of micro-chemistry, aided hy improved methods of cutting thin sections, and by the use of microscopes that, considered merely as optical instruments, are probably very near the state of per- fection, much progress has recentlj^ been made, more especially in the obscure department of histology dealing with the nature of proto- plasm, the structure of cells, and the phenomena of fecundation. As already stated, the earliest observers with the simple microscope undoubtedly saw some of the cellular elements of the tissues both of plants and of animals, but the first important step taken was by the botanist, Robert Brown, who, in 1831, made great j)rogress in the knowledge of vegetable cells. In particular, he directed special attention to the nucleus, previously observed by Fontana, and established the fact that it was a normal element in the cell. In 1836, Valentin discovered the nucleolus, and he described it as a little round body, a kind of second nucleus, in the interior of the first. As to the physiological significance of cells, Turpin, so early as 1826, was the first to attribute to them distinct individualities, and to make the generalization that plants were formed by an agglomeration of cells. Then came the announcement of the cell theory in 1839, first applied to plants by Schleiden, and then to animals by Schwann. A careful study of the history of this subject has convinced me that even before 1830, the cell theory was held generally by botanists, and was discussed more especially in the writings of Dutrochet, Mayen, Schleiden, and Yon Mohl ; but it was not put on a sure liasis until it was applied to animal tissues by Schwann. At this period then, 1839, the conception of a cell HISTORICAL INTRODUCTION. 203 was as follows — " A vesicle dosed hy a solid memhxme, containing a liquid in which floats a nucleus containing a nucleohis, and in tchich also one may find smcdl granular bodies." Further, it was held by the founders of the cell theory that all cells might originate in a structureless substance or kytoblastema. The cell theory itself was contained in the statements that the bodies of all plants and animals are formed originally of cells, and that it is by the evolution of, and changes in, these cells that all the tissues are formed (Fig. 71). Fir,. 71. — (1) Original conception of a cell, a, cell wall ; b, nucleus ; c, cell substance or contents ; d, nucleolus. (-I) Cell wall has dis- appeared, b, nucleus ; a, nucleolus ; c, cell substance or con- tents. (M) Modern view. Cell now consists of sjranular matter. Even the faint line, c, might be omitted ; no cell wall exists. The cell theory rapidly underwent modification under the influence of new facts and of philosophical speculation. Thus, in 1841, Henle showed that cells may multiply by budding, and in the same year Martin Barry observed that the reproduction of cells was accompanied by division of the nucleus. In 1845, Goodsir first promulgated the doctrine that cells never originate without pre-existing cells, a doctrine subsequently adopted by Remak and applied to pathological phenomena by Virchow. Then, in 1845, the botanist, Naegeli, showed that certain cells have no cell wall, and in 1857, Leydig defined a cell as a soft sub- stance containing a nucleus. Lastly, in 1854, Max Schultze described a non-nucleated amoeboid organism. Amoeba poirecta, that is a cell in the physiological sense, but destitute of either cell wall, nucleus, or nucleolus, nothing in short, but a little mass of apparently structureless matter. Thus, we come back to a structureless substance, and in this connection it is interesting to read the following quotation from Schwann, because it indicates the comprehensive view taken by him of the nature of living matter — " In the fundamental phenomena attending the exertion of productive power in organic nature, a structureless substance is present in the first instance, either around, or in the interior of cells already existing ; and cells are formed in it in accordance with certain laws, which cells become developed in various ways into the elementary parts of an organism."^ "^ For an interesting accovint of the earlier views regarding cells, see Dr. Drj-s- dale's Protoplasmic Theory of Life, 1874. Dr. Drysdale in particular refers to- •204 THE PHYSIOLOGY OF THE TISSUES. Now, we must retrace our steps to ascertain what were the earlier views held by naturalists regarding living matter. As early as 1839, Brisscau-Mirbel applied the term camUinn to the living matter in })lants, and a little later Schleiden called it mucilage or schleim. In 1835, the French naturalist Dujardin, in describing the Rhizopoda, first used the word sarcode to designate "a kind of mucus endowed with sj^ontaneous movement and contractility." Then a})peared the fomous Avord, ^^ro^o- j)lasm. According to Carnoy, this term Avas first used hy Purkinje, and he makes the following quotation from Reichcrt, Avho, in describing Purkinje's researches, Avhich were published in 1839 and 1840, says — " There is only, according to Purkinje, a decisive analogy between the two organic kingdoms in that relating to the elementary granules of the vegetable cambium and of the protoplasm of the animal embryo." Six years later, in 1846, Hugo von Mohl, in describing the tissues of plants, used the word and defined the appearance of protoplasm in such a manner as to entitle him to the credit of the first scientific use of the term. He A\Tites — " I am authorized to give the name of protoplasm to a semi-fluid nitrogenous substance, stained yellow by iodine, which is contained in the cavity of the cell, and which furnishes the materials for the formation of the primordial utricle, and of the nucleus." The Avord protoplasm, then, was first used to designate the living matter of plants, and although Dujardin, in 1835, described the properties of the living matter of animals under the name sarcode, it was a long time before naturalists recognized the practical identity of the two substances, the matter called sarcode being supposed to be peculiar to the lower orders of the invertebrates. It Avas not until 1861 that Max Schultze maintained the identity of the sarcode or protoplasm of the loAver beings Avith the living matter in the tissues of the higher beings, and it is Avorthy of notice that this identity Avas grounded not on structural but on physiological properties. Thus it Avas found that the liAdng matter in vegetable cells, as shoAvn by the Avell-known phenomenon of the streaming of the granules, and by the movements of the sexual elements of fungi, had properties identical Avith those of the sarcode of the loAver animals, and Avith those of the living matter in the higher the early speculations of Dr. John Fletcher in his work, published in 1835, -entitled Rudiments of Pliy.a J J a Kb, germinal vesicle ; Kf, germinal spot, and meltinS! awav of SOme of the (Method No. 9, Appendix.) '^ -^ epithelial ceils. We have now a vesicle filled with fluid, the Graafian vesicle, having a diameter of from •5 to 5 mm. The connective tissue forms the wall of the vesicle. It •consists of (1) a connective tissue covering the theca foUiadi, Avhich is formed of two strata, an outer (a) of fibrous tissue, tunica fibrosa, and ih) an inner tunica propria, rich in cells and vessels ; (2) a lining of stratified follicular epithelium, sometimes called the memhrana granulosa. This lining of epithelial cells forms a prominence at one side, called the citmulus ovigerus or discus proligerus, and the layer surrounding the ovum has been termed the tunica gramdosa. The space is occupied, as already mentioned, by the liquor f oil iculi. A mature human ovum measures in dia- meter from 1-1 25th to 1-1 50th of an inch = 180 /a. The germinal vesicle is about l-500th of an inch in diameter = 50 [x. It is important to note in this connection that the head of the spermatozoid is the 1-6 250th of an inch in length, and about l-8000th of an inch broad, so that the size of the male nuclear element is only about 1-1 2th part of that of the female nuclear element (Fig. 91). When the ovum reaches maturity, the Graafian vesicle is full of fluid, and, as already mentioned, bulges out from the surface of the ovary. The vessels in the neighbourhood of the vesicle become much congested, and at a given time the vesicle bursts and extrudes its contents. This is assisted by a rupture of some of the finer vessels, causing a haemor- rhage into the cavity of the vesicle. Coincident with these changes, by a reflex mechanism, the exciting cause of which is not known, the end of the Fallopian tube is applied to the ovary, and the oviun escapes into it and passes to the uterus. During its passage down the Fallopian tube it meets with the spermatozoids, and then fecunda- tion occurs. TEE ORIGIN OF TEE TISSUES. 223 Fig. 91. — Section of the Graafian follicle of a girl eight years old, 90 times magnified. Th, theca foUiculi ; Tf, tunica fibrosa ; Tp, tunica propria ; M, membrana granulosa — follicular epithelium ; C, discus or cumulus ovigerus ; E, ovum, with zona pellucida, germinal vesicle, and germinal spot. The clear space in the middle contains the liquor folliculi. (Method No. 10, Appendix.) 3. Influence of Spermatozoid on Ovum. — The question now arises, what is the influence of the spermatozoid upon the ovum ? It is interesting for a moment to notice the historical development of knowledge upon this question. The first step in this direction was made by Kolreuter in 1761, when he succeeded in artificially pro- ducing hybrids by the cross fertilization of plants. Soon after this Jacobi artificially fertilized the eggs of the trout and salmon, and in 1780, Spallanzani operated in the same way on the frog, the tortoise, and the dog. Curiously, however, he thought that the virtue lay in the fluid and not in the living particles. In 1824, Prevost and Dumas proved that the seminal fluid lost its effect after filtration Martin Barry observed spermatozoids in the ovum of the rabbit in 1843 ; Leuckart made the same observation in the frog in 1849 ; Nelson, in 1852, observed the spermatozoids in the egg of A scans mystax ; and in 1853, Keber observed the actual entrance of the spermatozoon into the egg of the common mussel. The subject also attracted the attention of Bischoflf, whose researches into the development of the dog were of 224 THE PHYSIOLOGY OF THE TISSUES. great value, and of the late Dx\ Allen Thomson. It was supposed that the spennatozoids entered the ovum, disappeared, and in some mysteri- ous way set up changes which resulted in the cleavage of the yolk, or more strictly speaking, in the formation of the first two cells of the embyro — the first two blastomeres. Opinion remained in this vague condition until 1872, when Biitschli made the remarkable observation of hvo nuclei in the fecundated egg of Bhahditis doUclmra, a nematode worm. As often happens in science, Auerbach, in 1874, made a similar observa- tion on the eggs of two other worms — Ascaris nigrovenosa and Strongylus auricularis — without kno'\\ang of Biitschli's description. Biitschli followed up his work in 1875, by discovering the same phenomenon in the eggs of the gastropods — Lymncea sfagnalis and Succinea Pfeifferi. He also noticed in the eggs of these molluscs a peculiar fibrillated appearance, such as occurs in connection vnth the division of nuclei. These investigations Avere quickly followed by those of 0. Hert"vvig, Ed. van Beneden, and Fol. It is to be observed that Biitschli did not explain the origin of the second nucleus which he had observed in the fecundated egg. This was accomplished by 0. Hert-\\ag, when, in 1875, he published his researches on the development of Toxojmeusfes lividus, an echinoderm. He has the merit of demonstrating that the second nucleus is the head of the spermatozoon. Further, 0. HertAvig at first supposed that the other nucleus was the germinal spot of Wagner set free by the destruction of the germinal vesicle. This error was pointed out by Van Beneden, and 0. Hertwig accepted the correction, with the resiilt that the other nucleus was soon shown to be, as Biitschli had previously conjectured, the germinal vesicle. The distinguished Belgian naturalist, E. van Beneden, early recognized the importance of these observations. In 1875, he described the fusion of the nuclei, and expressed the opinion that in the fusion of the two nuclei there was a phenomenon comparable to the conjugation of the Protozoa and Proto- phyta ; and in his magnificent monograph on the fecundation of Ascaris megalocephala (1883) he has largely advanced our knowledge of the subject. Although the entrance of the spermatozoon had been pre- viously observed, no specific changes were immediately noticed. This was reserved for Fol, a Swiss naturalist, who, in 1877, observed the entrance of the spermatozoid into the eggs of Asterias glacialis (a star- fish), and of various species oi Echinus (sea-urchins); and he studied and figured the phenomena that ensued. These observations have been confirmed by many naturalists (Fig. 92). THE ORIGIN OF THE TISSUES. 99.- ^ -kikfi v-a Fig. 92. —Fecundation of the eggs of Astenas glacialis. s-p, Sper- matozoids. The spermatozoid's are in the mucus covering the egg. At ^, a spermatozoid has reached the periphery of the yolk. At B, several are moving onwards towards a prominence in the surface of the yolk. At C, the heads of one or two sper- matozoids have united with and penetrated into this prominence. 4. The Formation and Expression of Polar Bodies. — Another series of phenomena has been brought to light. It is well known from the researches of Strasburger and others that, in the development of the reproductive organs of plants, certain portions of the matter that is devoted to the formation of spores, antherozoids, and oospheres (or ova) are not used for that purpose, but are extruded or othermse laid aside from taking part in the reproductive process. Thus, in the sporangium of many fungi, a part of the protoplasm, called the epiplasm, remains over after the formation of the spores. In the development of the zoospores of algse a part of the protoplasm, not used up, is extruded in the form of a vesicle from the sporangium, and Strasburger states that in some cases, just before the division of the spore-mother-cell, a mass of stuff is extruded from the nucleus, called the paranudeoliis. Similar phenomena have been observed in sexually- reproductive cells. The male element of mosses, the antherozoid, has an appendage attached to its posterior end. This appendage or vesicle has been found to be the " unused protoplasm of the mother-cell," along Avith actually a portion of the nucleus, which is thus excluded. In the angiosperms, during the development of the nucleus and the protoplasm of the pollen grain (the male portion) cell-like bodies are formed which are separated from the generative cell. Similar phenomena are noticed in the development of the female structures or gametes, that is to say, portions of protoplasm are extruded from the oogonium. Even in angiosperms nuclear divisions take place in connection with the I. P 220 THE PJ/rSIOLOGY OF THE TISSUES. (leveloimicnt of the oosphere or ovum. Thus, to quote from Dr. Sydney Howard Tines— " The nucleus of the young embryo-sac divides into two, one of which travels to each end of the sac ; each nucleus then divides, and each of the two nuclei divides again, so that there is a group of four nuclei at each end of the embryo-sac. Of those at the micropylar end, one becomes the nucleus of the oosphere, two the nuclei of the synergid^, and the fourth (polar nucleus), which is the sister nucleus of that of the oosphere, travels towards the middle of the sac, where it fuses with one of the chalazal nuclei, which has likewise travelled towards the middle of the sac, to form the definitive nucleus of the embryo-sac. It may be suggested that the division which leads to the formation of the nucleus of the oosphere, and of the so-called polar nucleus, is the one which we are seeking ; in that case the so-called polar nucleus would be the polar body." It appears, therefore, that in the development both of non-sexual spores and of male and female elements in plants a portion of the original nuclear substance is extruded or thrown aside and takes no share in the reproductive process. The portions thus laid aside or extruded have been called polar bodies. It is remarkable that analogous phenomena occur in the development of the ova and of the sperm cells of animals. The extrusion of minute particles of matter, or polar bodies, from the e*'" Avas first observed by Dumortier in the egg of Lymncea stagnalis in 1837, and the phenomenon was investigated by F. Miiller in 1848. No relation between these bodies and the germinal vesicle was noticed until the researches of Biitschli in 1875, to which I have previously referred. He observed a curious spindle-shaped structure passing between the terminal vesicle and the surface of the o\aim. In 1876, Ed. van Beneden detected the extrusion of two polar bodies from the egg of Asteracanthion rubens. The researches of Fol, Selenka, and 0. Hertwig not only corroborated these observations, but manj- details of the pheno- mena connected with their formation and extrusion were observed. Thus Calberla noticed that changes occurred in the germinal vesicle along wath the extrusion of the polar bodies, and that these bodies might be eliminated even before the egg reached its maturity. In the great majority of cases the formation of the polar bodies preceded the union of the sexual elements, and researches by Fol and others have shown that the genesis of these bodies is independent of fecundation. Let us now examine what actually occurs. When the o^nim is ripe it consists, as already described, of a cell wall enclosing contents, in which is embedded the nucleus or germinal vesicle, in which again is the nucleolus or germinal spot. Previous, then, to actual fecundation (although the spermatozoid maj' have penetrated the o^aim), a fusiform THE ORIGIN' OF THE TISSUES. 227 ■ioxvM-^^^^-i- ya iDody is seen (the " direction-spindle " of Biitsclili, and the " amphiastcr ■of rejection" of Fol), as was first de- scribed by Biitschli, stretching from the germinal vesicle (Fig. 93) towards the surface of the ovum, so that one end of ,,^...^. .„..„.„,,, „,,„..,,.,,. ^^.-q.^. ._., the spindle touches the surface whilst '°^°i^§i0MM'J6'^§^°^&§i^^o^' the other is in contact with the germinal vesicle. At the same time, or rather immediately before the appearance of the spindle, the wall of the germinal around the polar body ; c,yoik, vesicle becomes less and less distinct, and the protoplasm in its immediate ■vicinity assumes a radiated appearance. There is every reason to believe that a portion of the substance of the germinal vesicle forms part of the spindle. By-and-bye a small globule protruding from the surface of the ovum ajjpears at the external end of the spindle. This is the first polar body. In most cases a second spindle is formed, and there is the extrusion of a second polar body. Biitschli thought that the spindle was entirely extruded in the polar body, but Fol and Hertwig have Fig. 93.— Extrusion of ii polar body from the egg of a snail, Succineo, Pj'eifferi. a, polar body showing elongated filaments of nuclear matter ; 6, radiated appearance Fig. 94. — A, ripe ovum of Astenas glacialis with excentric germinal vesicle and spot. £, C, D, E, gradual metamorphosis of germinal vesicle and spot ; F, detachment of first polar body and withdrawal of remaining part of nuclear spindle within the ovum ; G, portion of living ovum with first polar body ; H, formation of second polar body ; I, after formation of the same, showinj;; the remaining internal half of the spindle in the foi-m of two clear vesicles ; K, ovum with two polar bodies and radial strise around female pronucleus ; L, expulsion of polar body {A — K, after Fol ; L, after Hertwig). shown that this is not the case. As to the genesis of the spindle itself there is still considerable difference of opinion. Biitschli conceived that the fibres of the spindle were formed by the coalescence of granules. Fol described them as due to a swollen condition of the " filaments bi- polair," meaning certain filaments passing from the germinal vesicle towards each pole of the egg. 0. Hertwig supposes that they arise from the fragments of the wall of the germinal vesicle and of the germinal spot ; and these views of Hertwig have been supported by Trinchese :and Blochmann. Considerable discussion has taken place as to whether these pheno- 228 rilE PHYSIOLOGY OF THE TISSUES. menu belong to the class of changes occurring in kary olcinesis. Flemming, in 1882, after a careful study of the egg by the methods followed in the- observation of karyokinesis, came to the opinion that the phenomena arc the same, with this difference, that in the egg the chromatin element does not l>ulk so largely as the achromatin constituent, the result being that in the karyoldnesis of cells the chromatin figures are the more apparent, Avhereas in the egg the achromatin figures arrest the attention. This A-iew is well illustrated by the figures of ordinary karyokinesis ■-■iven by Rabl (Fig. 95), in which the achromatin spindle-like figures are Mm n. L D. Fio. !I5. — DiaffTiini showing the mode of uucloar division. A shows strongly marked chromutiiT filaments with the spindle figure. At the ends or points of the spindle observe the star-.shaped or radiating arrangement of the protoplasm, and in the middle of the spindle the chromatin filaments. In the latter a longituhala. Fig. 9S. — The upper part of the Y- shaped figure has now reached the surface of the et^g, or stage prior to the genesis of the first polar body, which is formed by transverse cleav- age. Egg of Ascaris tnecjcdoce-phola. Fig. 09. — Formation of the first polar body in egg of Ascaris mepalocephala, indicating one of the last modifica- tions of the Y-shaped body. 230 THE PHYSIOLOGY OF THE TISSUES. tonally, just as if the limbs of the Y had opened out, and each limb had lieen cut in the direction of its length. In all cases of ordinary karyokinesis the cleavage always takes place transversely to the direction of the filaments of the spindle, and if Ave recognize the Y-shaped figure of E. A-an Bejiedon as being formed of two spindles, the importance of his observ'ation will be appreciated. The formation of the polar globules, cannot therefore be deemed an example of karyokinesis : at all events^ it is not of the usual type. After the removal of both the polar bodies^ the portions of the chromatic stars or discs, along A\dth the neighbour- ing portions of the prothyalosome, unite to form a body, which is in all respects similar to the polar bodies extruded. This he terms the- deuthjalosome. This body, the deuthj-alosomc, may be regarded, then, as formed of the remains of the germinal vesicle. The vesicle, b}' this elaborate process — Avhich occurs hefore fecundation, although it may be going on while the spermatozoon is in the egg — has throAvn off a portion of itself, and the body Avhich remains at the end of the process cannot be- regarded, morphologically or physiologically, as an ordinary cell nucleus. Xow Ave approach true fecundation. It has l)een ascertained that one single spermatozoon is sufficient for the process. Entering the ovum, either by a distinct aperture (in the OATim), termed the micropijle (Fig. 100), or, AA'here no special aperture has been observed, through the cell Avail, it quicldy undergoes chemical and physical changes. It becomes more susceptible to the action of colouring matters, and instead of being granular it assumes a homo- geneous apiDearance. The tail dis- appears, and the head becomes globular in form. E. A^an Beneden has obserA^ed in many eggs a re- fringent globule, similar in appear- ance to a body seen in the spermatozoid, and he has no doubt that this body has been throA\Ti off by the spermatozoid, and finally ejected from the egg itself, probably Avith the second polar body. This importo-nt obserA^ation is of profound Fig. 100. — A, blind end or cid-de-sac of the ovary of a star fish. B and C, development of the egg of Holulhuria Bohadschia, after Semper. B, young egg with the surround- ing epithelial cells detached ; at 'lU, the opening of the micropyle. 0, ripe egg, showing a thickened zona pellucida, and the epithelial cells much elongated ; m, micropyle, very narrow. D, egg of Unio, showing micropyle at in. THE ORIGIN OF THE TISSUES. 231 significance, as showing that the spermatozoid (as well as the germinal vesicle) gets rid of a part of its substance {seminal granule) before fecundation occurs.^ The body left in the egg after the formation of the two polar bodies is termed the female jpronucleus, while that formed by the head of the spermatozoid is the male pronucleus. Until the second polar body has been extruded, the spermatozoid remains inactive, but when this has occurred a series of changes ensues, which ends in the division of the first cell of the embryo into two cells (Figs. 101 and 102). Fig. 101. — Eipe egg of a sea urchin (Echinus), en., egg nucleus, or ger- minal vesicle. Fig. 102. —Fecundated egg of a sea urchin. The head of the spermatozoid, consti- tuting the spermatozoid nucleus, sp. n. or male pronucleus, is surrounded by radiated protoplasm ; en., egg nucleus, or female pronucleus. Fro. 103. — Fecundated egg of a sea ixrchin, sp. n. Spermatozoid nucleus, or Eiale pronucleus; en., egg nucleus, or female pronucleus. The two pro- nuclei are close together, and are both surrounded by a radiated mass of protoplasm . Fia. 104. — Eg<;- of a sea urchin immediately after fecundation. rn., fecundation nucleus. Both the male and female pro- nuclei have disappeared, and ai-ound the fecimdation nucleus the protoplasm has a radiated appearance. ^ Further, E. van Beneden and Ch. Julin have discovered the expulsion by the spermatomere of a minute globule, which does not therefore enter into the formation of the spermatozoid. La spermatogenese ches I'ascaride megalocephale. Bull, de VAcadimie Royale de Belgique, 1S84. The separation of a globule has also been described by J. E. Blomfield on The Development of the Spermatozoa, part 1, Lumbricus (Earth worm), Quart. Jour, of Micros. Science, 1880. See also on •232 THE PHYSIOLOGY OF THE TISSUES. The female pronucleus is now a nucleus containing two chromatin filaments, or loops, originally derived from the germinal vesicle of the egg. It is very curious to find that the number of loops seen in the various stages of these processes is very constant. Thiis, in the ger- minal vesicle there are two chromatin plates, each composed of four chromatin globules ; the same in the Y-shaped figure ; in the first polai- bod)' two chromatin bodies, each formed of two smaller ones ; in the deuthyalosome, two chromatin filaments, each dividing into two ; the same in the second polar body ; and now in the female pronucleus again two chromatin loops, each formed of two smaller portions. The male pronucleus is formed from the head of the spcrmatozoid, and it also contains two portions of chromatin. It is found near the lower pole of the egg. The two pronuclei approach each other, and ultimately unite. According to 0. Hertwig, Fol, and Mark, there is a complete fusion. This is fecundation, the union at last of the two elements (Figs. 103 and 104). E. van Beneden, in the Ascaris megalocephala has not observed com- plete fusion; on the contrary, he has been able to observe in the karyokinetic process which immediately follows, that the chromatin filaments supplied by the male and female elements remain distinct, so that in the two cells formed by the division of the first cell each new nucleus receives an ecpial portion of male and female elements. He divides the process into four stages. 1st. The four chromatin filaments increase in size and become coiled to a greater or less extent. It is to be remembered that two have been supplied by the male and two by the female pronucleus. 2nd. The Fig. 105.— Arrangement of chromatin fila- COntOUrS of the pronuclei becOme nients in the equator of the egg of .-isan-is , , ,. . mef/atncepkala. The filaments form the leSS and IcSS dlStmct; each chromatin dark irregularly-shaped bar. Only the t • t i ■ i middle portion of the egg is shown. loop dlVldeS traUSVersely lU the middle so that there are now eight loops, and the eight loops of chromatin move so that the shut end of each loop is directed towards the equator of the fecundation nucleus. Imagine an orange wdth eight loops of cord placed equatorially in it, the ends directed toAvards the axis passing from pole to pole. 3rd. Each loop is then divided longitudinally, thus giving rise to sixteen loops, and the two groups of eight are separated by a thin layer of this question Herbert H. Brown's paper on Spermatogenesis in the rat, Quart. Jour, of Micros. Science, 1885, in which Prof. Eay Lankester"s opinion is given as regards the globule in case of Earth worm, p. 20. THE ORIGIN OF THE TISSUES. 233 matter. 4th. Each of these groups of eight is now situated in what E. van Beneden terms sub-equatorial planes, parallel of course with the equator (Figs. 105, 106, and 107). Fig. 106. — Stage in the division of the egg of Escorts nugalocephala, showing the chromatin loops sepa- i-ating into two sets and retreating from each other. Pig. 107.— Stage of division of the egg of Ascaris inerjcUocephcUa, showing the breaking up of the chromatin threaJs into minute globules, and in some places a reticulated appearance from the crossing of short filaments. Thus it is evident that each loop has given a half of its substance to •each of the daughter nuclei, and as there was an equal number of loops from the male and female portions to start "vvith, it follows that each daughter nucleus must have male and female elements. The two sets of loops recede towards the poles of the nucleus, and then there is division of the body of the yolk, according to the ordinary method of karyokinesis already described. Further, the two cells thus formed, nourished by the fluids in which they exist, increase in size, and again divide karyoldnetically into four. The process is repeated over and over again so as to form the primitive cells from which all the tissues and organs of the body are derived ; and if the karyokinetic division is repeated so that portions of the chromatin derived from the male and female elements (multiplied and increased in quantity by nutrition, but the same in kind) are divided equally, it follows that all cells contain equal quantities of matter related to the male and to the female parent ; and, to crown all, according Fig. ids.— Egg of a sea urchin preparatory to cleavage. 234 THE PHYSIOLOGY OF THE TISSUES. to this view, each living cell is a herma^jhrodite being ^ (Figs. 108, 109, 110, and 111). Fig. 100. — Egg of sea urchin at moment of division, showing constriction of the protoplasm at ri;,'ht angles to the axis of the nucleus. Flo.llO. — Eju'-gof sea urchin after division. Observe the nucleus in each half. The streaked ap- pearance of the ijrotoplasm begins to be less distinct. Fio. 111. — Various stages of the cleavage of the egg to form embryonal cells. CH.iP. III.— THEORIES AS TO THE PHYSIOLOGICAL BASIS OF HEREDITY. In the previous chapter, we have discussed the origin of the cells and of the living matter that form our bodies. One is naturally inclined to ask, does a knowledge of these phenomena throw any light upon the Avell-known facts of hereditary transmission ? ^ Not a few theories of heredity have been advanced. The oldest of all is that the soul of an ancestor entered the body of a newly born being and made the body like that in which it had at one time existed. The theory of the trans- migration of souls may be traced to very remote times, and it exists ^ This view is associated with the name of Professor Sedgewick Minot of Har- vard. Science, vol. iv. 18S4. Also Proceed. Boston Society of Natural History, 1877. - Ch. van Bambeke, "Pourquoi nous ressemblons a nos parents." Bull, de VAcatUmie Roycde de Belgiqm. 3™''- serie, torn. x. 1885. This is an admirable resume of recent speculations. PHYSIOLOGICAL BASIS OF HEREDITY. 235 almost unmodified in the opinions of some of the so-called savage races of the present day, races probably representing, however, not the primitive type of thought and practice, but opinions modified in the course of ages, like those of the higher and civilized races. Stahl, in accordance with his mystical, views, held that the soul formed the foetus and shaped it according to the paternal or maternal type. Then, in our own day, came the doctrine of Pangenesis of Charles Darwin,^ which assumed the existence in all cells of small corpuscles which circulate free in the body. These gemmules are transmitted by the parents, and they are usually developed in the next generation, but may lie dormant for generations, and their evolution depends on their union with gemmules of a kindred nature, supplied from another organism. This theory is capable of explaining many of the facts, but it labours under the disadvantage that there is no vestige of proof of the existence of such gemmules. Haeckel ^ has endeavoiu"ecl to solve the problem by supposing that what is transmitted is not matter but a certain kind of molecular move- ment which the tissues acquire by constant repetition, and retain by a kind of unconscious memory. Heredity, according to this view, would depend on the more or less accurate transmission of sj^ecial kinds of molecular movement, while variability would be caused by the action of external agencies changing more or less the character of the movement so transmitted. The obvious criticism on this theory is, that we as yet know far too little of the character of molecular movements, to make our knowledge the basis of any such theory. Naegeli rejects both the theory of Pangenesis of Darwin and the theory of Perigenesis of Haeckel, because the one calls in the aid of hypotheti- cal germs and the other of hypothetical movements, and he endeavours to establish a theory on the basis of fact. Protoplasm, according to him, consists of two portions, one, fluid, called hygroplasm, and the other solid or insoluble, stereoplasm. The stereoplasm, however, is partly nutritive, but the other portion called idioplasm is the seat of all active changes. This idioplasm ramifies through the body, is in every cell and tissue ; and each tissue has its own kind of idioplasm, so that there is a ^ Charles Darwin, on the Variation of Plants and Animals under Domestication, 1867, chaps, xxxvii. and xxxviii. See also in this relation, Herbert Spencer's Principles of Biolo(jy, vol. i. chaps, iv. and viii. Also, Sir Richard Owen's work on Parthenogenesis, 1849. It is interesting also to read Professor Owen's criticism of Pangenesis in his Comparative Anatomy and Physiology of Vertebrates, vol. iii. p. 813. ^Haeckel, Die Perigenesis der Plastidtde oder die Wellenzeugung der Lebena theilchen, Berlin, 1876. -230 ^V//v' PIIVSlOLUaY OF THE TISSUES. vast variety of i(lioi)lasmi=;, differing, however, more dynamically than in any detail of structure. Reproductive cells contain idioplasm that has returned from the condition of somatic idioplasm to the state of the idio- plasm of the germ from which the organism sprang. Further, he sup- poses that the specific properties of the idioplasm depend on the grouping of more minute particles, the miceUa', and that this grouping is more complex in the idioplasm of the higher beings than it is in the lower. Suppose, then, that each parent transmits an equal portion of idioplasm, there -will be an equal transmission of the peculiarities of each parent ; and if one idioplasm predominates, the balance may lean to the side of the fiither or of the mother. This mass of idioplasm, as already said, extends through the body, having subsidiary characters peculiar to each kind of cell,l:)ut always carryingwith it the more special peculiarities which it had at first. During growth it retains all its specific characters, and the germ cell is simply a cell containing idioplasm, having chieflj' the primitive character. As, however, the somatic idioplasm is influenced by external conditions, to some extent, and as the retiirn of the somatic idioplasm to the state of germinal idioplasm is not always exact, the offspring never exacth^ resemble their parents. Hence arises variability. But while this theory is founded on the fact that there is an all-prevalent matter — the protoplasm, — it is liable to the same objection as Avas advanced to the other two theories — it is too conjectural. The question is, where is this idioplasm 1 If it is the reproductive matter, it must exist in many cells, more especially in those which yield the male and female elements. That it does exist in certain cells of plants is evident from the fact that cuttings produce roots, and Avhen a stem is cut across new shoots appear from the lateral buds. Almost any part of the leaf of a Begonia, if suitably planted, will give origin to a fully- formed plant. Similar phenomena may be observed in certain ani- mals — more especially in amphiliians. If the limb of a salamander be removed, the limb may be reneAved, that is to say, the cells forming the various tissues of the stump possess the power of producing cells of the same kind, and of rebuilding the limb. This has been aptly compared by Pfluger,^ to the growth of a crystal. A crystal of alum has a certain molecular structure, and if a small fragment of such a crystal is placed in a saturated solution of the same salt, the broken fraginent is repaired, so as to form a perfect crystal. This is more than an analogy. The molecular processes of the crystal are the cause of the deposition of the particles of alum in such an order as to renovate the crystal ; and, in like manner, the molecular processes in the cells of the outermost layer ^ Pfliiger, Ueber den Einfluss dtr Schirerkraft auf die Theilimg der Zellen und ail/die EntwicMunrj de-s Embryo. Archivf. Physiol, vol. xxxii. PHYSIOLOGICAL BASIS OF HEREDITY. 237 of the stump lead to the formation of new cells of the same kind out of the material furnished by the blood plasma in their vicinity. Seizing hold so far of Naegeli's idea, Strasburger ^ has modified it, and made the position more intelligible. Like him, he considers protoplasm as consisting of two substances, a nutritive hyaloplasm, and a formative hyaloplasm ; but he identifies the formative hyaloplasm ^viih the chromatin filament of the nucleus, which we have already studied. This substance he called nucleoplasm or nucleohyaloplasm. It is the active substance in the nucleus and even in the protoplasm around the nucleus, as Goodsir long ago stated. All the metabolic changes in the cell substance are controlled and directed by the nucleoplasm. Stras- burger states that the reproductive power of a cell depends on its being in the embryonic state, and in plants reproductive cells in this sense are not uncommon, seeing that many parts of plants are capable of vegeta- tive reproduction. This view throws light upon the phenomena of the expulsion of polar iDodies from the o\aim, and from the spermatozoid substance. These phenomena exjjress the return of the cells to their embryonic conditions. The late F. M. Balfour held that the extrusion of the polar bodies from the ovum, or rather the extrusion of a part of the germinal vesicle, was " requisite for its functions as a complete and independent nucleus," . . . "to make room for the supply of the necessary parts to it again by the spermatic nucleus. My view," he says, "amounts to the follow- ing, viz. : that after the formation of the polar cells, the remainder of the germinal vesicle, with the o\aim (the male pronucleus), is incapable of further development without the addition of the nuclear part of the male element (spermatozoon), and that if polar cells were not formed, parthenogenesis might normally occur. A strong support for this hypothesis would be afforded were it to be definitely established that a polar body is not formed in Arthropoda and Eotifera, since the normal occurrence of parthenogenesis is confined to these groups. It is certainly a remarkable coincidence that they are the only two groups in which polar bodies have not so far been satisfactorily observed." Further, he says, "The function of forming polar cells has been acquired by the ovum for the express purpose of preventing partheno- genesis." - ^ >Strasburger, JS^eiie Uiitersuchungpn fiber den Befruchtuiuiscorgang hei den Phan- erogamen als Grundlage fiir eine Theorie der Zeugung. Jena, 1884. - F. M. Balfour, Comparative Embryology, vol. i. p. 72. It is only right to note that this theory is strongly opposed by Carnoy, and that he especially disputes the correctness of the interpretation of the extrusion of the polar bodies, La Cyto- dierese de I'ceuf, La Cellule. Tome iii. fasc. i. p. 60. It is also asserted that- 238 T'iii^ PHYSIOLOGY OF THE TISSUES. This notion, which has been somewhat modified by various authors, and is refused by Carnoy as an explanation, is that the extrusion of the polar bodies from the nucleus of the ovum is the removal of the male idioplasm, so that what is left is truly female, and the extrusion of the l)article from the spermatozoid is the removal of the female idioplasm, so that what remains is truly male. The female idioplasm Avould thus transmit peculiarities of the mother, whilst the male idioplasm would send on those of the father. But, as Strasburger and Yon Kolliker acutely remark, the mother transmits not oidy her own peculiarities, but also those possibly of the maternal grandfather and of the maternal great- •■■randfather ; and, on the other hand, the father may send onward traits of the paternal grandmother and of the paternal great-grandmother. The maternal idioplasm cannot, therefore, be regarded as entirely female, nor the paternal as entirely male. Strasburger holds that the nucleoidioplasm filament may be regarded as made up of a number of segments derived from previous generations, and that in certain circum- stances one portion may influence the kytoplasm of the ovum more than the other, thus giving rise to a manifestation of some of the hereditary qualities of the particular generation to which the portion of nucleohyalo- plasm corresponded. It appears to me that here Strasburger passes entirely into the region of theory. The one statement of importance in his theory is the identification of the idioplasm of Naegeli, or, as he terms it, the nucleohyaloplasm Avith the chromatin filament. It is at this point that the important observations of E. van Beneden come in Avith great effect. If it be the case that the chromatin filaments from the male and those from the female remain distinct, and are communicated in equal amounts to the nuclei formed by the division of the fecundation nucleus, and if this process be multiplied indefinitely in the formation of the cells of the body, Ave see that each cell is representative of both father and mother, to a greater or less extent, according to the amount of maternal and paternal idioplasm present (Fig. 112). Further, it is necessary to assume that this idioplasm is capable by nutrition of being increased in quantity, but by the action of surrounding conditions it may be acquiring properties peculiar to itself, giving rise to the indiAaduality of Weismann has seen polar bodies extruded from the eggs of certahi crustaceans that are parthenogenetic (Weismann, Pdch.tun(jfe.r bei partlienogeneflschen Mem. Zool. Anz. 27th Septr. 1SS6). Ea^cu if this be the case it does not seem to me to destroy the A-alidity of Balfour's theory, but only to show that it is less extensive in its application. The extrusion of polar bodies by parthenogenetic ova may be the survival of an ancestral habit, and possibly after remoA^al of the polar body there may still be enough of the male matter left to alloAV of development going on, Avithout the entrance of a fresh spermatozoid. PHYSIOLOGICAL BASIS OF HEREDITY. 239 the person in whose body these changes are supposed to take place. A time comes, however, when cells lose the power of thus multiplying Fig. 112. — Different arrangements of the primary chromatin loops in the equatorial plane of the egg of Ascaris inegalocephaia. indefinitely by fission, the growth of the body is arrested, and the general characters of the individual are fixed. One can suppose, then, that if any cell in the body could get rid of its male portion of idioplasm, and if another got rid of its female portion, the two remaining parts might conjugate, and the process of cell division might start afresh. The stimulus would still be greater if the two idioplasms were more different than is likely to be the case if obtained from the cells of the same individual. The best efi'ect would be produced if a certain amount of difference existed, and this is attained by the idioplasm of two individuals being brought into contact. May not this be the key to the advantage gained by sexual difference, and may it not explain the infertility that results if two idioplasms from two individuals too closely related be brought into mutual relationship ? This rendering and expansion of Strasburger's theory I have reached after a careful consideration of the question in the light of the facts of modern investigation. The peculiarity of Strasburger's view is that the object of the extrusion of the polar bodies is simply the attainment of the embryonic condition, and that the reproductive matter — call it idioplasm or any name you choose — is formed by the conversion of somatic into repro- ductive idioplasm. In the latter statement Strasburger follows ISTaegeli. The essential distinction between Strasburger and Naegeli is that while the conceptions of the former are structural and material, those of the latter are dynamical. This supposed conversion of somatic into repro- ductive idioplasm is a difficulty, and a consideration of it has led Weis- mann^ to promulgate the theory of what is termed the continuity of the germ-plasma. This view is that heredity consists in the transmission of a nuclear substance of special molecular structure, not the idioplasm of Naegeli, nor the nucleohyaloplasma of Strasburger (identical mth chromatin), but a substance special and f)eculiar to the germinative cells. ■■■ August Weismann, Die continuitat des Keimplasma's als Grundlage einer Theorie der Vererbung. Jena, 1885. See also an account of this memoir by Professor H. N. Moseley in Nature, vol. xxxiii. p. 154, 240 THE rilYSlOLOGY OF THE TISSUES. This luiclear matter which he calls keimplasma, germ-plasma, is passed on from generation to generation without alteration ; it is therefore con- tinuous and inmiortal if individuals do not fail in propagating their species. This specific germ-plasma, derived from the male and female parents, is not entirely used up in the development of the offspring, but a portion of it is set aside to form the germ-plasma of the next generation. According to this view, the germ-plasma is in no way derived from the somatic-plasma ; each has an independent existence ; l)ut while the somatic-plasma cannot influence the germ-plasma, the portion of germ- l)lasma not laid aside to form the germ-plasma of the next generation exercises a potent influence over the development of the somatic-jjlasma, transmitting to it the characters of the parents. Professor Weismann supports this view with many striking illustrations, and he adapts the theory to the explanation both of the extrusion of polar bodies and of l)arthenogenesis. Thus the o^^^lm has two kinds of plasma in it, histo- genetic or somatic-plasma and true germ-plasma. In its early stages, the histogenetic plasma is engaged in forming the yolk and membranes, and as it is of no further use in the further development of the embryo, it is got rid of in the form of one or more polar bodies to make room for fresh histogenetic plasma, derived in turn from the germ-plasma of the spermatozoon. The polar bodies are thus ovogenous nucleo-plasma, got rid of to allow new nucleo-plasma to have free play. A similar explanation applies to the separation of particles from the male sperm cell. As to parthenogenesis, AVeismann supposes, in the first place, that the male and female elements in fecundation, that is to say, the germ-cell and the sperm-cell, are practically identical, and that after the extrusion of the polar bodies, development is started by the sudden addition to the so-called female element of the so-called male element. This sudden addition necessitates cleavage, and as nutrition goes on actively, for the same reason, cleavage goes on again and again to form the embryonic cells. In some ova, however (parthenogenetic ova), even after the extrusion of polar bodies, nutrition goes on so actively as to start the developmental process without the stimulus caused by the suddeii addition of the so-called male element. Pai'thenogenesis, according to this view, is thus merely a modification of the power of growth. The theory is not open to the objection urged against that of F. M. Balfour, namely, the undoubted extrusion of polar bodies from, at all events, some parthenogenetic ova. It appears to me, however, that this theory, so ably maintained by AVeismann, while it undoubtedly removes some of the difficulties, leaves the facts of hereditary transmission unexplained. If the portion of germ- PHYSIOLOGICAL BASIS OF EEREBITT. 241 plasma not laid aside influences the development of the body, conferring on each tissue and organ at least some of the proclivities of the parent, how does it do so ? The molecular mechanism by which this is accomplished is inexplicable. On the other hand, if this germ-plasma does not so influence development, how are the facts of hereditary transmission to be explained % It is, I humbly think, cpiite possible that AYeismann may be right in the conjecture that a portion of the original germ-plasma may be laid aside at the earliest period of development to constitute the germ-plasma of the next generation ; but surely this germ-plasma has received a molecular imprint from the parent in whose body it at one time existed so that it transmits certain of the character- istics of that parent. If the germ-plasma cannot be altered, how are we to account for the transmission of special characters of any kind ? AYeismann has admitted this difficulty by denying the alleged trans- mission of acquired characters by sexual reproduction ; but all characters must have been acquii'ed at one time or another, and any organism is what it is at the present moment, in consequence of this transmission of acquired peculiarities through countless generations. The germ-plasma cannot then remain the same, and therefore the only way in which we can suppose it to be modified is through the agency of the idioplasm throughout the body. This view Avill explain to some extent at least the transmission of acquired characters.^ In all these discussions, one aspect of the subject, as it relates to the higher organisms, and especially to man, is often omitted, namely, the influence Avhich the female parent exerts on the development of the off- spring, while in the uterus, in consequence of the intimate union that exists for a considerable period of time. It is a familiar observation that animals reared outside the mother's body, as in most invertebrates, in fishes, amphibians, and birds, are remarkably similar to each other. The individuals in a shoal of fish or a flock of sea gulls or sparrows can scarcely be distinguished. It is when we reach the stage of develop- ^ I have to express my admiration of Weismann's theory as a guide to an experi- mental investigation into this most difficult field of inquiry. I would further observe that it can hardly be expected that acquired characters in their full intensity ■will be transmitted to the immediately succeeding generation, seeing that the ova destined to become the individuals of that generation are found in the ovary of the mother during embryonic life. The influence of an acqiiired character might have a certain effect on the ova during the life of the mother, but not enough to reproduce the acquired character with any degree of strength in the ofispring. If the influences producing the acquired character acted also on the offspring, its effect on the offspring of the third generation would be intensified, and thus a pro- cess of accretion during several generations may be required to stamp acquired characters on offspring. I. Q 242 THE PHYSIOLOGY OF THE TISSUES. ment by placental connection that we begin to observe diversity of appearance, and if along with this we suppose that in consequence of a great development of the nervous system, the female becomes more and more susceptible to external impressions Avhich react on her body and through her body on her offspring in utero, an explanation, so far, of individual peculiarities of the human race becomes apparent. There can be no doubt that pregnancy makes a remarkable change on the mother, to such an extent that the characters of subsequent offspring may be influenced. Thus it is well knoA\'n to breeders of animals that a pregnancy induced by a certain kind of male, may deteriorate the characters of offspring that owe their origin to subsequent impregna- tions by other males. It is possible, therefore, that not a few maternal characters may arise in this way, and that if we could hatch human beings as we can hatch eggs, human beings might be so similar in appearance as to be practically indistinguishable.-' There is still one other view of the matter which I shall briefly notice. If the germinal vesicle be only the 1-5 00th of an inch, and the head of the spermatozoid be only the l-62.50th of an inch in diameter, dare we suppose, on purely physical grounds, that so small a particle of matter as is formed by the union of the two can contain a sufficient number of organic molecules to account in any way for the transmission of hereditary peculiarities 1 The late Professor Clerk Maxwell, in his profoundly interesting article on the Atom in the Encydopcedia Britan- nica, thus states the case — " The first numerical estimate of a diameter of a molecule was that made by Loschmidt, in 1865, from the mean path aud the molecular volume. Independently of him, and of each other, Mr. Stoney, in 1868, and Sir W. Thomson, in 1870, published results of a similar kind— those of Thomson being deduced not only in this way, but from considerations derived from the thickness of soap bubbles, and from the electric action between zinc and copper. " The diameter and the mass of a molecule, as estimated by these methods, are of course very small, but by no means infinitely so. About two millions of mole- cules of hydrogen in a row would occupy a millimetre, and about two hundred million million million of them would weigh a milligramme. These numbers must be considered as exceedingly rough guesses ; they must be corrected by more extensive and accurate experiments as science advances ; but the main result, which appears to be well established, is that the determination of the mass of a molecule is a legitimate object of scientific research, and that this mass is by no means immeasurably small. "Loschmidt illustrates these molecular measurements by a comparison with the smallest magnitudes visible by means of a microscope. Nobert, he teUs us, ^ A. Harvey. 1, Relative influence of male and female parents, Monthly Journal of Mediccd Science, 1854. 2, On the foetus in utero, Edinburgh, 1850. 3, Foetus In utero, Glasgow, 1859. PHYSIOLOGICAL BASIS OF HEREDITY. 243 •can draw 4,000 lines in the breadth of a millimetre. The interval between these lines can be observed with a good microscope. A cube, whose side is the 4,000th •of a millimetre, may be taken as the minimum visible for observers of the present ■day. Such a cube would contain from 60 to 100 million molecules of oxygen or of nitrogen ; but, since the molecules of organized substances contain on an average ■about 50 of the more elementary atoms, we may assume that the smallest organized particle visible under the microscope contains about two million mole- •cules of organic matter. At least half of every living organism consists of water, so that the smallest living being visible under the microscope does not contain more than about a million organic molecules. Some exceedingly simple organism may be supposed built up of not more than a million similar molecules. It is impossible, however, to conceive so small a number sufficient to form a being furnished with a whole system of specialized organs. Thus molecular science sets us face to face with physiological theories. It forbids the physiologist from imagining that structural details of infinitely small dimensions can furnish an ex- planation of the infinite variety which exists in the properties and functions of "the most minute organism. "A microscopic germ is, we know, capable of development into a highly organized animal. Another germ, equally microscopic, becomes, when developed, ■an animal of a totally different kind. Do all the differences, infinite in number, which distinguish the one animal from the other, arise each from some difference in the structure of the respective germs ? Even if we admit this as possible, we shall be called upon by the advocates of Pangenesis to admit still greater marvels. Por the microscopic germ, according to this theory, is no mere individual, but a Tepresentative body, containing members collected from every rank of the long- drawn ramification of the ancestral tree, the number of these members being amply sufBcient not only to furnish the hereditary characteristics of every organ of the body, and every habit of the animal from birth to death, but also to afford a stock ■of latent gemmules to be passed on in an inactive state from germ to germ, till at last the ancestral peculiarity which it represents is revived in some remote descendant. " Some of the exponents of this theory of heredity have attempted to elude the •difficulty of placing a whole world of wonders within a body so small and so ■devoid of visible structure as a germ, by using the phrase structureless germs. (See F. Galton on "Blood Relationship," Proc. Roy. Soc, June 13, 1872.) Now, ■one material system can differ from another only in the configuration and motion which it has at a given instant. To explain diflferences of function and develop- ment of a germ without assuming differences of structure is, therefore, to admit that the properties of a germ are not those of a purely material system." It is since that article was published in 1875, that most of the results ■detailed in Chap. II. (p. 214), have been reached. The opinions expressed by the eminent physicist require modification in the light of more recent research. Neither ovum nor spermatozoid is now to be regarded as destitute of structure, and the tendency of research is to show that the peculiarities of different ova depend on differences of molecular struc- ture. Further, small as the reproductive body formed by the fusion of the male and female elements is, it is still large enough to contain millions of organic molecules having a complexity of structure as great 244 THE PHYSIOLOGY OF THE TISSUES. as that of a molecule of albumin. The physiologist also lays stress on the fact that all the tendenc}- of his -work on these miiu;te structures is in the direction of detecting diffcrmces, not necessarily i)hysical in the sense of being discernible by an}' possible microscope, but differences of a chemical nature — in other words, molecular. Xor arc the tissues so- various, nor the individual peculiarities so numerous, as the phA'sicist is. apt to suppose. ]\Iuch depends on vegetative repetition, so that, given the peculiarities of an}' indiAndual, it is not improbable that a few thousand special types of cell formation, as regards molecular structiu-e,. would be sufficient to accoimt for the special peculiarities. There is. room enough for many such molecules in a cube of idioplasm having a side of l-500th of an inch. For bibliographical references to the subjects discussed iu the three preceding chapters, see a paper by the author, on " The Modern Cell Theory," in the Pro- ceedinrjs of the Philosophical Society of Glasgow, Vol. XX. Chap. IV.— FORMATIOX OF THE BLASTODERMIC LAYERS. Having considered the phenomena that immediately follow the union of the o\iim and spermatozoid, we shall next direct our attention to the earlier differentiation of the embryonal cells into the layers from which the tissues and organs are developed. This can be done Avithout discussing difficult embryological questions, and -wdthout entering much, into detail ; and it is ini})ortant to follow this course, because it will enable us to classify the tissues genetically and to observe relations- existing between tissues in the embryonal condition that would not be suspected when we contrast their characters as seen in fully formed tissue.^ As already explained in Chap. II. of this section, p. 214, the phenomena of fecimdation are followed by segmentation in which the ovum divides, into tAvo, then into four, eight, sixteen, etc., cells (Fig. 111). At the end of the segmentation process, the ovum consists of a sphere formed of primitive cells, or blastomeres, of uniform size, and these cells are arranged in a single layer to form a Avail around a central cavity. The Avail of the oAiim is noAV called the Uastoderrii, or germinal membrane,. and the caAdty in the centre is the segmentation cavity of Von Baer> This is the arrangement in the early development of Amphioxus, the most primitive vertebrate, in Avhich the stages are simple and easily understood. One side or wall of the blastosphere then becomes- thickened and invaginated, so as to give rise to a layer of cells forming a lining to the outermost layer. The embryo has noAv the form of a ^ Details as to the formation of organs and as to the formation of the embryonal vesicles will be given in discussing the reproductive process. It is not necessary to allude to these at present. FORMATION OF THE BLASTODERMIC LAYERS. 24^ sphere, the wall of which is formed of two layers, an outer, termed the epiblast or ectoderm, and an inner, termed the hypoblast or endoderm, and the cavity or space in the sphere, called the archenteric cavity or arch- enteron, communicates with the exterior by a small aperture, the blastopore. At this stage the embryo is termed a (jastnda. The embryo «oon becomes much elongated, so that the blastopore is at first at the posterior end, and a little later on is seen on the dorsal surface. A groove now forms on the dorsal surface of the elongated embryo by the development of two folds of the epiblast. The lips of this groove coalesce so as to form a canal, the neural canal, and the blastopore opens aato this canal by a foramen or passage, cancdis netirentericiis, seen in Fig. 113. This canal is closed at a later period. kri) 'pSC -e -- me Fig. 113. — A. Longitudinal section of Amphioxus. B. Transverse section of Amphioxus. ■ep, epiblast ; hyp, hypoblast ; me, mesoblast ; ps, first primitive segment ; psc, cavity in pi'imitive segment ; ic, archenteric cavity ; n, primitive neural canal ; en, cancdis nev/r- enteHcus. An, anterior ; P, posterior end of embryo. According to E. van Beneden, segmentation occurs in the ovum of the rabbit within one or two hours after the union of the male and female pronuclei, and the whole process is accomplished within seventy to seventy-five hours thereafter. In the segmentation process an early •differentiation of cells has been noticed. Even in the cleavage of the first cell the result is the formation of two cells, one, the upjDer, some- what larger than the other, the lower, and when eight cells have been formed, the four lower cells occupy a more central position than the four upper cells. Further, the upper or outer cells increase more 246 THE PHYSIOLOGY OF THE TISSUES. A. yc- MM g^ epi 'W'-. 4) I J'vp — L fi^ IE ti Fig. 114. — A. Longitudinal section through the blasto- derm of hen's egg at end of first hour. B. Transverse section through the same, epi, epiblast, outer layer ; hi/p, hypoblast, inner layer ; yc, yolk cells ; cf, cres- centic furrow. To see these layers in their natural posi- tion, turn the page so that the epiblast is uppermost. rai)idly than the lower or in- ner cells, so that the outer cells ultimately entirely en- close the inner cells. The in- vagination process producing a gastrula has not been seen in the mammalian o\'T.im, but there exists an aperture which Van Beneden regards as the- homologue of the blastopore. This aperture is soon closed by the growth of cells around it, and the o'saim of a mammal now consists of a dense mass of cells. The outermost layer is composed of cells of a pris- matic shape. The succeeding stages of de- velopment mil be best under- stood if we examine them in the first instance as they maj' be traced in the egg of the common fowl during incuba- tion. The yolk is surrounded by a thin membrane, the vitelline membrane, and at one point of its siuface a small white disc, about 4 mm. in diameter, may be seen. This is the blastoderm or cicatricula, corresponding to the layer of cells which entirely surrounds the ovum of the mammal. In the centre of the blastoderm there is a clear transparent portion, the area pellucida, in which the embryo is formed. A vertical section through the area pellucida of a fecundated egg, in which segmentation has. taken place, shows that the blastoderm is formed of two layers. The upper layer con- sists of somewhat elongated FORMATION OF THE BLASTODERMIC LAYERS. 247 cells, having their long axes vertical, and adhering by their sides. Those cells are of uniform size, about 9 /x, and each shows usually an oval nucleus. This layer constitutes the epiblast. The deeper layer of the blastoderm, termed the hypoblast, consists of somewhat larger cells, so highly granular as to conceal the nucleus. Between the blastoderm and the underlying yolk there is a space, the homologue of the segmen- tation cavity to which reference has already been made. See Figs. 114 and 115. After a few hours of incubation, the area pellucida loses its circular Fig. 115. — Three transverse sections through an egg in whicli the medullary ridge is beginning to form. The sections show the stages in the development of the chorda dormlis, and the formation of the two layers of the mesoblast. ep, epiblast ; hyp, hypoblast ; pi, parietal layer of mesoblast ; vl, visceral layer of mesoblast ; mp, medullary plates ; 7/i/, medullary folds ; c/i, chorda; 6c, body cavity or pleuro-peritoneal cavity. outline and becomes oval and then pear-shaped, and toAvards the narrow end of the pear-shaped area an opaque thickened portion makes its 248 THE PHYSIOLOGY OF THE TISSUES. appearance. This is the primitive streak or trace. A groove — the primitive groove — then appears on the iipper surface of the streak. Abont this period, that is to say, between the eighth and twelfth hour of incubation, a middle hxyer of the blastoderm — the wesohlast or 7nemderm — begins to be formed. The primitive streak is a mass of cells arising from proliferation of cells derived from the epiblast, and may be regarded as the commencement of the middle la3"er in connection Avith the epiblast. About the sixteenth hour, tAvo ridges or folds of the epiblast rise on each side of the primitive streak. These ridges, termed the dorsal ridges or medidlary folds, enclose plates known as the meduHary plates, and the ridges ultimately coalesce so as to form a closed tube — the neural canal — giving origin to the primitive cerebro-spinal axis. Below the medul- lary tube thus formed, the chorda dorsalis or notochord, an elongated cartilaginous rod, appears, and this becomes the centres of the bodies of the permanent vertebrae and the basis of the cranium. Some of these structures are seen in Fiais. 115 and 116. Fig. 1H3. — Two transverse sections through the embryo of a Xewt. A, Transverse section through the body, in which the neural canal is not yet closed and the primitive segments are beginning to separate from the ventral lamina;. B, Transverse section in which the neural canal is closed and the primitive segments are fully formed, mf, medullary folds ; Tii^), medullary plate ; n, nervous tube; ch, chorda dorsalis; ep, epiblast; pi, parietal layer of mesoblast ; vl, visceral layer of mesoblast ; ic, intestinal canal ; be, body cavity ; psc, cavity in primitive segment ; yc, yolk cells. FORMA TION OF THE BLASTODERMIC LA YERS. 249 As already pointed out, the mesoblast arises chiefly from the deeper layer of cells of the epiblast, but according to F. M. Balfour and His, it proceeds also in part from the hypoblast and from nuclei in the germinal Avail of the yolk at the outer surface of the latter. The mesoblast, in turn, divides into two layers, an outer, termed the somatic or joarietal mesoblast or somatopleure, which uniting with the epil^last gives origin to the osseous, fibrous, muscular, and tegumentary substance of the body- Fig. UV. — Diagram showing the development of the meso- blast and of the body cavity in the vertebrata. Trans- verse section through the blastopore of an embryo, bp, blastopore ; jnc, primitive intestinal cavity ; he, body cavity ; y, yolk ; epi, epiblast ; pi, parietal layer of mesoblast ; vl, visceral layer of mesoblast. Fig. lis. — Diagram of the aevelopment of the mesoblast and of the body cavity of a vertebrate. Transverse section through an embryo in front of the blastopore, i/ip, medullary plate ; ch, beginning of the chorda dorsalis ; ep, outer germinal layer or epiblast ; hyp, inner germinal layer or hypoblast ; pi, parietal layer of middle germmal layer or mesoblast ; vl, visceral layer of middle germinal layer or mesoblast; y, yolk ; ic, intestinal canal ; be, body cavity. wall and limbs ; and an inner, the visceral mesoblast or splanchnopleure, which, uniting with the hypoblast, forms the fibrous and muscular wall 250 THE PHYSIOLOGY OF THE TISSUES. of the alimentary canal, the hnnph and blood-vascular system, and the urinarj' and generative organs. Between the two layers of the meso- blast we have the general pleuro-peritoneal space or the body-cavity. The formation of these layers is well shown in Figs. 117 and 118. The mesoblast in amphioxus, instead of arising from the epiblast, originates in two diverticula or recesses constricted off from the archen- teric cavity, and the notochord is also developed from a third diverti- culum springing from the dorsal aspect of the archenteric cavity, a type of development met A\ath in various invertebrate forms. This is shown in Fig. 119. Pio. 119. — Transverse section through embryo of Amphi- oxus. ep, epiblast ; me, mesoblast ; hyp, hypoblast ; mp, merlullary or neural plate ; ch, chorda dorsalis ; ac, archenteric cavity ; be, body cavity. The vesicular form of the blastoderm of mammals, arising from the segmentation of the entire yolk instead of segmentation of only a por- tion of it, as occurs in the egg of the common fowl, causes certain modifications in the development of the layers. As already described, the covering of the ovum consists of a layer of elongated nucleated cells (Fig. 115), and in the interior there is a semi-fluid mass of broken down yolk substance and some of the primitive granular cells, or segmental sjiheres. The latter by proliferation form a layer adapted to the outer- most layer, and thus the wall of the vesicle soon has two layers. In the rabbit, about the fifth day, and when the inner layer of cells has spread over about one half of the vesicle, a discoidal thickening or opacity appears in the middle of the blastoderm. This is the embryonal area seen in Fig. 1 20, and in it the primitive streak and primitive groove appear as in birds, and then follows the development, in a similar manner, of the nervous system and of the chorda dorsalis. The formation of the three layers of the embryo having thus been traced, it remains to refer to these layers the genesis of the tissues and of the more important organs of the body. FORMATION OF THE BLASTODERMIC LAYERS. 251 1. The EpiUast gives rise to {a) the central nervous system ; from the infolding of the medullary plates to form the neural tube (see Fig. 116), the white and grey matter of the brain and spinal cord is formed ; Fio. 1120. — Views of the Blastodermic Vesicle of a rabbit on the seventh day, witliout the znna 'pellucida, or outer layer of the egg. A, From above ; B, from side, ag, area, iu which embryo appears ; ge, boundary of the hypoblast. and (Jj) the sympathetic nervous system and the peripheral nerves, both cranial and spinal, and ganglia, are also derived from the epiblast. (c) The ciliated epithelium in the central canal of the spinal cord and in the cerebral ventricles is epiblastic. (d) The epiblast gives origin to the epidermis and to all structures of epidermic nature, such as horn, hoof, nail, feather, etc., and also to the sebaceous and sweat glands. (e) The sensory expansions in the organs of special sense originate in the epiblast, either by involutions of the medullary or neural canal (retina and pigment epithelium of choroid), or by involutions of the ex-' ternal epiblastic layer covering the embryo (the sensory structures in the ear, in the nose, taste cells, touch corpuscles). (/) The crystalline lens is formed of infolded epiblast. {g) The epithelium lining the cavity of the mouth and anus, the epithelium of the glands and other structures related to these cavities, the enamel of tooth and the salivary glands are derived from the epiblast. (li) Lastly, the pituitary body belongs to this layer. 2. The Mesohlast gives origin to (a) the connective tissues ; (b) the muscles ; (c) the bones and cartilages ; (d) the heart, arteries, veins, capillaries, lymphatics, and serous membranes, with their lining cells or endothelium ; and (e) the embryonic blood and lymph corpuscles. From it also arise (/) the generative organs, their epithelium, and the genera- tive elements, ova, and spermatozoids ; (g) the urinary organs and. a. 252 THE PHYSIOLOGY OF THE TISSUES. \YAvt of the epithelium of the tubules of the kidney ; and {h) the mus- •cvilar, vascular, and coiuiective tissue elements in the wall of the alimentary canal and in the skin. The muscular fibres of the sweat glands of the skin are said to be epiblastic. (/) The spleen and other blood glands also spring from this layer. 3. The Hypoblast is the layer from which most of the epithelial struc- tures are derived, such as {a) the epithelium of the alimentary canal ; •(6) the epithelium of the resjiiratory organs, trachea, bronchial tubes, and pulmonary air cells ; {r) the epithelium of the Eustachian tube and tympanum of the ear ; { o J _ view. Then use the fine adjustment until complete distinctness of the object has been attained. Thereupon let the left hand hold the slide, and the right hand rest on the micrometer screw. As Ave see clearly only the points of the preparation lying in one plane, scrutinize the preparation during the delicate raising and lowering of the tube, i.e. during the slow turning of the micrometer screw. In addition to this, keep both eyes open during microscopic investigation. The delineation or sketching of microscopic objects is an invaluable aid to correct observation. Thus many details are shown which might otherwise have been completely overlooked. Even the most attentive contemplation cannot supply the advantages which drawing the objects METHODS OF MICROSCOPICAL RESEARCH. 259 commands. Endeavour also to sketch objects seen by low and high powers. In drawing, place the paper on the level of the stage, look into the microscope with the left eye, and with the right look on the paper and the point of the lead pencil. At first this occasions a little ■difficulty ; after some practice we rapidly acquire the necessary dexterity. When it is necessary to make an exact facsimile drawing of an object, a camera htcida should be used, of which by far the most ■convenient is that shown in Fig. 128. Fig. 128. — Abbe's Camera Lucida, made by Zeiss. A, Part screwed over the eyepiece. B, Mirror, reflecting light, coming from paper in direction S^ , at right angles in direction Sp — IF. This light is caught by the prism and reflected in direction of 0, the same direc- tion as that followed by the rays coming from the object in the direction AO. 2. Accessory Appliances.- sites for histological work — -The following are convenient requi- (1) A good razor, the blade of which has been flattened on one side. The iblade must always be kept sharp and must, previous to actual use, be passed over the strop. Let the razor be employed exclusively for the cutting of fine sections. (2) A fine ivhetdone. (3) A neat straight 2iciir of scissors. (■4) A delicate, easily shutting pair of forceps, with smooth or only slightly indented prongs. (5) Four needles with wooden handles. Let two of them be heated until they •can easily be bent ; let them be again heated, and then stuck into solid paraffin, by which they will again be hardened. In fine teazing, the needles must be sharpened and polished first on the whetstone, and then on the strop. The cataract needles of oculists are very useful. (6) A flat spatida, made of German silver, for lifting the sections from fluids on ito the slide. Instead of this, a broad-bladed knife may be used. One of those in & case of anatomical instruments suits quite well. (7) Pins, hedge-hog bristles, a small paint brush, a brush for dusting glasses. (8) Slides (of the usual shape) should be made of pure glass, and not too thick ((1-1 "5 mm.). (9) Cover-glasses of 15 mm. broad are for most objects sufficiently broad ; their thickness may vary from O'l to 0"2 mm. 260 THE PHYSIO LOG V OF THE TISSUES. (10) Ghi)^s phials to the number of a dozen, with a wide neck, of the capacity of 30 c.c. and upwards. (11) Some lar(jir preparation (jlasxi'^, with ground-glass stoppers; height, 7-10 cm. ; diameter, 6-10 cm. (12) A graduated cylindrical glaK-t, holding 100-150 com. (13) A glass funnel of 8-10 cm. at its upper diameter. (14) Pipette. The student can supply himself with pipettes if he draws off to- a lioint at one end, in the blow-pipe flame, a small glass tube about 1 cm. in diameter and 10 cm. in lengtli, and fixes on the other end a piece of india-rubber tubing, ft cm. long, tied with a strong packthread. (15) One dozen vatch glasses of 5 cm. in diameter. (16) One dozen, small reagent glasse.i of the capacity of 10 cm. in length, and of 12 mm. in width. (17) Glass rodx of 3 mm. thick, 15 cm. long, drawn to a point at one end. (18) Preparation dishes with glass lids of 10-12 cm. in diameter. (19) Txco sheets of blotting paper, a cork plate, large and s7naU gummed carder soft linen rags, a toirel. (20) A large earthenware pot for refuse. 3. Reagents. — It is not advisable to keep reagents in store in too- great qnantities, for many of them become decomposed in a compara- tivel)' short time. Reagents must be i)rocured shortly before use. Every flask must be provided with a large label indicating its contents. It is recommended that not only should the prescription of the parti- cular fluid be indicated on the label, but also the method of employing it. All the flasks should be closed tightly with corks or with good glass stoppers. The fluid must not reach to the under surface of the cork. ( 1 ) Distilled water, 3-6 litres. (2) Solution of common salt, 0'75 per cent. : distilled water, 200 c.cm. ; common salt, 1 "5 grms. The coi'k of the flask must be provided with a glass rod reaching down to the bottom of the Hask. The liquid decomposes readily, and must be frequently prepared anew. (3) Alcohol — (a) Absolute alcohol. Of this, it is necessary to have 200 c.cm. in,- readiness. The absolute alcohol of commerce is of the strength of 96 per cent., and it is, in the majority of cases, at ouce available for microscopic purposes. If,. however, anyone wishes to preserve in alcohol completely free of water, he must throw into the flask some small pieces (15 grms. to 100 c.cm. of alcohol) of dried sulphate of copper (CUSO4) that has been heated to a white heat. In the event of its turning blue, it must either give j)lace to a fresh supply, or be burned anew. Newly burnt chalk likewise serves a like purpose, only its operation is more pro- tracted, (b) Pure spirit (strength, 90 per cent, of alcohol), 3-5 litres ; may be called 90 per cent, alcohol, (c) 70 per cent, alcohol. 500 c.cm. are to be made by the mixture of 390 c.cm. of 90 per cent, alcohol with 110 c.cm. of distilled water. (d) Ranvier's third-part alcohol, or, shortly, Ranvier's alcohol. 45 c.cm. of 90 per cent, alcohol + 85 c.cm. of distilled water. (4) Acetic acid,, 50 c.cm. The kind used by chemists is 33 per cent, strong. METHODS OF MICROSCOPICAL RESEARCH. 2G1 (o) Acetate of iron (that purchasable in chemists' shops is 96 per cent, strong) must be inspected shortly before use (amount, 10 c.cm.). (G) Nitric acid. It is necessary to possess a iiask containing 100 c.cm. of con- centrated nitric acid of specific gravity, 1"18. (7) Pure hydrochloric acid, 50 c.cm. (8) Chromic acid. Make a 10 per cent, solution (10 grms. of newly procured •crystallized chromic acid to be dissolved in 90 c.cm. of distilled water). Of this, make (a) a solution of chromic acid, '1 per cent. (10 c.cm. of the strong solution to 390 c.cm. of distilled water), and (b) a "5 per cent, solution of chromic acid (50 c.cm, of the first solution to 950 c.cm. of distilled water). (9) Bichromate ofjyotash. Dissolve 25 grms. in 1,000 c.cm. of distilled water. It dissolves slowly (from three to six days). (10) Midler's fluid, 30 grms. of sulphate of soda and 60 grms. of pulverized bichromate of potash are dissolved in 3,000 c.cm. of distilled water. The solution is effected slowly at the ordinary temperature of a room (from three to six days), (11) Picric acid. Keep 50 grms. of crystals, and also a saturated aqueous solution <500 c.cm). The crystals must always be in a layer, from 2 to 3 mm. in depth, at the bottom of the flask. It dissolves easily. (12) Sulpho-picric acid (Kleinenbei-g). Into 200 c.cm. of satvirated solution of picric acid, pour 4 c.cm. of pure sulphuric acid ; a dense precipitate im- mediately forms. After the lapse of an hour, this mixture is filtered, and the filtrate is diluted with 600 c.cm. of distilled water. The residuum left behind in the filter is to be thrown away. (13) Osmic acid. Purchase 50 c.cm. of 2 per cent, aqueous solution. It must be kept in a dark place or in a dark glass, and, when well closed up, it keeps for many months. (14) Chromo-osmium acetic acid. Prepare a solution of chromic acid, 1 per cent, strong (5 c.cm. of a 10 per cent, solution to 45 c.cm. of distilled water), and pour into it 12 c.cm. of 2 per cent, solution of osmic acid, and add to it 3 c.cm, of acetic acid. This mixture need not be kept in the dark, and is available for use for a long time. (15) Nitrate of silver. Procure, a short time previous to using it, a solution of nitrate of silver, 1 grm. in 100 c.cm. of distilled water. The fluid must be kept in the dark or in a black bottle. It keeps for a long time. (16) Chloride of gold. Procure shortly before use a solution of 1 grm. of ■chloride of gold in 100 c.cm. of distilled water. Must be kept in the dark or in a black or dark brown flask, (17) Formic acid, 50 c.cm., for chloride of gold staining. (18) Concentrated (35 per cent.) caustic potash, 30 c.cm. The bottle must be •closed with an unvulcanized India-rubber stopper which is penetrated by a glass rod. (19) Glycerine. It is necessary to have at hand 100 c.cm. of pure glycerine, as well as a solution of 5 c.cm. of pure glycerine in 25 c.cm. of distilled water. As a safeguard against fungi, which speedily appear in this mixture, there may be added to it 5 to 10 drops of a pure 1 per cent, solution of carbolic acid. The cork of the bottle must be provided with a glass rod, as also in the case of — (20) Lavender oil, 20 c.cm. Oil of cloves serves the same purpose. (21) Dammar varnish. It can be bought in bottles, containing 50 c.cm., from drysalters, and, when too viscid, be diluted with pure oil of turpentine. It possesses the requisite degree of consistency when, if a glass rod be dipped in it, the drops fall off without dragging long threads after them. Dammar 262 THE PHYSIOLOGY OF THE TISSUES. varnish is to be preferred to the too strongly refractive Canada balsam (which is diluted with chloroform), but it possesses the drawback of drying very slowly, while Canada balsam dries quickly. The cork of the flask must be pro- vided with a glass rod. (22) Cemant for corer-r/last. Venetian turpentine may be diluted with sulphuric- ether until the whole forms a fluid trickling easily in drops ; next it is filtered while hot in a funnel surrounded by hot water, and the filtrate is thickened on the sandbath. The proper consistence has been attained when a drop of it placed by a glass rod on a slide immediately becomes stiffened to such an extent that it can no longer be compressed by the finger nail. It may be prepared in a chemist's- as a safeguard against danger from burning. Zinc-ivhite is also an excellent cement. A.^phalt-varjiish cannot be recommended. (23) Hcemafoxi/Un. (1) 1 grm. of crystallized hamatoxylm is dissolved in 10 c.cm. of absolute alcohol. (2) 20 grms. of alum are dissolved in 200 c.cm. of dis- tilled water, when warm, and filtered after cooling. On the following day, both solutions are mixed, and they are allowed to stand for eight days in a wide- mouthed vessel. The mixture is then filtered,^ and is ready for use. Diminution of transparency from development of fungi does not in the least injure its useful- ness. (24) Weigert's Juemafoxy/ht, for the demonstration of medullated nerve fibres of the brain and spinal marrow. 1 grm. of crystallized htematoxylin is placed in 10 c.cm. of absolute alcohol + 90 c.cm. of distilled water, and then boiled. After cooling, there is added to it 1 to 2 c.cm. of a solution — saturated when cold — of carbonate of lithium. The employment of this reagent demands previous treatment with (o) Saturated solution of neutral acetate of copper, 300 c.cm., and a subsequent- treatment with a {!)) Solution of ferridcyanide of jjotasdum aiul ofhorax. 2 grms. borax and 2i grms. of ferridcyanide of potassium are dissolved in 100 c.cm. of distilled water. (25) Neutral solution of carmine. 1 grm. of the best carmine is dissolved cold in 50 c.cm. of distilled water + 5 c.cm. liquor ammonite. The brilliant cherry-red fluid remains exposed until it no longer yields an odour of ammonia (two days) and it is then filtered. To be kept ready at hand. The odour of this solution may become very off'ensive, but its colouring power is not thereby injured. (26) Picrocarmine. Pour into 50 c.cm. of distilled water 5 c.cm. of liquor ammoniffi, and throw into this mixture 1 grm. of the best carmine. Stir round with a glass rod. After the solution of the carmine has been completed (in about five minutes), pour into it 50 c.cm. of a saturated solution of picric acid, and then let the whole stand for two days in a wide-mouthed vessel. Then filter it. Even abundant development of fungi does not injure the colouring power of this- excellent reagent. (27) Borax carmine. 4 grms. of borax are dissolved in 100 c.cm. of warm dis- tilled water. After the solution has cooled, 3 grms. of good caiunine are added during stirring, and then 100 c.cm. of 70 per cent, alcohol are poured in. After the lapse of twenty -four hours, filter the fluid, which drops very slowly, taking twenty- four hours and even longer in passing through the filter. The staining by borax car- mine demands siibsequent treatment with 70 per cent, hydrochloric-acid-alcohol,. 1 After the cooling of the alum, as well as after the haematoxylin-alum mixture has stood exposed for eight days, there are found (especially at a lower temperature ) alum crystals at the bottom of the vessel, which are not of further use. METHODS OF MICROSCOPICAL RESEARCH. 263 which is prepared by means of the addition of 4 to 6 drops of pure hydrochloric acid to 100 c.cm. of 70 per cent, alcohol. (28) Safranin. 2 grms. of the dye are to be dissolved in 60 c.cm. of 50 per cent, alcohol (33 c.cm. of 90 per cent, alcohol + 27 c.cm. of distilled water). Staining with safFranin requires subsequent treatment with absolute hydrochloric- acid-alcohol (8 to 10 drops of pure hydrochloric acid to 100 c.cm. of absolute alcohol). Both may be kept in readiness. (29) Eosin. 1 grm. of the dye to be dissolved in 60 c.cm. of 50 per cent, alcohol (33 c.cm. of 90 per cent, alcohol + 27 c.cm. of distilled water). (30) Vesuvin, or (31) Methyl- violet, B., may be kept in readiness in saturated watery solu- tions (1 grm. to 50 c.cm. of distilled water). 4. Preparation of Tissues and Organs. — The minutest organs of the animal frame are so constituted that they are immediately access- ible to microscopical investigation. They must possess a certain degree of transparency, which we effect either by dividing the organs into small portions — isolating the elements, or by cutting them into sections — dissecting them. But on the other hand the minutest organs may not possess a consistency Avhich permits the immediate prejmration of fine sections. They may be too soft (in which case they must be hardened), or too hard, from the presence of earthy matter (in which case they must be decalcified). As hardening and decalcification cannot be exercised on objects which are fresh without injuring their structure, both methods must be preceded by a process which renders possible a rapid harden- ing or stiff"ening and also a permanent hardening of the minutest particles. This process is termed fixing. The preparation of fine sections is accordingly possible, only after the previous fixing and hardening of the objects concerned (decalcification eventually following). But the sections also demand further manipulation. They may either be made transparent immediately by means of appliances for rendering- objects clear (which are even applied with success to fresh objects), or they m.ay be coloured or stained before clearing. Colouring matters furnish invaluable aids to microscopic research, as they admit of appli- cation to fresh, and even to living, organs. A great number of the most important facts have been discovered only by means of their helj). Colouring matters syringed into vessels (injected) show their mode of division and the course of the finest ramifications of the capillaries. 1. Nature of the Material. — For studies of the elements of form and of the so-called "simple tissues," we recommend amj^hibia, such as frogs and salamanders (the spotted salamander, whose elements are very large, being the best). On the other hand, the mammalia are re- commended for the studies of the organs. In many cases rodentia suffice (rabbit, guinea-pig, rat, mouse), or the tissues and organs of 264 THE rilY^WLOLlY OF THE TISSUES. young clogs, cats, etc., may be employed. Nevertheless no one should neglect any opportunity of acquiring the organs of the human sub- ject. Fresh material may be obtained from the surgical clinic. In general, it is advisable to collect the organs before the vital heat has departed. In order to do this Avith the utmost possible despatch, it is recommended to fill, first of all, with the suitable tiuid, the jars selected for the reception of the objects, and to pro- vide labels indicating, along Avith the object, the name of the fluid and the date. In the next place, let the instruments for dissection be arranged, and not until then should the animal be killed. 2. Killinfj and Disscdion of Animals. — In the case of amphibia, the operator should, with a strong pair of scissors, sever the verte- l)ral column at the neck, and destroy the brain and the spinal marrow by means of a needle thrust from the wound upwards into the cavity of the skull, and downwards into the vertebral canal. In the case of mammals, the operator should cut through the neck Avith a vigorous incision reaching to the vertebral column, or he may kill them with chloroform, which is poured upon a cloth and pressed against the noses of the animals. Small animals (4 cm. large), or embryos, may be thrown entire into the fixing fluid. After the lapse of six hours, open the abdomen and thorax hj incisions. It is easy with a strong pin to fix the bodies of small animals by the sole of the foot on to plates of cork or wax. The organs must be neatly removed, best with forceps and scissors ; squeezing and pressing of the parts, and taking hold of them by the fingers, are to be altogether avoided. The forceps should grasp the edge only of the object. Adhering im- purities — mucus, blood, and the contents of the intestines— must not be scraped off" with the scalpel, but must be removed by slowly swinging the part in the fixation-fluid used in the process. By the methods specitied in the sequel, it is unavoidable that scissors, forceps, needles, glass rods, etc., should be -wetted by the most diverse kinds of fluids — with acids for example. The instruments should be cleaned immediately after use by being rinsed in water, and wiped dry. The operator should avoid above everything dipping into another fluid a glass rod soiled, for example, with an acid or with a colouring material. Overlooking this circumstance often leads to the reagents being decomposed, or it may entirely nullify the success of the subsequent preparations. Glasses, watch glasses, etc., are easily cleaned when it is done immediately after use. If, on the other hand, the operator allows, for example, the residuum of a colouring material to dry up in a glass, the cleaning of the latter always entails a tedious consumption of time. The operator should never neglect to clean glasses immediately after use ; watch glasses at least may be throwTi into a vessel of water. All vessels in which the operator isolates, fixes, hardens, colours, etc., must be kept close (watch glasses may be METHODS OF MICROSCOPICAL RESEARCH. 265 covered with a second -watch glass if the duration of the manipulations lasts more than ten minutes), and should not be exposed to the sun. 3. Isolation. — We isolate either by teazing fresh objects, or by teazing, after previous treatment of objects with fluids which dissolve some elements of the tissue. Sometimes such fluids render teaz- ing altogether or partially unnecessary. It is a difficult under- taking to make a good preparation by teazing. Much patience and scrupulous fulfilment of the following directions are indis- pensable. The needles must be sharp at the point and quite clean; previous to use, they should be sharpened and polished on the moistened whetstone. The small object (at most 5 mm. broad) is now l^laced in one small drop upon the slide, and, if it is coloiu:'less, torn asunder on a black background, but if it is dark (coloured somewhat) the operation is performed on a white background. If the object is fibrous {e.g. a bundle of muscular fibres), the operator should place both needles on the one end of the bundle, and teaze, in the direction of its length, into two bundles ; one of these bundles is in the same way divided in turn into two bundles by means of placing the needles on the end, and so on, until very fine single fibres are reached. By observation of the (uncovered) ^preparation with a low magnifying IDOwer, we can ascertain whether the necessary degree of fineness has been reached. As isolating fluids there are to be recommended — (a.) For Eplthdia. Ranvier's alcohol is a useful fluid for isolation. Place sideways into 10 com. of this fluid a small piece, say of the mucous mem- brane of the intestines, of 5 to 10 mm. After the lapse of five hours (in the case of stratified pavement epithelium, after the lapse of ten to twenty-four hours or more), the small pieces are cautiously and slowly lifted out with a pair of forceps, and twice dashed gently against the slide, which has been covered with one drop of fluid. In consequence of the impact, many epitheliimi cells fall off' isolated, or in groups, wliich require only to be gently stirred round with the needle to secure complete isolation. Place a cover -glass over the preparation, and examine with the microscope. If the experimenter wishes to colour the object, he takes the small pieces en masse carefully out of the alcohol and places them into 6 c.cm. of picrocarmine. After two to four hours, the small piece is placed very carefully into 5 c.cm. of distilled water, and five minutes after is transferred to a slide and placed in a drop of diluted glycerine. Then put on the cover-glass. The preparation may be preserved. (h) For Muscular Fibres or Glands. These require 35 per cent, solution of caustic potash. Small pieces from 10 to 20 mm. broad are immersed in 10 to 20 c.cm. of this fluid. After the lapse of nearly an hour, the small pieces have fallen asunder into their elements, which are fished out with needles or with a pipette, and examined in a drop of similar caustic potash under a cover-glass. Diluted caustic potash operates in an altogether different manner. The elements are soon destroyed in dilute caustic potash. If the caustic potash is too old, isolation 266 THE PHYSIOLOGY OF THE TISSUES. does not succeed ; instead of it a pulpy softening of the small pieces sometimes occurs. For this reason, freshly-prepared solutions should always be employed. Even successful pi'eparations of this kind cannot be preserved. There is further required a mixture of chloride of potassium and of nitric acid. This is prepared by throwing into 20 com. of pure nitric acid such a quantity of chloride of potassium (5 grms.) that an undissolved sediment remains at the bottom. After the lapse of fourteen hours (sometimes earlier, often later), the object is sufficient!}' loosened, and it is now transferred into 20 c.cm. of distilled water, in which it remains one hour, although it may remain in it even eight days without injury. It is then transferred to a slide, where it may with ease be teazed in a drop of dilute glycerine. If the nitric acid has been well washed out, the preparations admit of preservation, and even of being coloiired under the cover-glass. Placing small pieces not yet teazed in picrocarmine does not succeed, as this colouring liquid makes objects brittle. (c) For (he Ducts and Ductlet>s of Glands. Place small pieces (from 1 cm. broad) into 10 c.cm. of pure hydrochloric acid. After the lapse of ten to twenty hours, the small pieces are immersed in 30 c.cm. of distilled water, which must be changed several times within twenty-four hours. The isolation is then success- fully accomi^lished by means of the cautious spreading out of the small piece with needles in a drop of dilute glycerine. The preparation may be preserved. 4. Fixing — General Hales. — (1) For fixing, there must always be em- plo3'ed a copious amount of fluid — more than 50 to 100 times the volume of the object to be fixed. (2) The liquid must be always clear; it must, immediately after it has become turbid, be changed, i.e. its place must be supplied by fresh fluid. The turbidity often, indeed, occurs one hour after immersion. (3) The objects to be fixed should 1)6 of the smallest possible size ; they should in general not exceed 1 to 2 c.cm. Should the preservation of the entire object be necessary, (say for subsequent adjustment, so that sections may be cut of the complete organ in its natural position) make in it several deep incisions five to ten hours after the first immersion. In the case of the fixing of delicate objects, the experimenter should place a thin layer of wadding (1 cm. in thickness) at the bottom of the vessel. 1. Ahiiolufe alcohol is very well adapted for glands, skin, and blood-vessels. It is used similarly for hardening. Objects placed in absolute alcohol may be cut after twenty -four hours.^ On this account it is specially adapted for the rapid mounting of preparations. The following directions must be observed— (I) The absolute alcohol must be changed after three or four hours, even if it is not turbid ; (2) the operator must take care that the immersed objects are not packed closely or allowed to stick fast to the bottom of the glass.- On this account we either suspend the objects in alcohol by a thread, or place a small pad ^ The working up of objects fixed in absolute alcohol should not be delayed too long, as the elements gradually deteriorate. Cut after the lapse of one to eight days. - The places in question, on being cut, appear strongl}- compressed. METHODS OF MICROSCOPICAL RESEARCH. 267 of cotton wool at the bottom of the glass ; in many cases frequent shaking of the glass suffices. Alcoliol not absolute (say 90 per cent.) operates differently, as it shrivels ob- jects, and cannot on that account be employed as a substitute for absolute alcohoL 2. Chromic acid is employed principally in two aqueous solutions — [a) as a solu- tion from O'l to 0"5 per cent. It is especially adapted for organs which contain much loose connective tissue. This powerful solution gives to connective tissue a strong consistency, but it labours under the disadvantage that it renders colour- ing difficult. Objects remain in it from one to eight days ; they are then placed from three to four hours under a water tap, or if that is impossible, they are placed for the same period in water, which must then be three or four times- changed ; they are then transferred for some minutes into distilled water, and lastly they are hardened, while daylight is excluded, in alcohol of gradually in- creasing strengths.^ {h) As 0'05 per cent, solution, which is prepared by diluting the O'l per cent, solution with an equal amount of distilled water. Procedure as with solution (a) ; objects may remain twenty-four hours in solution (6). Chromic acid solutions penetrate slowly ; consequently it is necessary, in a pro- cess of twenty-four hours' duration, that only small pieces (o to 10 mm. broad) should be immersed in them. 3. Kleinenherg's sidpho-plcric acid (p. 261). Delicate objects (embryos) are immersed for five hours in this fluid ; the more solid parts for twelve to twenty hours. Then, with a view to hardening, transfer icitliout the previous thorough washing in water to alcohols of increasing strength. 4. Al idler'' s fluid (p. 261). Objects are placed in large quantities (400 c.cm.) of this solution from one to six weeks. Thereafter, for four to eight hours, they are thoroughly washed in water (flowing when possible), rinsed for a short time in dis- tilled water, and lastly, daylight being excluded, they are placed within alcohols- of increasing strength. " He ivho does not ivith scrupulous conscientiousness follow the ge)iercd rules above given for hardening fails to attain success, for which failure the innocent Midler's solution is then made responsible even by otherwise experienced microscopists. " (Stohr. ) 5. Osmic acid solution (p. 261). By the use of this solution the operator avoids inhaling vapours very irritating to the mucous membranes. We fix, either by means of the immersion of very small (5 mm. broad) pieces in the acid (mostly employed in the 1 per cent, solution), which is to be diluted to the extent of one half with distilled water, and which is usually employed only in small quanti- ties ; or we fix by exposing the moistened object to the vapour of osmic acid. To attain this, pour 1 c.cm. of 2 per cent, solution into a small test-tube to the height of 5 cm. ; add the requisite amount of distilled water, and fasten the object with hedgehog bristles to the lower surface of the cork with which the test-tube must be firmly closed. After the lapse of ten to sixty minutes (always according to the size of the object) the small piece is taken out and at once thrown into the liquid contained in the small glass. In both cases the objects continue for twenty-four hours in the acid ; at the same time the glasses must be well closed and kept in the dark. The objects are then taken out, rinsed for a couple of minutes in distilled water, and hardened in alcohols of increasing strength. ^ By this term is understood first, Ranvier's third-part alcohol ; then 70 per cent, alcohol ; then 90 per cent, alcohol, and lastly, if necessary, absolute alcohol. 268 TJIE PHYSIOLOGY OF THE TISSUES. 6. Chroiiio-O'iminm acetic arid (p. "2(51) is an excellent fluid for the fixation of the parts of the nuclens. Place small pieces (2 to 5 mm. broad), quite fresh, and still warm irilh rifal heat, into 4 c.cm. of this liquid, in which they remain one day ((two days are preferable), but in which they may continue even longer. The small pieces are then thoroughly washed for the space of one hour or longer iu flowing water (where possible), rinsed in distilled water, and hardened in alcohols of increasing strength. The I'lqnixhonce uHcd for fixation are of no further use : they should be thrown awai/. o. Hardening. — The best fluid for hardening is alcohol of gradually increasing strengths. Here also the rule that Ave should employ liquid co])iously holds good — as Avell as that of changing alcohol Avhich has be- come turbid or coloured.^ The exact process is as follows — After the objects have been fixed (in one of the fluids above enumerated) and thoroughly washed in Avater,- they are transferred for twelve to twenty hours into 70 per cent, alcohol, and after the lapse of this period they are placed in 90 per cent, alcohol, in which the hardening is completed iifter the further lapse of twenty -four to forty-eight hours ; objects may continue in this alcohol for months. The 90 per cent, alcohol that has been employed for hardening may be afterwards used for the hardening of the liver (to be used as an embedding substance to facilitate the cutting of sections), or for burning in a spirit lamp. 6. Decalcification. — Objects to be decalcified must not be immersed fresh in the decalcifying fluid ; they must, on the contrary, be previously flxed and hardened. For this end, place small bones (up to the size of the metacarpus) and entire teeth, and pieces saAved off" from the larger bones (from 3 to 6 cm. long), into 300 c.cm. of Miiller's fluid, and thence, after the lapse of tAvo to four Aveeks (and after prcAdous thorough Avashing), into 150 c.cm. of alcohol of gradually increasing strength. After the small bone has continued for three days or longer in 90 per cent, alcohol, it is transferred to the decalcifj^ing .solution, Aaz., diluted nitric acid (pure nitric acid, 9 to 27 c.cm. added ^ The pieces that have been fixed in chromic acid and in ]M tiller's fluid giA'e ofi' €veu while in alcohol, substances which, by the simultaneous influence of daylight, appear in the form of precipitates. If, on the other hand, the alcohol is kept in the dark, no precipitates are produced, but the alcohol assumes a yellow colour, remaining free from tm-bidity. On this ground the exclusion of daylight has been recommended ; it is sufficient to place the glasses in a dark part of the room. The 90 per cent, alcohol also, so long as it still remains intensely yellow, must be changed once every day. - Objects fixed in sulpho-picric acid are excepted ; these are directly transferred from this fluid into 70 per cent, alcohol. In this case the 70 per cent, alcohol must be changed several times during the first day. METHODS OF MICROSCOPICAL RESEARCH. 2G9 to 300 c.cm. of distilled water). Here also, large quantities (300 c.cm. at least) must be employed, which, at the beginning, must be changed daily, but, subsequently, every four days until the decalcification is accomplished. We hasten the process by means of punctiuring mth an old needle, and incisions with a scalpel.-^ Decalcified bone is flexible, soft, and admits of being easily cut. Foetal bones, heads of embryos, etc., are decalcified in nitric acid of a weaker kind (1 c.cm. of pure acid to 99 c.cm. of distilled water), or in 500 c.cm. of saturated acpieous solution of picric acid. The process of decalcification requires several weeks in the case of thick bones ; in the case of foetal and small bones, three to twelve days. AVhen decalcification is completed, the bones are thoroughly washed, from six to twelve hours, in Avater, and again hardened in alcohols of increasing strengths. It not unfrequently happens, in the case of beginners, that the bone is placed in alcohol when the decalcification is yet incomplete, and then turns out to be use- less for histological investigation. In such cases, the entire process of decalcification must be repeated. If objects lie too long in the decal- cifying fluid, decomposition is induced. 7. Sections.- — The razor must be sharp ; the success of the prepara- tions depends on the sharpness of the blade. In section-cutting, the blade must be moistened with alcohol (not with water, which moistens the blade only to an imperfect extent). For this end, dip the blade, before every third or fourth section, into a flat glass dish filled with 30 c.cm. of 90 per cent, alcohol, which serves also for the reception of the prepared sections. The blade must be held in a horizontal position, lightly grasped, with the thumb pressed against the edge, the fingers against the hinder part of the blade, and the back of the hand directed upwards. First of all, cut a slice from the object so as to expose a smooth siurface, and then sej)arate from the object, with one .stroke, a piece of suitable thickness. Now begins the making of successive sections ; this must always be clone as smoothly as possible, with symmetrical thinness, and A\dth one light, not too rapid, stroke.- We then place the dish on a black background, and pick out 1 The needle and the scalpel must be carefully cleaned immediately after use. " The blade is not to be pressed through an object, but to be drawn through it. It is wise to make always a very large number (10 to 20) of sections, which are then to be transferred into the glass dish with a needle or by the immersion of the knife. Fine sections, which either have not been stained, or have already been stained throughout, can best be cut and floated off by the edge of the razor being mclined directly on the slide. 270 THE PHYSIOLOGY OF THE TISSUES. the best sections. The thinnest sections are not alimijs the most useful ; for many prc])arations, e.g. a section through the coats of the stomach, thicker sections are more to lie recommended. For specimens intended for general inspection M-ith low powers, let the operator produce large, thick sections ; for small structures suitable for high powers, he must cut thin ones. For high poAvers, the very smallest fragments of 1 to 2 mm. broad, obtained by means of the superficial manipulation of the knife, or marginal parts of somewhat thicker sections, often suffice. If the object to be dissected is too small to be held by the fingers, it ought to be imbedded. The simplest method is that of imbedding in liver. Take either ox's liver or, what is better, human fatty or amyloid liver (to be ob- tained from the pathological department), and cut it into thick pieces (3 cm. in height, 2 in breadth, and 2 in thickness) ; these are immediately thrown into 90 per cent, alcohol, which must be changed the next day. After three to live days longer, the liver possesses the requisite hardness. Now, cut one of these pieces from above down to the half of its height, and squeeze the object to be cut into the fissure so produced. If the object be too thick, the operator can, with a small ;scalpel, cut furrows in the liver, into which the object is fitted. Most objects may be cut in liver, and very delicate sections can thus be obtained after some practice, regularly carried on for a few weeks. 8. Colouring, Staining. — The particular solution of the colouring matter must always be filtered before use. Small funnels are made of -double thicknesses of filter paper (5 c.cm.), and fastened in a cork frame which has been prepared by means of the excision of a jiiece 2 cm. broad out of a cork plate 5 cm. Ijroad. The frame of cork is fixed on four long pins. Such funnels can be used several times. Funnels and frames are to be employed only for one and the same fluid. 1. NxLcleus Htainhuj ^cith hamatoxijl hi (p. 2Q2). Filter .3 to 4 c.cm. of colouring solution into a watch glass and introduce the sections into it. The time in which the sections acquire colour is very various. Sections in alcohol of fixed and hardened objects acquire colour in one to three minutes. If the fixation has been efi'ected with Mtiller's fluid, the objects must be immersed someM'hat longer (five minutes) in the fluid. [Sections of objects fixed in strong chromic acid, or of those not entirely free from acid, often take on colour very slowly, and sometimes not at all. We can remedy this inconvenience either by means of protracted preservation for two or three months in 90 per cent, alcohol, to be changed two or three times, or by immersing such sections, before they are brought into the hfematoxylin, for five to ten minutes, in a watch glass, along with 5 c. cm. of distilled water, to which three to seven drops of 35 per cent, caustic potash are added. We then transfer the sections for one to two minutes into a watch glass with pure distilled water, and thence into the hccmatoxylin. Such sections also become coloured after five to ten minutes.] METHODS OF MICROSCOPICAL RESEARCH. 271 The sections must next be transferred from the colouring matter into a watch glass containing distilled water, rinsed, i.e. moved somewhat with the needle, in order to free them from the surplus coloiiring matter, and, after the lapse of one to two minutes, transferred into a large dish filled with 30 c.cm. of distilled water. The sections must remain here at least five minutes, during which their purple colour gradually passes over into a beautiful dark blue, which becomes all the more pure the longer (twenty-four hours) we allow the sections to be in the water. The sections at first appear entirely blue. About five minutes after, in many cases only after the lapse of hours, the difi"erentiation is first effected, so as to admit of many details being perceived even with the naked eye. The colouring matter which has been used is again poured back through the filter into the bottle containing the htematoxylin solution. The watch glass must be immediately cleaned. It is recommended to beginners to leave the sections in the dye for a time, ranging variously from one, three, to five minutes, and then to watch what length of time is adapted for successful staining. In the case of hsematoxylin colouring, the principal thing is careful washing. 2. Diffusive staining — for the staining of protoplasm and of the intercellular substances, (a) Sloio colouring. A small drop of neutral solution of carmine (p. 262) is placed by a glass rod in a dish filled with 20 c.cm. of distilled water ; at the bottom of this dish there lies a small piece of filter paper. ^ The sections must lie in the fluid over night. The moi'e rose-tinted the fluid is, the longer the colouring process lasts, and the brighter the section becomes. The beginner is always inclined to regard the pale-rose tint as unlikely to produce a good stain, until, on the next day, the dark-rose sections turned to red teach him a better lesson. But this mode of colouring is seldom applicable for its own sake ; on the other hand, it is to be recommended for compound staining. Colour first of all with carmine solution, then with hsematoxylin. (b) Bapid colouring. Pour 10 drops of eosin solution (p. 263) into 3 to 4 c.cm. of dis- tilled water. The sections remain therein one to five minutes, they are then for a short time "rinsed out" (as in the case of hfematoxylin colouring) in a watch glass with distilled water and transferred for ten minutes into 30 c.cm. of distilled water. The colouring may be employed alone or combined with haema- toxylin. The entire process of hsematoxylin colouring is to be completed first, and the eosin colouring next. 3. Staining of the chromatic substance in nuclear division. The objects are placed, from sixteen to forty-eight hours (the longer the better), in only 3 c.cm. of solution of safi'ranin (p. 263). Then the solution, together with the sections, is poured out into a plate containing distilled water ; entirely opaque sections are lifted out with the needle and, in order to change their colour, placed in 5 c.cm. of hydrochloric-acid-alcohol. If the section does not yield much colour (after a half to two minutes), it is transferred into 5 c.cm. of pure absolute alcohol, and after a minute cleared up and mounted. Too long continuance in hydrochloric-acid-alcohol (just as in absolute alcohol) — may lead to a too great change of colour of the preparation. We employ the safi'ranin colouring only after precedmg fixation with chromo-osmium acetic acid. 4. Staining in mass. Colouring of the nucleus in entire pieces of organs pre- vious to their separation into sections. Fixed and hardened objects are, when small (5 mm. broad), transferred for twenty-four hours to 30 c.cm. of borax 1 If this is neglected, the sections acquire colour only on one side. 272 THE FJlYiSlOLOUV OF THE TJS.'SUE^. carmine ; when larger, for two to three days ; they are transferred out of this directly into 25 com. of hydrochloric-acid-(70 per cent.)-alcohol (p. 263) — (the borax carmine, after being used, is poui-ed back into the flask). After the lapse of a few minutes, the alcohol is red, and must now be replaced by new hydrochloric-acid-alcohol. After nearly a quarter of an hour, the alcohol is again changed, and this clianging is continued without intermission until the alcohol is no longer coloured.^ The piece is then transferred into 00 per cent, alcohol, and if it has not, after twenty-four hours, become hard enough for cutting, it is trans- fen-ed for twenty-four hours or more into absolute alcohol. 5. Picrocarminc Double dyeing, nuclei and connective tissue red, protoplasm yellow. Filter o c.cm. of the fluid into a watch glass. The period of time in which picrocarmine operates varies according to the tissue, and can only be approximately given. After the colouring has beeu completed, the colour is again filtered back into the flask, and the object transferred for ten to thirty minutes into 10 c.cm. of distilled water. It, of course, loses colour under the cover-glass. If the object, e.g. a section, is to be placed in absolute alcohol, it must not remain there long (one to two minutes), as the alcohol extracts the yellow colour.- Picrocarmine is especially employed in investigations of fresh objects. If the solution is good, we secure a beatxtiful colour, which comes out brilliantly, especially with the subseqiient application of glycerine acidulated with a few drops of acetic acid. 6. Coloimiuj of nuclei icith aniline dyes. The best aniline dyes for this purpose are vesuvin and methyl-violet B. Filter 4 c.cm. of the fluid into a watch glass, and place the sections in it. They become, after two to five minutes, coloured quite dark ; they are then, for a short time, washed in 5 c.cm, of distilled water, and transferred to a watch glass containing absolute alcohol, where they lose much colour. After a few minutes (three to five), when the sections have become lighter, we can recognize individual parts {e.g. the glands in the case of the skin) with the naked eye. The sections are now transferred into a second watch glass with 5 c.cm. of absolute alcohol, and, after two miniites, cleared up and mounted in dammar varnish (p. 2G1). The resiilt is a very beautiful colouring of the nucleus. Vesu^^n should be kept in glycerine. A disadvantage lies in the large consumption of absolute alcohol. 7. Silver staining — for the representation of cell boundai-ies and colouring of cement substances. The use of metal instruments is to be avoided ; use a glass rod. Instead of pins, use hedgehog bristles. The object is immersed in 10 to 20 c.cm, of 1 per cent., or weaker, solution of nitrate of silver (p. 261, par. 15). After half a minute to ten minutes (always according to the thickness of the object) it is taken out of the fluid — which has meanwhile assumed an almost milky hue — by a glass rod, rinsed, and exposed to direct sunlight in a large white dish (a porcelain plate) containing 100 c.cm. of distilled water. After the lapse of a few minutes, a slight browning will supervene, the sign of the suc- cessful reduction of the silver. Immediately on the object becoming a dark 1 This may occupy one to three days ; during the first day, change every two hours ; dui'ing the subsequent time, every four hours. If the operator wishes to be economical, he may slowly, with a needle, push the object out of the layer of the red fluid in which it lies, and bring it to another uncoloured part of the fluid. 2 This deprivation of colour can be avoided, if we throw into the watch glass a small crystal of picric acid along with the absolute alcohol. METHODS OF MICROSCOPICAL RESEARCH. 273 reddish-brown (usually after five to ten minutes), it is taken out and transferred to a watch glass containing distilled water, to which a couple of grains of common salt are added, and, after five to ten minutes, it is put for preservation in 30 c.cm. of 70 per cent, alcohol, in darkness. After three to ten hours, substitute 90 per cent, alcohol for the alcohol of 70 per cent. The substance is im- mersed in the solution of silver in the dark. The reduction, on the contrary, must be accomplished in the sunlight.^ If there is no sunshine, lift the object (after it has been taken out of the silver solution and washed thoroughly for a short time in distilled water) into 30 c. cm. of 70 per cent, (later on into 90 per cent. ) alcohol, in the dark, and expose it, while in the alcohol, to the light at the first blink of sunshine. 8. Gold staining — for demonstrating the terminations of the nerves. Steel instruments must not be immersed in the gold solution. All manipulations in the gold solution are to be undertaken with glass rods or with small wooden rods. Heat in a small test-tube, to boiling, 8 c.cm. of 1 per cent, solution of chloride of gold + 2 c.cm. of formic acid (p. 261). The mixture must boil up thrice. In the cooled mixture, very small pieces of tissue (at the most 5 mm. broad) are immersed for the space of an hour, and kept in the dark ; they are then for a short time thoroughly washed with distilled water in a watch glass, and exposed to the light (not necessarily sunshine) in a mixture of 10 c.cm. of formic acid with 40 c.cm. of distilled water. The reduction by which the small pieces become dark violet on the outside is accomplished very slowly (often after twenty-four to forty-eight hours at the earliest). The pieces are then transferred to 30 c.cm. of 70 per cent, alcohol, and, on the following day, to 90 per cent, alcohol, in which, for the prevention of further reduction, they must remain for eight days. 9. Injection. — The filling of the blood vessels and of the lymphatic vessels ■vvith coloured substances is a peculiar art, which can be ac- quired only by means of much practice. Knowledge of the many little artifices employed can scarcely be acquired by lectures or even with all the help of copious directions. Practical instruction is here indispensable. Anyone wishing to inject must have a syringe, working well, and provided with an easily movable piston and nozzles of various diameters. As injection material use Griibler's Berlin blue, 3 grams, dissolved in 600 c.cm. of distilled water. Begin >vith the injection of single organs, e.g. the liver, which possesses the advantage that even an incomplete filling of its vessels yields useful results. Place the injected object for two to four weeks in Miiller's fluid, and harden it in alcohol. The sections must not be too thin. 10. Mounting and Preservation of Preparations. — The prepared sections, etc., are now, with a view to microscopical investigation, transferred to ^ The reduction doubtless succeeds also in ordinary daylight, but it is efl'ected slowly, and yields less distinct forms. I. S 274 THE PHYSIOLOGY OF THE TISSUES. a slide and placed under a cover-glass. The media in which the sections are examined are either (1) Avater; or, if it is desired to brighten up and preserve the sections, (2) glycerine; or (3) dammar solution. Transference to the slide is effected thus — the investigator places first of all a small drop of the requisite fluid on the middle of the slide ; he then lifts the section with the spatula and lays it on the slide. It is a good method to lift very delicate sections with the end of a glass rod, and to lay them on the slide hy rolling it round. When the section lies smoothly, place a cover-glass on it.^ The latter must be grasped by the edge. In the act of covering, the cover-glass is placed on the slide, resting on the left edge, and then it is slowly lowered on to the preparation, while the under surface of the cover- glass is supported by a needle held in the right hand. It is still simpler to place a drop of the requisite fluid on the lower surface of the cover-glass and then to allow the cover-glass to fall gently on the preparation. The fluid in which the section happens to be must fill up the entire space between the cover-glass and the slide. If a sufiiciency of fluid is lacking (which is recognizable by large air bubbles appearing under the cover-glass), place, with the point of the glass rod, one drop more of the fluid on the rim of the cover-glass. If there is a sui^erabundance of fluid — and this usu.ally occurs, especially with be- ginners — the liquid which has oozed out from under the rim of the cover- glass must be absorbed by filter paper. The upper surface of the cover-glass must always he dry. Remove small air bubbles under the cover-glass by means of frequently carefully raising and lowering it with the needle. 1. Never neglect to examine uncoloured as well as coloured sections in water or in solution of common salt, as in these many details of structure, e.g. connective tissue formations, distinctly emerge to view, whilst the same, under the brightening influence of glycerine or of dammar varnish, almost entirely elude observation. Objects mounted in water or even in a solution of common salt cannot be pre- served. 2. Preparations mounted in glycerine may be preserved. In order to prevent removal of the cover-glass, fix it with cover-glass cement. As a previous condition of success, the rim of the cover-glass must be completely dry, for cement adheres only to dry surfaces. To dry the slide round the cover-glass, first absorb with filter paper the glycerine oozing from under the rim of the 1 Investigations with low magnifying powers without a cover-glass are only admissible in the most superficial examinations, as, for example, to ascertain if object has been thoroughly teazed out. In all other cases, the cover-glass is indispensable. In order to be persuaded of this, let anyone examine an uncovered section, then let him cover it with a cover-glass and examine it again. Many a good preparation, the covering of which has been neglected, appears unfit for use. Investigations with high powers without a cover-glass are in general inadmissible. METHODS OF MICROSCOPICAL RESEARCH. 275 oover-glass, and then with a cloth moistened with 90 per cent, alcohol (the cloth being drawn over the point of the finger), carefully wipe the slide all round the cover-glass, without touching the latter. Then heat a glass rod and plunge it into the cement,^ and bring four drops on to the four corners of the cover- glass. Then lead the cement round the margins of the cover-glass, so as to cover the cover-glass on the one hand and a portion of the slide on the other, to the breadth of 1 to 3 mm. Finally, smooth with the heated rod the upper surface of the layer of cement. Preparations preserved in glycerine are often beautifully transparent on the second or third day. Hsematoxylin and other colouring matters grow dim in glycerine after some time ; on the contrary, picrocarmine and carmine retain their colour. 3. Mounting objects in dammar varnish is an excellent method of preservation. Dammar varnish has, as opposed to glycerine, the advantage of retaining colours, but with the drawback that it renders objects more transparent than dilute glycerine and frequently causes delicate structures to disappear. Sections which happen to be in water or alcohol, cannot be placed at once into dammar varnish ; they must he previously entirely freed from water. To this •end, the sections are transferred to a covered watch glass containing 4 c.cm. of absolute alcohol, in which they may remain for two minutes in the case of thin sections, and for ten minutes in the case of thicker ones.^ Next we fish out the sections with the needle (very delicate sections with spatula or section lifter) and transfer them, with a view to clearing up, into a watch glass containing 3 c.cm. of lavender oil or oil of cloves.' Place the dish on black paper so as to observe the sections becoming transparent. Avoid breathing into the watch glass, as immediate turbidity of the lavender oil is the result. If indi- vidual portions of the sections do not become transparent after two or three minutes (in this case the portions appear a dirty white when the light falls on them and dark brown when the light passes through them), the section has, in that case, not been freed from water and it must once again be transferred to absolute alcohol. After the clearing up has been completed, the section is transferred to a dry slide and the superfluous oil * carefully wiped off with filter paper, or with ^ Glass rods crack very easily when heated, and metal rods are preferable, but they cool too rapidly. We can to some extent avoid the cracking by heating the ^lass rods, while they are being continually turned round, up to a red heat. Glass rods heated for only a short time crack immediately on being plunged into the cement. 2 Beginners should transfer the sections out of the water, first of all into 4 c.cm. of 90 per cent, alcohol, and then quickly into the same quantity of absolute alcohol. ' We can also bring the section directly out of the absolute alcohol on to the slide, wipe off the superfluous alcohol and place upon it a drop of lavender oil. At first the oil always trickles off from the section and it must be guided back again by a needle ; after complete clearing up, which we can observe under the microscope with a low power, the oil is as soon as possible wiped off and a cover- glass with dammar varnish placed over it. If we examine the uncovered section in oil, section and oil may become obscured by the breath ; in such cases, let the turbid oil trickle off and substitute a drop of fresh oil. ■* The oil in the watch glass used in clearing up may again be poured back into the bottle. 276 THE PHYSIOLOGY OF THE TISSUES. a linen duster wrapped round the forefinger, and a cover-glass is placed on it,. to the under surface of which a drop of dammar varnish has been attached.. When several sections are brought under one cover-glass, in the first place arrange the sections in close proximity by means of the needle, then with the^ glass rod spread the dammar varnish over the under surface of the cover-glass,. in a layer proportionately thin throughout, and place the cover-glass over the sections. Large air bubbles are got rid of by placing a small drop of dammar varnish near the rim of the cover-glass. It not unfrequently occurs in the case of beginners that the varnish becomes- turbid and finally renders the entire preparation, or parts of it, opaque. The reason is, that the section has not been freed from water. In the case of slight turbidity (which reveals itself under the microscope as consisting of minute drops of water), slight heating of the slide may be a remedy. In the case of denser turbidity, place the entire slide in oil of turpentine, carefully lift oflf the cover-glass half an hour afterwards, place the sections in oil of turpentine for two minutes to dissolve the adhering varnish, and lastly place it, with Or view to the complete removal of the water, into 4 c. cm. of absolute alcohol, which must be changed after five minutes. The lavender oil and dammar varnish pro- cess is then repeated. Dammar varnish dries very slowly, and therefore the slides must not be placed on their side. The successive processes through which a fresh object has to pass until it is pre- served as a finished stained section is a veiy long one. When, for example, it is prescribed in the appendix detailing special methods thus — " Fix in Miiller's fluid fourteen days, harden in gi'adually concentrated alcohol, colour the section in car- mine and hifimatoxylin, mount in dammar varnish" — the process is as follows: — The fresh object, 1 c.cm. in size, is placed in 200 c.cm. of Miiller's fluid,^ which, immediately after turbidity has set in (usually after one hoiir), is changed. After twenty-four hours the fluid is again changed ; the object now remains in it for a period of fourteen days. After the expiration of this, it is washed in flowing water for one to four hours ; then placed in 20 c.cm. of distilled water for fifteerr minutes ; then in 50 c.cm. alcohol (70 per cent.) and in the dark for twenty-four hours ; then bx 50 c.cm. of 90 per cent, alcohol for twenty -four houi's, when the 90 per cent, alcohol is changed. The fixed and hardened object may now be cut after a suitably long time, during which the 90 per cent, alcohol is perhaps once more changed. The section is- taken out of the alcohol and transferred into 30 c.cm. of dilute carmine solution for twenty-four hours ; then into 5 c.cm. of distilled water for fifteen minutes ; then into 3 c.cm. of hcematoxylin for five minutes; then into 20 c.cm. of distilled water for ten minutes to two hours ; then into 5 c.cm. of absolute alcohol for ten minutes ; then into 3 c. cm. of lavender oil or oil of cloves for two minutes, and lastly the section is mounted in dammar varnish. 11. Investigation of Fresh Objects. — This is placed at the end of all the* other methods because it is the most difficult, and presupposes a some- what practised eye. Experience is most easily acquired by the investi- ^ The directions for size are calculated only for one piece 1 c.cm. large. When. the piece is larger, or when several pieces are submitted to the process, more fixing; and hardening fluid must, of course, be employed. METHODS OF MICROSCOPICAL RESEARCH. 277 gation of objects dlredidij prepared (hardened, coloured, etc.). If anyone las once distinctly seen and studied details of structure seen in stained specimens, it is not so difficult to make out these even in fresh objects. That which now follows recjuires attention. Slides and cover-glasses XQUst not be greasy. Clean them with alcohol and dry them with a clean ■cloth. Then place one drop of '75 per cent, solution of common salt on the slide, next place in it a small piece of the object to be investigated, and cover the latter with a cover-glass. In this process, all pressure must be carefully avoided. In the case of very delicate objects, lay two small strips of paper on the slide so that the cover-glass "will rest on them without pressing the object itself. If the object requires no further manipulation, the observer should run round the edge of the cover-glass s. thin layer of paraffin to prevent evaporation. Melt on an old scalpel Si large piece of paraffin, nearly as large as a lens, and let it run, not from the point, but from the edge of the scalpel, on to the rim of the cover-glass. Gaps, here and there, may be filled up by applying the heated «nd of the scalpel. It is easy to subject fresh objects to the operation of certain reagents (acetic acid, caustic potash, colouring matters) while Tinder the microscope. One may remove a part of the medium in which the object lies (as, for example, the solution of common salt), and substitute another fluid in its place. To do this, place a drop, e.g. of picrocarmine, with a glass rod on the right rim of the cover-glass. If the drop does not reach entirely to the rim of the cover-glass, do not incline the slide to it, but guide the drop with a needle to the margin of the cover-glass. We see now that a little of the colouring matter mixes with the solution of common salt, but that a regular flow of the colouring fluid does not take place. In order to secure this, place a little bit of filter paper on the left rim of the cover-glass,^ and the picrocarmine ^vill soon occupy the entire under surface of the cover-glass.^ Lay the filter paper aside, and allow the stain to operate. When the colouring las been completed — a fact which can be always observed under the microscope — place on the right rim of the cover-glass a droj), e.g. of _glycerine, to which add, in cases of colouring by picrocarmine, as much acetic acid as will drop from a needle dipped once into it (thus a small drop), and then lay the filter paper again on the left side of the rim. In this manner, we can conduct an entire series of fluids under the cover- glass, and test their action on the tissues. Certain of the fluids, picro- 1 1 cut out a piece 4 cm. long, 2 cm. broad, notch it transversely, and place the paper roof so formed on the slide in such a manner that it touches the left rim of -the cover-glass with the rim entirely and straightly cut, and 2 cm. broad. 2 When the first drop has penetrated, place (always in proportion to what is 3ieeded) two or three further drops on the right of the cover-glass. 278 THE PHYSIOLOGY OF THE TISSUES. carmine for example, must, after preceding fixation A\ath osmic acid, remain very long in contact -vnth the objects. Evaporation is prevented by leaving the preparation in a damp chamber. Moist chambers are made by means of a porcelain plate and a small glass lid, of 9 cm. diameter at the least.^ Poiu" water to the height of 2 cm. into the plate, then place in the middle a small glass basin, or a cork plate standing on four wooden feet. The slide, along Avith the preparation, is placed on this, and the whole is covered with the glass lid, the free edge of which is immersed in water. 12. Preservation of Preparations. — Finished preparations must immedi- ately be labelled. Do not employ gummed paper tickets, but those of pasteboard 1-2 mm. thick, which are fixed on the slide by means of the cement called water-glass.- Special boxes for holding specimens are thus superfluous. The slides may be placed over each other Avithout pressiure on the preparations. The labels should be as large as possible (2 cm. broad in the case of slides of the English size), and they shoidd have recorded on them the name of the animal, the organ, and, where possible, a short indication of the method. Use boxes or cabinets which allow the slides to lie flat, not those in which they stand on their sides. Chap. VI. —MICROTOMES AND SERIAL SECTION CUTTING. A microtome is an instrument by which extremely thin sections of organs and of tissues may be cut. There are many varieties of such instruments, and most of them have certain points of excellence that commend them to their inventors, or to those who acquire dexterity in their use. I shall onh' refer to those of which I have had special experience. There are essentially five methods of imparting to tissues the proper consistence for section cutting by means of microtomes : (1) embedding the tissue or organ in parafiin or in a mixture of white wax and parafiin ; (2) infiltrating the tissue Avith gum and then freezing it by the application of freezing mixtures ; (3) freezing the tissue directlj^, and often in the fresh state, by the use of ether Avhich, by its evaporation, produces the requisite degree of cold ;. (4) infiltrating the tissue A^th paraffin dissolved in chloroform or other soh^ent ; and (5) holding the hardened tissue firmly by means of celloidin or other substance capable of giA^ng it sviflicient resistance to the pressure of the knife. ^ A pot, a larger preparation glass, etc., serve the same purpose. ^ It is a liquid of the Adscidity of syrup, to be had at all druggists, and must be- preserved in a well-stoppered vessel. MICROTOMES AND SERIAL SECTION CUTTING. 279 1st Method. — Paraffin" and Wax Embedding, The microtome used in this method consisted of a strong brass cylinder fixed into a horizontal brass plate having a smooth upper surface. The bottom of the cylinder consisted of a flat plate exactly fitting the cylinder, and attached underneath to a thick screw having a very fine thread. A well about 30 mm. in depth, and 45 mm. in diameter, was thus formed, the bottom of which was moved upwards by a slight turn of the screw. The tissue, previously hardened, was immersed in melted spermaceti, and then in a mixture of melted white wax and paraffin, or in paraffin alone. When the mass became solid, by turning the screw it was pushed upwards, and a section was made by pressing a razor or broad knife over the surface of the broad and smooth plate, in the centre of which the well opened, the tissue and razor being kept moist with alcohol. This method had many disadvantages, as, for instance, the shrinkage of the tissue from the paraffin, so that it became loose ; but it did good service in its day, especially in the hands of such a man as the late Mr. A. B. Stirling, assistant curator of the Anatomical Fig. 129. — Rutherford's Microtome, arranged for freezing with ice and salt. Museum of the University of Edinburgh, who prepared histological specimens with great skill and success. It is now almost entirely abandoned. 280 THE PHYSIOLOGY OF THE TISSUES. 2nd Method.— The Ice Freezing Microtome of Professor Rutherford AND Allied Instruments. In 1871, Professor Eutherford/ of the University of Edinburgh, invented and described this instrument, and it is not too much to say that it revolutionized the teaching of histology in this country. By means of it, 400 or 500 thin sections can be cut in the course of an hour. This has made it invaluable in the teaching of large classes of students. Its simplest form is seen in Fig. 129. It consists of a stage or plate B, in the centre of which is a Avell of considerable depth, the bottom of which is movable by the screw D having a fine thread. An indicator i^ is a little spring having a small point on the inner aspect of its lower end which, by sliding with a turn of the screw into the small holes, serves to make the turns of the screw equal in amount and consequently secvues sections of uniform thickness. Surrounding the well is the ice box C, having a tube H for the escape of water when the ice melts. The bit of hardened tissue is immersed in a strong solution of gum arabic (■with a few drops of a solution of carbolic acid added to prevent the growth of fungi in the gum) for several days, so that the gum may thoroughly soak through it. Some strong gum solution is poured into the bottom of the well, finely pounded ice and salt are placed in layers or mixed in the freezing box, and when a thin layer of ice has formed round the interior of the well, the bit of tissue is placed in position. A sufficient amount of gum solution is poured in to fill the well, the mouth of the well is then closed with a bit of gutta-percha sheeting, and the whole apparatus is wrapped in a piece of thick flannel cloth, and left until freezing has been thoroughly established. The wrappings are then removed, the bit of gutta-percha taken from the mouth of the well, and Avith the knife held firmly so that it glides imiformly on the plate B, the mass of frozen gum at the mouth of the well is cut otf. The screw D is then turned and another section cut until the tissue is reached. It is then easy to cut numerous sections quickly after each other, turning the screw D to the requisite amount betAveen each movement of the knife. When a sufficient number of sections accumulate on the knife (or Avith each section Avhen great care is necessary), they are AAdped off" the knife by a camel-hair brush into a flat A^essel containing Avater. The Avater dissolves the gum. After some time, the sections are gently collected on a straight needle, trans- ferred to another vessel of Avater, and from that into a third ; at the end of half an hour, they are gathered in the same Avay and transferred to a ^ William Rutherford, Journal of Anatomy and Physiology, 1871 ; see also The Lancet, 3rd January, 1885. MICROTOMES AND SERIAL SECTION CUTTING. 281 stoppered bottle containing methylated spirit, where they may be kept till required. The most useful form of knife is one "vvith a blade 200 mm. in length and 30 mm. in breadth. The back of the knife should be 6 mm. in thickness to prevent bending of the blade, and the side of the blade which touches the microtome should be ground flat, while the upper surface is bevelled. Another form of freezing microtome is that of Williams, made by Mr. Swift of London. It is a circular box of considerable capacity, having a metal pillar in the centre bearing on its upper end a brass plate 30 mm. in diameter. This box is packed with powdered ice and salt, and then the lid is pressed on, so that the brass plate just men- tioned passes through a hole in the centre of the box and. is on a level with the upper surface of the lid. The upper surface of the lid is made of glass. The bit of tissue, soaked in gum, placed on the brass plate, soon becomes frozen. The sections are then cut by a razor mounted in a frame supported on three fine screws passing through it, and by lowering the edge of the razor by turning the screw placed beyond the edge of the razor, and pushing the frame, carrying the razor, somewhat Fig. 130. -Rutherford's Microtome, arranged for freezing with ether spray. obliquely across the frozen tissue, thin sections can be readily made. This form of freezing microtome is serviceable for private use, and for cutting sections of small organs. 282 THE PHYSIOLOGY OF THE TISSUES. 3rd Method. — Freezing by Means of Ether. The production of cold by the evaporation of ether has been applied to many microtomes. The first microtome in -which it Avas used was the invention of Dr. Bevan Lewis in 1877.^ Dr. Eutherford^ has applied the method to his own microtome, as seen in Fig. 130, and he gives the following directions as to its use — " The tissue, which should not be more than a third of an inch thick, is laid on the roof of a zinc box Z and covered with gum. Ether, ■which must be anhydrous, is then blown from the bottle by the elastic bellows y against the lower surface of the zinc plate. The condensed ether flows down through the tube P, and is collected in a vessel. The spray -producing tubes T can be readily pulled out of the slot under Z for examination. The tissue is soon frozen, and it remains frozen for about five minutes in a cold room without any further production of spray." A very convenient form of microtome in which ether spray is used as the freezing agent is that invented by Professor Charles S. Roy, shoAvn in Fig. 131. "With this instrument, the consumption of ether is small, and the mechanism works Anth a;reat smoothness. Fig. 131. — Roy's Microtome, arranged for ether spray, as made by the Cambridge Scientific Instrument Company. 4th Method. — Ixfiltkatiox with Paraffin. The principle of this method is to infiltrate the tissue "nath paraffin, so that the minute cellular elements may be so supported as to remain in their natural position even when they have been cut in section. One of ^ Journal of Anatomy and Physiology, \%11, vol. xi. - Lancet, Jan. 3, 1885. MICROTOMES AND SERIAL SECTION CUTTING. 283 the simplest methods is the folloT^ang — The bits of tissue to be em- bedded, after having been fixed and hardened according to any of the common methods, are thoroughly dehydrated with absolute alcohol. On a suitable water bath, the temperature of which can be kept uni- form by a mercurial regulator governing the gas flame underneath, the student should have a few shallow vessels, each capable of holding 40 CO. of fluid. Chloroform., to which a little sulphimc ether has been added, is poured into one of these, and one or two bits of tissue are transferred from the absolute alcohol to the chloroform. This is then gradually heated to the melting point of the paraffin employed. Two kinds of paraffin should be at hand, one melting at 50° C, and the other at .36° C, and paraffin masses having difl"erent melting points can be readily made by mixing these in various proportions. Suppose the temperature of the water bath is regulated to stand at 38° C, then pieces of paraffin are by degrees added to the chloroform, until the bubbles at first given off cease to appear, indicating that the paraffin dissolved in chloroform has thoroughly penetrated the object. In this gradual infiltration there is scarcely any shrinkage. The paraffin is- kept at its melting point for some time, so that the chloroform is com- pletely driven ofi". The mass is allowed to cool, and then sections may be cut in the dry condition, either with a razor or by means of a suit- FiG. 132. — Microtome for Cutting Objects in ParafBn. able microtome. The sections are j^laced on a slide, and the paraffin is dissolved by pouring over the slide a little turpentine, benziii. 284 THE PHYSIOLOGY OF THE TISSUES. naphtha, or x3"lol. The section may then be stained, and ultimately mounted in Canada balsam or dammar according to the usual methods. Various forms of microtomes arc now employed for cutting sections thus infiltrated with paraffin. One of the most convenient of these is shown in Fig. 132. The paraffin block is held firmly by the clip D, and the knife L is moved by the block K, somewhat like a carpenter's plane, cutting a thin section at each stroke. With each section, the paraffin block is graduall}' raised to an extent varying according to the required thick- ness of the section. This is done by pushing the block E along the inclined plane C. In other microtomes, the paraffin block is raised automatically by the same movement as determines the horizontal movement of the knife, and thus a series of sections may be cut in quick succession. 133 A microtome of this construction is shown in Fig. Fig. 133. — Bivet's Miorotome, as modified by Reichert. In this instrument, the clip d holding the paraffin block is moved .slightly upwards by means of the toothed wheel Z with each horizontal MICROTOMES AND SERIAL SECTION CUTTING. 285 movement of the knife M, and there are arrangements by which the amount of upward movement, and consequently the thickness of the section, may be determined. For certain kinds of work, such as cutting sections of embryos or sections of particular parts of the brain and spinal cord, it is often important to secure a consecutive series of sections, so that a number may be mounted on one slide for comparative examination. Various contrivances have been designed to secure this end. The most service- able and simplest of these is shown in Fig. 134. Fh3. 134. — The Rocking Microtome of the Cambridge Scientific Instrument Company. The pai^ffin block is fixed to the end of the horizontal bar. This bar moves in a V-shaped pivot on another rectangular bar placed underneath, which also moves on a V-shaped pivot. A glance at the diagram shows that the rectangular bar is connected with a screw bearing a disk having a toothed edge, whilst a cord is attached to the end of the upper bar and passes round a pulley connected with the knobbed arm seen at the end of the instrument. When we move this knobbed arm, the toothed disk is pushed round a little by a small catch, the horizontal rectangular bar is slightly raised, and the upper bar is also moved so as to bring the paraffin block down on the edge of the razor. " The distance between the centres of the two pivoted systems is 1 inch, and the distance of the screw from the fixed rod is 6^ inches. The thread of the screw is 25 to the inch ; it follows that if the screw is turned once round, the object to be cut will be moved forward by tjV of k or xi^ inch. The feed can be varied from 286 THE PHYSIOLOGY OF THE TISSUES. to /v of a turn, hence the thickness of the sections cut can be varied from a minimum, depending on the sharpness of the I'azor, to a maximum of /w of 7,^ of it chaynber is very convenient. It is shown in Fig. 141. The cham- ber consists of a cylindrical glass, A, narrow at the top, cemented to the slide, B C. Into the upper and narrow end of the glass chamber, the lower end of the body of the microscope, bearing the objective, passes, and the glass and end of the microscope are bound together by india-rubber sheeting, as shown in the figure. Fig. 141. — Von Recklinghausen's Moist Chamber. 2. Cells. — Many tissues consist almost exclusively of one species of cell. Such are termed simple tissues to distinguish them from other tissues, in the structure of which various species of cells are concerned ; in the latter case, the term complex tissues is employed. By the term simple tissue, epithelial tissue, connective tissue, muscular tissue, and nerve tissue are represented. By the term complex tissue, we desig- nate the structures resulting from the union of various simple tissues- structures which receive the more appropriate term of Organs. It must also be borne in mind that in point of fact the simplest tissues of all are composed of several tissues. For example, muscular tissue consists of muscular cells, of connective tissue, and of vessels and nerves, which, in their turn, are themselves composed of different tissues. (1) General Themj of Cells.— The collective organs of the animal body consist of cells and of substances formed from cells as, for example, the intercellular substances. By the term cell is understood a morphologi- cal element limited in space, which is, under certain conditions, in a position to receive nourishment, to grow and to propagate itself By reason of these powers, the cell may bear the designation of an Elementary Organism. PROTOPLASM AND CELLS. 293 The morphological properties of cells have already been described, but a brief recapitulation may here be given. The essential constit- uents of a cell are — (1) protoplasm, a fine granular, yielding substance, which, insoluble in water, is easily capable of a flowing motion, and •consists principally of albuminous bodies, water and salts ; and (2) the nucleus, an almost clear, sharply defined vesicle, which is confined within a thin membrane, the nuclear membrane, and contains a network of minute threads of diflferent degrees of fineness. Thickenings of these fibres are termed nuclear corpuscles or nucleoli. In the meshes of the reticulum there exists fluid, viz., the nuclear fluid. The reticulum and the nucleoli are readily coloured by means of many dye stufis, on account of which the substance composing them is termed chromatin. The nuclear fluid is incapable of taking on colour. The nuclei also consist of albuminous bodies, of water and salts. To these there is also added a substance peculiar to nuclei, nuclein. The majority of cells contain one nucleus ; only isolated cells possess several nuclei (giant •cells). Cells without nuclei (the horny cells of the epidermis, the coloured blood corpuscles of mammals) originally possess one nucleus, l)ut lose it in the process of development. As unessential constituents of cells may be mentioned — the cell membrane or cell wall, which is wanting in many cells, and in cases in which it is present, it is either the firm peripheral layer of protoplasm or it appears as a thin, almost structureless, cuticle ; as constituents of secondary importance, may also be classed minute grains of pigment and drops of fat, of watery and mucous moisture, which occur in the protoplasm of individual cells. In A'ery young cells sometimes the cell wall possesses a certain amount of elasticity so as to mould itself upon the surface, when the included protoplasm changes its form, but at other times it is stiff and rigid. It is permeable by water and by aqueous solutions of acids, bases, and salts, but it will not permit the pas- sage of oils and fatty substances. Its chemical constitution is not the same in the animal and vegetable kingdom. In plants it is formed of cellulose, a non-nitrogenous substance ; but in animals it is always nitrogenous. It contributes only to the life of the cell by its physical property of permitting the passage of fluid by osmosis, but it is not connected in any other way with the vital phenomena of the cell. Occasionally, it may become hard and impermeable by the deposit in it of calcareous salts and of silica. (2) Form of Cells. — The form of cells is very various. It may be: (1) Spherical, which is the form of all cells in the embryonic period, but in the case of the adult, the white blood corpuscles, for example, are spherical ; (2) Disc-shaped, as, for example, the coloured blood corpuscles; 294 THE PHYSIOLOGY OF THE TISSUES. (3) Polyhedral, the cells of the liver; (4) C3'liiidrical, the epithelial cells of the small intestine ; (5) Cubical, the epithelial cells of the capsule of the lens; (6) Flattened, for example, endothelial cells; (7) Spindle- shaped, such as many connective tissue cells ; (8) draA\Ti out into long fibres, for example, smooth muscular fibres ; and (9) those which are star- shaped, such as many ganglion cells. The form of the nucleus is in most cases adapted to the form of the cells. It assumes an oval shape Avhen surrounded by cylindrical, spindle-shaped, and star-shaped cells ; it is spherical when siurrounded by round and cubical cells. Irregularly shaped or polj^moi'phic nuclei occur in the case of leucocytes (white blood corpuscles) and giant cells. The form would appear to depend largely on the amount and direction of the pressure to which the cell is subjected. The most important physical character of the active cell is its permeability to fluids. If ceUs are placed in distilled water, they swell out by imbibition ; on the other hand, if placed in a fluid of greater specific gravity than that of their contents, they may shrivel and become irregular in form by the passage of a portion of their contents into the surrounding fluid. (3) Size, of Cells. — The size of cells fluctuates from microscopically small forms, 4 /a in diameter (certain coloured blood cells), up to bodie.s visible to the naked eye, such as the ova of birds, amphibia, etc. (4) Nutrition of Cells. — The nutritional changes of the cell consist of assimilation and disassimUation. By assimilation the cell receives from the medium Avhich surroiuids it certain materials which it converts into its own proper substance, or which it may utilize in various ways. There appear to be two phases of the assimilative process : (1) one in which the cell transforms the matter which it receives, and the other (2) in which the substances transformed become an integral part of the cell. The first phase is Avell marked in the life of the vegetable cell, but is not so distinct in the animal cell, which in a manner lives upon materials previously formed by the plant; the second phase, on the contrary, exists to an equal extent both in the animal and vegetable cell. The assimilating process in the K-vdng portion of the cell has received the name of metabolism, while the chemical change by Avhich matters taken up by the cell may be converted into other matters is called metastasis. Thus a cell may, by metabolic processes, convert dead matter into living- matter like itself, or it may, by metastatic processes, com^ert dead matter into another form. The disassimilative processes occurring in the living ceU consist of chemical changes in the substance of the cell itself, or of materials in contact with the cell. Disassimilative processes con- stitute a marked feature in the life of animal cells. Certain cells have the property of separating or forming a special kind of material. Thus PROTOPLASM AND CELLS. 295 some cells form fatty matter, others pigments, and a third, one or other of the substances existing in bile. This power has been termed elective affinity. Certain materials are separated from cells by a process which may be termed cellular excretion, and other cells may store up certain materials in their interior, a process called cellular secretion. (5) Irritability of Cells. — By this term we mean the aptitude which a cell has of responding to a determinate stimulus, and which is an important condition of vital phenomena. The stimulus may be mechanical, chemical, physical, or vital. The same kind of stimulus may produce different results, varying according to the nature of the cell. Thus the response to a stimulus of a muscular cell is contraction; of a glandular cell, secretion ; of an epithelial or connective tissue cell, cell multiplication ; and of a nerve cell, some kind of activity resulting in sensation, perception, volition, or one or other of the stages of intellectual acts. It may be laid down as an axiom that no activity in a cell ever occurs without an antecedent stimulus. (6) Movement of Cells. — One of the most evident phenomena in the life history of many cells is movement. This is seen in the form of amoeboid motion, of ciliary motion, and in the contractions of certain fibres (mus- cular fibres). Amoeboid motion is the most important. Widely spread, it has been observed in nearly all kinds of animal cells. In marked cases, in that of leucocytes (white blood corpuscles) for example, the protoplasm of the cells stretches out finer or coarser extensions, which sub-divide, again flow together, and produce in this manner many diverse forms. The extensions (pseudopodia) may be again retracted, or they fix themselves to some particular spot and draw to themselves in some measure the remaining part of the body of the cell. Thus there are changes of situation, which are termed the migrations of the cells, and which play an important part in the economy of the animal frame. The expansions of the protoplasmic body may flow round grains or small cells, and thus enclose them within the body of the cell (Fig. 155). Amoeboid motions in the case of ^, warm-blooded animals are very """^ i 2 2/2 slow, and can only be detected by careful observation with the aid of the hot stage. They may be readily studied by watching the move- ments of the leucocytes, or white blood corpuscles, in the blood oi 3% s g s m Minutes. amphibia (frog or newt), when Pig. 142.— Leucocytes in frost's wood. X560d. IP i 1 • Changes of form observed during ten minutes. such forms as are represented m 0, beginning of observation, then at intervals TTi- -\ A n 11 1 of i, 1, 2 minutes, etc. (Method No. 11, Fig. 142 may be observed. Appendix.) 296 THE PHYSIOLOGY OF THE TISSUES. A remarkable example of movement in cells was described by Lister as occurring in the pigment cells of the skin of the frog. These cells contain fine molecules of a black pigment. Under the influence of light, these molecules collect towards the central part of the cell, leaving light-coloured areas around each dark mass of molecules. In these circumstances, the frog's skin, to the naked eye, is pale. In darkness, the molecules pass out towai'ds the circumference of the cells, and con- sequently the skin of a frog, from which light has been excluded for some time, appears dark, if examined before light has had time again to make it pale. VonAVittich found in the tree-frog {Hyla arhorca) that contraction of the pigment cells followed mechanical irritation of the skin or irritation by electricity or bj'' turpentine, and that the same result followed irritation of the motor nerves passing to the limb. Undoubtedly, in this case, the movement of the protoplasm in the cell is due to a reflex nervous influence, as division of the nerves passing to the limb causes the skin to appear dark, and the darkness does not then disappear on exposure to bright light. The motor impulse passing to the pigment cells must therefore travel from the nerve centres along the ordinary motor nerve trunks. On the other hand, the sensory impulses which affect the centres so as to cause the motor impulses to pass out, travel from the retina along the optic nerves to the nerve centres, as it has been ascertained that, after blindness, exposure to light no longer causes the skin to become pale. Thus, whether the play of light and shade in the skin of such amphibians is due to a streaming of the molecules to and from the centre of the cell, or to a contraction of the protoplasm in which the molecules are embedded, [there is here an automatic mechanism by which the tint of the animal's skin can be brought into harmony with that of its surroundings, a fact of great general significance. There is yet another phenomenon of movement which is observable not only in the living but also in the dead cell. It is the so-called molecular motion or Bro"\^Tiian movement, an oscillation of the minutest granules in the cell, which is the result of fluid molecular currents. It is readily Fig. 143.— Drop of saliva, observed in salivary corpuscles (Fig. 143). squamous' epithelial cells • (7) Fo^-matioii and RepwducHon of Cells. — A dis- and b, salivary corpus- .... . .. , ,. , -.-, cies, in which the tiuctioii was at One time dra^vn between two kinds oi Brownian movement may ,, „ ,. • /-i \ c n p ,• / ,• be observed. Cell lormatiou, VIZ., (1) tree cell lormation [generamo cequivoca) and (2) the origination of cells through division of pre-existing cells. According to the theory of free cell formation, the cells were supposed to have originated in soft granular matter adapted for the purpose, called hjtohlastema. This theory has been totally abandoned ; we now know only one kind of cell formation, viz., the formation of cells through the division of cells already existing. Omnis cellula e cellula. In the division of a cell, the nucleus plays an imjDortant part. The division is efiected, not often in the simple way of the division, first of the nucleus, and then of the protoplasm into two equal parts, termed direct division or karyostenosis ; PROTOPLASM AND CELLS. 297 but it occiirs as a complex process, termed indirect division or karyo- kinesis. This latter process has already been fully described at p. 213. As special modifications of cell division, the so-called endogenous cell formation and the process of budding furnish good examples. Endog- enous cell formation is seen in cells which possess a firm covering, such as ova (Fig. Ill, p. 234) and in cartilage. (See Cartilage.) The pro- cess of division is exactly the same as that above described, with this difference that the daughter cells, originating from one cell (the mother cell) through continued division, remain enclosed within a common covering. The process of cell multiplication is termed gemmation or budding, when a cell sends forth shoots or buds which gradually separate from the parent and grow into independent cells. Young cells always exhibit the character of the mother cell. The development of connec- tive tissue cells, for example, from the division of an epithelial cell never occurs. (8) Phenomena of Secretion in Cells. — This process is manifested by differences seen in the form and contents of the gland cell when it is in a state of rest and when it is in a state of activity. In many, as, for example, in the cells of a serous gland, these differences are shown in a small bulk and a dark appearance of the cells in the state of rest, and an increased bulk and clearer appearance in the state in which the process of secretion is active. In the case of other gland cells, such as those of mucous gland cells in man, the formation of the secretion admits, on the other hand, of being more exactly described. We begin with the secretionless w Fig. 144. — Secreting Epithelial Cells. From a thin section through the mucous membrane of the human stomach, x 560 d. -p. Proto- plasm ; s, secretion ; a, two cells in a resting state. The cell situated between these shows the beginning of the mucous metamorphosis, e, The upper wall of the cells on the right has burst, the contents have escaped, the granulated protoplasm has again increased, and the nucleus has again become round. (Method No. 12, Appendix.) condition in which the cylindrical cell contains a granular proto- plasm, and an oval nucleus placed almost in the middle of the cell. The formation of the secretion now appears on the free side of the gland «ell, and is shown by the alteration of the granular protoplasm into a clear mass (Fig. 144, 5, s). As the process advances (c) larger masses of protoplasm are converted into secretion, and the nucleus and the remainder of the granular protoplasm are pressed against the base of cell, whereby 298 THE PHYSIOLOGY OF THE TISSUES. the nucleus gradually becomes round or even flattened. The entire cell, filled A\ath secretion, has now become larger. Finally, the wall of the cell ruptures on the free upper surface. The secretion issues out gradually, while the protoplasm re- generates itself and the nucleus re- sumes its i^revious oval form. The cell thus passes back to the state of rest. Several of the stages of the pro- cess of secretion are illustrated in Fig. 145. The majority of gland cells do not perish in the act of secre- tion, but they are in a position to repeat the same process. The cells of the sebaceous glands are ex- cepted from this rule, as secretion is. formed in these by the cells falling in pieces. (See also Secretion.) (9) Gh-owth of Cells.— The growth of cells is determined by the activity of the protoplasm contained in them. Seldom do they grow equally in all directions, so as to preserve the original form of the cell. As a rule, disproportionate growth occurs. The original form of the cell is thereby changed, and it becomes distended, or flattened, or put out of shape, etc. Most cells are soft and liable to change their shape under mechanical influences. Thus, the cylindrical epithelial cells in the empty urinary bladder assume flattened shapes when the bladder is distended. (10) Evolution of Cells. — Each cell may be regarded as an organism which has a determinate period of existence. The duration of life is very variable. Some, such as epithelial cells, may pass through exist- ence in from twelve to twenty-four hours ; those of the mammary gland may have a more transitory existence; whilst such cells as those of cartilage probably exist during the lifetime of the individual. Cells die in various ways. They may be mechanically removed from the superficial surfaces, as is the case with epidermic cells ; or they may undergo chemical transformations of such a nature as to be inconsistent with the vitality of the cell. For instance, as frequently happens in pathological conditions, the cell may become infiltrated -with calcareous, fatty, or amyloid matter. Sometimes, also, cells may break down,, molecule by molecule, undergo liquefaction, and be absorbed. Fig. 145. — Section of sub-lingual gland of man, x 240 d. Opposite 2, six cells are seen full of secre- tion, s, fj ; two cells, containing no secretion, s, I, are thrust away from the centre of the pouch of the gland to form a half-moon or crescentic mass. This figure will be repeated and described fully in treating of salivary secretion. STRUCTURE OF CELLS AND CELLULAR TISSUES. 299 Chap. VIII.— STEUCTURE OF THE VARIETIES OF CELLS AND OF CELLULAR TISSUES. From the morphological point of view, all the tissues are composed of cellular elements, although in the fully developed condition of certain tissues it may be difficult to detect the cell form. Thus, while epithelial and cartilaginous tissue and the corpuscles in the blood show the usual cell type, so-called muscle and nerve fibres have become so much altered as to conceal the fact that they also are composed of cells. Modern physiology, however, attaches so much importance to the doctrine that all tissue elements are formed of cells — a doctrine also having a direct bearing on many pathological questions — as to make it desirable, in the first instance, to take a general survey of the cell- elements, leaving the functions of these cell elements to be afterwards . discussed.^ 1. Leucocytes are cells having no cell wall, and consisting of a granulated, glutinous protoplasm, and of one or more nuclei. A de- finite form cannot be ascribed to them, because, during life, they show amoeboid motion ; in a state of rest they are globular (Fig. 146, 8) ; their size fluctuates between 4 and 14 /i. Leucocytes are found in the lymph and chyle vessels (lymph and chyle corpuscles), in blood-vessels (white or colourless blood corpuscles), in the marrow of the bones (marrow cells), in adenoid tissue, and lastly, scattered in the connective tissue and between epithelial and gland cells, whither they have wan- dered by their amoeboid motions. On this account they are sometimes called wandering cells. 2. Coloured Blood Cells (coloured blood corpuscles, Fig. 146) are soft, ductile, very elastic stirictures, possessing a smooth surface. In man and the mammalia they have the shape of almost flat circular discs, ^ which are hollowed out on each side, and consec[uently resemble biconcave lenses. The llama and the camel are exceptions to this rule, as their blood corpuscles are oval. In the case of man, their surface diameter is, on an average, 7'5 [jl ; their thickness 1"6 /^. The coloured blood corpuscles of our domestic mammalia are all smaller ; the largest are those of the dog (7 '3 /x). The coloured blood corpuscles consist ^ Histological details regarding the various tissues shortly described in this chapter, which are of special physiological interest, will be given when we con- sider the special functions. For example, the structure, etc., of the blood corpuscles will be again referred to in the description of the characters of blood. ^ Besides these, a few spherical coloured blood corpuscles are to be found (Fig. 146, A 7) ; they are smaller (5 fi) than the ordinary red corpuscles. 300 THE PHYSIOLOGY OF THE TISSUES. of a stronica (protoplasm), which is brought into close relation with the colouring matter of the blood. Haemoglobin gives to the coloured blood corpuscles their yellow or A . H If Ji greenish-yellow hue. Thev ^ O ^^ ^ h* have no cell wall nor nu- ^^S OO ^ ■''W % "^^^^-^^ cleus. The coloured blood ^ %JS // ^j^^' corj^uscles of fishes, am- (;x^' ^ ^'^ i^^ /^ phibia, reptiles, and birds °*^ Q-^, % ^1^ ^mj ^^® distinguished from ^%>' "' '^ m ^^ those of mammalia by ^ ^ o°o their oval and biconvex T, ^,r T,^ A 1 . r T, r X, r shape, by their large size Fig. 146.— Blood corpuscles — A, of man ; B, of the frog, . 560 times magnified. 1-6, disk-shaped coloured blood (in the Case of the froo" corpuscles. 1, Deep focusing ; 2, high focusing of the ^ objective; 3 and i, edgeways; 5, become stellated by 22 11 long, 15 M broad), aS evaporation ; 6, after the addition of water. 7, Spherical i coloured blood corpuscles. S, Colourless blood corpuscles. WCll aS by the ])resence of 9, Small blood plates. 10-13, Coloured blood corpuscles of the frog. 10, Entirely fresh, nucleus little distinguish- a rOUlld Or OVal UUCleUS. able; 11, some minutes later nucleus distinctly visible ; ^ ■, , IS, seen on the side ; 13, after addition of water. lU, in Other rCSpectS, they Living. 1,7, Dead colourless blood corpuscles. (Method t i , • • ••> No. 13, Appendix.) display properties similar to those of the mammalia. The size of the coloured corpuscles varies greatly in diiferent animals. Gulliver has given the following measurements in fractions of an inch — Diameter. ^an, ^^ Elephant, - - ^i^ Musk Deer, u aVu Long Short Diameter. Diameter. Camel, - ^^ ^Vt Ostrich, ^^ ^ij^ Pigeon, ^i„ ^\^ Humming Bird, ^^ ^.^^^ ^^og. ttW t^Vt Crocodile, --.-.. ^^^ ^^ Proteus, ^^ ^ Pil^e, - - ^^ ^^ Sliark, ^,1^ ^^1^ Earth-worm, ^i^ ^^i^^ Leech (after addition of water), - - -j-^ g-^Yr The size of the coloured corpuscles bears a relation to the calibre of the ultimate capillaiy vessels, which are just large enough to admit of their passage in single file. Injections, therefore, of the blood-vessels are more easily made in reptiles than in any other animals. The addition of icater causes the coloured corpuscles to lose their colour, to swell out, and become globular. Syrup, gum, solutions of albumin, and dense saline solutions, render them flaccid, misshapen, irregular in outline, contracted, puckered, etc. Acetic acid appears at first wholly to dissolve mammalian cor- STRUCTURE OF CELLS AND CELLULAR TISSUES. 301 puscles, but their form may be faintly recognised on adding to them tincture of iodine. But on the oval corpuscles of birds, reptiles, and fishes, the effect is simply to dissolve, or render very transparent the protoplasm, while the nucleus is unaffected, and even becomes more distinct. Astringent solutions, and especially a solution of nitrate of silver, cause puckerings and folds to appear in the cell. This is well seen in the corpuscles of the newt, where also a solution of boracic acid develops the nucleiTS in the form of an oval body, from which processes radiate outwards. A solution of magenta causes a minute molecule to appear on the external margin, as pointed out by Sir William Roberts. The same observer, also, was the first to describe the effect of a dilute solution of tannic acid, which causes one, and sometimes two, protrusions to take place from the corpuscle. A stream of carbonic acid passed into frog's blood is quickly followed by the appearance of the oval nucleus, which may again disappear under the action of air, or of oxygen. 3. Epithelial Cells are sharply-defined cells consisting of proto- plasm and a nucleus. A cell membrane is frequently awanting, but it is often represented by a more consolidated portion of the peri- pheral layer of protoplasm. Most epithelial cells are soft, and readily adapt themselves to pressure brought to bear upon them; hence the numerous forms taken by epithelial cells. Two princii^al forms may be distinguished — (a) the flat, and (b) the cylindrical or jDrismatic. There are numerous transitional forms from the one kind to the other. (a) Flat Epithelial Cells, flat cells, pavement cells, seldom jDossess regularity of shape. Pigment epithelium alone (see Fig. 150) consists of tolerably regular hexagonal cells ; in the majority of cases the con- tour is very irregular. (h) Cylindrical Epithelial Cells, cylinder cells, looked at from the side, are distended elements, whose height surpasses their breadth ; looked at from above, they appear polygonal ; they are thus in reality prismatic. Epithelial cells, which are as long as they are broad, are Fig. 147. — Epithelial cells of the rabbit, isolated, X 560 diameters. 1, pavement cells from the epithelium of the mucous membrane of the mouth ; 2, cylindrical cells, corneal epithelium; 3, cylindrical cells, with cuticular edge, s, intestinal epithelium ; 4, ciliated cells — h, cilia, bronchial epithe- lium. (Method No. 14, Appendix.) termed cubical cells. Many cylindrical cells bear on their upper surface a striped border (Fig. 147, 3), which is a product of the cell — a kind of •302 THE PHYSIOLOGY OF THE TISSUES. cuticular structure. Other cylindrical cells have on their free surface delicate cilia, which during life show oscillating motion in a certain direction. We call these ciliated or Jiagellate cdls.'^ Epithelial cells are so bound together that they either touch each other with their smooth surfoces {i.e. through the interposition of an intermediate or cement sub- stance present in very small c[uantity), or they are united by variously shaped prolongations. There are also included under such prolongations the deli- cate bristles and filaments Avhich are visible on the upper surface of certain epithelial cells (Fig. 148). These are connecting threads which penetrate the cement substance between two epithelial cells, and effect a connection Avith bouring epithelial bristles and cells ; the bristles '^iw-^ •'■^^jjinll^' neigh- cells. Cells proAdded with filaments are termed bnstle themselves have been indi- FlG. 14S. — Spinous, bris- gricli tie, or furrowed cells. '"^^^ a, from the undermost layers of the human epi- dermis; 6 a cell from a catcd of late by the appropriate term, intercellular papillary tumour of the '' l r r j human tongue. bridges (Fig. 149). ^ ^V Fig. 14f). — From a perpendi- cularsection through the stra- tified pavement epithelium of the stratum mucosiim of the epidermis, x 560 d. Seven pavement epithelium cells bound to each other by inter- cellular bridges. (Method No. 15, Appendix.) Fig. 150.— Simplepave- ment epithelium (pig- ment epithelitim of the retina) of man, x 560 d. (Method No. 16, Appendix.) Fig. 151. — Simple cylindrical epi- thelium (intestinal epithelium) of man, x 560 d. c, Stripe-shaped cuticular edge, cylindrical cell ; tp, tunica propria, from small intestine. (Method No. 17, Appendix.) Varieties of Epithelial Layers. Epithelial cells are arranged sometimes in simple, sometimes in com- pound, layers. We accordingly distinguish — (1) Simple Pavement Epithelium. — This variety is observed in the pigment epithelium of the retina (Fig. 150), epithelium of the alveoli of the lungs, of the coat of the stomach, of the rete vasculosmn Hcdleri of the skin and of the lining membrane of the labyrinth. The epithelium formed of one layer of cubical cells constituting the covering of the plexus choroidea, and the epithelium on the inner surface of the capsule of the lens, in ^ The specially differentiated sense epithelium cells will be described in treating of the senses. STRUCTURE OF CELLS AND CELLULAR TISSUES. 303 the thyroid gland, and in most other glands, also belong to this class of epithelium. (2) Simple Cylindrical Epithelium, Fig. 151, such as the epithelium of the intestinal canal and of many ducts of glands. (3) Simple Ciliated Ep)ithelium exists in the most delicate bronchise, in the uterus, in the Fallopian tubes, in the nostrils, and in the central canal of the spinal cord. (4) Stratified Pavement Epithelium,. — In this variety, the cells are not all pavement cells, as the deepest layer consists of cylindrical cells. Above this, there are several layers of differently shaped bristle cells (mostly ^ ^ J ^^^ "^--^ in the form of irregular polygons. Fig. 152), and on _^_^^^^-J^r:'^'.- these are placed cells more and more flattened toAvards - '- the surface of the epithelial layer. Stratified pave- - - :; ment epithelium is found in the mouth, in the cavity of the throat, in the oesophagus, on the vocal cords, on the conjunctiva, in the vagina, and in the female urethra. The epidermis is also covered over ' with stratified pavement epithelium ; but the latter is characterized by the cells of the layer nearest to ^-l. "\ the surface being changed to small horny scales, 'w while they have lost their nucleus. On the nails no. i52.-stratifiedpave- and hair also, horny, but in this case nucleated, S'manf x^fo" d ^""l^y. scales are found. _ s^J^fl^af^./eilk'^iMei^od^ (5) Stratified Cylindrical Epithelium is, in the case of ^°- ^^' Appendix.) man, found only on the conjunctiva palpeh'arum. The arrangement of the layers is the same as in the next form. (6) Stratified Ciliated Epithelium. — The cells next to the surface are cylindrical and they bear minute hair-like processes, cilia; in the deepest layers, roundish cell elements are met with, and in the middle layers, spindle-shaped ones occur. Stratified ciliated epitheliimi is found in the larynx, in the trachea, and in the large bronchiee, in the nostrils, and in the upper part of the throat, in the Eustachian tube, and in the testicles 1 (Fig. 153). 4. Connective Tissue Cells are recognized as such only when found in the tissues, that is to say, an isolated connective tissue cell is indistinguishable from a leucocyte. In form they are very variable. Connective tissue cells are either flat, polygonal, bent, or twisted in various directions; or they are roundish, oval, spindle or star-shaped. (See Connective Tissue.) Their arrange- 1 The embryonal origin of epithelial layers has already been indicated, p. 251. 304 THE PHYSIOLOGY OF THE TISSUES. ment is the reverse of that of epithelial cells, inasmuch as the 'rule is that they are separated from their neighbours by means of consider- 'J:.v.-4 Fig. 153. — Stratified ciliated epitlielium: 1, oval ; 2, spiudle shaped ; 3, cylindrical cells. X 500 d. From the mucous mem- brane of the nose {ra- gio respiratoria) of man. (Method No. 19, Appendix.) Fig. 154. — Endothelial cells on the serous covering of the diaphragm. (Some of the ragged openings are probably accidental.) able masses of connective tissue. Endothelial cells are an excep- tion to this as they have a great resemblance to simple flat ej^ithelium and can be distinguished only by their anatomical position (in the cavities of the joints, in the sheaths of tendons, in the bursce mucosce, and on the inner surface of the heart, blood-vessels, and lymphatics). (Fig. 154.) The protoplasmic nucleated body of the cells of connective tissue may contain granules of colouring matter. The cells are thereby changed into pig- ment cells, vrhich occur in man only in the eye, but in many of the lower ani- mals they are widely diflPused. Other mrttefi"n~theii'pTOtopksml'*^a?A^^^^^^ Connective tissue cells may contain small Td^d i^H^ranofLf oTSr^am^; clrops of fat, which, Avheu they become :^:^^t^^^^^^^^C^^ la^ge. give a spherical shape to the ^^^^^l^lT^tS^ cell. Such are termed fatty cells. (See e, another with granules of melanin. pjg_ 1 5 5^ f/. ) Jn fatty CcUs, the protoplasm forms only a narroAV border, situated on the periphery of the cell. The flattened nucleus is found at the side of the cell. The border is fre- quently so thin that it sinks out of sight. (See Fig. 155.) 5. Fat or Adipose Tissue Cells are nearly akin to the fatty cells above described, but they are nevertheless distinguished from the latter by the fact that they are united without the interposition of an intermediate substance, so as to form in certain parts of the body a tissue interpenetrated by numerous blood vessels, lymph vessels, and STRUCTURE OF CELLS AND CELLULAR TISSUES. 805 nerves, namely, fatty or adipose tissue, whicli plays a very important part in various physiological processes. In extreme emaciation. Fig. 156. — Fat tissue cells from the axilla, x 240d. y4,of an individual only a little emaciated ; 1, on focusing the objective on the equator of the cell ; 2, objective slightly raised ; 3, U, cells put out of shape by pressure ; p, traces of protoplasm situated in the neighbourhood of the nucleus. B, of a highly emaciated indivi- dual ; k, nucleus ; /, small drops of fat ; t", blood capillaries ; b, bundle of connec- tive tissue. (Method No. 20, Appendix.) we find, in individual fat tissue cells, the fat shrunk to small drops, and its place occupied by a pale protoplasm, mingled with mucous fluid. The cell is no longer spherical, but has become flat. Such cells are termed serous fat cells. 6. Muscle Cells. — The fibres of the muscles appear in two forms, which we term the smooth and the transversely striated. Both are cells, of which the body is greatly extended in a longitudinal direc- tion. (1) The smooth muscular fibres (Fig. 157) (contractile fibre cells), are spindle-shaped, cylindrical, or slightly flattened cells, having pointed ends. Their length fluctuates between 45 and 225 /x ; their breadth between 4 and 7/x ; in the pregnant uterus, still longer, smooth muscu- lar fibres, measuring ^ mm., have been found. They consist of a homogeneous protoplasm, containing a rod-shaped nucleus. ^ This nucleus is characteristic of smooth muscular fibres. The fibres are imited firmly to each other. Smooth fibres are found in the intestinal canal, in the smaller bronchial tubes, in the gall bladder, in the pelvis, in the kidney, in the ureter, in the urinary bladder, in the sexual organs, in blood and lymph vessels, in the eye, and in the skin. As this kind of fibre is not subject to voluntary control, it is often called involuntary muscular fibre. (2) The transversely striated muscular fibres are to be recognized as ^ In individual smooth muscular fibres a longitudinal striation of the protoplasm has been observed. I. U 806 THE PHYSIOLOGY OF THE TISSUES. cells only by studying their development. As the result of a colossal growth in length, of continued division of the nucleus, as well as of peculiar difterentiation of their protoplasm, they have attained to Fig. 1.07. — Two smooth muscular fibres from the small intestine of a frog. Isolated by means of 35 per cent, solution of caustic potash. The nuclei have, by action of the caustic potash, lost their characteristic form. (Method Xo. 21, Appendix.) highly complicated forms. They have the shape of long cylindrical fibres, which are usually rounded off at the ends, or obtusely pointed, but in certain cases (muscles of the eye and muscles of the tongue) the fibres are branched (Fig. 158). Their length, only in rare cases, exceeds 4 cm. ; their thickness fluctuates between 15 and 50 /x ; and the muscles of the face have finer fibres than the muscles of the trunk. The following table gives certain measurements of muscular fibres in fractions of an inch. (Allen Thomson.) Muscle of Diameter of Fibres. Distance ok Transverse Stri^. Greatest. Least. Average. Birds, - ^"5(r TTi7TF 1 TTTT-OD Mammalia, TTTJ TT"iJ"S" -e\-(i TO Man, TTS" 1 <>16 T-ou TF^uU Reptiles, TXTU 1*00 -^ TTioT) Fishes, - 1 "B5 7T^ 1 TTTTnr Insects, - 1 1 ^h> ITsW Each transversely striated fibre consists of the foUoAving parts — (1) A structiu-eless covering or membrane, termed the sarcolemma, which surrounds the fibre like a sac. (2) Oval nuclei, placed parallel to the axis of length of the muscular fibres, Avhich nuclei lie, in the case of the mammalia, between the sarcolemma and muscle substance ; but in other vertebrates, in the muscle substance itself, in Avhich they are surrounded by a very small amount of protoplasm. This protoplasm is the remain- der of the original cell protoplasm not spent in the up-building of the transversely striated muscle substance, and it is accumulated especially at the poles of the nuclei. The granulated accumulations of protoplasm are termed interstitial granules. (3) The muscle substance. It is trans- STRUCTURE OF CELLS AND CELLULAR TISSUES. 307 versely striated, i.e. it shows alternately dark and narrower, and clear and broader transverse bands. The substance of the dark transverse bands is doubly refractive (cmisotrojpk substance) ; that of the bright transverse bands is singly refractive {isotropic substance). Under strong- magnifying powers, double transverse stripes {transverse lines) may be perceived (Fig. 164), which divide the clear transverse bands into two ■equal parts ; in the case also of the dark transverse bands, another set of transverse stripes, the so-called middle discs, has been described. The collective stripes and bands (with the exception of the middle discs) penetrate the entire thickness of the muscular fibres, and they are thus in reality discs. Besides the transverse striation, there is also to be observed a more or less pronounced longitudinal striation of the muscular fibres. Certain reagents, for example, solution of chromic acid, cause this longitudinal striation to manifest itself still more distinctly, and •effect even a falling asunder of the muscular fibre lengthwise into delicate similarly stri2)ed threads, which are termed fibrils. Other reagents, for example, solutions of hydrochloric acid, bring the transverse striation more distinctly into prominence, and they can, moreover, cause £i falling asunder of the muscular fibres transversely into discs. Fibrils iind discs can fall asunder into still smaller, roundish-edged, anisotropic portions, which are termed the primitive muscle particles or the sarcous elements. Fig. 158. — Portions of isolated transversely striped muscular fibre of a frog, X 50 d.' 1. Effects of water; s, s', sarcolemma. At x the muscular sub- stance has been torn asunder, its transverse striation is not visible, but its longitudinal striation, on the contrary, is distinctly seen. 2. Action of acetic acid; k, nuclei. The delicate puncturing corresponds to the inter- stitial granules. 3. Action of a concentrated solution of caustic potash ; ends rounded off, the numerous nuclei appear to have flowed into a vesicle or swelling e ; the transverse striation of the substance of the muscle.'? is in 2 and 3 invisible under this magnifying power (Method No. 22, Appendix). 4. Branched muscle fibre from the tongue of a frog (Method No. 23, Appendix) 308 THE PHYSIOLOGY OF THE TISSUES. Fig. 15fi. — Polarizer for microscope. A, Nicol's prism ; B, brass case ; D, end next mirror ; C, plano-convex lens — which is not necessary. Examination of muscn/nr Jibre by polarized liijhi (see p. 66). — As already described, when examined by ordinary light, a muscular fibre is seen to consist (1) of a substance which forms the sarcous elements, highly refracting, and black M^hen seen by trans- mitted light ; and (2) of an intermediate or fundamental substance, ■which is much less refractive, and is clear by transmitted light. Suppose now that arrangements are made by which the fibre, whilst on the stage of the microscope, can be placed between crossed Nicol's prisms. This is accomplished by placing; one]^ Nicol's prism below the stage {the {j}o/arize7; Fig. 159), and another in the eyepiece of the microscope (the analyzery Fig. 160). Either or both of these pripms are capable of rotation, and they may be so rotated with reference to each other that when we look through the microscope the field is quite dark. It will then be found that if a striated muscular fibre is in the dark field, the sarcous elements (represented by the dai'k transverse bands seen with ordi- nary light) shine very distinctly, while the clear band seen with ordinaiy light is now invisible. The observation is more striking if the slide containing the muscular fibre is placed on a thin plate of selenite. As only doubly refractive substances thus shine out in the dark field of crossed Nicol's, it follows that the sarcous elements are anisotropic or doubly refractive, while the substance which unites the contiguous discs is isotropic, that is, singly refractive. According to Engel- mann, contractility is manifested exclusively by the anisotropic portion. Each sarcous element behaves as a doubly refractive body having only one positive axis,^ of which the optical axis is parallel to the long axis of the muscle. As they change their form during contraction, becoming shorter and thicker, they cannot be regarded simply Fig. 160. — Analjv.er for microscope. A, Nicol's prism ; B, eyepiece ; C, field yrlass ; D, eyeglass. Other references of no importance. 1 A body having its molecules uniformly arranged in all directions afi'ects light only by simple refraction, that is, the incident ray gives rise to one refracted ray, and the crystal possesses one refractive index. When, on the other hand, the molecular structure of a body possesses parts of different density in certain direc- tions, it manifests double refraction, that is, a ray of light traversing it is divided into two rays, one of which is bent more out of its course than the other. Common glass is simply refractive, and is said to be isotropic ; but if it be compressed in one direction, it may become doubly refractive or anisotropic. _i_i::li_ ?;^j~'jv~Ti' 1 1 1 -y}-_\ ... 1^— - Ij — I— |— |— ) , my--^ r'' h STRUCTURE OF CELLS AND CELLULAR TISSUES. 809 as double refractive bodies like crystals, but, as suggested by Briicke, they must consist of a great number of small doubly refractive particles, to which he has given the name of disdiaclasts, and which he supposes are irregularly distributed through the fundamental substance (Fig. 168). The latter, which may be termed inicsde-plasma, is a viscous liquid capable of absorbing water freely, while the disdiaclasts are more dense and are less capable of taking up water. As has been remarked by Schafer, Briicke himself pointed out that in living muscle at rest the whole of the muscular substance appears doubly refracting, and that it is only in contraction that the alternate stripes appear singly refracting.^ Some authors have claimed the fibrils, others the discs, and others again the sarcous elements, as the primary form elements of the fibres of the muscles. In the latter case, we would have to represent the building up in such a manner that the fibrils are formed from sarcous elements placed so as to form a row and the discs from the sarcous elements being placed side by side, in both cases assuming that the sarcous elements cohere. Various theoretical views have been advanced re- garding the ultimate structure of striated muscular fibre, and the question must still be regarded as unsettled.'^ A view, regarded with favour for some ^ ' = _ Fig. 161.— Showing time, was that the muscular fibre might be regarded hypothetical views - "^ ° regarding the struc- as built up of small caskets (see Fig. 161), bounded ture of striated ■^ _ ^ . muscle. Four fibres at the two ends by a thin membrane or band in the side by side. «, ni7-ir-)7- T7- clear bands or discs, centre of the clear band, called Dome s line or Krause s each formed of two clear bands or discs, membrane, c, and laterally by the sarcolemma. in this termed the lateral ,. 11/P11 discs of Encjdmann, casket there were supposed to be (irom above down- separated by a thin wards in Fig. 161)— (1) a thin clear disc, the lateral disc known as dou/s of E^igelmann; (2) a disc of dark substance, the sarcous membrane: 6,' two element of Boivrnan ; (3) in the centre of this disc, an ill- stance°constitutiDg J n -I 1 1, j.-j.j.-j.T_ 7' 7- the sarcouis elements defined granular substance constituting the median disc of Bomaan, having of Hensen, h ; and (4) another lateral disc of Engelmann. lu-defined^band or Further, the dark portions forming the sarcous elements J^",®' of^HenTenTc, of Bowman are the anisotropic or doubly refractive part, ^opic Vr'singVre- while the clear portion, formed of two discs of Engel- f ^'^an^fsotropic^" or mann separated by Dobie's line, is singly refractive or ^"^^J^ refractive isotropic. These appearances have undoubtedly been figured and described, but they are to be regarded as optical expres- ^ Briicke, Physiologie, p. 465. ^ An excellent account of this subject is given by A. van Gehuchten, in Etude .sur la Struchire Intime de la Cellule Muscidaire Striee. — La Cellide, tome ii. '1^ Fascicule. 310 THE PHYSIO LOO Y OF THE TISSUES. sions of layers of substances acting differently on light, and also some- times superposed, so that the structure of one fibre shines through, and affects the appearance of, the one above it. There is also the further difficulty that if a living nnxscular fibre is examined under the micro- scope, especially A\ath high powers, the amount or degree of relaxa- tion or of contraction affects in a marked Ava)^ the optical appearances, so that the fibre may present a different appearance M'hile at rest from Fig. 1C2. — Living muscular fibre from Geotrupes ster- coranus. Fig. 163.— The same fibre seen by polarized light, with crossed Nicol's prisms. Avhat it shows in a condition of contraction. Thus, a fibre may show the appearance represented in Fig. 162, in which the clear bands are narrow, Dobie's line dark and well defined, and the dark band very wide : or a fibre may, in another condition, show more details of structure, as in Fig. 164. In Fig. 163 the same fibre as is represented in Fig. 162 is seen as it appears in the dark field of crossed Nicol's prisms, with polarized light. It will be observed that the dark anisotropic portion of Fig. 162 is clear and brilliant in Fig. 163, while Dobie's line, and the part on each side of it, is dark. I have recently convinced myself, after a study of muscular fibre with the use of chloride of gold, that the appearances shown in Figs. 162, 164, 165, and 167, can be seen, and they support the view that the muscle sub- stance is composed of rod-like structures, as first described by Professor E. A. Schafer.^ Whether these rods, in turn, are only the optical expressions of a fibrillar net-work Avith elongated meshes, as advanced by Marshall, is in my opinion more doubtful. I have not been able to satisfy myself upon this point, and especially have I failed in detecting appearances favoiu-ing the vieAv that there is a net-Avork Avith irregularly formed ^ As to Schiifer's views, see Philosophical Transactions, 1873, and Quain's Anatomy, vol. ii. p. 123, et seq. Fig. 164.- •'Muscular fibre from Hydi-ophilus pisccus (large water-beetle), show- ing all the details of struc- ture represented in the diagram. Fig. 161. STRUCTURE OF CELLS AND CELLULAR TISSUES. 311 meshes in the clear disc. The conception of a muscular fibre as an elongated cell in which the fibrillar net-work assumes a regular arrange- ment is one of much significance, as it brings muscular fibres into the general category of cells. Fig. 165.— Muscular fibre of Geotnipes stercorarius, living, showing rod-like structures passing longitudinally in the dark substance, and rod or some- what square-shaped points or dots in the clear substance. Fig. 160. — Same fibre as in Fig. 165, seen with polarized light. Dark bands of Fig. 165 are the clear bands of this Figure. Fig. 167. — Muscular filire from Geophilus, showing rod - like structures running longitudinally with dots or points at their ends. Fig. 16S.— Same fibre as in Fig. 167, seen with polarized light. Observe that the rods and the dots of Fig. 167 are clear, and are therefore doubly re- fractive or anisotropic. Striated muscle fibres are found in the muscles of the larynx, of the extremities, of the eye, and of the ear. They are also found in the tongue, in the pharynx, in the upper half of the oesophagus, in the larynx, and in the muscles of the genital organs and of the rectum. In the case of many animals, e.g. the rabbit, two different sorts of transversely striped muscles are found ; red (for example, the semitendinosus and the soleus), and white (for ex- ample, the adductor magnus). The red muscles contract slowly, the white ones quickly, and they also showmicroscopic differences, inasmuch as the fibres of the red muscles possess a some- what irregular transverse striping, a more distinct longitudinal striping, and a larger number of roundish nuclei than the pale variety. In some animals, the two kinds of muscular fibre appear to be separated from Fig. 169. — B. Portion of a human muscuhir fibre, X 560 d. (a) Anisotropic and (i) isotropic transverse bands ; (5) transverse line ; (k) nuclei. (Method No. 23 a, Appendi.x;.) A. The end of the mus- cular fibre of a frog, x 240 d. Splitting into fibrils (/) ) W nucleus. (Method No. 24, Appendix). :312 THE PHYSIOLOGY OF THE TISSUES. lar fibres, a, low degree I), a higher ; and, c, the highest degree. one another in particular muscles ; hut in others, as in man, the two kinds of fibres are sometimes mixed in the same muscle. The con- traction of the transverse striped muscles is quick and under the control of the Avill, and hence this kind of muscle is often termed voluntary muscle. When the fibre contracts, the contraction occurs in the broad, dark, transverse band, which, when contracted, increases in breadth, while the clear discs and the narrow, dark, transverse band play a purely mechanical part. Muscular fibre is sometimes the seat of fatt}' changes, in which the striae disappear, and the muscle- substance becomes crowded with small molecules of fat (Fig. 170). The smallest blood-vessels, or capillaries of muscle, form a fine network of elongated meshes between the fibres, but not extending within the sarcolemma, as Fio. 170.— Fatty degen- <5}ir,TVTi in Fi'a T71 ci-ation of human musou- SnOWn lU J^lg. i/i. (3) The muscular fibres of the heart occupy a special position. They show transverse striation, but it is not so distinct as in ordinary striated muscular fibre. A consideration of their development as well as their behaviour under the microscope show that the mus- cular fibres of the heart are only modified contractile fibre cells. In animals low in the scale {e.g. the frog) they are spindle-shaped, provided with a large nucleus, the protoplasm is distinctly striped trans- versely as well as longitudinally (Fig. 112, A); in the mammalia the fibres take the form of short cylinders, whose ends are often irregularly dentate (Fig. 172, B). The protoplasm is differentiated into transverse striped fibrils, between which the remains of non- differentiated protoplasm may still be seen. Longi- tudinal striation is often very distinct. Around the simple or duplicated nucleus, there lie masses of homogeneous protojDlasm, and granules exist in veiy large quantities. The sarcolemma is absent. The connection of the irregularl}^ cubical-shaped fibres by means of short, crooked, or transverse processes is characteristic of the muscular fibres of the hearts of Fio. 171.— Capillary net- the higher aiiimals (Fig. 172, B). work of striated muscu- -.^ ii-i 77 7 i t-t.t larfibre. a, arteriole ; 6, Muscular fibres are developed, as already indicated, venule: c and d, capil- p ,i i ,• o\&i,f; the two other cells are surrounded by a covering containing a nucleus, h. b, spindle- shaped ; c, multipolar ganglion cells from the spinal cord of an ox, X 80 d. (Method No. 28, Aj)pendix.) d, multipolar ganglion cells (those of Purkinje) from the cortex of the cere- bellum of man. (Method No. 29, Appendix.) d x SO d. JO, poles of protoplasm ; ax, axis cylinder pole. The nuclei of 6, c, and d, have, by the method employed in preparing them, lost their characteristic form. A connection of the ganglion cells occurs in such a manner that a network of fibrils is formed by the protoplasmic poles of several ganglion cells. Closely related to nerve cells are the nerve fibres. These may be here described, although their cellular nature is still the subject of controversy. 8. Nerve Fibres occur in tAvo forms, designated as (a) medul- STRUCTURE OF CELLS AND CELLULAR TISSUES. 315 apparently homogeneous, faintly lated, and (b) non-medullated nerve fibres. The two forms are not to be regarded as sharply separated locally or generically. It is quite a usual phenomenon for one and the same nerve fibre to be, in the beginning of its course, medullated, and towards the end of its course non-medullated. (a) Medullated Nerve Fibres are shining fibres, from 1 to 20 /x in thickness. The most important part is a fine elastic cylindrical fibre traversing the axis of the fibres. This is called the axis cylinder of Purkinje, or the band of Remak (Fig. 175). Delicate longitudinal stripes, which are sometimes observable on it, are the expression of the fact that the axis cylinder may be com- posed of fine fibrils ; a very notice- able transverse striping (Fig. 176), which becomes visil)le after treat- ment with a solution of nitrate of silver, has not yet been clearly explained. The axis cylinder is sur- rounded by the sheath of the marrow, or the tvhite substance of Schtoann, This consists of a fluid, strongly-refractive, fat-like substance called A la B c -Tc Fig. 175. — Nerves, c, ordinary-sized nerve fibre, showing axis cylinder surrounded by white substance ; d, smaller nerve iibre, with white substance scarcely visible ; /, varicose nerve fibre, from grey matter near surface of cerebrum ; o, nerve fibre, stained by perosmic acid, showing one of the nodes of Kanvier, or complete hiterruptiou of the white substance ; b, nerve fibre, showing nucleus and node of Ranvier (the axis cylin- der is blackened by the action of perosmic acid) ; g, non-medullated nerve fibres from sympathetic, having no white substance, and nucleated at intervals : c, smallest fibre. 2 .? Fig. 176. — Medullated nerve fibres, from the sciatic of the frog, x 240 d. i, f, 3, Fresh with solution of common salt. (Method No. 30, Appendix.) 3, fibre with constrictions, r ; U, nerve fibres under the inflijence of water. (Method No. 31, Appendix.) 5, nerve fibre treated with absolute alcohol. (Method No. 32, Appendix.) »>., coagulated marrow ; a, axis cylinder. B and C, medullated nerve fibres cif the rabbit, X 5(i0d. 6, Fresh ; c, cylin- dro-conical segments. 7, S, Hardened. (Method No. 33, Appendix.) «, Axis cylinder ; 6, biconical swelling ; r, constriction ; w, white substance coagulated and lifted off from s, Schwann's sheath \'k', nucleus of the endoneurium. :M{y THE PHYSIOLOGY OF THE TISSUES. Fig. 177.— MeduUated nerve fibres of the frog treated with .sohition of uitrate of silver, x 5(30 d. 1. -/•, nodes; a, axis cylinder blackened only to a slight extent ; 6, bicoiii- cal turgescence caused by the axis cylinder being squeezed in the isolation process. 2. a, ax is cylin- der in situ blackened only to a slight extent. 3. axis cylinder with transverse striping. The axis cylinder is not visible in the case of 3. (Method No. 34, Appen- dix.) myelin. Under favourable circumstances, we perceive that the \vhite substance is not continuous, but is divided at somewhat irregular dis- tances into cylindro-conical segments by means of oblique incisions or grooves {Lan- termann^s Notches, Fig. 176, Z»). The centx'al band, which, during life, is quite homogeneous, experiences, after death and when different reagents have been added to it, a structural metamorphosis. At the begin- ning, the nerve fibre possesses a double con- tour ; at a later period, the central band or marrow may be broken up into small globular masses. The sheath of the white substance is a delicate structureless cuticle, known as Schuxinn's sheath or neurilemma. On the inner surface of the sheath, prolate spher- oidal nuclei may be seen surrounded by a small quantity of protoplasm. At regular dis- tances, constrictions are observed where the white substance is wanting, so that the axis cylinder and Schwann's sheath come into contact. These constrictions are known as nodes, or the nodes of Ramier. The axis cylinder shows a biconical swelling in the neighbourhood of the nodes. Treatment with solu- tions of nitrate of silver shows also cement substance on the nodes (Fig. 177, r). Every medullated fibre is provided Anth nodes, which, being ar- ranged at regular intervals, divide it into interannular segments. Medullated nerve fibres occur in the roots and branches of the cerebro-spinal nerves, but they are also present in the sympathetic nerves. At a subsequent " y . ' stage, they appear in the brain and spinal cord, Avhere they lose Schwann's Fig. 17S.— Teazed preparation of the sym- pathetic nerve of a rabbit, x 240 d. 1, sheath. ihc thickness 01 a nerve fibre Non-medullated ; 2, thin meduUated nerve , . . - fibres ; 3, ganirlion cells ; the characteristic AVarrantS nO COllclUSlOn With respect tO appearance of the large nuclei has been lost . , , ,., . ^ , by the action of the osmic acid used in its motor or seusory qualities ; On the ne^cViv«tlssurfibres!^5°"deUcate''conLcti?e Other hand, it is established that the tissue fibres. The protoplasm surrounding r.-, • • xi. • i the nuclei of the pale nerve fibres is not fibres increase in thicloiess in propor- r^faSis:*jiivti.„_ Fig. 195. — From a C'orsoplantar longitudinal section of the great toe of a human embryo of four months. Two thirds of the first phalanx are indicated, x 50 d. r, centre of ossification ; li, cavities of the cartilage en- larged, containing several cartilage cells. The cells cannot be recognized here under a feeble magnifying power, but only their point- shaped nuclei, g, calcified matrix of cartil- age ; K, hyaline cartilage ; k, growing cartilage, we perceive the cartilage cells arranged in groups of three to four cells ; each group is produced by the repeated division of one cartilage cell ; o, osteogenous, or bone forming tissue; P, perichondria! bone. (Method No 58, Appendix.) Fig. 196. — Fi-om a dorsopalmar longi- tudinal section of a finger of a four months' human embryo. Two thirds of the second phalanx are shown, X 50 d. il/, primordial medullary space containing, h, cartilaginous mar- row and blood-vessels ; 7i, enlarged cartilage cavities ; j', osteoblasts by this time arranged in a layer ; the two uppermost osteoblasts x are already surrounded to the extent of onehalf with bone matrix. -F, jjeriosteum. (Method No. CO, Appendix.) 340 THE PHYSIOLOGY OF THE TISSUES. the peripheiy ; but with the gradually increasing thickening of the perichondrial strata of bone the channels are closed up (/t'), so as now to become canals containing vessels. These are the Haversian canals. By the activity of the osteoblasts enclosed in the Haversian canals, new bone strata, termed the later Haversian lamellae, are formed. b h oIk -^-^on\:\ Fig. 19S.— Portion of a transverse section through the diaphysis of the humerus of a human embryo of four months, x SO d. P, periosteal trabeculaj having osteoblasts, ob, on their margins ; h, h, h, small Haversian canals shut in by bone formation ; h', small Haver- sian canal closed ; E, encbondrial trabeculfe of bone likewise covered with osteoblasts and containingr the remains of calcified carti- lage matrix, g ; z, marrow cells; h, blood vessels. (Method Xo. 61, Appendix.) Thus a bone has been formed out of a cartilage by the dis- solution and removal of the cartilage and the supplying of its place by bone (encJiondrial ossification) and by vY/^ — ^ - e bordtr;T ^^ ^^^^^^^ necessary to replace by diffusion a unit of weight of the dissolved body (1 gramme), is •called the endosmotic equivalent of the body. This equivalent depends (1) on the chemical nature of the body ; and (2) on the degree of con- centration of its solution. The follomng table, given by Jolly, shows the equivalents of certain substances — Fig. 202.— Endosmometer. A, irlass cylinder coustnicted so that an organic membrane (piece of bladder), a, b, can be tied over its lower end by the OSMOSIS IN RELA TION TO TISSUES. 849 Name of Substance. Endosmotic Equivalent, Chloride of sodium, ------- 4-0 Sulphate of soda, - - - - - - - - 11 '0 Sulphate of potash, ------- 12-0 Sulphate of magnesia, - - - - - - - 11 '5 Sulphate of copper, ------- 9-5 Sulphui-ic acid, -------- 0"3 Caustic potash, -------- 200 '0 Alcohol, -.---.._. 4-3 Sugar, ---------- 7-2 That is to say, suppose the endosmometer to be filled Avith solutions of the substances in the above list and to be placed in distilled water, 4 grammes of water would pass through the membrane into the endos- mometer for 1 gramme of chloride of sodium, 1 1 grammes of water for 1 gramme of sulphate of soda, and so on. Thus, by this method of comparison it can be shown that the endosmotic equivalent of chlorides is small, of nitrates greater, of bases very great, of acids small, and of acid salts much smaller than of neutral salts. As Jolly, in these experiments, took no account of the degree of con- centration of the fluids, and as he always employed dried membranes, these figures do not show the quantities of these substances which would pass through organic membranes in the living state. Hofmann has shown also that the amount of water of hydration or of crystalliza- tion, even in the same salt, influences to a, remarkable extent the endosmotic equivalent. When more water passes towards the salrae solution than the amount of the latter which enters the water, the diffusion is said to be iwiitive, and negative when the reverse is the case. With alkalies positive endosmosis is strong ; with acids, negative endosmosis is the rule, while salts are positive and range between the two extremes. When endosmosis is positive, the equivalent increases with the degree of concentration ; when, on the contrary, it is negative, the equivalent dimmishes as the concentration increases. Thus, in the diffusion of sulphuric acid with water, more acid will pass into the water as the acid becomes concentrated ; but, on the contrary, in the diffusion of potash with water, more water will pass into the potash as the latter is concentrated. According to Ludwig, sulphate of soda is an exception amongst bodies showing a positive endosmosis, as its equivalent diminishes by concentration. Diffusion occurs with a constant raindity so long as the solution has the same concentration, and as the temperature remains the same. (a) Effects of the Degree of Concentration. — The rapidity, however, does not depend only upon the endosmotic equivalent, but also upon the solubility of the substance and its chemical composition. It increases with concentration. When saline solutions diffuse into water, the rapidity with which the salt passes towards the water, as well as that with which the water passes to the salt, increases with the degree of concentration, but not at the same rate. The rapidity with which the water passes to the salt becomes greater, whilst the rapidity with which the salt 350 'I'lil'^ PHYSIOLOGY OF THE TISSUES. passes to the water remains near-ly proportional to the degree of concentration. Thus it follows that the more a solution approaches saturation, the greater is the quantity of water which passes in the same time. [h) Effects of Ttmptralnre. — As the temperature increases, the rapidity of the diffusion also increases, and the rapidity of diffusion increases more rapidly as the temperature rises. This will be seen in the following table, given by Eckhard, showing the rapidity with which common salt passed through the fresh peri- cardium of an ox in the same time with an increasing temperature : — Temperature in Quaiititj' of Common Degrees C. Salt which passed. 8-0, 0-303 9-6, 0-364 13-8, 0-396 18-3, 0-474 22-5, 0-549 •26-0, 0-628 (c) Effects of a Mixture of Saline Matters. — When a solution dififuses through a membrane not only with water, but with a solution of the same or of a different substance, that is, where two solutions of the same substance or of difl'erent sub- stances, are on opposite sides of the membrane, the diffusion depends partly on the degree of concentration of the two solutions, and partly upon the chemical properties of the two bodies dissolved. Suppose two solutions of the same substance, biit of unequal degrees of concentration ; the more concentrated solution will diminish, while the more dilute will increase in density. In this case, the endosmotic equivalent has a constant value. On the other hand, the rapidity of diffusion will be in the inverse ratio of the difference of concentration of the two fluids present ; that is to say, as the initial difference of concentration of the two fluids diminishes, the rapidity of difi"usion will also become less. (d) Nature of Siibstance. — All colloidal substances in solution pass with great difficulty through organized membranes. Such bodies attract water, so that, when their solution diffuses with this fluid, there is a positive current of water. The endosmotic equivalent of these substances is very high, but on the other hand the rapidity of their diffusion is low. Albumin in solution has a stronger endosmotic affinity for saline solutions than for water, and the cui'rent of albumin increases very rapidly with concentration of the saline solution. When a mixture of colloidal bodies in a fluid along with some crystalloids is permitted to diffuse into water, none of the colloidal matters passes through the membrane. Thus from a solution of gum, albumin, and sugar, none of the first two will pass, but the sugar will pass through with great ease. There is thus a kind of mechanical separation of the substances. To this general rule, however, there is one exception, namely, when by the diffusion of a substance mixed with colloidal matters, there is pro- duced on the other side of the membrane a liquid toward which the colloidal matter has a great tendency to diffuse. Thus when a mixture of albumin with common salt is exposed to diffusion into water, the salt alone at first passes through the membrane, but when the water on the other side of the membrane contains a certain amount of salt, the albumin then dififuses with considerable intensity. Von Wittich and Funke have studied, as regards diflfusion, the differences of solutions of various albuminoids, and they have found that of these bodies, peptones possess the property of diflfusibility to the greatest extent. OSMOSIS IN RELA TION TO TISSUES. 351 (e) Effects of Electric Currents. — A continuous electric current passed through a fluid, in a diffusion apparatus, affects diffusion ; the quantity of fluid situated on the side of the negative pole increases, whilst that on the side of the positive pole diminishes. The mass of fluid moves in the direction of the positive current, and the quantity of fluid carried away is always greater as the fluid is easy to move and as the galvanic current is more intense, and the amount is independent of the nature of the surface and of the thickness of the porous plate. When water is allowed to diffuse with a saline solution, and a galvanic current is directed through the two liquids from the water to the salt, more water will pass to the saline solution than would have passed if the current had not been there. If now the direction of the current be changed, so as to pass from the salt to the water, more salt will then pass towards the water, or, in other words, the osmotic action has been inverted by the galvanic current. Albumin is found in the body, combined with alkalies, as albuminate of soda or of potash, and it behaves in these compounds as a feeble acid. Suppose that albumin and saline matters are submitted to diffusion with water, and that at the same time a current is passed through the solutions, it will be found (1) if the positive current be directed from the solution of albumin towards the water, salts will pass from the side of the water, and albumin will remain near the positive pole ; (2) if the current pass from the water to the albuminous solution, water will pass to the albuminous solution, and albumin will pass through the membrane into the water, and will be deposited near the positive pole. The albumin then be- haves as acids do, having passed from the negative towards the positive pole. These interesting facts, ascertained by Von Wittich, indicate the possibility of the physical phenomena of nutrition being affected by a continuous galvanic current, and they suggest many researches of therapeutical importance. (/) Eature of Orgcmic Membrane. — The nature of the membrane affects os- motic action. Dry membranes have always a higher endosmotic equivalent than those which are fresh or wet. In comparing organic membranes of different struc- ture, it is found that the dimensions of their pores have an important influence, just as is known to be the case in diffusion through plates of clay. Buchheim has shown that for a membrane with large pores, the endosmotic equivalent of a salt is smaller as the affinity of this salt for water is greater, while, on the other hand, for very dense membranes, the equivalents are proportional to the affinity of the salts for water. The following table by Harzer shows the variations of endosmotic equivalents for different membranes — Ox Bladder. Pig's Bladder. Ox Peri- cardium. Collodion Membrane. Chloride of sodium, - Chloride of potassium, Sulphate of soda, - - - - Sulphate of potash. 6-460 5-601 18-764 13-908 4-335 12-231 11-700 4-000 10-200 3-891 13-632 8-915 6 097 8-181 4-147 1 Of all of these membranes, that formed of collodion is the most dense ; the bladders of the ox and the pig are both less, and the pericardium of the ox occupies a mean position. The sulphates have a much stronger affinity for water than the alkaline chlorides. All the phenomena of osmosis may be attributed to the following causes : (1) to a force of attraction of two fluids, the one for the other ; 352 THE PHYSIOLOGY OF THE TISSUES. ( 2) to a relative attraction that the substance forming the membrane exercises upon the two liquids in dift'usion — a force which determines the mode in which liquids pass, and the rapidity with which they pass, through small porous canals ; (3) to the narrowness of the pores through which the liquids pass ; and (4) to the diminution of adhesion of the liquid to the wall of the porous canals, by reason of elevation of tem- peratiire. Briicke was the first to attribute the phenomena of diffusion to an attraction betAveen the walls of canals and water. The importance of applying these facts regarding filtration and os- mosis to the phenomena of the nutrition of living tissues is becoming more and more recognized. In the present state of our knowledge, however, it is impossible to follow all the stages of the process, and we can only make general statements. The connective tissues are surrounded on all sides by fluids such as blood, serous transudations, and lymph. These fluids may be regarded as saline solutions of albuminous matters, or as solutions containing both crystalloids and colloids. The fluids are imbibed by the connec- tive tissues just as they would be taken up by porous substances, by a process which may be called capillary imbibition. But when the fluids reach the ultimate tissues in the form of protoplasmic masses, or of protoplasm more or less modified, we have to deal with a homogeneous structure containing no pores. Here an interchange occurs between the fluid and the tissues by a kind of molecular imbibition, a process somewhat similar to that by which a membrane, as above stated, allows osmotic phenomena to occur. Molecular imbibition possesses the two following characters : (1) When a tissue imbibes a fluid, it usually increases in volume, but this increase does not always correspond to the quantity of the fluid imbibed, and in some cases actual diminution in size of the mass of tissue may occur after imbibition ; and (2) tissues imbibe more distilled water than water containing saline substances, and consequently the fluid which is imbibed by a membrane \nll be less concentrated than the fluid in which the membrane is immersed. This probably explains why serous effusions are in general less concentrated than the plasma of the blood. (Beaunis.) The passage into the tissues of part of the plasma of the blood through the thin walls of the capillaries, under the influence of blood pressure, is an example of filtration. The greater the amount of pres- sure exerted in the vessels, the greater will be the amount of fluid forced through their walls. A similar phenomenon is seen in the separation of water and saline matters from the blood in the Malj)ighian bodies of the kidney, where the blood passes under considerable pres- sure through a complicated arrangement of minute vessels. Colloidal OSMOSIS IN RELA TION TO TISSUES. 358 matters, such as albumin, in these circumstances pass with difficulty, and only under strong pressure, whereas, on the other hand, crystalloids pass more readily, and under feeble pressure. Any circumstances, therefore, which increase beyond a certain limit the pressure in the vessels, may be attended by the appearance of albumin in the urine. It is to be especially noted that in the living body we rarely find the conditions of osmotic phenomena so simple as to consist of a fluid on each side of a membrane, and each under the same pressure. Almost invariably one of the fluids is under a greater pressure than the other, and thus the interchange that takes place must be due partly to filtra- tion and partly to osmotic action. In 'addition it is possible that there may be some kind of attractive influence exerted by the living tissues themselves, and thus the process by which they obtain fluid pabulum becomes more complicated. A special influence of this kind, to be remembered in studying secretion, by which certain matters are removed from the blood, is the selective activity of ej)ithelial cells, of which examples will be given in treating of secretion. 3. Absorption of Gases by Moist-Organized Membranes, and by Fluids. Difiusion between gases and liquids is not modified in its essential points by the interposition of a wet organic membrane. The coefficient of absm-ption of a gas is the volume dissolved by one volume of water at 0° C. and 760 mm. pressure. The quantity of gas exchanged between two gases on opposite sides of a membrane, one of them being dissolved in a fluid, depends partly on the coefiicient of absorption of the gas and partly on the pressure of the gases on opposite sides of the membrane. A gas which possesses a large coefficient of absorption, such as carbonic acid, is absorbed by a wet membrane in greater quantity than oxygen, hydrogen, or nitrogen. The amount of absorption diminishes with elevation of temperature and with diminution of pressure. Absorp- tion of gas by a liquid depends upon the pressure of the gas on the surface of the liquid ; the greater the pressure the greater is the amount of gas absorbed by the fluid. When a state of equilibrium between the tension of the gas in the fluid and the pressure of the external gas is attained, absorption ceases. If the pressure of the external gas diminishes, and the tension of the gas in the fluid increases, a portion of the gas dissolved in the fluid will pass into the exter- nal gas until a state of ec[uilibrium of pressure is re-established. Again, if a mixture of gases be exposed to a liquid, each gas is dissolved independently of the others, in proportion to its partial pressure. There I. z 354 THE PHYSIOLOGY OF THE TISSUES. is thus a state of continual gaseous exchange established between fluids of the body in Avhich gases are dissolved and the external atmosphere. This process, as we shall hereafter see, is the essential phenomenon in respiration, as it occurs in the ultimate air cells of the lung. It is highly probable that the same physical explanation may be given of the interchange of gases Avhich constantly occurs between the gases dis- solved in the blood and those set free in living tissues, as one of the ultimate chemical products which are the result of their vital activity.^ ^ Consult, regai'ding the laws of absorption and of diffusion of gases, Wundt's Physique Medicate, p. 201. 355 SECTION IV. THE CONTRACTILE TISSUES. acl'L The phenomenon of contractility is exhibited by various cells, such as the colourless corpuscles of the blood, connective tissue corpuscles, the ■corpuscles of lymph, and the corpuscles of pus. These amo3boid cells, when examined T\dth sufficiently high power, manifest a slow circulation of the granules lying in the protoplasm, and also slow changes of form, such as have been already described in treating of that substance. They have been seen to take up into their substance small particles of pigment, such as carmine, indigo, and aniline, and even globules of milk. Contractile bodies may wander through the interstices of the tissues, a phenomenon termed the migration of cells. Contractility, however, is more especially manifested by muscu- lar fibre, which is arranged to build up the chief portion of the organs termed the muscles, and whichalso exists in the coats of the hollow viscera and of the vessels. Our knowledge of the physio- logical properties of muscles has been largely derived from the study of the living muscles of the frog. These retain their vitality long after the death of the ani- mal. The physiological student should make himself familiar Fig. 203. — Muscles seen on the anterior aspect of a frog's hinder limb, s, sartorius ; adJ, adductor longus ; v.i., vastus intemus; ex., extensor cruris ; with the general anatomy of the *•«•> tiWalis anticus ; f.t., flexor tarsi ; t.p., tibiaUs muscles of the hinder limb of the frog, as shown in Fig. 203. posticus ; g.e., gastrocnemius ; r.i'., rectus internus major or gracilis, or adductor magnus ; r.i"., rectus intemus minor or cutaneus ; ad'", adductor mag- nus : ad" adductor brevis. 356 THE CONTRACTILE TISSUES. Chap. I.— THE SPECIAL STRUCTURE OF MUSCLES AND THEIR RELATIONS TO NERVES. In the lower forms of invertebrate beings, the contractile elements consist of filiform processes of cells situated in the endoderm or in the ectodeiTa. Such cells, first described by Kleinenberg, are termed myo- epithelial cells (Figs. 204 and 205). The epithelial portion of the cell Fig. 205.— Myo-epithelial cells of Hydra_ Fio. 204.— Muscle cells from Lizzia Kollikeri. The ">, vi, muscular fibres. elongated upper cell is from the circular fibres of the subumbrella ; the two lower cells are myo-epithelial cells from the base of a tentacle. may become so much reduced in size as to leave only the nucleus sui'- rounded by a thin layer of protoplasm, but the elongated process or fibre remains, and thus a layer of muscular tissue may be formed immediately below the surface. According to F. M. Balfour^ the muscular elements of the higher groups, including mam- mals, also belong to the myo- epithelial type. Embryonic muscle cells are at first epi- thelial cells; they become spindle-shaped ; part of the protoplasm is diflerentiated into striated muscular fibres, and the undifi'erentiated part seen around the nucleus is the epithelial element of the cells. By further division, the number of fibrils in each cell increases, so that a primitive bundle is formed, which is surrounded by a sheath or sarcolemma. The chief part of the muscular sj'stem of the trunk is formed from the mesoblast, which, as- \^- Fig. 206.— Portion of a transverse section of a muscle of the thigh (adductor) of a rabbit, x 60 d. P, peri- mysium internum, containing at g two blood-vessels cut across. //;, muscle fibres ; they have in many places separated from each other so that we can perceive p, the perimysium oi individual muscle fibres. At :r a transverse" section of a muscle fibre has fallen out. (Method Xo. 65, Appendix.) MUSCLES AND THEIR RELATIONS TO NERVES. 357 already shown, is derived primarily from the epiblast, and thus the epithelial origin of muscle becomes apparent. The structure of muscular tissue has already been described in treating of cells, but before considering the functions of the tissue, we shall give an account of the mode of union of the fibres so as to form muscles, the mode of connection of the fibres with tendons, the arrangement of the blood-vessels, and the manner in which the nerves terminate in muscle. (1) Formation of a Muscle. — The muscular fibres nm side by side, and they are held together by sheaths of loose connective tissue. Xeigh- bouring muscular fibres never come into direct contact, but each individual fibre is surrounded by its o^\^\ sheath, the sarcolemma. A bundle of such fibres m.ay be enclosed by a sheath of connective tissue, termed the perimysium intermim, and a number of such bundles constitutes a muscle, which is surrounded by a thicker layer or sheath of connective tissue, the perimysium externum^ The internal peri- mysium communicates with the external at many places, and thus a transverse section of a muscle shows the appearance depicted in Fig. 206. The perimysium consists of fibrillar connective tissue and elastic fibres. It occasionally contains fat cells, and it is the bearer of the nerves, blood- vessels, and lymphatics. The perimysium inter- num, has ramifying upon it only capillaries and the delicate terminations of nerves. (2) Connect'wn of Muscular Fibres icith Tendon. — The perimysium of individual bundles of fibres be- comes continuous with the connective tissue fibres forming the tendon, and the sarcolemma ends at the termination of the striated muscular fibre fig. 207.— Portion of a longi- ._,. nA^7\ tudinal section of the gas- vill be a difference of potential between the zinc and the liquid, then a smaller difference of opposite sign between the liquid and the copper, and finally a dif- ference between the copper and the zinc. The algebraic sum of these differences is not zero, and there results, in consequence of the ten- dency of electricity to flow from ■' . "^ . Fig. 214. — Diagram of a Voltaic Element. points of high to points of low potential, a current of electricity which, by a common convention, is 364 THE CONTRACTILE TISSUES. said to How from the copper to the zinc throixgh the external circuit or ■wire M, and from zinc to copper through the liquid. This flow of electricity would reduce the Avhole circuit to the same potential were it not for the fact that the passage of electricit}^ through the liquid de- composes it, and l^y so doing generates electricity, which serves to keep the current flowing. The energ}'^ required to keep the current flowing through the circuit is supplied by the chemical action, the result of which is the production of sidphate of zinc at the expense of the zinc plate and the liberation of a corresponding amount of hydrogen from the sulphuric acid (H^SO^ + Zn = ZnSO^ + H.,). The hydrogen is set free at the copper plate. The arrangement just described is called a roltaic clement; the copper and zinc plates being commonly termed the plates, and the liquid the electrolyte. The copper plate is called the positive pole, and the zinc plate the negative pole, and the Avires forming the external circuit, or joining the two poles of the cell to any piece of apparatus, are called the electrodes. The algebraic sum of the contact differences of potential above re- ferred to is called the potential of the cell, but Avhen the circuit is closed and a current caused to floAv, it is more commonly called the electro- motive force (e. m. f.) of the circuit, i.e. the electrical force or pressure which causes the current to flow. The electromotive force in the circuit of a voltaic cell producing a cvuTent is the same as the iet{sm, or the unit magnetic pole, is that quantity of magnetism which, when placed at a distance of one centimetre from an equal quantity of magnetism, repels it with the force of one dyne. 2. U7iit Magnetic Field is such a magnetic field that when unit quantity of magnetism is placed in it, it is acted on by a force of one dyne. The unit magnetic field is thus the field produced by unit quantity of magnetism at a distance of one centimetre. 3. Unit current of electricity is such a current that when made to flow round a circle of one centimetre radius, it produces a magnetic field of as many units intensity at the centre of the circle as there are centimetres in the length of the circumference of the circle. The unit current flowing in a circle of one centi- metre radius would therefore act on unit quantity of magnetism placed at the centre of the circle with a force of 2ir dynes. 4. Unit quantity of electricity is the quantity conveyed by i;nit current in one second. 5. Unit difference of potential is the diflference of potential between the ends of a conductor of one centimetre length, when it is held with its length at right angles to the direction of magnetic force in a magnetic field, and kept moving uniformly with a velocity of one centimetre per second in a direction at right angles to its own length and the direction of the magnetic force in the field. 6. Unit electromotive force is produced in a closed circuit, if one centimetre of its length is held in the manner and moved in the dii'ection and with the velocity described in the last definition. 7. Unit resiatance is the resistance which, when cted on by unit electromotive force, allows unit current to flow. 8. Unit capacity is the capacity of a body which requires unit quantity of elec- tricity to raise its potential by units. In practice, the magnitudes of the units above defined were found inconvenient and certain multiples and sub-multiples have been adopted. The units of any im- portance for physiological purposes are the unit of current, the Ampere ; the unit quantity of electricity, the Coulomb ; the unit electromotive force, the Volt ; the unit resistance, the Ohm ; and the unit capacity, the Farad or Microfarad. The Ampere is equal to one tenth of the c. g. s. unit of current, or approxi- mately the current of an ordinary Daniell through an Ohm ; the Coulomb to one tenth the c. g. s. unit of quantity, or the quantity of electricity conveyed by an Ampere in one second ; the Volt to 100,000,000 times the c. g. s. unit of e. m. f., or approximately the e. m. f. of a Daniell's cell ; the Ohm to 1,000,000,000 times the c. g. s. unit of resistance, or the resistance of a column of pure mercury at 0° C. 1 mm. square and 1,050 mm. in length; the Farad to tuo Ou^ouuo ^^ ^^^ c. g. s. unit of capacity ; and the Microfarad, which is the common practical unit, is the millionth part of a Farad. ^ ^ The terms Ampere, Coulomb, Volt, etc., are often written without use of capital letters, thus ampere, coulomb, volt, etc. APPARATUS EMPLOYED IN STUDY OF MUSCLE. 369 2. Voltaic Elements. Many varieties of voltaic elements may be employed for physiological purposes. The following are the most common — Fig. 216.— Daniell's Element. (1) Daniell. There are various forms of this convenient element. The one shown in Fig. 216 consists of a glass jar containing a solution of sulphuric acid, 1 to 7 of water ; in this is placed a roll of amalgamated zinc, Z ; within the roll there is a porous earthenware jar containing a rolled piece of copper, C, immersed in a saturated solution of sulphate of copper. Sulphate of zinc is formed by the action of the sulphuric acid on the zinc and hydrogen is set free. The hydrogen passing through the porous vessel reduces part of the sulphate of copper, depositing copper on the copper plate, and the sulphuric acid thus liberated passes into the solution of the acid in the outer jar, and thus makes up for the loss of the acid caused by the continuous formation of sulphate of zinc. Thus, the current is very constant so long as an ample supply of crystals of sulphate of copper is placed in the inner compartment and so long as the zinc plate in the outer vessel lasts. The e. m. f . is = 1'072 volt. The positive electrode is connected with the copper and the negative electrode with the zinc. This element is useful when it is desirable to have a tolerably constant current. (2) Bunsen. This element consists of a vessel containing dilute sulphuric acid, 1 to 7 of water, in which is a roll of amalgamated zinc ; in this is a porous earthen- ware vessel containing a rectangular block of carbon in strong fuming nitric acid (Fig. 217). The hydrogen set free in the outer compartment by the action of the sulphuric acid on the zinc is absorbed by the nitric acid, and thus polarization is to a large extent prevented. Its e. m. f. is 1 -9 volt. The positive electrode is con- I 2A 370 THE CONTRACTILE TISSUES. nectecl with the carbon and the negative with the zinc. This element is incon- venient for ordinary physiological purposes on account of the acid fumes given off when it is in action. (3) Grove, This element, of which a small and convenient form is shown in Fig. 218, is similar to Bunsen's element, except that a strip of platinum,?, takes the place Fig. 217. — Bunsen's Element. of the carbon in the fuming nitric acid. Its e. m. f. is 1 '96 volt. The positive electrode, K, is connected -vnth. the platinum and the negative electrode with the zinc, Z. Fig. 21S.— Grove's Element. (4) LeclancM. This element, of which there are various forms, consists of a glass vessel or jar, containing a saturated solution of sal-ammoniac in water, in APPARATUS EMPLOYED IN STUDY OF MUSCLE. 371 which is immersed a roll of amalgamated zinc, Z (Fig. 219). In this is placed a jar T, containing a plate of carbon, K, surrounded with small pieces of carbon mixed with black oxide of manganese. Its e. m. f . is 1 '48 volt. The positive electrode, B, is connected with the carbon and the negative electrode with the zinc, C The Leclanche is a very convenient element, united in series, for medical purposes. (5) Gaiffe. This is a small and convenient form of element much used in the ■construction of batteries employed by physicians. It consists of zinc (not amalgamated) in a 5 per cent, solution of chloride of zinc, and of silver surrounded Fig. 219. — Leclanche's Element. by chloride of silver. Its e. m. f . = 1 •02 volt. The positive electrode is connected with the silver and the negative electrode with the zinc. (6) Sniee. Smee's element consists of two plates of amalgamated zinc, having a plate of platinized silver between them, the whole being immersed in a solution of sulphuric acid, 1 to 7 of water. The positive electrode is connected with the platinized silver and the negative electrode with the zinc. The e. m. f. is "4" volt. (7) Marie-Davy. This is an element much used in the construction of medical batteries. It consists of amalgamated zinc in slightly acidulated water and of car- bon in a paste of mercurous sulphate. Its e. m. f . is 1 '52 volt. The positive electrode is connected with the carbon and the negative electrode with the zinc. 372 THE CONTRACTILE TISSUES. (8) Grtnet or Bichromate. This is an element of great convenience. It is formed of one plate of amalgamated zinc placed between two plates of carbon, and the whole is immersed in a fluid having the following composition— Water, 100 parts ; snlphurio acid, 25 parts ; and bichromate of potash, 10 parts. The zinc plate attached to the rod, B, may be pulled up out of the fluid when the element is not required, so that the fluid does not require to be changed frequently. Immeiliately after changing with fresh fluid this element may give an e. m. f. of 2"03 volts for a few seconds, but it rapidly becomes weaker, and falls to about 1 "8 volt. The positive electrode, E, is con- nected with the carbon and the negative electrode with the zinc, D (Fig. 220). (9) No(i-Ddrffel Thermo- Electric Battery or Pile. This consists of a number of thermo-electric junctions concentrically ar- ranged. One of the metals is German silver, the other is an alloy of antimony and zinc, bhe exact composition of which is known only to the manufacturers of the element. One of [the metals of each junction is ex- panded to form a cylinder, so as to secure a uniform temperature at one junction. The other junction is heated by placing the apparatus over the flame of a spirit lamp. The elements are arranged in series to form a batteiy, as- shown in the figure. Each element has an e. m. f. of ^V volt, and a battery of 20 elements has therefore an e. m. f. of 1"25 volt. It is remarkable that such an ar- rangement transforms into- electricity less than one per cent, of the heat energy given out by the source of the heat used in generating the current. For physiological purposes this thermo-electric battery is very convenient, as it is readily put in working order and there is no risk of injury to sensitive structures like nerves from acid fumes (Fig. 221). The positive electrode- Fig. 221.-Noe-DorffelThermo-Electric Battery. .^ connected with the alloy of the antimony and zinc, and the negative with the German silver. Fig. 220.— Grenet's Element. APPARATUS EMPLOYED IN STUDY OF MUSCLE. 873 (10) Storage Cell. If there is a dynamo machine available, the physiologist may abandon the use of all kinds of batteries (except, perhaps, a Grenet and a Noe- Dcirffel thermal pile) and employ a small accumulator, which may be charged from time to time.^ The late Professor Fleeming Jenkin thus summarized the probable sources of weakness in a battery — " When a battery does not give the expected results, one •of the following defects is to be looked for : (1) Exhausted solutions — for example, in a Daniell battery, the sulphate of copper worked out, leaving the solution colourless, or nearly so ; (2) bad contacts between the electrodes and the wires, •oxidized or badly screwed-up binding screws, etc. ; (3) empty or partially empty cells ; (4) filaments of metallic deposit causing short circuiting between the battery plates ; (5) creeping or deposits of salts forming short circuits either between the plates or from cell to cell. Shaking the cells increases their e. ni. f. temporarily by disengaging the gases adherent to the plates. Floating filaments and broken plates give rise to false contacts, which cause the current given by a battery to vary suddenly when it is shaken. " 3. Induction Coils. The form of induction coil most commonly employed by physiologists is the sledge indudorium of Du Bois-Eeymond, seen in Fig. 222, pro- vided with Neefs Interruptor, represented diagrammatically in Fig. 223. ^■^'U'en.-^^i Fig. 222. — liiductorium of Du Buis-Reymond : o, primary coil ; b, secondary coil ; c, bunch of wires in centre of primary coil, for increasing intensity of induction current ; d, binding screw, for attachment of wire from galvanic element. The current passes up the pillar d, along steel spring to e, from there to the screw the point of which touches the back of the spring at e. Frora / through wire of primary coil to i, along the two pillars of soft iron i, which it renders magnetic and thus draws down the head of the spring k. This interrupts the current at e, by breaking the contact of the spring with the screw point. When the current is thus interrupted, the spring flies up by its elasticity and again establishes the circuit at e. Thus the current is opened and closed automa- tically, and each time it is opened and closed there is an induction shock from the secondary coil 6. The intensity of the induced current becomes weaker as ■we withdraw b from a along the graduated board o. The automatic apparatus was applied by Neef. The binding screw a is connected by a wire to /" ; the bind- ing screw h by a wire to i. When the wires from the battery are connected with g and h, Neefs arrangement is out of the circuit, and an opening or closing shock may then be obtained by opening or closing a key (Fig. 224) interposed in the circuit. The interrupter was originally invented by Philipp Wagner, ^ As to the structure and uses of accumulators, see Hospitaller's Electricians' Pocket Book, p. 210. :37-i THE CONTRACTILE TISSUES. The descriptions appended to the figures explain the mechanism of the instrument. Neef s interruptor woi^ks automatically, and thus a rapid series of shocks is transmitted to the nerve or muscle. Fig. 223. — Diagi-ammatic view of the arrangement of Neef's interruptor at the end of the indnctoriuni, seen in Fig. 222. The arrows indicate the direction of the current. A, wire connected with the + pole of battery ; and IJ, with the - pole, c, primary- coil ; and i, secondary coil. The secondary current induced in the secondary coil at the moment of opening is in the same direction as that of the primary, while that of closing is in the op- posite direction. Thus, the direction of the currents in the secondary is reversed with each momentary opening and closing of the primary, "With such an arrange- ment, it is found that the opening shock acts more powerfully than the closing shock. This is due to the fact that, at the moment of the closing shock, an extra current is induced in the primary coil by the action of each coil of wire on all the other coils. The extra current so induced in the primary, being in the opposite direction to the chief ci;rrent in the primary, weakens the latter, so that the induced current occurring in the secondary coil at the moment of dosing the primary is thus weakened. When the circuit of the primary coil is, on the other- hand, opened, no extra current occurs in the primary, and its effect on the secondary coil is therefore greater, because the primary current at the moment of CLOSING OPENING Opening has not been weakened. Thus, the opening shock is considerably stronger than the closing shock. The relation of the opening and closing- shocks is clearly shown by the diagram in Fig. 224, in which I refers to the cur- rent in the primary, and II to the current in the secondary coil, and A A is the basement line of the upper, and h b the basement line of the lower cui-ves. On closing the primary circuit, the current in the primary coil in place of rising to its maximum, indicated by the dotted vertical line A B, attains this maximum slowly as shown by the curved line 1 . This is owing to the production of the Fio. 22-1. — Diagram showing the effects of the extra currents on induction currents. APPARATUS EMPLOYED IN STUDY OF MUSCLE. 375 extra current in the reverse direction. "When the primary circuit is formed, there is a secondary induced current, represented by curve 2, placed below h b, because it is in the opposite direction to I. On opening the primary circuit, its current suddenly ceases, as shown by the vertical line 3 3, and is unaffected by any extra current. When the primary circuit is opened, an induced current is generated in the secondary coil, and this induced current at once reaches its maximum, as shown by the vertical line 4 4, and then falls off gradually, as shown by the curve 4 4'. To equalize the two currents. Von Helmholtz invented the arrangement shown in Fig. 223. A loop of wire, g, is carried from the pillar g (the first pillar on the left) in the direction of the arrow to the screw/', and/' is screwed up so that it does not come into contact with the back of the spring h, but /is screwed up so that with each vibration of h, the under surface of the spring touches the screw point /. The current from the + pole enters at A, and at first passes through the loop wire g to /', thence to the primary coil c, and thence to the electromagnet b. When the soft iron core of the latter is mag- netized, the hammer h is pulled down, and the under surface of the centre of the spring touches the top of the middle pillar at the screw /. This short circuits the primary, and the current then returns to the battery by passing down the pillar a, and out by the wire B to the - pole. But the closure of the short circuit so weakens the current flowing through the electromagnet b. that the hammer is released and the short circuit being broken at /, the current must pass by the long circuit, and thus the current in the primary coil c is never opened, but the opening shock in the secondary coil i is due to the weakening of the current in the primary at the moment of short circuiting. By this method, an extra current is induced in the primary at opening (or rather at the moment of weakening the current by short circuiting), and as this is in the reverse direction to the chief current in the primary, the opening shock is reduced in strength, and becomes nearly equal to the shock from the secondary coil at the moment of closing. The weakening of the primary circuit thus produced is shown in Fig. 224 A' A", in its diminished distance from B. When the short circuit is broken, as already described, the strength of the primary is at once increased, as shown by the curved dotted line, 1'. This increased strength of the primary produces an induced current in the secondary, as shown by the dotted curve 2', below b II. The weakening of the primary by the re-establishment of the short circuit is pictured by the dotted curve 3 '. This in turn produces an induced current in the secondary coil, represented by the curved line 4'. Thus, with the original arrangement, the strength of the closing induction shock is represented by the curve 2, and that of opening by the curve 4 4 4, while these values, with the arrangement of Von Helmholtz, are shown by the curves 2' and 4', and it will be seen that these curves are very similar in appearance. When the arrangement of Von Helmholtz is employed, there is an extra current in the primary coil, both at the moment of opening and of closing the primary current, and the extra current may be employed as a stimulus by connecting the positive and negative poles of the battery with the pillars g and a, and the wires for carrying off the extra current to the tissues are connected to /' and /. The primary coil should consist of at least 432 windings, hav- ing a resistance of 1"27 ohm, and the secondary should have 14,680 windings, with a resistance of 1362 ohms at 16° C. Professor Bow- 376 THE CONTRACTILE TISSUES. Jitch has also improved the iuductorixim by having the secondary coil mounted on a rotating disk, so that it may be placed \vith its axis forming an angle with that of the primary. Thus, the strength of the secondary current may be modified according to the amount of angular deviation, and this may be read off from a gi'aduated arc at the side of the base of the secondary coil. In using the inductorium for physiolo- gical purposes, the observer should always state (1) the battery em- ployed, so as to indicate the e. m. f. ; (2) the number of windings and the resistance of the primary coil ; (3) the number of windings and the resistance of the secondary ; (4) the distance of the secondary from the primary ; (5) Avhether or not Von Helmholtz's loop "svire was used ; and (6) the amount of angular deviation of the primary and secondary coils. 4. Accessory Appliances. For the purpose of opening and closing currents, various instruments may be employed. One of the most convenient is termed a keij, seen in Fig. 225. It consists of a rectangular wooden frame, by which the key may be screwed to the table. On the top is a square block of vulcanite, a, bearing two rectangular bars of brass, h and c, which may be joined by the handle, d, carving a horizontal piece of brass. The key is closed when the arm is horizontal, as in the figure. On moving the handle, d, backwards and to the right (see Figure), the brass arm is raised, and of course the contact between h and c is broken. The key is then said to have been opened. Wires are attached by binding screAvs to h and c. If the key is interposed in the course of the wire leading from one of the electrodes of a battery or inductorium, the circuit will be broken when the key is opened, and closed, or formed when the key is closed ; but if the positive and negative j^oles are connected re- spectively with h and c by the two inner bind- ing screws, while the two outer binding screws have wires passing from them to a nerve or muscle, or to a galvanometer, or other electrical apparatus, it is clear that when the key is closed t)ie battery current will be short circuited, Fig. 225. — Du Bois-Reymond's friction key. APPARATUS EMPLOYED IN STUDY OF MVSCLE. 377 and that when the key is open, the current will then pass onwards. Thus, the key may be used (1) for opening and closing a current, and (2) for short circuiting. For rapid opening and closing of a current, an arrangement like Neefs Interrujptor may be employed, or a vibrating metallic sprwg may be interposed in the circuit, so arranged that, as it vibrates, a needle at the end of the spring will make and break contact by dipping into a small vessel containing mercury.^ By varying the length of the spring, its vibration period may be increased or diminished. An ordinary metronome, fitted up as represented in Fig. 226, is also useful for making Fig. 226. — Metronome, arranged for making and breaking an electrical current. and breaking currents. As the pendulum swings backwards and for- wards, it makes and breaks contact by dipping into the small vessels containing mercury. These vessels are interposed in the circuit by wires dipping into the mercury, and it is clear that when the fork- shaped wire of the metronome also dips into the mercury the circuit will be formed. By moving the slider of the metronome up or doAvn, the number of interruptions may be regulated. ^ A beautiful and most convenient spring of this description is made by the Cambridge Scientific Instrument Company, and is very useful for many purposes. 378 THE COXTRACTILE TISSUES. When tolerably strong currents are used, interrupters consisting of a point dijiping into and out of mercury are \qtx ti'oublesome on account of oxidation of the surface of the mercurj\ This difficulty is removed by the use of the ingenious arrangement of Hugo Kronecker, shown in Fig. 227. By allo-n-ing a stream of water, or, still better, of alcohol, to Fig. 227. — Kronecker's arraugemeiit for securing clean raercurial contact. flow through the tube c, the surfecc of the merciu^y in o is kept clean at the place where the current is closed and opened by the vibrator g, which establishes the current passing from g to d. It is sometimes necessary- to reverse the direction of a current. This is readily accomplished by the use of a simple apjjaratus, termed PoliVs Commutator, seen in Fig. 228. Fig. 228. — Pohl's Commutator. For description, see text. It is a disk of wood A, haviug six little pits or depressions at regular distances, to each of which a binding screw is attached. From the holes / and Ti two wires APPARATUS EMPLOYED IN STUDY OF MUSCLE. 379 proceed, which are attached at their outer ends to the ends of the binding screws,, but loosely, so that they may move from side to side. These wires pass into a piece of glass or vulcanite n, but without their inner ends touching. Thus, a- bridge is formed ; but if a current entered the end of the bridge by the binding screw marked + to the right, it coitld not pass across to the binding screw having the wire — attached on the left. Two curved wires k m and q i are soldered at their centres to the wires / and h, but with the ends free. As the bridge n can rock backwards and forwards, the ends i m may dip into the troughs of mercury d c, or the other ends k q may dip into the troughs g b. By this arrangement, when the wires dip into c d, as in the Figure, suppose a ciirrent entering at + on the right, it would pass along h i into d, and out by the curved wire x, round the circuit, back to the curved wire y, into vi, thence by 1 into /, and thence back to battery by -ware - on the left. On the other hand, if k q dipped into g h, the current would go out from b and return to g. Thus the current, by simply reversing the bridge, could be sent to a circuit x y, or to one represented by iv v. Now suppose the cross bars o p inserted so that p joins the troughs c b, and o joins d g ; then, if a current enters h, it will pass by i into d, and then out by x to circuit, back to y, then into vi, then by 1 into /, and back by wire - to battery. If, however, the bridge be reversed so that k q dip into g b, when the current enters by h, it will not pass by i into d, because the circuit is broken there ; but it will go by q into b, then by cross wire -p over to c, then out by the wire y, round the circuit to x into d, then by cross wire o to g, then by k up to 1 , and then back to battery by wire - . Thus, by the use of the cross wires o ;;, if the current enters by the binding screw + on the right, x wUl be + pole if the bridge is in the position seen in the Figure, and y will be the + pole if the bridge is reversed so that k q dip into g b. The cross wires thus reverae the direction of the current. The most convenient arrangement for stimulating a nerve is an apparatus, termed the Polarizable Electrodes of Du Bois-Reymond, seen Fig. 229.— Polarizable Electrodes of Du Bois-Eeymond. a, upright brass rod r b, screw for tightening the rod c on a ; above screw a; is a ball and socket- universal joint ; d is a curved brass rod bearing A, a piece of vulcanite, through which two wires pass, having binding screws e e' at one end, and rectangular platinum points//' at the other. in Fig. 229. The nerve is laid across the platinum points / /', Even with such electrodes, irritable nerves might be stimulated by iiSO THE CONTRACTILE TISSUES. •currents produced by polarization caused hy the action of the animal fluids on the metallic substances. In certain refined cxi)eriments the nerves are stretched across Non-polarizaUe Electrodes, shown in Fig. 230. This form may be used not merely for stimulating nerves, but, and more especially, for conveying currents from nerve and muscle into the circuit of a galvanometer. (See the Chapter on the Electrical Phenomena of Muscle.) The electrodes here shown are those used by Professor Burdon-Sanderson, but for the purpose of stimulating a nerve they may be of much smaller size. They consist of, a, a brass stand -carrying a horizontal bar of vulcanite h, which in turn sui)ports the •electrode c. Each electrode is a curved glass tube d, closed at one end by a small piece of sculptor's clay e, moistened with saliva or "vvith a Fio. 230.— Xou-iiolaiizable Electrodos of Burdon-Sanderson. '75 per cent, solution of conuuon salt, and containing a saturated solu- tion of pure sulphate of zinc. Into the other end of the glass tube, there is dipped a small rod of amalgamated zinc. Such an arrangement, care- fully prepared, is absolutely non-polarizable, so that if a nerve or muscle were laid across two such electrodes, and a current from a battery or inductorium were passed through the nerve or muscle, the latter would be stimulated by the current from the electrical apparatus, and by nothina; else. APPARATUS EMPLOYED IN STUDY OF MUSCLE. 881 Several electrical appliances are shown in Fig. 231, and reference is made to the description of the Figure. Fig. 231. — Apparatus for electrical experiments on muscle and nerve. T. Electro-magnetic signalling apparatus for recording seconds on a revolving cylinder ; a, bobbins covered with wire ; h, brass rod carrying pencil or pen ; c, steel spring. II. and III. Brass forceps for holding leg of frog ; o, forceps ; b, binding screw for wire. IV. Platinum electrodes for stimulating nerve ; a, jDiece of vulcanite carrying glass plate c c, on wliicli are the copper wires terminating in rectangular platinum points ; i, universal joint. V. Pohl's commutator for reversing the direction of electric currents ; d and e are connected with the battery or ind\iction coil ; & c and a f are binding screws for attaching wires in different circuits ; g, bridge of copper wire, divided and insulated in the centre by glass tube, for the purpose of sending the current entering by d e, either in the direction of 6 c or af. VI. Du Bois-Reyrnond's key, con- sisting of a piece of vulcanite on which there are two rectangular pieces of brass a b, each having two binding screws. The two pieces of brass are connected by an arm of brass, the handle of which is seen at c. VII. and VIII. Two forms of apparatus for stimulating nerve, consisting of vulcanite troughs, into which are fixed platinum wires, which may be attached to the wires coming from the battery by binding screws at a a a a. Chap. IV.— THE GRAPHIC METHOD. Many movements are too rapid to be followed in all their phases by the unaided eye. For example, the prongs of a tuning-fork are in active periodic motion during the time that a tone is emitted by the fork, but the prongs may appear to the eye to be stationary-. Again, the movements of a bird's wing or of the limbs of a quickly-trotting horse are too rapid to be followed by the eye. The motion of the living heart exposed in the living animal seems so irregular and tumul- tuous as to make one with no experience of scientific methods doubt the possibility of tracing its various movements. Even when the isolated muscle of a frog's limb is caused to contract by irritating the nerve supplying it, the eye cannot tell whether the contraction occurs in a shorter time than the relaxation, and still less whether the con- 382 THE CONTRACTILE TISSUES. traction is at a uniform rate in time, or -whether it contracts faster at the beginning and more slowly towards the end, or the reverse. Sup- pose the muscle were hung vertically, and that it was connected at its lower end to a lever, and that the point of the lever, bearing a pencil, ^\'ere brought against a sheet of paper also placed vertically, if the paper were moved horizontally from right to left, a horizontal line Avould be drawn upon it by the pencil so long as the nuiscle did not contract. On the other hand, suppose the paper to be stationary and the muscle to contract, the pencil at the end of the lever would draw a vertical line, the height of which, making allowance for the am})lification of the movement by the lever, wovdd be a measure of the amount of muscular contraction, or, in other words, of the shortening of the muscle. Finally, suppose that, while the muscle shortened by contraction, the sheet of paper moved at a uniform speed from light to left, then the pencil would describe a line passing obliquely upwards, and to the right (Fig. 2.32, from below the line B, in direction a, c), so long as the muscle contracted, and a line obliquely down ward, and also to the right, during the relaxation of the muscle. Thus, by a series of contractions, the zig-zag line would be drawn. If the motion of the contracting-muscle were more uniform, then a line like the curve FiQ. 232 -Diag.-am illustrating the graphic A would be described. This illus- method. No muscle would actually record such curves. trates the essential principle of the graphic method, by which movements are recorded on a surface, which may be either stationary or moving Avith a uniform A^elocity, a method which has been of great value in all sciences dealing with movement, and not least to physiological science. An excellent illustration of the graphic method is seen in the tracing of harmonic motions. Suppose two jiendular motions acting on the same point, and that the times of the motions were as 1 to 1, or as 1 to 2, or as 2 to 3, or as 3 to 4, and that the surface over Avhich the point travelled was stationary, then the beautiful figures seen in Fig. 233 will be described. If, however, the recording surface be moving from right to left, then the forms of the curves described by the com- bination of the same motions will be those shown in Fig. 234. This illustration shows that, hoAvever complicated may be the move- ments of a point, these movements may be recorded, and that the character of the tracing on the recording surface will vary according as the recording surface is stationary or moving. The method of the graphic registration of movement is not entirely THE GRAPHIC METHOD. 383 modern. In 1734, the Marquis d'Ons-en-Bray described an anemometer which recorded its movements on a sheet of paper, rolled round a cylinder, moved by clockwork. Magellan, in 1779, made designs for an instrument he called a " Meteorograph " for recording automatically many meteorological phenomena. It was then a dream to attempt to Fig. 233.— Lissajous' figures of liamiouic motions. A, 1 : 1, unison ; B, 1 : 2, oatave ; C, 2 : 3, fifth ; and D, 3 : 4, fourth. The figures 1, 2, 3, 4, and 5 in each row represent the phases of the harmonic motions. record buch phenomena, but it has become a reality in these latter days. A thermometer was described by Eutherford in 1794, by which the curves ot fluctuating temperatures were recorded on blackened paper. About'1800, Thomas Young showed how time can be measured on the surface of a cylinder moving at a uniform speed. The celebrated James Fig. 234. — Figures of harmonic motions, with recording surface moving from right to left. A, Unison ; B, 1 : 2, octave. Watt, at a date I have been unable to fix, devised a method of tracing the movements of the indicator of his engine on a cylinder rotated by the engine itself. Thus, he obtained a curve representing variations of 384 THE CONTRA CTILE TISSUES. steam pressure at different times. ^ A method of recording the oscil- lations of mercury in a manometer was invented by Ludwig, and resulted in the kymograph, or recorder of blood pressiu'e, and during the past tAventy or thirty years numerous ingenious instruments have been invented by physiologists for recording movements. No physiologist has done more in this direction than Marey, to whom we are largely indebted for the development of the graphic method to its present condition of precision and convenience. I. Measurement of Time or Chronography. As many of the phenomena of nervous and muscular actions are of such short duration as to make it imj)ossible to observe their phases with the unaided senses, it is neces- sary to construct apparatus for the measurement of minute intervals of time. Accordingly, in recent years, much ingenuity has been expended in the construction of chronographic or time-measuring instruments, of which the following are examples — (a) Recording Cylinder. — This is a cylinder revolving at a uniform rate by means of clockwork. Sup- pose the surface of the cylinder to be divided by sixty lines, parallel Avith its axis, at equal distances from each other, and that the cylin- der makes one revolution in a second, the distance between tAvo of the lines will represent the gV Fig. 235.— Original Chronometer devised by part of a SCCOnd, aud any phcuO- homas Young for measuring time, a, Cylin- . der rotating on vertical axis ; 6, weisrht acting nieuon rCCOrded OU the movillg as motive power ; c, d, small balls for regulat- . "" ing by centrifugal action the velocity of the cylinder between the tWO lllieS mUSt cylinder ; e, marker recording a line on the _ - . . . - cylinder This illustration is given to show have happened dunug that interval the form of the first apparatus used for „ . t • • i , i_i j_ i chronographic tracings. 01 time. it IS evident that by means of such an arrangement, seen in Fig. 235, first suggested by 1 Marey, La Methode Graphique dans Its Sciences Experimentales, p. 113. For a view of Watt's Indicator used with the steam engine, as constructed by Watt, and improved by M 'Naught and Richards, see Clerk Maxwell's Theory of Heat, third edition, p. 154. THE GRAPHIC METHOD. 385 Thomas Young, ^ intervals of time, even to the y-oV-o of ^ second, may be measured T\dth accuracy. The difficulty in such graphic measurements of time is to cause the cylinder to revolve at a uniform rate. This is accomplished by means of regulators, of which the simplest is that of Foucault, now attached to all revolving cylinders used for physiological purposes. To obtain, on a revolving cylinder (the time occupied by a revolution of which is not known) continuous registration of minute intervals of time, it is necessary to make use of a chronograph. (b) Chronographs.- — Thomas Young was also the first to devise the method of inscribing upon a rotating cylinder all the vibrations of a metallic rod bearing a very light style or marker. ^"\Tien these vibra- tions are isochronous, each of the undulations traced upon the cylinder corresponds to a regular interval of time. Duhamel was the first to apply to one of the limbs of a tuning-fork a small marker, which traces Avith great regularity the vibrations of the tiuiing-fork, and in this way, if the surface receiving the tracing is mo\dng with sufficient rapidity, intervals of the -g^-g- of a second, or less, may be readily measured. An example of tracings thus obtained is seen in Fig. 236. Fig. 236. — Tracings of the vibrations of a tuning-fork, 10 vibrations per second, a, b, cylinder moving rapidly ; c, d, cylinder moving slowly. It is difficult to apply the vibrating limb of a tuning-fork to a re- volving cylinder or other moving surface, more especially if other recording apparatus is adjusted to the cylinder at the same time. To record more easily vibrations indicating time, an arrangement consisting of a marker, vibrating in unison "with a tuning-fork, which is kept in action by the interruptions of an electric current, is much employed. The apparatus, as applied to a revolving cylinder, is seen in Fig. 237. The arrangement of apparatus consists of three parts— a battery, an interrupting tuning-fork, and the chronograph. The latter, seen in Figs. 238 and 239, consists of a very fine stylet fixed at the extremity of a steel spring, and armed with a small mass of steel, somewhat wedge-shaped, which fits in between two small keepers, b i, of the 1 Thomas Young's Lectures on isatiiral Philoioiijhy ; Lecture xvii., on Time- keepers. Plate XV. Fig. 19S. I. 2b 386 THE CONTRACTILE TISSUES. electro-magnets, a a. The tmiing-fork interrupts the current from the battery. This it does automatically. When the iron of the electro-magnet between the limbs of the tuning-fork becomes magnetic, the limbs are approximated, and a small piece of platinum wire affixed to one of them is removed from contact Avith a platinum surface (Fig. 237, i), so as to Fig. il37.— Chronograph applied to revolving cylinder, a, Grenet's element; b, wooden stand bearing taniug-fork, vibrating 2u0 times per second ; c, electro-maguet between limbs of the fork; <(, e, positions for tuning- forks of 100 and 50 vibrations per second ; /, tuning-fork lying loose, which may be applied to d; (j, revolving cylinder; A, chronograph vibrating s3mchronously with the tuning-fork. Current is interrupted at i. Foucault's regulator is seen over the clockwork of the cylinder, to the right of ij. break the circuit. On the circuit being thus broken, the electro-magnet ceases to act, the limbs of the tuning-fork recede from it, so as to bring the platinum ^vive again into contact with the platinum siu-face, and thus again to complete the circuit. The chronograph thus vibrates in Fig. 23S. — Side view of chronograph, a a, coils of wire ; & b, keepers of electro-magnets ; c, vibrating style fixed to steel plate e; d, binding screws for attachment of wires; +, from tuning-fork ; — , passing to battery. imison with the tuning-fork. The great advantages of this apparatus are its accuracy and facility of ready adjustment, and it can frequently be appKed where it would be extremely difficult to bring the ttming-fork into direct contact Avith the movina; surface. THE GRAPHIC METHOD. 387 N- A very convenient form of interrupting tuning-fork, made by the Cam- bridge Scientific Instrument Company, is slio'vvn in Fig. 240, and it is here in- troduced to indicate the position of the electro-magnet between the limbs of the fork. Along with the interrupting tuning-fork, the same makers supply a time-marker (Fig. 241) for recording on a, surface of smoked paper, and a time- marker (Fig. 242), having at the end a kind of syphon pen, for recording in ink on a long continuous band of white paper. The latter arrangement is especially useful where it is necessary to obtain a 1 ,- i • rrn 1 • Fig. 239. — Front view of clironoffrapli, to lOna; COntmUOUS tracmg. i he mechanism show positton of vibrating marker. Same of both instruments is seen on inspecting the figures. description as given of last figure. Fig. 240. — Interrupting or Chronographic Tuning-fork of Cambridge Scientific Instrument Company. Fig. 241. — Time-marker of Cambridge Scientific Instrument Company for recording on a surface of smoked paper. (c) Electrical Signals. — It is often of great importance to determine the moment of the commencement or termination of a phenomenon. 388 THE CONTRACTILE TISSUES. This is readil}' done h\ means of electro-magnetic arrangements, such as are seen in Fig. 243. The apparatus consists of two electro-magnetic /11 Fig. 242. — Time-marker for recording with ink when a tracing is taken on white paper. bobbins, which, the moment the current passes, attract a steel plate placed above them, and draw down the "\n4ting stylet, so as to make a lower horizontal line. "When the current is interrupted, the spiral Fig. 243. — Aijparatus for recording electro-magnetic signals ou a revolving cj'linder. a a, bobbins covered with wire ; b, steel plate fixed in a frame, which is pulled downwards when the iron cones of the bobbins become naagnetic ; c, steel spring, by the elasticity of which the plate is drawn quickly iiiiwards when released from the bobbins ; d, rod, bearing a marker, recording on the cylinder/ at e. spring elevates the lever, which traces an upper horizontal line imtil th& current is again closed. The current may be opened or closed by means of the seconds pendulum of a clock, by a tuning-fork (Fig.' 240),. THE GRAVHIC METHOD. 389 or by a metronome (Fig. 226). By such an arrangement for closing or opening the circuit, the instant of the commencement or termination Fig. 244. — 1, Line drawn by marker of apparatus shown in Fig. 243 ; 2, ' vibrations of tuiiiug-fork, iO vibrations per second ; 5, signal, the rise in tracing 1 indicates the interruption, and the fall shows the formation of the current, whilst the cj-linder was rotating quickly; 3, 4, 0, show similar tracings, but with cylinder going raore slowly. of any phenomenon will be recorded. An example of a tracing obtained by such an apparatus is given in Fig. 244. Fig. 245. — Signal of Deprez, as made by the Cambridge Scientific Instrument Company. A delicate form of apparatus for recording signals, similar in principle to the chronograph shown in Figs. 238 and 239, but much smaller, is Fig. 246. — Tracing taken with the signal of Deprez, acted on by a current in- terrupted by a tuning-fork, vibrating 500 times per second. ■called the signal of Depre~. It is represented in Fig. 245, and an •example of a tracing is given in Fig. 246, 2. The Direct Recording of Movement. Movements may be recorded if we attach a lever to the moving structure, as near the fulcrum as possible, and bring the other end of the lever into contact with the recording surface, or the lever may 390 THE CONTRACTILE TISSUES. simply be laid on the moving part, so that it may rise and fall with each movement. In the case of muscular movements, special registering in- struments have been invented termed Myograplis. The first myography invented by Yon Helmholtz, consists of a brass framework, Fig. 247^ Fir,. 247. — Myograpli of Von Helmholtz, shown in an incomplete form, a, forceps for holding femur of frog ; b, gastrocnemius muscle ; c, sciatic nerve ; d, pan for weights; e, marker recording on cylinder g; f, counterpoise, by moving which the weight of the framework may be reduced. movable round a horizontal axis, and kept in equilibrium by a counter- weight. The tendon of the muscle is fixed by a hook to the middle of the frame, and a balance for the purpose of carrying weights is attached underneath. From the other extremity of the brass framework there hangs a marker, w^hich traces uj^on any surface the movements of ascent or of descent of the muscle. The registering surface may consist either of a stationary plate of smoked glass, as in the arrangement of Fick (see Fig. 15, p. 2S), or of a vertical rotating cylinder (Fig. 256). When the tracing is obtained by Fick's method, it consists of a series of vertical lines, as seen in Fig. 16, p. 29. A convenient form of myograph is the spring inyograph of Du Bois- Reymond, sho^\Ti in Fig. 249. This consists of a rectangular glass plate,, h, moving horizontally along two slender steel wires, d. It may be impelled hoxdzontally by the recoil of a steel spring, c, when a check is set free at the other end of the apparatus. Thus applied, the muscular contraction will produce a curve. (See Fig. 248.) By removing the rod THE GRAPHIC METHOD. 391 carrying the steel spring, the smoked glass plate may be slowly moved in front of the stylet by means of a long screw attached to the plate, the handle of which is seen a little below c. The spring myograph is sho^\Ti on a larger scale in Fig. 248. See description of Figure. Fig. 248. — Spring Myograph of Du Bois-Reymond. A B, metal pillars to which the wires, k k, are attached, and along Jc k the frame carryinif the glass plate slides in the direction of the arrow when the spring b is released ; d, short bar of metal projecting from lower surface of frame which opens the key h when the frame is carried across by recoil of spring 6. Thus the primary circuit of an inductorium may be mechanically interrupted. In Von Helmholtz and Fick's arrangement, whether applied to a cylinder or to a moving glass plate, the muscle must be placed in a Fig. 249. — Spring Myograph, showing Chronograph applied to it for the purpose of ascertaining the velocity with which the blackened glass plate 6 is drawn across by the recoil of the spring c. vertical position and have a certain weight to bear. Nor can the apparatus be conveniently applied to the muscle while in situ in the body 392 THE CONTRACTILE TISSUES. of the animal recently killed. There are thns a want of sensitiveness and a want of convenience, hoth of which are to a great extent obviated by the myograph of Marcy. The arrangement of this apparatus for an experiment is seen in Fig. 250. The principal piece of the apparatus consists of a horizontal brass plate supporting the axis of a registering lever, which moves in a hori- zontal plane. Consequently the lever registers upon a cylinder moving horizontally. A thread attached to the tendon of the gastrocnemius muscle of the frog is fixed by a small button to the lever. Fixed to the brass plate, and upon the same plane, there is a flat piece of cork on which the pithed or decapitated frog is laid (in which consequently all sensation has been abolished). Fig. 250. — Arrangement of apparatus for experiment with the Myograph of Marey. a, recording cylinder; b, railroad carrying the myograph c; d, galvanic element ; e, induction coil ; /, key. A view of the apparatus is shown in Fig. 251. Attached to the side of the cork plate, there is a brass support bearing the electrodes for stimulating the nerve or muscle. When the muscle is stimulated elec- trically, it contracts and moves the horizontal lever, which traces a curve upon the moving cylinder. If it be desirable to have a series of tracings from a muscle for a considerable period of time, these may be obtained by placing the entire apparatus upon a support, moved by clockwork upon a little railroad (see Fig. 251) parallel with the register- ing cylinder. Marey has also devised a double myograph, which differs from that just described only by having a second lever, so that the two gastrocnemei of a frog may be attached each to a lever ; the two levers THE GRAPHIC METHOD. 393 are superposed, and the tracings are close together but still distinct. By such an arrangement it is possible to study graphically the forms of the curves obtained by the contractions of two muscles under different conditions, say the one poisoned and the other normal. Fig. 251. — Jlarey's myograijh. A, pillar ; B, horizontal metal plate ; (', screw for adjustinfj the height of the marker, so that it may, E, descend ou the surface of a horizontally moving cylinder, by moving the lever, D ; //, pulley for thread ; F, thread from gastrocnemius to lever ; &, /, spring for bringing lever back to original position during relaxation of muscle. The great advantage of Marey's method is that it registers the curve of muscular contraction so as to give its different phases of movement. This will be appreciated upon comparing the two tracings seen in Figs. 252 and 253, which show the changes in muscular contraction under Fig. 252. — Myographio tracing obtained by the method of Fiok : changes in the amplitude of contractions of a muscle under the influence of a gradually increasing temperature, a, &, c, individual contractions. To be read from left to right. the influence of a gradually increasing temperature. It maj- lie observed in both that the rise of temperature increases the amplitude of the con- traction, but the vertical lines do not indicate whether or not any change has taken place in the duration of the contraction. It mil be 394 THE cos TRACTILE TISSUEIS. seen, however, l)y studying Fig. 253 that heat not only changes the amplitude, but causes the contraction and relaxation to occur in shorter h'lQ. i'j;!— Myograpbic tracing obtained bj' the metliod of Maroy, corre- sponding to a g7-adiial heating of a muscle, as in Fig. 'Ib'l. U will be noticed that not only does the amplitude of the contractions change, but also their form and their duration. To be read from left to right. intervals of time. This is a good example of the value of the graphic method of resiistration. 3. The Transmission of Movement. One of the chief difficulties in the registration of animal movements is that of attaching directly to the body a marker which will inscribe on a blackened surface the phases of the movement. It is therefore neces- sary to have means of transmitting the movement to a distance, or, in other Avords, transferring to the i"ecording surface the movement we Avish to examine. This Marey has accomplished by the use of tambours, or drums, united by tubes containing air. One of these tamboiu's is seen in Fig. 254. It consists of a shallow metallic case, or drum, the upper surface of which is formed by a thin indiarubber membrane su])- Fiour are in opi)osite directions : when the mem- brane of the one is drawn downwards, that of the other is forced upward. When it is desirable to obtain the movements of the levers in the same direction, one of the tambours must be reversed. What lends liTeat value to this method of transmission of movement is that Fig. 257. — Arraiigemeiit of iqiparalus lor tiaiiMmbSiuu ot uuisciilar move- ment by tambours, a, voltaic element; b, primary coil; c, secondary coil of inductorium ; even although for convenience \ in taking a tracing the plate fj g \ may be moved up or down by f a rack and pinion movement h c, I the plate g' g', which is of the ; same weight as g g, may be moved in the reverse direction. I When used, the pendulum is 1) pulled to the left towards ff, ■> and it is held in position by the j spring catch e ', which is turned I outwards. On the pendulum I making a swing, it is caught ' at the extreme limit of its ; swing by the catch e ', which is I also turned outwards. The 1 under surface of the frame carrying the glass-plates has two little projections//', each moving on a delicate hinge, and when the pendulum swings across, these strike the arm b of a break-mechanism placed immediately below //' in the position occupied by these in Fig. 264. This break-mechan- ism is shown in Fig. 265. In newer forms of the myograph than that shown in Fig. 264, the break-mechanism slides on a horizontal bar, so that it can be brought into operation at any PHENOMENA OF MUSCULAR CONTRACTION. 409 -point in tlie swing of the pendulum. The break-tnechan- ism is simply a little arm 6, Fig. 265, which is knocked aside by //' in Fig. 264. The primary circuit of an inductorium passes from the binding screw d, Fig. 265, through pillar rj to a, thence through h (when in contact with a), and then back to the battery, and J is insulated from the rest of the instriiment. When b and a are in contact, the primary circuit is formed, but when b is knocked away from a by ^ in Fig. 264, it is opened. The opening shock of the inductorium is sent to the nerve-muscle preparation connected with a myograph placed in front of the front glass- plate, the muscle contracts, and a curve is described on the glass-plate, such as is shown in Fig. 266. As -t\as instrument has been of great service in measuring Fig. 265. — Break-Apparatus of the Pendulum Myograph. the rate of the transmission of the nerve current, it will be further described when we consider the func- tions of nerve in Vol. II. The pendulum makes a •complete swing from one catch e. Fig. 264, to the other •catch e' in one second, and the time of any part of the ■swing may be recorded by means of a chrono- graphic tracing, as shown in Fig. 266. The pendulum myograph is usually provided with two "break" mechanisms, similar to that shown in Fig. "265, but so arranged that the one is opened by the swing of the pendulum a little sooner than the other. Thus, by having two circuits, a nerve may be stimulated by one induction shock, and then by another happening the x^th of a second later, and the muscle -curve may then be similar to that shown in Fig. 276, p. 416. The instrument has also been used by Burdon-Sanderson as a rheotome, that is an instrument for opening and Kilosing two independent circuits, at very short periods 3 Y 41() THE CONTRACTILE TISSUEiS. of tiiiic.^ Thus the time relations of various electrical phenomena may be investigated. For purposes of comparison, it is often important to obtain a series of ciirves in juxtaposition. We may get this by using the travelling stage of Marey (Fig. 267), so that the shock is sent to the muscle a little later in each revolution of the cylinder. ^ Burden-Sanderson. On the electromotive properties of the leaf of Dion£ea. Phil. Trans. Part I. 18S2. PHENOMENA OF MUSCULAR CONTRACTION. 411 It can easily be arranged that the recording cylinder carries a Avire attached to its axle, through which the current passes when the vnxQ dips with each revolution into a cup of mercury interposed in the circuit Thus each revolution of the cylinder makes and breaks the Fig. 268. — Arraugement for recording consecutive contractions of a frog's gastrocnemius muscle, each contraction occurring a little later in time th;iu the one immediately jire- ceding it. current passing through the primary coil of an inductorium, either at the same moment, or a little later, mth each successive contact. Thu^ arrangement for the latter purpose is shown in Fig. 268. By this method, tracings may be obtained, such as those shown in Fig. 269. — Tracings of muscular contractions produced by successive single induction shocks, to be read from left to right, thus — a, b, c. Fig. 269, and Fig. 283, p. 429, an insj^ection of which at once elicits- facts of physiological interest. 412 THE CONTRACTILE TISSUES. The duration of muscular contraction varies in different species of animals ; it is very short in birds, somewhat longer in mammals and fishes, and still longer in reptiles. The amplitude of the curve, indicating the ammint of nmscidar contraction, increases Avith increase of the stimulus, at first rapidly, then more and more slowly, and Avhen a maximum is reached it remains constant, as indicated by the amplitude of the curve remaining the same. Fatigue diminishes the amplitude and increases the duration of the contraction, as seen in Fig. 283. Stoppage of the ■circulation and cold has the same, whilst the gentle application of heat has the reverse, eff'ect. The duration of muscular contraction is in- creased in diabetes, jaundice, cerebral hemiplegia, and in some cases of sclerosis of the spinal cord. The rapidifAj of muscular contractions is very great in man and in the mammalia. Thus, Landois found that each contraction of the muscles in writing the letter n lasted only -0564 of a second. A striking illus- tration of the rapidity of muscular contractions is seen in the rapid movements of the fingers of a violinist or pianoforte player, or in the rapid movements of the muscles of the larynx when a singer trills in the execution of vocal music. If a fatigued muscle has been strongly stimulated, it may contract, but it may not immediately relax when the stimulus has ceased to act, so that it may require a weight to stretch it to its original length. This condition of temporary contraction is called the contraction remainder. It may be seen to some extent in muscles not greatly fatigued. B. — Pkopagation of a Wave of Contraction, When a living muscle is stimulated, it not only contracts, but becomes thicker. The curve of the movement of thickening has been found to be generally the same as that of contraction. This may be shown by the arrana;ement seen in Fia;. 270. Fig. 270.— Diagram showing apparatus for recording the curve of thickening, a b, Two light levers joined at the point on the right and resting on the muscle, c d. The muscle is caused to contract by sending an induction shock through it. The trac- ing is recorded and compared with that obtained by a myograph. As has been already mentioned, if a stimulus is applied to one end of a living muscular fibre, a wave is seen to propagate itself towards the other end. This has been termed the ivctve of contraction, and Marey has PHENOMENA OF MUSCULAR CONTRACTION. 41.5 measured its rapidity by grasping it at two places with instruments- called myographic pincers (Fig. 271, 1, 2). Each of these is connected with a transmitting tambour. The recording tambours, 1', 2', trace the curves on a cylinder. When we stimulate one of the ends of th& Fig. 271. — Arrangement for tracing the •wave of muscular contraction. The muscle is diagrammatic. muscle, the thickening which accompanies the contraction, as it passes- from one end to the other, acts through the pincers upon the two- receiving tambours successively, and a tracing is obtained such as is seen in Fig. 272. It will be seen that the two curves do not coincide, and the distance between their summits gives the rapidity of propagation of the muscular- wave, which is from 1 to 3 metres per second. The wave ""in the^ Fig. 272. — Tracing of the propagation of the muscular wave. Chrono- graphic tracing, 100 vibrations per second underneath. (Marey.) muscle of the heart travels slowly, being only at the rate of 10 to 15^' mm. per second, and in the muscles of the lobster it progresses at the speed of 1 metre per second. In living human muscle it is very rapid, 10 to 13 metres per second — so rapid that some have denied its exist- ence. The wave-length has been estimated at only 206 to 380 mm^ Cold diminishes the rate, as shown in Fig. 273. 414 THE CONTRACTILE TISSUES. Fatigue, the tendency to coaguLition of the myosin so as to i)roduce ji(j(yr mortis, veratrin, and sulphocyanide of potassium also dela}^ the pro- _gression of the Avave. The wave of contraction excited in a muscular fibre is limited to it alone, and is not transmitted to neighbouring iibres. A muscle contracts as a Avhole only Avhen all its fibres have l)een simul- taneously excited. When the nerve stimulus acts, as there is a nerve fibre for each muscular fibre, and as the motor end-plate is near the Fio. 273. — Curves showing the rapidity of the transmission of the wave of contraction in the muscle of a rabbit. A, muscle in normal condition ; B, when the muscle was cooled by ice. Observe that in B the wave has travelled more slowly. centre of the fibre, a wave of contraction proceeds to each end of the fibre from the end-plate. Each fibre is from 30 to 40 mm. in length, so that even with this comparatively short length it can scarcely be said to contract instantaneously as a Avhole. Practically, a muscle contracts as u whole when it receives the nerve stimulus, but in many muscles, and in long muscles especially, the distance of individual muscular fibres from the main nerve trunk will vary so that they will not receive the nerve stimulus precisely at the same instant. Chap. VIII.— THE GENESIS OF TETANUS. If a rapid series of induction shocks be sent to the muscle of a frog from Du Bois-Reymond's inductorium, seen in Fig. 222, the muscle will l)ass into a state of strong contraction, in which it will remain a consider- able time, so long as induction shocks are transmitted to it. This .«tate of strong contraction is termed tetanus or cramp, and is quite different in character from the short contraction caused by a single shock, which we have termed a twitch. In the first place, it may be •observed that the shocks sent by the apparatus, when the primary •circuit is automatically closed and opened, may be regarded as instan- taneous excitations. AVhen excitations, by induction shocks, amount in number to three or four per second, the muscle contracts more strongly, and the contraction continues for a longer time than if the muscle had received a single shock of the same intensit3\ The height THE GENESIS OF TETANUS. 415 of a contraction caused by a single shock is usually from a third to a half that caused by a rapid series of shocks, or tetanus. We may readily make this observation by interrupting the current of the induction coil by means of a metronome or by a vibrating spring, so that the num- ber of shocks sent per second may be varied at pleasure. It will then be found that the opening shock is more powerful, as shown by the amount of muscular contraction, than the closing shock, and an explana- tion of this phenomenon has already been given (p. 374). When the shocks succeed each other regularly, at the rate of about fifteen per second, and in such a way that the duration of the interruption is short compared with the duration of the shock, the muscle remains persist- ently contracted. The phases of this state of permanent contraction must be carefully studied. When a muscle is stimulated by an inductorium, it draws a curve on the smoked surface of a revolving cylinder which is different in character from the curve produced by a single shock, described in the last chapter (Figs. 263 and 266). The curve of tetanus is shown in Fig. 274. Fio. 274.— Tetanus produced by numerous shocks from an inductorium. The interrupted current acted a little before a, and after a period of latent stimulation, the muscle began to contract so that the lever rises rapidly, and at b the muscle reaches the maximum of contraction. The contraction lasts until c, when the current is shut off and the muscle slowly relaxes. It is important to note that when a muscle is tetanized by an induction current, it contracts suddenly, as indicated by the rapid rise of the lever, that tetanus is a condition which lasts for a considerable time, and that the muscle relaxes slowly. The curves in Figs. 274 and 275 should be compared. When a series of induction shocks is more slowly transmitted to the muscle in contact with Marey's myograph, the primary current being interrupted by a metronome, a tracing is obtained, as in Fig. 275, A number of distinct oscillations will be observed in the ascent of the curve. Each oscillation is somewhat shorter than the one immediately preceding it. Three cases in which a muscle is excited by successive shocks present themselves for consideration. 1. When a second excitation acts after the termination of the con- traction occasioned by the first, it produces a second muscular 410 THE CONTRACTILE TISSUES. contraction having the characters of the first, and so on, as regards successive excitations, until the muscle becomes fatigued. 2. If the second excitation acts during the period of latent stimulation following the first, the shortening is not greater than for one excitation, and the curve of contrac- tion is the same. 3. If the second excita- tion acts during the two last portions of the pre- ceding contraction, the shortening corresponding to the second excitation is added to that of the first. The curve thus produced is seen in Fig. 276. If other excita- tions quickly follow, each capable of causing a partial contraction, there is then a summation of the effects of the individual excitations, and a state of permanent contraction or tetanus is produced, which is the fusion of the partial contractions resulting from the succes- sive shocks. This is well illustrated by varying the number of Fio. 275. — Tnicing of a muscle passing into a tetanic state, when the primary current of an inductorium is rapidly made and broken by a metronome. The first shock was transmitted to the nerve at a, the second an instant after 1, the third an instant after 2, and so on. It will be observed that with each succeeding shock the muscle becomes shorter, though the amount of shorten- ing at each shock is less. Pig. 276. — Tracing of a double muscle curve. While the muscle was en- gaged in the first contraction (whose complete course, had nothing inter- vened, is indicated by the dotted line), a second induction shock was sent to it, at such a time that the second contraction began just as the first was beginning to decline. a,b, first contraction; c,d, second con- traction ; /', chronographic tracing. excitations transmitted to the muscle. Thus, we may obtain a curve like that shown in Fig. 276 if the second excitation has been sent just when the muscle was beginning to relax from the effects of the first excitation, and like those in Figs. 275 and 277, if the excit- ations followed faster, but not too fast, so that the contractions caused by individual excitations appear like teeth on the tracing, and, finally, like Fig. 278, when the excitations follow in very rapid succession, and individual contractions cannot be seen. Tetanus may be maintained for a considerable time, which varies according to the degree of vitality THE GENESIS OF TETANUS. 417 of the muscle, but by degrees it passes off, and the muscle slowly returns to its original length, under the influence of fatigue. A study of the genesis of tetanus clearly shows that it is a condition produced by a fusion of individual contractions. Fig. 277. — Curve showing the genesis of tetanus, a to h, individual con- tractions ; 6 to c, muscle now tetanic, but wavy line still indicating indi- vidual contractions — the slope of the line from i to c showing that the muscle is becoming fatigued ; c, indicates time when induction shocks were stopped ; c, rf, slow relaxation of muscle. The number of shocks required to cause tetanus varies. From 16 to SO^per second are sufiicient for the gastrocnemius of the frog, while 10 per second may cause tetanus of the frog's hyoglossus muscle. Kro- necker and Stirling found incomplete tetanus of the red muscles of the rabbit to be caused by 4 shocks per second, while 10 shocks per second caused complete tetanus. On the other hand, the pale required over 20 per second. It is curious that, according to Marey, the muscles of Fig. 27S.— Curve showing genesis of tetanus, a to 6, result of first shock ; then observe the cumulative effect of the successive shocks, as shown by- gradual ascent of curve from 6 to e ; stoppage at e of induction shocks ; «, /, gradual relaxation of muscle. birds are not tetanized by even 70 per second. If shocks are sent to a muscle at the rate of from 220 to 360 per second, instead of tetanus, there may be only a momentary contraction. This result was obtained by Bernstein, but Kronecker and Stirling, by using an ingenious instrument invented by them, termed a tone-indudorium,'^ produced tetanus by shocks so rapid as 24,000 per second. The study of tetanus becomes of great interest when we remember that many, if not most, of the muscular movements occurring in the body are of this character. A simple muscular movement, such as ^ See Landois' and Stirling's Physiology, vol. i. p. 717. I. 2d 418 THE CONTRACTILE TISSUES. flexing the arm, throws certain muscles into a condition of normal tetanus. This is proved hy Kronecker's experiment of placing two needles, one in a human muscle and the other in its tendon, and con- necting both with a telephone. If, then, the muscle be tetanized by induction shocks coming at the rate of about 100 per second, a sound will be heard. The sound is a proof of vibrating motion. The pheno- mena of shivering of muscles is an example of incomplete tetanus. Chap. IX.— MODES OF EXCITING MUSCULAR CONTRACTION. In addition to the normal stimulus transmitted by a nerve, muscular contraction may be excited by electrical, mechanical, thermal, and chemical stimuli or excitants. 1. Electrical Stinmli. — An electrical stimulus acts most effectively on muscle when the current passes in the direction of its filires. We have already seen that a single inchidion shock of sufficient strength will cause a single contraction or twitch, and that a number of shocks, com- ing at short intervals in succession, will cause tetanus, spasm, or cramp. As already explained, p. 374, the opening shock produces a more powerful eflPect than the closing shock, but the two may be made nearly equal by the use of Yon Helmholtz's arrangement. "When a current from a battery of voltaic elements is passed through a muscle, no effect in the way of contraction can be observed, except at the moments of opening and of closing the cvurent, or Avhen the ciurent is very suddenly and largely increased or dimin- ished in strength. If the muscle forms part of an electrical circuit and the cturent is opened and closed, the resulting contraction shows a muscle curve differing from that produced on imtating the nerve, by the contracted muscle relaxing slowly, so that the time of relaxation is very much longer than the time of contraction. The passage of the current, however, causes electrolytic changes, and no doubt there Avill be also the production of heat owing to the resistance opposed by the muscle as a conductor to the passage of the electrical current. Siippose a muscle to be traversed by a voltaic current, it is stimu- lated only near the negative pole when the circuit is closed, and near the positive pole when the circuit is opened, and if the motor end-plates have been paralyzed by curara, a wave of contraction may be observed starting from the negative pole on closing, while it starts from the positive jjole on opening. (Eutherford.) It is important to observ-e that a muscle, wdth paralyzed nerve- endings, responds much more readily to the opening and closing shocks MODES OF EXCITING MUSCULAR CONTRACTION. 419 of a voltaic current than to the shocks of induced or Faradic currents. This is probably owing to the muscle-protoplasm not responding to the very short and sudden stimulations of induced cuirents, while, on the other hand, the motor end-plates and the nerves themselves readily respond to sudden shocks of this character. It would appear that all kinds of protoplasm, except that of nerve and nerve-endings, are more susceptible to the more sluggish opening and closing shocks of a voltaic current. 2. Mechanical Stimuli. — The effect produced by a mechanical stimulus depends upon the intensity of the stimulus and the rapidity with which it acts. Pressure upon a niuscle, if slowly applied, may not excite a contraction, but if the pressure be made suddenly, contraction may be the result. When a muscle is stimulated by any mechanical excitant, the result is not only a contraction passing from the excited point through the whole of the muscle, but a permanent coriti"action at the point touched. This contraction, termed by Schiflf, its discoverer, idio-muscular contraction, may be observed in the living man when a blow IS struck with a blunt body across a muscle, and perpendicularly to the direction of its fibres. This produces a local contraction like a weal, or ^s if there was a bit of whip cord beneath the skin. There is then a contraction which extends the whole length of the muscle, and when this contraction has disappeared, there remains at the point of excita- tion a transverse swelling, which lasts for a certain time. A muscle may pass into a state of tetanus, if the nerve supplying it is irritated by ■successive mechanical shocks. 3. Thermal Stimuli. — The sudden passage of a muscle from one tem- perature to another considerably higher or lower than the first may ■excite muscular contractions, and as the temperature is raised from 28° C. to 45° C, the individual contractions become stronger. About "60° C, tetanus may be produced. Above this limit, the myosin or other albuminous constituent of the muscle coagulates, and the muscle becomes stiflT. The effect of heat on a muscular structure is strikingly illustrated by the action of heat on the heart of a frog, an experiment which may be readily performed by means of the arrangement shown in Fig. 279. In this case, however, the heat acts not merely on the muscular fibre, but also on the intrinsic nervous mechanism or ganglia of the organ. (See Heart.) 4. Chemical Stimuli. — A great many chemical agents cause ■contractions by their influence on the chemical composition of the imiscle or of the nerve supplying it. Amongst the agents which •excite contraction are the fixed alkalies, the mineral acids, acetic, oxalic, tartaric, and lactic acids, alcohol, ether, creosote; the neutra,! 420 THE CONTRACTILE TISSUES. alkaline salts, such as chloride of sodium, and sulphates and carbonates^ of the alkalies ; metallic salts, such as those of iron, zinc, copper, silver, and lead, and concentrated solutions of urea, sugar, and glycerine. These excite contraction when used as weak solutions, and \\\ the case- of several, the solution must be considerably stronger before it has any effect on the nerve of the muscle. A Aveak solution of bile also stimu- lates muscular fibre. Neutral alkaline salts act to the same extent on muscle and nerve. The vapour of hydrochloric acid causes single con- FiG. 279. — Arrangement for studying the action of heat on a frog's heart. A copper plate, carrying at one end two delicate levers, 6, moving on a joint at d ; the heart is placed underneath the levers near d, one on the auricular and the other on the ventricu- lar portion, and the movements of the levers are recorded on the cylinder e. When heat is applied by the spirit lamp, /, to the end of the copper plate, the heart beats faster and faster until it passes into a state of tetanus, from which it may recover if the copper plate is cooled with a piece of ice. tractions, while chlorine gas produces tetanus. It is said that the vapour of bisulphide of carbon stimulates the nerves and not the mus- cular fibres. Very dilute solutions of caustic soda or caustic potash first stimulate and then kill both nerve and muscle. Certain solutions cause rhythmic movements in muscle. The most efficient fluid for this purpose is called Biedermann' s fluid, prepared by dissolving 5 grammes of chloride of sodium, 2 grammes of alkaline phosphate of soda and "5 gramme of carbonate of soda in 1 litre of water If the sartorius muscle of a froa;, to which curare has been MODES OF EXCITING MUSCULAR CONTRACTION. 421 given so as to paralyze the motor end-plates, is immersed in this solu- tion so that about one half of the long thin muscle dips into it, the muscle Tjegins to contract rhythmically in a manner analogous to the pulsations of the frog's heart. This is an interesting experiment, as suggesting that rhythm may, to some extent at least, depend on the chemical composition of the fluids circulating through the rhythmic organ. All of these substances possibly excite either the nerve or the muscle by absorbing water from them, as the contractions resemble those follo"vving rapid drying of a muscle or nerve by exposure to the air. Distilled water injected into the blood-vessels of a muscle excites the contraction ■of individual fibrillae, producing the local quiverings called fibrillar con- tractions ; but it rarely excites contraction when applied directly to the nerve or muscle. Chap. X.— THE PRODUCTION OF HEAT BY MUSCLE. The apparatus required in investigating the thermal phenomena of muscle consists of a galvanometer of small resistance, and a thermo- -electric pile, or thermo-electric needles. A thermo-electric needle is com- posed of an iron wire, to each end of which is soldered a copper or •German silver wire. Several such needles are passed through the anuscle so that one set of junctions of iron -with copper, or iron with German silver, is embedded in the muscle, and the other set is exposed to the air. The adjacent ends of the needles, composed of copper or silver, are in connection with two wires which go to the galvanometer. This arrangement is not very delicate, and for more refined investiga- tions a thermo-electric pile is necessary. A pile consists of short bars of bismuth and antimony, having each end of a bar of bismuth soldered to the end of a bar of antimony, the bars lying parallel. Thus there are two sets of alternate junctions, one of which may be applied to the body giving off heat, while the other is kept at as near a uniform tem- perature as possible. In these circumstances, if the one set of junctions becomes hotter than the other, a current of electricity is generated which deflects the needle of the galvanometer. By such arrange- ments, it will be found that there is a deviation of the galvano- meter needle when the muscle contracts, owing to the development of a thermo-electric current, from the metallic junction in, or applied to, the muscle being raised to a higher temperature than the other junction kept at a constant temperature in a vessel of boiling water or in melting ice. Heidenhain has shown, by the use of extremely delicate arrangements of this nature (Fig. 280), that the instantaneous excitation 422 THE CONTRACTILE TISSUES. of the nerve supplying a muscle is attended by a rise of teniperatnrc iir the muscle. The development of heat in a muscle depends — (1) ;ipon its tension ; (2) on the work done ; and (3) on the state of fatigue of the muscle. 1. Tenmiu unci Heat. — The more a muscle is stretched, the greater will' 1)0 the amount of heat developed when it contracts. According to- Beclard and Heidenhain, a muscle develoj)s its maximum amount of heat, supposing the intensity of the stimulus to remain the same, when it is so stretched as to be unable to contract at all. This occurs- Fio. 280. — Heidenhain's arrangement for studying the heat-phenomena of muscle, a, gastrocnemius muscle of a frog having a small thermo-electric pile, 6, in contact with it. The fifrure f> in the upper left-hand comer shows the surface of the thenno-electric pile, consisting of \T< junctions, c, Is a frame bearing the pile ; d, support of the frame c, with a counterpoise e attached to it ; /, j\ two glasses containing melting ice to give a uniform tem- perature to the other thermal junctions, {/, h. in tetanus of a limb when the antagonistic muscles oppose each other, a fact which accounts for the high temperature observed in cases of tetanus. 2. Work Done and Heat. — As to the relation between the heat produced in, and the work done by, a muscle, when the muscle remains con- tracted supporting a weight sufficient again to stretch it, no effective work is done, and energy appears only as heat. Energy appeared as-' motion during the contraction, Ijut this portion disappeared when the THE PRODUCTION OF HEAT BY MUSCLE. 423 weight restored the muscle to its original length. Again, the energy of a contracted muscle appears as heat, and that of a contracting muscle either as heat or mechanical work, or both. If we add the energy appearing in the form of heat in a contracting muscle to the energy liberated as mechanical work, the sum is the total amount of energy expended during the contraction. When a muscle lifts a series of gradually increasing weights up to a given height, the amount of heat increases with the work done up to a maximum point peculiar to the condition of the muscle at the time. If the weight be still increased, it will not be lifted so high, but the work done will be greater and the amount of heat generated in these circumstances will be less. It follows that the maximum pro- duction of heat by a given muscle is reached sooner than its maximvim amount of work. The greater the heat, the smaller will be the mechani- cal work, and vice versa. The heat, therefore, produced during work is, in the same time, inversely proportional to the amount of work. Several large contractions liberate more heat than a greater num- ber of small contractions, even although the amount of work done" be the same in both cases, or in other Avords, large contractions cause greater changes in the muscle substance than a greater number of small contractions. Lastly, suppose a muscle does work by a number of small contractions in a given time, in each case lifting a weight, more heat will be produced than if the muscle kept up the same Aveight for the same time by being in a state of tetanus. 3. Fatigue and Heat. — With regard to fatigue, it may be stated that as a muscle becomes exhausted by successive excitations, or by the approach of death, both the amount of work done and the production of heat simultaneously diminish. The two quantities, however, do not diminish equally : heat diminishes more rapidly than work, so that it is possible to have a muscle capable of performing a small quantity of mechanical work, but at the same time producing no appreciable amount of heat. Heat to a small amount is also produced by the relaxation of a muscle, indicating that this is not merely a passive process, but that it also in- volves a small amount of metabolism. The venous blood, coming from a muscle in a state of activity, is cceteris paribus warmer by '6" C. than that flowing from a muscle at rest ; and it has been ascertained that the gastrocnemius of a frog, deprived of blood, will show an increase of about "001° to •005° C. for each contraction, until it becomes much fatigued. Tetanus of the same muscle for two or three minutes will produce a rise of temperature of -014° to -018° C. Billroth and Fick determined the heat produced by tetanus of the muscles of mammalia whilst the blood was circulating as equal to a rise of 5° C. Leyden also foimd a rise of temperature during 424 THE CONTUACriLE TISSUES. tetanus in animals, and Wiindcrlich obsei'ved the same phenomena in I)atients suffering from tetanus ; and, further, that the maximum temperature was not attained, in these cases, until after death. The heat produced by a contracting muscle is a physical expression of the metabolic changes occurring in it. The chemical energy set free by these metabolic processes appears as heat and mechanical work, and the general law regulating the relation of these is that, within certain limits, the greater the resistance off'ered to the contraction the greater will be the Avork done. In these circumstances, as much as one fourth of the total energy may appear as Avork, the remaining three fourths appearing as heat — a remarkable result when compared Avith that of the best constructed engines, AA'hich yield as AA^ork only one ninth part of the total energy supplied to them by the oxidation of the fuel, while the remaining eight ninths are manifested as heat. Chap. XI.— THE WORK DONE BY MUSCLE. The amount of Avork, W, done by a muscle Avhen it contracts is measured by the product of the load, I, and the height, h, through Avhich it is lifted. Thus, W = Z x h. Hence if / = o, that is if the muscle has lifted no weight, no Avork has been done ; and again if the muscle be so over loaded that /i = o, there Avill be no Avork, as the muscle has not contracted. The Avork done by a healthy muscle in lifting a Aveight will vary according to the strength of the stimulus and the Aveight to be lifted. A feAv experiments made Avith Von Helmholtz's myograph (Fig. 15, p. 28) in Avhich the muscle is stimulated by single induction shocks of equal intensity, will show this clearly. a. The Contraction as a Function of the Stimulus. — Suppose the gastroc- nemius muscle of a frog is loaded AAdth say a weight of 10 grammes, and the apparatus is arranged for a single induction shock. Removing the secondary coil of the induction machine to a considerable distance from the primary, let a single shock be sent to the muscle. If no contraction folloAvs, let the secondary coil be moved nearer the primary until the first visible contraction is obtained and recorded. This point has been termed the point of liminal intensity of the stimulus. Then advancing the secondary coil ouAvards definite distances nearer the primary, let each contraction be recorded as an ordinate on the abscissa line at distances proportional to the distances the secondary coil has been moved. Such a method shoAvs that the amount of contraction increases Avdth the increase of stimulus, at first rapidly, then more sloAvly, until a maxi- mum is reached ; the maximum remains for some time, and afterwards TEE WORK DONE BY MUSCLE. 425 the amount of contraction may become less owing to the fatigue of the muscle from repeated stimulations. As will be seen in the course of this ■discussion, the amount of contraction alone is no measure of the amount of muscle work. This depends, as already pointed out, on the product of the amount of the contraction and the load lifted. /?. The Contraction as a Function of the Resistance. — Suppose in this experiment the strength of the stimulus is the same, while the load which the muscle has to lift is gradually increased. Let a contraction be recorded when there is no load to the muscle at all. Then load successively with 10, 20, 30, etc. grammes, and record the several con- tractions at proportionate distances along the abscissa line. It can thus be shown that as the weight is increased from zero upwards, the con- traction increases ; as the weight continues to be increased, contraction .also increases, but more slowly, and beyond a certain point increase in the weight is followed by a diminution in the amount of contraction. y. The Contraction as a Function of the Time between Successive Stimulations. — In a third experiment, it is interesting to observe the effect of varying the time between successive stimulations, keeping the load or resistance and also the strength of the stimulus the same. This may be done by sending shocks to the muscle at intervals of a second, half a second, a third of a second, and so on. The result shows that if a certain amount of time is allowed to elapse between successive shocks, the same amount of work may be over and over again repeated, but when the shocks come too quickly, the work quickly diminishes, or, in other words, the muscle becomes fatigued. 8. Measurement of the Work Done. — The amount of work done by a muscle at a given time will depend on the strength of the stimulus, the amount of the resistance to be overcome, and the condition of the muscle as regards fatigue, and no doubt there will be for a given muscle, after a period of rest, a maximum amount of possible work corresponding to a •certain strength of stimulus and a certain load to be lifted, or resistance to be overcome. As already pointed out, the work done is ascertained by multiplying the actual amount of shortening of the muscle by the weight lifted. Suppose, for example, a muscle lifts 10 grammes 20 millimetres in height, the work done is 200 gramme-millimetres. This may also be shown diagrammatically by drawing an abscissa line, marking off upon it the distances proportionate to different weights, and drawing as ordinates the actual work done in the case of each weight. A line drawn through the summits of the ordinates will give the curve of the work done with the same stimulus and increasing loads. Such a line would of course slope upwards. The heights, however, of the marks made on the smoked glass-plate of the myograph would diminish 42G THE CONTRACTILE TISSUEiS. as the amount of work increased. Thns, supp.ose the diagram in Fig. 281 represents the result of an experiment of a muscle lifting gradually increasing Aveights, The figures at the side indicate millimetres, and the figures along the liase line indicate grammes. It is clear that the Fig. 281.— Diagram to show the mode of measuring muscle work. Avork done as indicated by the first line on the left is 10 x 5 = 50 gramme- millimetres ; the next, 20 x 5 = 100 gramme-millimetres, etc. ; Avhile the last on the right, 100 x 3 -= 300 gramme-millimetres. Weber has given the foUoAving figures with reference to the work done by the gastrocnemius muscle of a frog : — Weisrht Lifted in Grammes. • Height in Millimetres. Work done in Gramme- Millinietres. 5 15 25 30 27-6 251 1 1 -45 7-3 138 376 286 219 This table shows that the work increases with the weight up to a certain maximum, after which a diminution occurs, more or less rapidly according as the muscle is fatigued. To maintain a muscle in a tetanic state, successive stimuli are required. The first few stimuli cause the muscle to contract, and then work is done, but no work is done during the tetanic state. The chemical changes set up by the successive stimulations required to keep u]> tetanus find expression as heat and as continued contraction. A tetanic contraction will do more work than will be done by a single contraction caused by a stimulus of the same strength as the individual stimuli producing the tetanus, but the tetanus will of course quickly produce fatigue in consequence of the rapid metabolism taking place in the muscle. Static Fwce of a Muscle. — In charging a contracting muscle Avith gradually increasing Aveights, there arrives a time Avhen the elongation due to tension of the muscle exactly compensates the contraction of the muscle. When this is so, the muscle is in a state of equilibrium, and the Aveight expresses what Weber termed its static force, or absolute farce. The force is stated usually as the number of grammes per square centi- THE WORK DONE BY MUSCLE. 427 metre.-^ This force for frog's muscle has been found to be 2,800 to 3,000 grammes, and for human muscle, to be from 7,000 to lOjOOO" grammes, that is to say, a strip of frog's muscle having a transverse section of a square centimetre would, when stimulated, itndergo no change in length with a weight of say 3,000 grammes attached to one end. The force of individual muscles in the human body may be measured by means of instruments, termed di/n- amometers, one of Avhich, ad- apted for testing the muscles of the arms and hands, is seen in Fig. 282. On com- pressing this instrimient with both hands, a strong man may force round the index to 70, representing kilogrammes of pressure. The muscles of women are feebler than those of men. With another kind of dynamometer, a man, by pulling, may move the index to 150 kilogrammes. Fig. 2S2. — Dynamometer, a, b, strong steel spring ; c, scale. Chap. XII.— THE MUSCLE SOUND. When a stethoscope is firmly applied over a powerfully contracting muscle, such as the biceps in a muscular man, a deep tone is heard, which is produced by about twenty (19-5 ?) vibrations per second. This sound was first detected by AVollaston. It may also be heard during profound stillness, as in the middle of the night, if we stop the ears and powerfully contract the muscles of mastication. This- tone is the same in pitch as the resonance tone in the ear. The muscular sound, according to Von Helmholtz, is produced by the varia- tions of tension which occur during the fonuation of a continuous- muscular contraction. It is probable that the sound heard is not that due to twenty vibrations per second, which number of impulses on the ear would not produce the sensation of a tone, but to forty vibrations per second, or the first overtone or harmonic of the fundamental tone. The existence of the muscular tone renders it probable that, to secure the contraction of a voluntary muscle in man during life, about twenty impulses per second are transmitted from the nerve-centres to the ^ To find the mean transverse section of a muscle— (1) Ascertain tlie volume by dividing the absolute weight of the muscle by its specific gravity, 1'058 (C.G.S.), and then (2) divide the volume by its length. This gives the mean transverse section. (Landois.) 428 THE CONTRACTILE TISSUES. muscle along the motor nerves. Of course it is understood that the contraction is not merely a twitch, as Avhen one quickly flexes and extends the arm, but such a contraction of the muscle as is necessary to support a weight for a short time. This condition of contraction may be regarded as a kind of normal tetanus, and no doubt the great majority of voluntary contractions are of this nature. Irritation of a motor nerve or of the spinal cord produces a muscular sound of the pitch above mentioned, but it is remarkable that if tetanus is produced in a muscle by rapid interruptions of the j^rimary circuit produced by a vibrating spring, the pitch of the muscular sound so produced is exactly that produced by the number of vibrations of the spring, and by increasing or diminishing the number of vibrations of the spring, the pitch of the resulting muscular sound can be raised or lowered in the scale. Thus, a tone may be heard corresponding to a pitch of from 700 to 1000 vibrations per second, when the number of vibrations of the interrupting spring reaches those amounts. If the nerve is irritated, the sound is not so loud. Chap. XIII. —THE PHENOMENA OF MUSCULAR FATIGUE. A muscle becomes fatigued after continuous work. Fatigue means a diminished power of work. Up to a certain point, the substances pro- duced in a contracting muscle are eliminated as quickly as they are formed, and at the same time new materials necessary for the repair of the muscle are supplied. There is thus, as it were, a balance between the two processes. When, however, the muscle is excited to very fresoh, shown in Fig. 286. It consists of a thick copper cylinder, A (Fig. 286), in the interior of which a magnetized ring (Fig. 285) is suspended from the top of a glass tube by a single filament of silk. From the ring A, in Fig. 288, an aluminium rod passes up to B, a frame which holds a circular plane mirror. Plugs of copper are used to close up the ends- of the chamber in which the circular magnet swings, whilst the chamber containing the mirror (not shown in Fig. 286, but placed a little above A) is closed by a brass cover canying a thin circular piece of glass, so that the mirror can be seen. On each side of A (Fig. 286) the coils of wire B B slide in a wide slot so that they can be brought close to or even over the copper chamber A. Each coil contains about 30,000 turns of very fine wire, and the resistance is equal to 7,000 ohms for each coil.^ The copper chamber damps the oscilla- tions of the magnetic ring, because the oscillations of the latter set up induction currents in the opposite direction in the copper, and these react on the ring, so that it quickly comes to rest. The ring magnet (corresponding to the needle in an ordinary galvanometer) is rendered astatic by the arrangement shown in Fig. 287, called Haiiy's bar. It consists of an accessory magnet, S N, placed in the magnetic meridian, and therefore parallel to the needle. Its north pole should be pointing north, as is that of the needle. It is supported on the bar e f, which is in the same direction as the axis of the coils, and the magnet can slide up and down the bar, which is graduated into centimetres. The magnet may be moved from a distance by the cord m n, passing from the wheel k to the arrangement of pulleys 1 c, shown in the figure, and thus the oscillations of the ring magnet are qnite under control. An image of a scale placed in front of the Galvanometer. A, Mag- mirror may either be watched by the observer through a net; B, mirror; C, hook ■' jo for suspending by silk short focus telescope, or a lamp may be placed in front of the mirror so that a beam of light is reflected from it on to a scale placed at a suitable distance. The advantage of this instrument is that it- is practically an aperiodic or dead heat galvanometer, that is to say, when in- fluenced by a current, the magnet swings slowly and comes to rest without oscil- lation, and when the current is withdrawn it swings back again to zero and then comes to rest. 2. Thomson's Reflecting Galvanometer. — The other form of galvanometer is Sir ^ The instrument is also provided with two coils of low resistance, 1 ohm, suit- able for thermal currents (p. 421). Fig. 288.— Magnet and mirror of Wiedemann's THE ELECTRICAL PHENOMENA OF MUSCLE. 443 William Thomson's reflecting gal- vanometer, seen in Fig. 289, an instrument more sensitive than the one above described and more stiit- able for refined investigations. This instrument is upon the same prin- ciple as the ordinary galvanometer, but it is modified by having the needles constituting the astatic system very short and very light, by having the coils of wire in the l)obbins brought as close to the needles as possible, and by having a small silvered mirror attached to the uppermost group of needles. A lamp is placed in front of the little mirror, and a ray of light is reflected by the mirror iipon a scale placed at a convenient dis- tance in front of the instrument. The galvanometer made for phy- siological purposes has a resistance of 8,498 ohms at 18° C. One Daniell's element gives through a circuit of 254,970,000 ohms resist- ance a deflection of 130 divisions on the scale, and throvigh a resistance of 33,146 meg-ohms^ a deflection of 1 division. It is F'O- 2S9.-Reflecting galvanometer of Sir William Thomson, a, Upper, and b, lower bobbm of nne wire ; therefore an instrument of great a small mirror is attached to the upper group of mag- „•+„„„„ „„j „f Q-,7+„QTv,Q ^qK netic needles in the centre of (t; c, brass rod, bearing d, resistance and of extreme deli- ^ ^^^^^^^ ^^^^^^ ^^^ regulating the position of the cacy. needles underneath ; e e e e, binding screws. 3. Lippmann's Capillary Electrometer. The action of the substances set free at the electrodes in electrolytic decomposition, and the energy shown as motion in these circumstances, are strikingly manifested by the behaviour of a drop of mer- cury in dilute sulphuric acid, when the positive pole of a battery is put La con- nection with the mercury and the negative dips into the acid. The mercury extends towards the negative electrode during the passage of the current, becoming covered with a film of sub-oxide, which dissolves in the acid, leaving a bright surface. By making and breaking the current a series of oscillations is set up. Movements in the mercurial electrode and adjacent acid have been observed by many phj'sicists, and have received various explanations. Erman, in 1809, was the first to observe that when a drop of mercury was placed on a grooved surface between the electrodes it moved towards the negative pole ; and he also observed that " a drop of mercury in a horizontal tube, with dilute acid on both sides, moved at the passage of the electric current through the tube toward the negative electrode."- . 1 A meg-ohm = 1,000,000 ohms. 2 For an historical account of this subject, see Lippmann, Annale'< de Chemie et de Physique, 1875, p. 540. 444 THE CONTRACTILE TISSUES. This latter phenomenon was fully investigated by Lippmann,* aiul led, along with researches by Quincke, not only to a theoretical explanation of electro-capillary action, but also to the construction of the capillary electrometer. It is now known that these phenomena, both as seen in the experiment with the globule of mercury and in the capillary tube, are due to a change in the surface tension pro- duced by the electrical polarization of the surface of tlie mercury. Lippmann's form of the electrometer, shown in Fig. 290, consists of a tube of •ordinary glass, A, 1 metre long and 7 millimetres in diameter, open at both ends, and kept in the vertical position by a stout support. The lower end is drawn to a capillary point, until the diameter of the capillary is '005 of a millimetre. The tube is filled with mercury, and the capillary point is immersed in dilute sulphuric acid (1 to 6 of water in volume), and in the bottom of the vessel, B, containing the Fig. 2P0.— Lippmann's Capillary Electrometer. acid there is a little more mercury. A platinum wire, a, is put into connection with the straight tube having the capillary point, and another platinum wire, )3, ^ Lippmann, Comptt.-i Rendus, 1873, p. 1407 ; Annal. de Chem. et dt Phys. op. cit, p. 494 ; Poggendorf's Annahn, cxlix. p. 547 ; also Phil. Mag. [4], xlvii. p. 281. THE ELECTRICAL PHENOMENA OF MUSCLE. 445 enters the wide tube, B. Finally, arrangements are made by which the capillary point can be seen with a microscope, M, magnifying 250 diameters. The appearance of the tube under the microscope is shown in Fig. 291. Such an in- strument is very sensitive ; and Lippmann states that it is possible to determine a difference of potential so small as that of the tttJttt of a Daniell. Lippmann made use of the arrangement shown in Fig. 290 for bringing back the mercury to the zero- point when it had been pushed up the capillary tube by the action of a constant electro-motive force in the direction of j3 to a, and for measuring by means ^ig. 291.— Appearance of the of a manometer, H, the pressure in mm. of mercury capillary tube in Lippmann's . electrometer, required to bring the mercury back. In the appar- atus, T is an India-rubber bag or tube capable of being compressed between plates- approximated by the screw E, so that pressure is exerted on the upper end of the long vertical tube, and the amount of this pressiire is measured in mm. of mercury by the manometer, H. Various simpler forms of the capillary electro- meter can be easily made, in which the capillary tube rests in the horizontal position on the stage of the microscope, and in which, also, the tube may be so' fine as to bear examination with a magnifying power of 400 diameters.^ The special value of the capillary electrometer lies in the quichiess with luhicJi it responds to any sudden variation in the e. m. f. As observed by Burdon-Sanderson,. with reference to changes in potential in the injured heart of the frog while alive,, it is striking to notice " that the movements of the mercurial column not only correspond closely in time to the actual changes which they represent, but express^ with very great accuracy the difi"erences of potential which actually exist in each successive phase of a variation." The oscillations of the column of mercury in the capillary tube may be photographed on a moving sensitive plate, when a picture such as is seen in Fig. 292 will be obtained, which shows at a glance gradual or sudden changes in potential. - Fig. 292. — Changes in potential caused by the induction currents from the secondary coil of an inductorium at the moments of closing, c ; and of opening, r ; r, the primary cur- rent. From a photograph by Marey of the oscillations of the mercury in the capillary tube of Lippmann's electrometer. 4. The Telephone. By this instrument sounds may be heard on making and ^ See author's paper on Lippmann's capillary electrometer, Proceedings of the Philosophical Society of Glasgow, vol. xiv. p. 37 ; Burdon-Sandersou, on the electromotive properties of the leaf of Dionsea in the excited and unexcited states, Phil. Trans, part i. 1882 ; Loven, Nordiskt Medic. Arkiv. vol. xi. No. 14. As to a form made by Prof. Dewar, see S. P. Thomson's work on Electricity. - Thus Burdon-Sanderson has photographed the oscillations caused by changes- of potential in Dionsea and in the frog's heart. Royal Institiition Lecture, 1882. 446 THE CONTRACTILE TISSUES. breaking the current from a muscle at rest. The current shown by a muscle in -iction cannot be so detected. The telephone has not been of any special service in the investigations of currents from animal tissues. Method of Making Experiments with Galvanometer. — The very sen- sitiveness of the galvanometer is one of its chief sources of error. If the ends of the galvanometer wires are connected with two wires, say of copper, the ends of which are immersed in a conducting fluid, a deflec- tion of the needle at once indicates a powerful current, because the two wires are not in an absolutely similar electrical state ; or if the wires are •directly connected with the tissue to be examined, another powerful deflection occurs, because electricity is evolved at the point of contact of the Avires and the moist tissue. Further, if metallic conductors, say composed of zinc, coming from the galvanometer, were brought into connection with living muscle, little or no current Avould be obtained, and even if there were a current, it might be due to contact of the metallic conductors Avith the living tissue exciting electrolytic decomposition. Hence it is necessary to have .a fluid interposed between the metal and the animal tissue, as, for •example, the zinc "wire or plate forming the terminals of the galvano- meter must be immersed in a saturated solution of sulphate of zinc. But as sulphate of zinc solution would have the eff"ect of irritating the living muscle, it is necessary to have an inactive substance between the tissue and the sulphate of zinc solution. All these conditions are fulfllled by the arrangement of Du Bois-Reymond, which is that usually employed, and which may conveniently be termed the non-polarizable electrodes. Many modifications of this apparatus have, from time to time, been emjDloyed for particular purposes, but the form most convenient for demonstrating the principal electrical j^heno- mena of nerve and muscle is what is here described. It consists (Fig. 293) of two troughs made of zinc, mounted on insulating plates of vulcanite. The inner surfaces of the troughs are carefully amalgamated, and they are filled nearly full of a saturated solution of sulphate of zinc. Into each trough is then placed a small cushion of clean blotting or rfilter paper, Avhich Cjuickly becomes permeated by the solution. Finally, a small thin film or plate of sculptor's clay, or kaolin, moistened with a half per cent, solution of common salt, or still better, with saliva, is laid on each paper pad. These clay-pads are for guarding the tissue T'lG. 203. — Non-polarizable elec- trode of Du Bois-Reymond. c, zinc trough ; v, vulcanite base ; s, vulcanite shield to keep pad 6 of blotting paper in position ; p, moist clay ; ;enieiit of the " Fall Rheotome." The falling weight F having injured the muscle in the way shown in Fig. 303, is then made to strike against keys a, X, O, by which the galvanometer circuit can be closed and then opened, or other effects produced speedily, the one after the other. THE ELECTRICAL PHENOMENA OF MUSCLE. 459 The Current in a Livimg Man. — Such is a general statement of the facts offered by Du Bois-Eeymond and Hermann for and against the theory of an electrical current in normal living muscle. If Du Bois- J?,eymond were correct it "vvas natural to suppose that the muscles of a living man would show a considerable current, and a negative variation on voluntary contraction. But Du Bois-Reymond completely failed to detect a current from the living muscles of man at rest. If, however, one dips both hands into ^ailcanite troughs filled with salt solution and connected with the galvanometer, and then voluntarily contracts the muscles of one arm, the needle is deflected to one side ; if the muscles of the opposite arm are contracted, the needle swings in the opposite direction. This Du Bois-Reymond accounted a negative valuation. But Hermann declares it to be no muscle current at all, but to be due to a secretion stream directed from Avithout inwards on the irritated side — a skin current. In his experiments, stimulation produced sweating and the electric current, but when atropia Avas given both disappeared. " A man," he says, " paralyzed by curare would show the Du Bois- 'Reymond current in spite of the absence of contraction, and a man under the influence of atropia would not show the current in spite of the occurrence of contraction." ^ That is, in the former case, stimula- tion could not produce contraction on account of the curare, but it would produce sweating, and a current — a secretion stream — would appear ; in the latter case it would produce contraction, but on account of the atropia there could be no sweating and there would be, therefore, no current. To the Du Bois-Reymond theory of polar particles, or the pre- existence theory, Hermann, therefore, opposes what he calls a difference theory because it refers all electromotive effects of muscle to two kinds of physiological change. The first part of the theory is that ihe dying poiiion of the substance behaves itself negatively to the living, and the electro- motive force has its seat in the demarcation zone between the living and the dying. To this he adds a rider that not only death but irritation as well causes the affected substance to become negative to the unaffected portion. Hermann enunciates his views in the following four propositions — (1) "Localized death in continuity of protoplasm, whether caused by injury or by metamorphosis (mucous, horny), renders the dead part negative electrically to the unaltered part ; (2) Localized excitation in continuity of protoplasm renders the excited part negative electrically to the unaltered part ; (3) Localized waiming in continuity of protoplasm renders the warm part positive, localized cooling the cold part negative to the unaltered part ; (4) protoplasm is strongly polarizable ^ Hermann, Handbuch der Phys., Bd. I. Th. I. .s. 224 u. 225. 4G0 THE CONTRACTILE TISSUES. on its limiting surfaces (first shown as regards the protoplasm enclosed in tubes of muscles and nerves) ; the polarization constant decreases on excitation and on dying." ^ Chap. XIX. -SUMMARY OF THE PHENOMENA OF A LIVING MUSCLE. When the energy which, for want of a l)etter term, is called nerve force reaches a muscle fibre, it acts on the motor end-plate, probably setting up molecular changes in it which are propagated to the con- tractile substance of the muscle. These molecular changes raj^idly pass towards each end of the fibre and they occur simu.ltaneously with, and are the source of, the action current or negative variation. According to Du Bois-Reymond, this wave of negative variation is due to the polarization of hypothetical electromotive molecules, while the rival theor}' of Hermann assumes that the result of the nerve stimulation is to produce negativity at the point stimulated, and that this negativity (as regards other points) passes from point to point along the muscle, thus producing a current, but it is important to notice that the fad of the propagation of a wave of electrical disturbance has been established and is independent of the theories offered as to its explanation. The short period during which the action current passes through the fibre does not exceed the y^^- of a second, and for the longer period of the yi-„ of a second the fibre is apparently inactive. This is the period of latent stimulation, and during this period molecular phenomena are occurring which are antecedent to contraction. It is highly probable also that during the period of latent stimiilation energy is liberated as heat, but ii portion of the heat of muscular tissue is probably evolved during the succeeding phase of active contraction. At the end of the latent period, the fibre contracts, and during the contraction of a number of fibres existing together in the organ we term a muscle, a sound is produced — the muscle sound. The muscle fibre then relaxes and the katabolic products of the nervous stimulus are then quickly removed and the muscle substance is built up anew. Another consequence of the molecular dis- turbance excited by the nervous stimulus is the increased production of carbonic acid and probably of other waste products and the increased consumption of oxygen. This resum^ embraces the principal phenomena, but others have been mentioned in the preceding pages. The student should make himself master of the physiological changes in muscle as they illustrate in a striking way the anabolic and katabolic processes occurring in living matter, and he will see that many of the processes ^ Burdon-Sanderson's Translations, op. cit. p. 328. TEE PHENOMENA OF THE ELECTRIC FISHES. 461 happening in other tissues, such as in glands, in the sense organs, and even in the central nervous organs are analogous to those of muscular contraction. Chap. XX.— THE PHEISrOMENA OF THE ELECTEIC FISHES. The phenomena manifested by electrical fishes have in recent years attracted the attention of various phj^siologists as bearing on the mode of action of muscles, and especially of nerves. The indefatigable labours of Sachs, Fritsch, Ranvier, and many others, by which they have been able to describe the histological structure of electric organs, and the elaborate researches of Du Bois-Reymond and of Sachs, and more recently, in this countr}', of Burdon-Sanderson and Gotch, on the electrical phenomena manifested by these organs, have brought to light many details of one of the most remarkable structiu'es in the whole realm of nature. To do justice to this subject would lead me beyond the space at my disposal, but I will attempt to give a brief account of the chief facts, because a general knowledge of these cannot fail in being both interesting and suggestive to the physiological student. About fifty species of fishes are known or are believed to possess. electrical organs, and to have the power of communicating electrical shocks. This may possibly be an over estimate of the number, and at all events, the electrical properties "of not more than five or six have been investigated. From one point of view, it is remarkable that any animal should possess an electrical apparatus, but yet when one considers the value of such an organ to the animal, as a weapon of offence or of defence, it may appear strange that an electrical organ has been found only in a few fishes. The electrical fishes best known are Torioedo Galvani, or T. marmorata, and some other species of torpedo found in the Adriatic and Mediterranean seas ; Gymnotiis eledricus, an eel living in the lagoons in the region of the Orinoco in South America ; and Malapterurus eledricus, the ra" ash, or thunderer fish of the Arabs, a native of the Xile, the Xiger, the Senegal, and other African rivers. In addition to these, we know of Mwmyrus longipinnis, M. doi'salis, and M. anguilloides, fishes allied to the jjike family, found in the Nile ; Rhinobatus eledricus, a ray from Brazilian seas ; and Tridiiurus eledricus, a ribbon-like fish found in the Indian Ocean. The common skate of our own coasts, Fucia batis, also possesses, an electrical organ, first described by Stark in 1844, afterwards, in 1847, by Robin, and the electrical properties of which have been recently investigated by Burdon-Sanderson and Gotch. It will be 462 THE CONTRACTILE TISSUES. observed that the electrical fishes do not liclont;- to one class or group, and that some are found in fresh water, while others inhabit the ocean. Electrical fishes have been known from early times. Aristotle describes the benumliing shocks of the torpedo, as recognized by fisher- men ; and, in a recent work on the malapterurus, Fritsch figures an ancient Egyptian carving, executed 3,000 years before the Christian era, in which fishes, like malapteruri, are distinctly seen. It was not until 1773, however, that Walsh showed experimentally that the shocks of the toi'pedo are electrical in their nature. Since that period, many investigations have been made, both as to the structure and as to the functions of the electric organs. 1. Torpedo. — The electric organs, two in number, are large, flat, kidney-shaped bodies, placed on each side of the head and gills. The organ is composed of a number of hexagonal prisms, placed vertically Jietween the dorsal and abdominal integuments, and each prism is divided by a series of delicate membranous plates or diaphragms nttached by their edges to the aponeurotic sheaths separating the })risms (Fig. 305). The plates are separated from each other by a jelly- like albuminous fluid. About 30 plates occur in each mm., linear measurement, of a prism, and a, i)rism of medium height contains about 615 jjlates. Each organ contains say 800 prisms, and thus there are roughly about 500,000 plates in each organ, or 1,000,000 in both organs. This powerful electric bat- tery, thus divided into compartments is richly supplied with large nerves. These are (1) a large branch from the trigeminal, and (2) four branches from the vagus which spring from a large lobus dectricus between the corpora higeniina and the medulla ■ohlongata. According to Pacini, the nerves enter the laminte at the points of their attachment to the prisms, and are disti'iljuted to their under surface, and in the fluid between that surface and the next lamina They ramify here in a very vascular nucleated tissue. Each Via. 305. — Torpedo Galvani, showing the prisms fi-oni the dorsal surface, and the gi-eat nerve trunks, b, ending in the smaller nerves a, distributed to the prisms. THE PHENOMENA OF THE ELECTRIC FISHES. 463 prism presents a structure somewhat analogous to that of a voltaic pile. The piles are vertical and the plates horizontal. Savi, in 1844, observed that the nerves ended in the electric plates in the form of a rosette. According to Ewalcl, one of the most recent writers on the subject, the electric nerves pass through the gills and enter the electric organ as four large branches and one smaller branch. They then divide freely, and run between the prismatic columns, always ending near the middle of the column. From these smaller branches still smaller ones originate, each consisting of three or four fibres, and these spread out obliquely over the lateral siu-face of the column. Each nerve fibre divides into from 12 to 25 delicate nerve filaments, called by "Wagner, their discoverer (1847), nerve tufts (nervenbiischel) (Fig. 306). Each nerve tuft gives off as many fibrils as there are plates in the column, each jDlate being supplied Avith one fibril. Fig. 306. — Xerve tuft in tor- pedo, showing numerous little branches coming off from the larger nerve. Fig. 307. — Terminations of nerve on the plate of the prism of a torpedo, seen after the action of osmio acid, x 800 d. Fig. 308.— Diagram of prism of torpedo, showing 11 plates, each supplied by its nerve fibre, issuing from the chief nerve A, so as to form one of Wagner's tufts. The main nerve reaches the centre of the column, and the branches given off from it to the individual plates lie almost in a straight line or plane parallel to the axis of the column. These fibrils do not pass to the plates by the shortest course, but only the shortest fibres go directly 464 THE CONTRACTILE TISSUES. to the plates, the majority making a hook-shaped bend, and then each reaches its appropriate plate after a course of greater or less length. This arrangement is seen in Fig. 308. When a fibril reaches a plate, it divides dichotomously again and again so as to form an expansion, as seen in Fig. 307, and it is remarkable that the arrangement of the divisions of the fibril on one plate corresponds almost exactly Avith that on neighbouring plates. The final little twigs of nerve end by forming an anastomosis with adjoining fibrils, and thus produce what may be called an electrical end-plate. The fact that a single nerve fibre, by splitting into fibrils supplies from 1 2 to 25 plates, shows that all those plates may be thrown into electrical activity at the same time by one nervous impulse. Eanvier describes the structures between each pair of plates of the torpedo as consisting from below upwards of (1) a first layer, or nervous layer, formed of two portions — (a) a superficial portion containing the plexus of nerve fibrils above described, and {b) a deeper layer showing peculiar finger-shaped projections called electric hairs by Ranvier, and lying side by side so as to give the appearance of a palisade (Remak) ; (2) a second, or intermediate layer, also consisting of two parts — (a) a superficial or ventral portion, finely granular, next the palisade of elec- tric hairs, and (Ij) a deeper portion, more coarsely granular than a, and containing nuclei ; (3) a thin clear layer, called by Eanvier lamella ; and tive tissue, Above this, or nervous and so on. in Fig. 309 the dmsal of connec- partition. (4) a layer , forming a we again find the first layer above described, These layers are shoAvn according to Eanvier. This remarkable organ is now known to be related to muscular tissue. Fritsch, by studying the development of the torpedo at Naples and Villa- franca, has found that, as previously shown by De Sanctis, the torpedo, in its ontological history, passes through a squaliform stage, a rajform stage and a torpedo stage ; in the first resembling shark-embryos in Fig. 309. — Attachment of the electric plates of torpedo to the sheath of the prism, c/, sheath of jsrism ; v, ventral or nervous plate ; \ rfT^"-' cC^^ Fig 310. — Yei'tical section through one of the plates of the electric organ of gymnotus, about l.SOO diameters (?) ; serui-diagrammatic, to show all the structures. P, Pacini's line ; 'p, anterior papillse ; p', posterior papillae ; st, bacillary layer ; p", thorn i^apilla ; 6, amoeboid cells in thorn papilla ; observe the nerve fibre entering the apex of this papilla, a, nuclei of connective tissue ; el, peripherical electrical nerve ; c, c, nuclei of nei-ve fibres ; d, blood corpuscles in A'essel ; V, posterior surface of lamella ; tm, mucous tissue between the anterior papUlaj and the next lamella ; s', aponeurosis between the plates; v blood-vessel. which a few cells like those of connective tissue are interspersed. The middle of the plate is trans- parent and homogeneous in a fresh condition, but soon after removal from the body there appears a thin dark line, called FacinVs line, dividing the plate into two parts. The posterior surface of the plate is also thrown into papillse (posterior pajnlke) of Aery various sizes, constituting the hacillary layer, some being merely short rounded projections, Avhile others called tJm-n papilla.'' are long, conical in shape, and reach nearly to the posterior partition of connective tissue. These have been supposed to be supporting pillars, but they are regarded by Fritsch as really parts of the electric plate, in which the electric nerves terminate. This "vdew is clearly borne out by Fig. 310 copied from one of Fritsch's drawings. The ultimate nerve fibrils pass along the anterior surface of the partitions and shortly before en- tering the papillae, and on the posterior surface of the electric plate, they lose the white sub- stance of Schwann (medullary sheath), the axis cylinder alone entering the electric plates and mersfino; into their substance. The medullary sheath breaks up into fibres containing nuclei here and there, analogous to the nuclei of Eanvier's nodes in a nerve-fibre. These small fibrils pass to the pos- terior surface of the electric plate. THE PHENOMENA OF TEE ELECTRIC FISHES. 467 The microscopical examination of the electric organ of the gymnotus leads to the conclusion that the plates arise from embryonic muscle, each plate being equivalent to a number of primitive bundles cemented together. In some preparations, the plates may be found broken into parts corresponding to the individual bundles, or rather to the primitive •cylinders or fibres of eml^ryonic muscle, a longitudinal cleavage taking- place like what may be seen in muscle. Sometimes, again, the plates •cleave transversely, along Pacini's line, a division analogous to that of muscle into Bowman's discs (p. 309). While the nerve cells in the central nervous system of the torpedo are located in one organ, the electric lobe, those of the gymnotus occur throughout the greater part of the length of the spinal cord. This organ in the gymnotus is not specially large, „^ and it is of the usual shape. In the anterior l^art of the body of the fish it is somewhat flattened, it is rounder about the middle of the body, and is again flattened towards the tail. No special cells connected with the electric nerves occur in the encephalon. In the spinal cord, however, special strands or columns exist, one in each lateral half of the cord. These ganglion cells are so situated in the grey matter of the cord as to surround the central canal like a cylinder, open in- feriorly, supposing the cord to be placed in the natural position of the fish, with the back upwards and the belly downwards. The cells are seen in Fig. 311, which repre- Cuo J a S.T. Fig. 31 1.— Kight half of spinal cord of a small gymnotus, seen in trans- ients one half of a transverse section of the "^^^^^ section, x 30 d. sr, substantia reticularis anterior ; sr' , substantia •cord. Broad axis cylinders arise from the reticularis lateralis ; sr", substantia 11 1 • reticularis posterior ; /a, fasciculus poles 01 these cells and run out into the anterior ; cmt, commlssura trans- . versa ; ne, a fe'w small nerve cells ; anterior roots 01 the spinal nerves, and pass '''<-'-e, anterior root of spinal nerve; •■ , . , . >' J.1 1 i • 1 Observe the large ganglionic cells ine roots 01 these electric nerves run along- in the grey matter, of which one is side the ordinary motor roots as far as the ^ "^"^ ^"^ '^' intervertebral foramina. The remaining poles of the ganglion cell ■ some of them larger than others — run into the neuroglia, and disappear in the direction of the lateral columns and of the commissure. The electric nerve-cells are of a roundish form (Fig. 312), and they consist of highly-granular protoplasm, from which very distinct poles {axis cylinders) emerge. The ordinary multipolar motor cells, charac- teristic of the spinal cord (Fig. 173, p. 313), become the fewer the more 468 THE COS TRACT HE TISSUES. numerous the electric cells, and the motor cells lie exclusively in the anterior coruua or horns of grey matter. The shape of these motor cclls is irregularly ])olygonal, often Avith concave borders ; there is less protoplasm, and the poles are not so distinct nor so broad as is the case with the electric cells. According to Fritsch, cells transitional from the motor to the electric cells may be observed. The more rounded the cell, the more granular the pro- toplasm, and the more distinct the poles, the more does the cell become of the electric and the less of the motor type. Such transitional cells occur in the inner group of the motor cells in the anterior cornua, and they are more especially present in a mass of grey matter in the cervical region, which, from its position, is analogous to the vesicular column of Lockhart Clarke, seen in the spinal cords of the highest vertebrates. The cells in this- grey mass are few in number and small in size,, but they show the characters of electric cells, and they send their poles into axis cylinders, passing- out of the cord by the anterior roots. Towards the posterior end of the cord the electric cells become fewer in number, and gradually their characters merge into those of motor cells. 3. Malapterums eledricus. — The electric organ in this fish forms a layer beneath the skin, enveloping the whole body, Avith the exception of the- head and fins. A layer of fat separates it from the subjacent muscles. The ultimate structure shows numerous lozenge-shaped spaces or loculi filled with fluid, having a peculiar layer of electric tissue on two sides of the lozenge-shaped space, as shown in Fig. 313. The electric tissue- consists of a layer of granular protoplasm, containing niiclei (often double), more especially towards each margin. Both borders, and more especially the inner border, show a delicate striation, which really con- sists of tubular pores, represented in the diagram. Around each nucleus there is a halo of clearer protoplasm. The broad firm tubulated zone is distinct from the more slimy inner one. Each space is sur- rounded by a well marked fibrous memljrane. From the general ap- pearance of the electric tissue, Fritsch regards it as consisting of a layer of modified epithelial cells forming giant cells. The inner free surface is bathed in a mucoid fluid. In the epidermis there are peculiar club- shaped cells, having double nuclei of a glandular character, which are forms transitional into those constitutins; true electric cells. In some= -Electric fi'om the middle region of the spinal cord of gymno- tus, X 314 d. a, sheath of neuroglia ; b, smaller nerve poles anastomizing with those of adjoining cells ; c, chief nerve pole, passing into axis cylinder of an electric nerve. THE PHENOMENA OF THE ELECTRIC FISHES. 409 parts of the body, as towards the tail, the lozenge-shaped spaces may be found without any electric tissue, and transitional conditions also •occur. Although quite different in character from the plates or ■discs in the torpedo and the malapterurus, being epithelial instead of muscular, the lozenge- :shaped arrangements may also be ■called electric discs. The num- ber of these discs is enormous. One cubic centimetre of the elec- tric organ of a middle-sized malapterurus contains 14,000 ; a transverse section through the thickest part of the body exposes -3,000, in a row from head to tail 1,600 are found, and Fritsch •computes that the total number in one fish amounts to 2,000,000. On tracing the nerve into the electric organ, it is found that ■only the axis cylinder enters at •one side or angle of the space, and that it insensibly merges into the substance of the electric tissue. (See Fig. 313, n.) The finest nerve fibrils running be- tween the discs have an ex- tremely thin axis cylinder sur- rounded by a delicate medullary .sheath (white substance), and they show the nodes of Eanvier. In larger nerves no longitudinal fibrillation of the axis cylinder can be seen under the action of osmic a,cid, and the apparent thickness of the nerve is due to the thick- ness of the medullary sheath. Xot only is the medullary substance thicker, but the nerve fibril is surrounded by concentric layers of connective tissue, between Avhich even delicate capillaries and nerve fibrils may run. When the nerves are traced inwards to the spinal cord, they are all found to spring from one single nerve, the electric nerve, the axis cylinder of which enters the cord and ends in a process or pole of a gigantic nerve cell. This nerve cell has numerous proto- w " -, ^ M 2 > O ^ « ^ ^ 470 THE COS TRACTILE TIS,^UES. I)lasmic processes which coalesce here and there so as to form a per- forated plate, in the meshes of which capillaries and nerve fibres may l)e seen. A ganglionic cell of similar size and appearance lies in the adjacent half of the spinal cord, and both cells arc connected by a commissure passing across the median line. Each of these giant nerve cells measures -21 mm., and we have here a unique example of an organ being supplied by one nerve cell. The axis cylinder springing from one of the poles of the perforated nervous plate is only moderately thick, but as it ultimately supplies a nerve fibril to each electric disc, the sum of the transverse sections of these idtimate fibrils must greatly exceed that of the parent nerve. If we call the transverse section of the parent nerve 1, the total trans- verse section of the chief larger branches Avill be represented by 2, and the increase goes on so rapidly that the sum of the transverse sections- of the ultimate fibrils reaches the enormous amount of 364,000, showing the remarkable fact of a gradual increase in the amount of matter form- ino- the axis cylinder as we pass from the spinal nerve cell to the ultimate nerve fibril. It would appear that a similar increase is seen in the nerves of other animals, a fact not yet sufficiently recognized in theories of nervous conduction. (See p. 316.) The malapterurus has a complete lateral nervous system, similar tO' that of silurus, and Fritsch states that the electric nerve is a portion of this system Avhich, though originating from the trigeminus nerve,. is in other and non-electric malapteruri connected with the vagus. The fresh electric organ of malapterun^s was found by Du Bois- Reymond to have a neutral reaction. It became acid on the third day after death. After immersion for five minutes in Avater at 40° to 50° C. it gave an acid reaction. Thrown into boiling water, pieces of electrical organ became acid, thus behaving like the tissue of the central nervous organs, and not like muscle. 4. Baia hutis, or Common Skate. — Professor Burdon-Sanderson and Francis Gotch have recently investigated the structure and functions of the electrical organ in the skate, and have supplied me with the following short sketch — ■" On each side of the spinal column from the caudal fin to- about half way up the tail of this fish, partly in contact ^^dth the skin, and partly enveloped in the so-called sacro-lumbalis muscle (Goodsir) there is an elongated fusiform body, consisting of a number of longitudinal series of discs (Fig. 314). It is divided by septa of connective tissue into tubes, in which the discs are Fig. 314.— Diagram showing the geiieml arrangement in the skate (liaia) of the discs and iinmcular fibres as seen in a longitudinal section. THE PHENOMENA OF THE ELECTRIC FISHES. 471 suspended transversely at distances of about 'S mm. One surface of the disc is directed forwards, and is flat ; while the other looks backwards, is slightly concave, and shows a number of little alveolar depressions. One of these discs is shown in section in the sketch in Fig. 315. Upon the flat surface of the disc is the nervous membrane which in structure corresjjonds to a nerve-endplate in muscle. The alveoli on the posterior surface are occupied by capillary l)lood-vessels, but these do not pene- trate the disc. The substance of the disc, underneath the nerve membrane (electric membrane), consists of fine laminae, parallel to its surface, which are seen in section as striae. But towards its posterior surface it consists apparently of structureless material, beset -with numerous nuclei." Fio. 315. — Semi-diagrammatic view of a disc from the electric organ of the skate, o,, a, connective tissue with capillaries ; b, nerve layer, nerve endings branching dichotomously, the terminal branches all tendintr in the direction of the arrow ; e, striated layer, which corresponds to the bacillated substance of g5'miiotus ; d, d, processes of transparent struc- tureless material, containing numerous nuclei, 'corresponding to the thorn papillae of a gymnotus. The electric organ in the skate thus resembles that of the gymnotus. The organs are supplied by nerves coming from the ventral or motor roots of the spinal nerves, and these run along with the motor nerves going to the muscles of the tail. I am not aware of the existence of special nerve cells in the spinal cord. 5. The General Electrical Properties of the Electric Fishes. — The general phenomena of the electric fishes may be illustrated by considering in the first place some of those manifested by the torpedo. The shock emitted by the torpedo is weak compared with that of the malapterurus, but as pointed out by Du Bois-Reymond the strength of the shocks of the torpedo is much diminished by the good conductivity of the sea water by which the fish is surrounded. The shock of the torpedo is weaker because less current passes to the person touching it. When a livin"- 472 THE CONTRACTILE TISSUES. malapterurus was placed in salt solution its shocks became much weaker, indeed Du Bois-Reymoiid states " it apparently lost its electrical pro- perties." When the living torpedo is touched it may or it may not emit a shock, and it soon becomes evident that the discharge is to a certain extent under the control of the will. If the brain be removed without injuring the electric lobes, irritation of one or other of these lobes will cause an electric discharge limited to the side corres})onding to the organ irritated. The electric lobes are thus undoubtedly the electric centres. These may be also excited by reflex action, that is to say, an irritation of the skin may cause a reflex discharge, the impression being carried to the nerve centres from the part of the skin irritated by the sensory nerves supplying that part, and on reaching the electric lobe charges are set up therein which resiilt in the transmission of an excita- tory change along the electric nerves to the electric organ. Finally, irrita- tion of the electric nerves themselves causes shocks, but these are weak in comparison with those obtained by directly irritating the electric lobe. The reflex "discharge" of the fish Avas found by Marey to consist of a succession of shocks. He led the current of the organ through a delicate chronograph, and during the reflex activity of the organ this vibrated, thus recording the rate of the shocks making up the discharge. In fresh fish the rate varied from as many as 200 a second to 70 a second : but it soon sank to 20, and finally to only 1 or 2 per second, owing to the exhaustion of the nervous centres. The latent period of the dis- charge is at least y^", as shown by recent experiments of Gotch upon the discharge in curarized torpedoes. Each individual excitatory state of the organ, the repetition of which makes up the discharge, may be evoked in a cut-out portion of organ by mechanical, chemical, or electrical stimulation of its nerve. There is a distinct interval of time between such nerve stimulation and the electromotive response of the organ. This is partly accounted for by the slow transmission of the excitatory process from the jjoint of stimu- lation down the nerve trunk, the rate of transmission being only 5 to 7 metres per second. Besides this, however, there is a true latent period, the duration of Avhich is dependent upon the temperature, etc., of the organ, but which, ixnder the most fiivourable conditions, is not less than two"- The response after this interval begins very abruptly, develop- ing a high electromotive force, and then subsides at first rapidly, and subsequently more slowly, its total duration amounting to about tw"- The discharge is therefore analogous to tetanus of a muscle and the indi- vidual shocks composing it to the individual short ' twitches ' of a muscle. Physical conditions aff"ect the organ : thus, heating to 22° C. stimulates the organ Avhile cooling makes it less active. The administration of strychnia THE PHENOMENA OF THE ELECTRIC FISHES. 473 to a torpedo causes the fish to emit a rajDid series of shocks, by which it soon becomes exhausted. It is remarkable that the response of the organ to excitation of its nerve is not in any way affected by curare. This was shown by Moreau in 1862, and the recent experiments of Gotch (ProceediiH/s of Physiological Society, 1888) fully confirm his conclu- sions. The reaction of the organ at rest is neutral but it becomes acid by activity. Moreau has succeeded in charging a Leyden jar by the shocks of a torpedo. In a recent research on the electrical organ of torpedo/ Mr. Francis Gotch arrives at the following conclusions — In the active state, the ventral surface of each plate becomes negative to the dorsal surface, and the efifect is summed up so that the dorsal end of a column becomes positive to the ventral end. The electromotive activity of the organ may be produced in at least three different ways, and then shows itself (a) as the response of the organ to excitation of its nerves, whether reflex or direct, this nerve-organ response being characterized by a short period of delay, a very rapid development, and a less rapid decline ; [h) as a prolonged after-effect following the passage of a strong cixrrent through the substance of the organ, and which therefore follows a powerful response ; and [c) as a prolonged electro- motive change following mechanical or thermal inji^ry along the length of the columns of the organ, this latter being the electromotive expression of a prolonged local excitatory process occurring in the immediate neighbourhood of the injury. According to Pacini, the nerves are always distributed to the side of the electric plate which becomes negative in the discharge. This law has been confirmed by all later researches. In the torpedo, therefore, as the nerves are in the lower plate, the shocks pass from the belly to the back ; in the gymnotus the posterior surface of the j^late is negative and therefore the discharge is from the tail to the head, and in the malapterurus the negative side of the organ is anterior and thus the current passes from the head to the tail. An electric shock may be obtained from the gym,notns by touching the fish with one finger or with a conducting substance interposing a resist- ance similar to that of animal tissue, such as a bit of wet wood or wet leather. Such a shock is slight and the full effect is obtained only when the body forms part of a complete circuit through which the greater part of the current passes. The wider apart the points of con- tact and the better the conducting medium, the more severe is the shock. Dr. Sachs having one naked foot in contact with the head and the other foot with the tail of a gymnotus received a series of shocks which caused him to shriek with pain and stand erect as if petrified, and sometimes a man may be thrown on the ground by a single shock. Fishes and frogs ^Fhil. Trans. 1887, p. 478. 474 THE CONTRACTILE TISSUES. ill the water along with a gymnotus are killed by the shock, and the electric eel, in attacking its enemies, throws itself into an arch and touches them with head and tail, and thus gives them the strongest possible shocks at its command. If the current is sent through a man's head, he sees a bright flash of light. As already stated, the current passes in the fish from the tail to the head and any point of the surface of the fish is positive to any posterior, and negative to any anterior point. The fish cannot alter the direction of the current, but it can limit the amount of the electric organ throAvn into action. If a metallic circuit is made, a spark may be obtained on brealcvu/ the circuit while a current is passing, but a spark is rarely obtained between the two poles of an open circuit. The ciUTcnt will not illuminate a vacuum tube. It will magnetize a needle. It is remarkable that the discharge of a gymnotus will not pass through flame, and that it can scarcely be perceived with dry copper handles. If a section of the electric organ of a gymnotus be laid on a pair of non-polarizable electrodes connected with a galvanometer, a slight cur- rent is obtained. The organ may be excited to action by mechanical, chemical, or thermal stimuli. The organ at rest is alkaline in reac- tion ; after electrical tetanus or that produced by strj^chnia, it becomes neutral, and after exjDosure to the air for an hour or two, it gives an acid reaction, from the formation of lactic acid. The time of the electric discharge is about equal to that of a muscular contraction. The gymnotus does not become so readily fatigued as the toi'pedo. Sachs mentions that an eel which in the course of an hour had given about 150 discharges, was still able to send a strong shock through a circuit or chain of eight persons, the first of whom, touched the head and the last the tail. Du Bois-Reymond gives a very interesting account of the malapterurux. Generally the fish gives one or more discharges on being touched, but occasionally it maj- not give a shock, even in escaping from the hand. He has no doubt that some of the discharges are voluntary and others reflex. Malapteruri quickly kill other fishes by electrical dis- charges. A frog brought into contact with the skin is tetanized. As regards the strength of the shock, Du Bois-Reymond writes — ^ "In comparison with its size, the shock of the malapterunis is surprisingly violent. If the head and tail of a powerful fish are touched with the fore-fingers in the water, the shock does not extend beyond the knuckles. If it is seized with ^ Du Bois-Reymond, " Observations and Experiments on Living Malapterurus." Burdon-Sanderson's Biological Memoirs. Translation by Miss Edith Prance, p. 387. THE PHENOMENA OF THE ELECTRIC FISHES. 475 hands thoroughly wetted, a se\'ere shock is felt up to the elbow. If it is touched with one hand, a pricking sensation is experienced in the skin, a burning one in wounds, and a painful shock is felt in all the joints of the submerged parts. The best way to take the shock is to hold with wet hands ordinary metal handles, which are connected with the linings of a leading-otf cover, and to let an assistant put this on the fish. As one is accustomed to test electric shocks in this way and is not disturbed by anxiety to get proper hold of the fish without hurting it, or by the feeling of repulsion at laying hold of it, one can better judge of the sensation caused by the shock. The shock does not seem so sharp as that of a Leyden jar. but has a somewhat swelling character. Several maxima may be frequently dis- tinguished in it. By sending opening shocks of an induction apparatus, with two Grove's in the primary circuit, through the water of an experimental tub by means of copper plates plunged into it, having first approximated the secondary coil to the primary as closely as possible, the shock which I experienced when my hands were immersed in the water was certainly not stronger than that of a vigorous fish. An openmg shock with the coil quite pushed in, and one Grove in the circuit, taken directly through the handles, has about the same strength as such a shock." Mr. Francis Gotch, in a communication to the Physiological Society,^ states "that a malapterurus gave to the fingers a smart shock which became un- pleasantly severe when both the head and tail were touched, and these seemed comparable wnth the break shock of a Du Bois-Reymond coil, with three Daniell cells in the primary circuit and with the secondary coil pushed quite up. A shock could also be obtained by placing the fingers in the water surrounding the fish and then exciting the animal mechanically." The discliarge of the malapterurus effects the electrolysis of iodide of potassium but it does not decompose water. Xo sparks could he obtained from the discharge although the interval between the metallic conductors were as small as '01 mm. Du Bois-Reymond also made slits in tinfoil not wider than -0033 to 'OOS mm., and no spark across the slit was noticed. On the other hand the secondary current of the in- duction coil passed over this slit when the secondary was 90 mm. from the primary coil, and yet these induction shocks could not be perceived by the tongue. The difference between the behaviour of the fish and of the inductorium is due to the fact that, in the case of the fish, most of the current is short-circuited, and the shock is only obtained by deriva- tion. Du Bois-Reymond - puts the matter thus : Suppose two ecjual currents, la and Ih, flowing into two conductors, A and B, with resist- ance A. Suppose also the end of conductor B be connected to a side conductor \, while A forms part of an unbranched circuit. It can then be shown that if the resistance A of the two conductors be increased by the same amount, Ih will lose more in strength than la ; or to state it in another form, as the actual resistance in both circuits increases, the difference in favour of the current in the conductor A (that is, the un- ^ Proceedin(js of the Physiological Society, 12th December, 1885. - Du Bois-Reymond, oj). cit. p. 394. 47G yV7£' CUSTllACTILE TISSUES. liranched circuit) increases. Hence, in experimenting with the fish, it is important to have the resistance of the external or ex{)erimental tircuit as small as possible. To obtain sparks, the circuit must be suddenly opened when the shock is strongest. Du Bois-Eeymond .-succeeded in passing the discharge of the fish through the primary coil of a Ruhmkorff's inductorium, so that a shock AA'as emitted by the secondary coil. Three shocks of a malapterurus magnetized needles 37 mm. long by •? mm. thick, when placed in the central cavity, 8 mm. wide, of a coil of 735 turns of copper ware -4 mm. thick. Any point of the organ nearer the tail is 2:)ositive to any point nearer the head, and the intensity of the shocks increases according to the distance at which electrodes leading off a current are applied. It is remarkable that the shocks emitted by the electrical organ pass through the body of the fish, and that the accumulated discharges strike the central cerebro-spinal system vertically to its axis. This of course leads to the c|uestion of how far the nervous organs of the fish arc -sensitive to electric shocks. Du Bois-Eeymond found that induction shocks from an inductorium, ha^dng two Grove's elements in the primar}' circuit, killed or rendered unconscious such fishes as tench, perch, chuli, pike, and silui'us, and the same current tetanized frogs, l)ut a malapterurus was apparently unaflfected. When the induction •shocks were made much stronger, the fish noticed them, Imt, " If it came in the ueighboui-hood of the electrodes, where the current densitj' was greatest, it withdrew hastily, gave a shock or two, and sought with correct instinct that position iii which its axis of length cut perpendicularly the least dense current curves, as if it knew the laws of the distribution of current in non-prismatic conductors." -^ A constant current from 30 Grove's cells did not appear seriously to inconvenience the fish, but here also "it sought that position which is theoretically the most protected." These experiments clearly show- that malapteruri have a certain immunity from electric shocks, and that they do not suffer from their own discharges. Xow, if Ave consider that sensor}' nerves require a certain strength of stimulus to excite them, and that electrical currents produce peculiar effects on nerves, it is clear that the lower limit at which excitation occurs is higher for the nerves of malapteriu'us than for the nerves of other animals. When the electric nerve is directly irritated, a number of rapid shocks are emitted, so that if the nerve of a nerve-muscle i^rejiaration (Fig. 261, p. 401) is laid on the organ on the side corresponding to the electric nerve irritated, the nuiscle of the preparation is tetanized. 1 Du Bois-Eeymond, op. c'lt. p. -400. THE PHENOMENA OF THE ELECTRIC FISHES. 477 Du Bois-Eeymond. failed to get evidence of a current from the electric nerve when it was placed on the cushions of the galvanometer (Fig. 294, p. 447), nor did he observe any negative variation when it was irritated, but the nerve Avith which the experiment was made was in a very exhausted condition (removed 2| hours after the circula- tion had ceased), and he states that "it is conceivable that a more efficient electrical nerve of the malapterurus might show traces on the galvanometer of the nerve-current and of its negative variation." Mr. Francis Gotch,^ in his observations on a malapterurus, in which he used both the galvanometer and the capillary electrometer, made the important discovery that the discharge on electrical excitation of the skin is not of a reflex character, but is the result of a direct excitation of the electrical organ at the point irritated by a break induction shock with 1 Daniell's element in the primary circuit and the secondary coil 80 mm. or less from the primary. In these circum- stances the latent period is extremely short. Further, the molecular disturbance set up at the point irritated is propagated through the electrical organ at the rate of about 2-5 metres per second. When the fish was irritated by a single induction shock on " the dorsal surface between the eyes," a long discharge took place, causing tetanus of a nerve-muscle preparation in contact with it, the individual contractions- of which were at the rate of 10 per second. As regards the electrical phenomena of the skate, Dr. Burdon- Sanderson has communicated to me the following facts — The direction of the discharge is towards the tail, that is to say, it is such that at the moment of activity the ends of the electric nerves become positive to the trunks, in accordance with the law of Pacini. The discharge may be induced in the living animal by mechanical stimulation of the surface of the body, and then consists of a rapid siiccession of similar instantaneous eifects. In the separate organ the anterior end is negative to the posterior, that is, there is an "organ current" in the same direction as the discharge. This difference of potential is increased enormously by increase of temperature or by injuring the surface. The electromotive force of the discharge relatively is far inferior to that of torpedo in its most active state. The discharge can be heard by the telephone, but cannot be felt by the fingers. - 6. Special Electrical Phenomena of the Electric Fishes. — Du Bois- Reymond and Sachs, and more especially the former, have discovered a number of phenomena which are of profound interest, but some of ^ Francis G-otch, Proceeding-'i of Physiological Society, op. cit. xxviii. ■^ Goodsir communicated to Du Bois-Reymond the suggestion that the electrical organ of the skate is active only during sexual excitement. 478 THE COi^TltACTlLE TISSUES. which, from their very nature, are difficult of explanation. Thus ])u Bois-Keymond has investigated the distribution of the current in the torpedo, and has shown that the shock })enetrates the hody of the fish and attains a greater density in the brain and spinal cord than else- where. The currents floAving in the back from the bordei's of the organ to the mesial line, and in the belly from the mesial line to the borders, pass through the brain and cord, as this " is the shortest path between the most active portions of both organs." The greater the length of a, column in torpedo (the number of plates being constant in a unit of length) the greater is its electromotive force. As the columns arc longest in the median region of the organ, the electromotive force of these columns is greater than that of the lateral columns. Hence, in air, a current flows in the back from the middle of the organ to the borders, and in the belh^ in the reverse direction. In salt Avater, however, the currents of the higher cohmms have nearly the same strength as those of the shorter ones. If all the cohimns in both organs were of the same length, and if the organs Avere united in the middle plane, then the most positive part avouIcI be " the middle of the median line" on the back, and the most negative the corresponding part on the ventral aspect. When the organs are separated, then the most positive part is nearer the inner than the outer border, in con- secpience of the thinning of the organ toAvards the sides. Hence the inner border is most positive dorsally, and most negative ventrally, and therefore a current floAvs in the l:)ack from the outer border to the middle line, and in the belly in the reverse direction. Further Du Bois-Reymond has shoAvn that the curves of the direction of the currents passing out from the dorsal surface through the Avater and back to the ventral surface are inclined outAA^ards, and also that, in consequence of the greater inclination of the median columns, by making the current curves still more oblique, the dorsal surface of the fish Avhen it lies half buried in mud at the bottom of the sea is more protected electricallj- than the ventral surface which does not require protection. By the organ current is understood a current existing in the organ during rest and passing in the direction of the shock. Such a current exists in all electrical fishes. Thus, in the torpedo, on applying the cushions of the galvanometer to the dorsal and ventral sides, after the electric lobe of the brain had been destroyed, so as to remove from the animal the poAver of giving voluntary electric shocks, a current Avas readily passed through the coils of the galvanometer. By another method, Du Bois-Reymond AA^as able to examine bundles of columns of the length of 29 mm., and Avhcn these Avere placed on the electrodes so that one electrode touched the end and the other the side of the column, THE PHENOMENA OF THE ELECTRIC FISHES. 479 a current was obtained. The current thus obtained from bits of the organ of torpedo was remarkably small, "considerably less than the force of the nerve-current in frogs." ^ Sachs obtained from bits of the organ of a gymnotus 40 mm. long, an e. m. f. of from '01 5 to -030 (mean, •0225) of that of a Daniell's element. With strips of the organ of torpedo 29 to 12 mpi, (mean, 20 mm.), Du Bois-Reymond obtained •0085 of a Daniell's element. Double this would have been -017, thus a little less than that of a gymnotus. He also shows that the e. m. f. of a single plate of gymnotus is about -00006 of a Daniell's element, while that of a single plate of torpedo is only -0000117 of a Daniell's element, or about 3-3 times less than that of gymnotus.'^ The torpedo is a sea-water fish, Avhile the gymnotus inhabits fresh water, and it is remarkable that the ratio of the e. m. f. in the two fishes is almost the same as that of the resistance of fresh water to salt water. (Christiani.) Each plate of the fresh-Avater fish has a larger e. m. f. than that of the sea-water fish because it has a greater resistance to overcome. The organ current decreases as the organ dies, but in the cold it may remain for 24 or 48 hours after the death of the fish. Strips of the electric organ of a malapterurus have, in the hands of Du Bois- Reymond, yielded no organ current, but, as he suggests, this may be owing to the weakness of the animals under investigation. Du Bois-Reymond has made a number of remarkable observations on the polarizing effects produced by passing currents through the organ of the torpedo. In this way polarization currents are produced. If the polarizing currents are in the direction of the natural shock-current of torpedo, as it runs in the body of the animal, that is from belly to back, they are termed homodromous, and if in the opposite direction to that of the natural shock-current, that is from back to belly, he calls them heterodromous. " Polarization eff'ects from belly to back are absolutely positive, and, according as they follow an absolutely positive or negative polarizing current, they may be considered relatively posi- tive or negative." The character of the polarization efiect seems to depend on the length of the time of closing. Thus, with homodromous currents, a long closing time gives a polarization relatively negative, while a short closing time gives a polarization relatively positive. As functional activity diminishes, positive polarization disapj^ears and then ^ Du Bois-Eeymond — Burdou -Sander-son's Translations, o^). cit. p. 441. 2 The e. m. f. between the longitudinal and transverse section of the gastroc- nemius of a frog is about -03 to '08 Daniell, and that between the longitudinal and transverse section of a sciatic nerve of a frog about -02 Daniell. Burdon- Sanderson found, on an average, an e. m. f. between the lobe of the leaf of JDiomea muscijnda and the mid rib of '006 Daniell. 480 '^iiE COS TRACTILE TISSUES. only negative polarization remains. With homodromous currents polarization may be in two directions, first relatively negative and then relatively positive. Since shocks of very short duration polarize posi- tively, the strength of the normal shock of the organ may be itself in- creased. From these and other observations Du Bois-Reymond infers that "the strength of the homodromous current is so much gi-eater than that of the heterodromous that the relatively positive heterodromous polarization is constantly masked by the relatively negative." As early as 1857, Du Bois-Reymond observed this law in experimenting "wdth fresh strips of the organ of malapterurus " in which positive polarization appeared in full force in the direction of the shock, the descending current (homodromous in malapterurus) Avas always greater than the ascending (heterodromous) in the relation of 100 to 112, 116, or 125, and he inferred from this the existence of a polarization current which was added to the current of the battery passed through the organ." In some of his more recent experiments with torpedo he found the strength of the homodromous current of 30 Grove's elements to be more than tAvice that of the heterodromous current. The e. m. f. of this addi- tional polarization current must be assumed to be high when the difference between the effects of homodromous and heterodromous currents is so great. But a difference of current strength depends not only on unequal electromotive forces, but on unequal resistances, and hence the explanation of these facts may be, not the generation of a new c. m. f. bv the homodromous ciuTent, but that the organ conducts better in the natural direction of the shock cm-rent than in the reverse direction. That is to say, the resistance is greater, in the organ of torpedo, in the doAvnward than in the upward direction, or the resist- ances are irreciprocal. This irreciprocity, or inec(uality of resistance in the two directions, may depend on some peculiarity of the organic molecules during life as it gradually disappears as the organ slowly dies, or it may at once be removed by plunging the organ into boiling water. In later researches, Du Bois-Reymond found that the irrecipro • city increases, but not proportionally, with the cuiTent density, or, in other words, when the distance of the secondary coil from the primary of the inductorium, by which shocks Avere transmitted through the oro-an, was diminished to zero, the irrecipi^ocity became greater than when the secondary stood 100 mm. from the primary. At 100 mm. the resistance to a homodromous current to that of a heterodromous current was as 1-33 to 27-66, giving an index of irreciprocity of -0481 (that is 1-33 -r 27-66), while at zero the ratio was as 264*7 : 477, or an index of irreciprocity of -5549, while if the irreciprocity had been in direct proportion to the current density, the index woidd have been -8293. THE PHENOMENA OF THE ELECTRIC FISHES. 481 This apparent irreciprocity exists in each transverse lamella of the organ. A careful inquiry into the conducti-vity of the organ of torpedo, enables Du Bois-Eeymond to state that — " Even in the homodromous direction, in which the organ conducts best, it conducts scarcely half as -well as frog muscle parallel to the fibre, and 7 "5 to 12 times worse than the sea water of the aquarium. The ratio would be still more unfavourable with sea-water from the Mediterranean, which conducts nearly 150 times better than tap water. But in the heterodromous direction the organ con- ducts even 20 to 58 times worse than sea water." In summing up the results of his investigations on the torpedo, Du Bois-Eeymond endeavours to show the important part played by irreciprocal conduction, and his opinion is that it takes the place of insulating septa, which were assumed by the earlier physiological Tig. 316. — Diagram showing the current curves in the electrical dis- charge of torpedo. electricians either to exist permanently around the organ or to be called into existence at the instant of the discharge. His view will be under- stood with the aid of the diagram in Fig. 316. Suppose a sino-le column of the organ of a torpedo surrounded by a conductino- mass. The current curves mil issue from the upper end and pass throuo-h the conducting mass to the lower end, and these curves will be symmetri- cally arranged around the axis of the column. Suppose also the column divided into an upper and lower portion by a transverse plane. The part above the transverse plane, 0-0, Fig. 316, ^rill be the positive dorsal I. 2 H 482 . THE CONTRA CTILE TISSUES. end of the column, and the part l>elow 0-0 will be the negative ventral end. All the curves issuing from the dorsal end will pass perpendicularly through the transverse plane, so that the tension of this plane will be zero. In the column, currents will flow from bell)- to back, those near the axis of the column nearly ])arallel to it, while those nearer the lateral surfaces are more divergent, and some of the most divergent will cut the lateral surface. On the left, the figure (Fig. 316) shows the outermost system of curves around one column cutting the transverse plane. If now a second column be placed alongside of this one, the current curves on the two halves of the cohmins in apposition will be compounded, and so with a third, a fourth, or an}' number of columns placed side by side. Thus the density of the current in 1 will be doubled by that of 2, trebled by that of 3, and increased 7(-times by the addition of n^^ column. Each column Avill be met as it Avere l>y the currents of the others, homo- dromously, that is, in the direction of its own current. Now the torpedo has two symmetrical organs, as shown in Fig. 316, in which D V, the sagittal plane, may be regarded as insulated. Looking at the left of the figure, imagine the strip "with transverse marks to represent one column, it is clear that the currents from this column pass heterodromously through all the other columns. The currents issuing from the axis of the column pass out and meet the small fish seen in the diagram. The little fish Avill receive a shock which Avould be stronger if the column under consideration were insulated. This is shown in the right of the diagram, when the shaded portion indicates an imaginary arrangement for insulation. It will be seen that a larger number of current curves strike the little fish on the right side than on the left. The insulation also compels currents which, on the left side, pass through part of the organ, to pass on the right side, round the edges of the organ. Thus insulation Avould increase the action of an electromotive column on any part of external space. In torpedo, however, the columns are not insulated, but the same eff"ect is obtained by the fact that all the currents passing throiigh the organ are heterodromous, in which, as we have seen, the resistance is much greater, and consequentlj^ the same effect is produced as if the columns were insulated. That is to say, hy increasing the resistance in the downAvard direction, the density of the total current into external space is increased. Each column conducts its own homodromous current but offers resistance to the heterodromous currents of all the others, and consec[uently the heterodromous currents are forced to pass round the edges of the organ as if it were composed of a non-conducting substance. The part of the animal, however, be- tAveen the median borders of its tAvo electric organs, that is its cerebro- spinal L!,xis, is traversed by the strongest floAV of current, and, Avith THE PHENOMENA OF THE ELECTRIC FISHES. 483 reference to this, the importance of the fact that the torpedo and -electric fishes in general have a kind of immunity from the effects of electric shocks is obvious. These remarkable results of Du Bois-Reymond, to explain which he lias advanced the beautiful theory above set forth, have not been un- questioned, and in a communication made to the Royal Society by Mr. Francis Gotch,^ the fact of the existence of irreciprocal conduction in the torpedo has apparently been disproved. Du Bois-Reymond's method depended on observing the deflections of a galvanometer when the polarizing homodromous or heterodromous currents were sent through .a strip of the or-gan and also through the circuit of the galvano- meter. Gotch discovered that intense currents of short dui'ation, such as Avere used by Du Bois-Reymond, always cause " an excitatory response " in the tissue of the electric organ ; that the intense current du.e to this response is added to the homodromous current, and the result is that the latter must be stronger than the heterodromous. Thus, calling the homodromous cixrrent a, and the heterodromous h, and the " excitatory response current " of Gotch c, when a short strong cur- rent passes upwards (homodromous), the result is a + c; but if the short strong current passes do^vnwards (heterodromous), the result is h — c. By means of a fast moving rheotome, Gotch Avas enabled to " switch in " the induction currrent only, and then, on passing it in ■either direction, upwards or downwards, the resistance is found to be the same, that is to say, there is no irreciprocity. The apparent irreciprocity, therefore, is an excitatory phenomenon, not due to a cliff"erence in resistance, but to the adding or subtracting of an addi- tional electromotive force. Mr. Gotch has also made the remarkable discovery that the organ may be excited in a secondary way by its own discharge. Strong healthy fish, in the summer, when excited by one irritation, gave not •only a first discharge, but also in quick succession a second, third, fourth, and so on at intervals of about —^ of a second, each succes- sive shock becoming weaker than the one before it. Thus, as clearly .stated by Mr. Gotch — " The response of the isolated organ to nerve excitation is multiple ; :a primary, secondary, tertiary response following the application to the nerve of a single stimulus. Since all these responses produce currents similarly directed through the columns of the organ, each column during ^ Francis Gotch, M.A. Oxon., B.A., B.Sc. Lond. Further observations on the ■electromotive properties of the electrical organ of torpedo marmorata. Eead before the Royal Society, March 9th, 1888. 484 THE CONTRACTILE TISSUES, its activity must reinforce by its echoes the force of the primary explosion, both in its own substance and also in that of its neighbours." The brief account I have given of the electric organs indicates that such investigations Avill probably throw light on the obscure relations that exist between nerves and muscle and other terminal organs. In the case of torpedo, gymnotus, and raia, the nerve termination is evidently analogous to a motorial end-plate in muscle (Fig. 317)^ and in malapterurus to the terminations of nerves in the cells of glands (Fig. 318). The nerve stimulus, or, as it may be put, the molecular disturbance transmitted along a nerve causes changes in its end-organ, and these are propagated to the surrounding substance. These changes are associated vnth a change of potential, and the part becomes negative. A wave of negativity passes through the organ, and then there is. probably a physiological result according to the kind of organ in Avhich the changes may take place. If it be a muscle, the chief expression of the change is a variation in form or FiG. 317.— Pictorial end-plate in muscle, n, nerve. Fig. 318.— Mode of nerve-terminations in glands, according to Pfliiger. I. Xerve ending in a group of cells, but not apparently uniting -with any of them. II. Nerve sending branches between cells. III. Nei-ve fila- ments apparently continuous with substance (nucleus?) of cells. IV. Ganglionic nerve cell, with process passing to seci-eting cell. Pfluger's observations have not been confirmed. Kupfifer describes a similar arrangement in the salivary gland of insects, and JIacallum believes he has traced nerve-endings into the cells of the liver. TEE PHENOMENA OF THE ELECTRIC FISHES. 485 ■contraction ; if it be a gland cell, the chief expression of the change is the formation of certain matters or metabolism ; and if it be an electrical •organ, it is an electrical discharge. In all these, however, similar phenomena occur, but to varying amounts. Thus, call contraction a, ■electromotive phenomena h, and metabolic changes c. In a muscle, a is large, and h and c relatively small ; in a gland, a probably does not •occur as an active contraction, although the cell may slowly change in form and bulk ; h is also small, but c is relatively large ; and in an electrical organ, there is no evidence that a occurs, h is largely shown, and c is relatively small. Thus, all these phenomena are linked together, and as our knowledge advances into the molecular processes ■of the one, it is likely soon to shed light on the other two. Finally, I cannot help remarking that in few departments of physio- logical science can more striking examples of internal adaptation be found than in the construction and place in nature of the electric fishes. As "v^Titten by Du Bois-Eeymond, we have in them " surprising instances of that organic adaptiveness, which is an ever-new source of wonder even to the strict adherent of mechanical casuality." It seems to me, — again using a phrase of the distinguished Berlin physiologist, — that if any human imitation of the electrical mechanism of a torpedo or gymnotus could have been the outcome only of the "profoundest reflection of a clever brain," even so we can account for such adapta- tions only by assuming an intelligent consciousness acting behind natiu-al phenomena. Consult as to the older views of the nature of electric organs : — John Goodsir's Anatomical Memoirs, vol. II. p. 289 et seq. As to the more modern histology : — E,ANViER, Histologie du Systeme Nerveux, vol. II. p. 157 et seq. ; Carl Sachs, Unterstichungen am Zitteraal (Gymnotus electricus), 1881 ; Gustav Fritsch, Das Gehirn unci RiicJcenmarh des Gymnotus electricus, 1881 ; Gustav Fritsch, Die elelctrischen Fische, erste Abth. Malapterurus electricus, 1887; August Ewald, ilher den Modus der Nervenvtrhreitung im elelctrischen Organ von Torpedo, 1881. As to electro-physiology, see the Translations of Du Bois-Reymoxd's Memoirs in Burdon-Saxderson's Biological Memoirs, 1S87 ; also, Paper by Du Bois- Keymond in Carl Sach's volume above mentioned ; Papers by Burdon-Saxder- SON and GoTCH, communicated to the Royal Society, already referred to, 1887-88 ; and Paper by Gotch, at the end of which is an excellent bibliography, on the electromotive properties of torpedo marmorata, Phil. Trans, vol. 178 (1887) B, pp. 487-537. 487 APPENDIX I. Regarding Reagents, see p. 260 ■ Modes of Isolation, p. 265 ; Modes of Fixing, p. 266; Hardening, p. 268; Decalcification, p. 268; Staining, p. 270 ; and Investigation of Fresli Objects, p. 276. By the following methods preparations may be made similar in character to those repre- sented in the page or pages referred to under each method — SPECIAL METHODS OF MAKING HISTOLOGICAL PREPARATIOXS. 1. Nuclei, Method No. 1, p. 206. The reticulum in nuclei can be readily seen in the cells in the skin of the larvae or tadpoles of amphibia. The larvse of the triton or newt are easily procured in the months of June or July. [Dr. Macallum, of University College, Toronto, informs me that the phenomena may be readily studied in the epithelial cells of the skin of Kecturus.] Fresh specimens, 3 to 4 cm. long, are thrown into 20 c.cm. of chromo-osmium-acetic acid (p. 261), in which they quickly die. After one or two days, cut from the tail a portion, 1 c.cm. long, or peel off a bit of skin from the tail with the forceps. Scrape off with a scalpel some of the epithelium; the remainingthin portion of skin is hardened in 50 c.cm. of alcohols of gradually increasing strengths (p. 267), colour in saffranin (p. 263), and mount in dammar varnish. Striated muscles of the tail, and involuntary muscle from the intestine, also give beautiful results by the same process. See also p. 271, par. 3. 2. Karyokinesis, Method No. 2, p. 213. Place for two days portions of the anterior segment of the eyes of young newts in chromo-osmium-acetic acid (p. 261), wash thoroughly in water, and harden in alcohol of gradually increasing strengths (p. 267). Two days later, cut out the cornea, and pull off with the forceps a thin lamina of corneal tissue. Colour in saffranin (p. 263), and mount in dammar varnish, so that the convex side of the cornea is directed upwards. The nuclei are stained red, and with high powers karyokinetic forms will be seen. 3. Testis, Method No. 3, p. 215. Divide into two equal portions by a trans- verse cut the testis of a young human subject, and fix both portions in 50 c.cm. of Kleinenberg's sulpho-picric acid (p. 261), then harden in 30 c.cm. of alcohols of gradually increasing strengths (p. 267). Colour thick transverse sections with carmine (p. 2C2) and with hematoxylin (23, p 262), and mount in dammar varnish. 4. Ttjbuli Seminiferi, Method No. 4, p. 216. fix portions, 2 cm. broad, of fresh testis of ox in 20 c.cm. of Mliller's fluid (p. 261), and at the end of fourteen 488 APPENDIX L days transfer them to alcohol of gradually increasing strengths (p. 267). Colour very thin sections with ha?inatoxylin (p. 2(52), and mount in dammar. The smaller canals, containing developing spermatozoids, are coloured intensely blue, and the nuclei of the peripheral cells are often stained more deeply than the nuclei of the cells lying near the lumen. 5. Spermatoblasts, Method Ko. 5, p. 218. Fi.x: small portions, 5 mm. broad, of testis of ox newly killed in 10 c.cm. of chromo-osmium-acetic acid (p. 261). At the end of one or two days, wash the preparations under the water tap for one hour, and then harden in 20 c.cm. of alcohol of gradually increasing strengths (p. 267). Colour thin sections with safiVanin, and mount in dammar. 6. Elements of testis. Method No. 6, p. 218. Place a portion, 1 c.cm. in size, of fresh testis of ox in 20 c.cm. of Ranvier's alcohol (p. 260), and at the end of five hours teaze out in a drop of the alcohol. Colour while under the cover- glass (p. 277) with picrocarmine (p. 262), and preserve in diluted glycerine. (a) To see liriiKj spermatozoids, place on a clean slide a drop of the milky fluid issuing from the cut surface of a fresh testis, add a drop of salt solution (p. 260), put on a cover-glass, and examine with a high power. After a little time, allow a drop of distilled water to pass under the cover-glass (p. 277), and the movements of the spermatozoids will cease. Observe the different forms, oval or round, of the head. Immature spermatozoids carry a little behind the head small bits of protoplasm. To preserve spermatozoids, allow semen diluted with water to dry on a slide, place a cover-glass on it, and fix with cement. (6) To detect semen staiius on linen, cut a bit of the stained portion, 5 to 10 mm. broad, moisten in a watch-glass with distilled water, for from five to ten minutes, and then teaze out a small portion. Then mount the teazed fragment in water, put on cover-glass, and examine with a magnifying power of 500 diameters. Examine the edges of the linen fibres, as the spermatozoids adhere to these. Sometimes the heads of the spermatozoids break off. The isolated heads may be recognized by their peculiar brilliancy, and in the semen of man, by their very small size, about 4 )x, or the -g-g'-u-ij of an inch. (c) Spermatozoids of the frog. The male frog is recognized by well-developed warts on the fore feet. Kill the animal ; open the abdomen. The testes are two small kidney-shaped bodies which lie at the sides of the vertebral column. Cut one testicle by a transverse incision, squeeze out a drop of fluid on a slide, dilute with a drop of salt solution (p. 260), put on cover-glass, and examine. The sper- matozoids are then seen to have very long slender heads, and the tails so thin as almost to escape notice. Immature spermatozoids lie in clusters. 7. Ovary, Method No. 7, p. 220. Ovaries of small animals ai'e to be fixed as a whole. The ovary of one of the higher animals, or of a woman, should have a number of transverse and longitudinal cuts made in it before throwing it into from 100 to 200 c.cm. of Kleinenberg's sulpho-picric acid (p. 261). It is afterwards hardened in 100 c.cm. of alcohol of gradually increasing strength (p. 267). Ova often fall out of the larger Graafian vesicles, and many sections may have to be made to secure a good specimen. Colour with h.-ematoxylin (p. 262) or with borax carmine (p. 262), and mount in dammar. 8. Ovary, Method No. 8, p. 221. See Method No. 7. 9. Ovary, Method No. 9, p. 222. See Method No. 7. 10. Ovary, Method No. 10, p. 223. See Method No. 7. (a.) Fresh ova. Obtain the fresh ovaries of a cow from the slaughter house. The large Graafian vesicles are about the size of a pea. Cut one or more out of METHODS OF MAKING HISTOLOGICAL PREPARATIONS. 489 the ovary by means of scissors. Lay the isolated vesicle on a slide, and prick it with a needle, making the puncture as near the slide as possible. The liquor folliculi flows out, and among cells of the cumulus ovigerus, the ovum must be sought for with the aid of a magnifying glass. On finding it, place it in the middle of a slide in a drop of salt solution, surround it with a little ring of paper, and then lay the cover-glass gently on, so as to avoid pressure. This is an opera- tion much easier to describe than to perform. (6) Ova of frog. Lay on a slide a bit of fresh ovary of a frog, and puncture all the black ova, so that the contents flow oiit. Place the remainder in a watch- glass, in distilled water, and wash by moving the contents to and fro with needles. Place the watch-glass on a black background, so as to see distinctly the unpig- mented follicles. Remove a bit of the unpigmented portion to a slide, cover with a, drop of salt solution, put on a cover-glass, and examine. Such ova have a very large germinal vesicle, the germinal spot disappears early, and usually cannot be seen. The dark patch seen is the yolk nucleus. Often on the circumference of the ovum a delicately striated membrane may be seen. This is the theca folliculi with a single layer of epithelial cells. 11. Leucocytes, Method No. 11, p. 295. Clean a slide and cover-glass carefully with alcohol. Place on the slide a small drop of frog's blood, lay on the cover- glass, and fix it with a layer of paraffin (p. 277). Coloured and colourless cor- puscles are seen. Examine the more granular colourless corpuscles carefully with high power. The movements occur slowly, and the most effective way of being convinced that they do occur is to make drawings of the same leucocyte at intervals of one or two minutes. 12. Secreting Epithelial Cells, Method No. 12, p. 297. To see the changes connected with secretion, use the stomach of a dog or cat that has received no food for a day or so. The stomach of the rabbit is not suitable, as the chief cells are small. Portions of mucous membrane, 1 cm. broad, are placed in 10 c.cm. of absolute alcohol, and the alcohol is changed in half an hour. Then examine. The human stomach, if obtained soon after death, shows the gland structure even better than that of the dog or cat, as the gland tubes are wider apart. Cut very thin sections. For further details, see Stomach, Vol. II. 13. Blood Corpuscles, Method No. 13, p. 300. Carefully clean with alcohol a slide and a small cover-glass. Then puncture with a clean needle the tip of the finger. The drop of blood first coming to the surface is wiped away and the second drop is caught on the cover-glass and the cover-glass is placed on the slide. Seal up the preparation with a little paraffin. Examine with high power, observe rouleaux and coloured and colourless blood corpuscles. The jagged edges of many corpuscles are produced by evaporation. After removing the paraffin from the rim of the cover-glass on the one side, place a drop of water on the edge of the cover- glass, thus discolouration of the blood corpuscles is noticed, while the water be- comes yellowish, the blood corpuscles also become globular, and they appear as pale spheres which disappear entirely at the close of the experiment. Then study carefully one corpuscle. [a) Small blood plates or plaques are obtained if, previous to the puncture of the finger, we lay on the tip a drop of a filtered mixture of 5 drops of an aqueous solution of methyl violet (p. 263) along with 5 c. cm. of a solution of common salt (p. 260), and then prick the finger through the drop. The blood issuing out of it mixes with the methyl violet ; a drop is laid on the under surface of the cover-glass and examined with high powers. The plaques are coloured intensely 490 APPENDIX 1. blue, of peculiar brilliancy, disc-shaped, and not to be confounded with the white corpuscles likewise coloured. Their quantity varies in individual cases. Avoid confounding the blood plaques with gi-anulated impurities which may be even in the filtered colour solution. {})) Coloured blood corpuscles of animals (frog, etc.) should be taken from the newly-killed animal and treated according to Method No. 13. (o) Blood stains. Moisten small portions of the stain in 35 per cent, solution of caustic potash on the slide, teaze small portions of blood-stained linen in a drop of the caustic potash. Although the coloured blood corpuscles of our domestic mammalia are smaller than those of man, it is nevertheless impossible to decide, from the size of the blood corpuscles, the question whether the blood originally belonged to man or to some other mammal. It is, on the other hand, easy to distinguish the oval blood corpuscles of the other vertebrates from disc-shaped ones of the mammalia. {d) Blood crystals (pp. 119 and 129). (a) The production of the hs'min crystals is easy (p. 129, Fig. 53). Cut a small flap, 3 mm. broad, of blood-soaked, diy linen, and place it along with a morsel of common salt, the size of a pin's head, on a clean slide. Add a large drop of glacial acetic acid and pound, with a blunt glass rod, the salt and the linen until such time as the acetic acid assumes a brownish hue. Do this rapidly otherwise the acetic acid evaporates. Then gently heat the slide over the flame until the fluid boils up 07ice. The small bit of linen is now removed and the dry brown spaces investigated on the slide with high powers. We occasionally perceive, even without a cover-glass and without any preserving fluid, hroirn crystals of hsemin near numerous fragments of white crystals of common salt. For preservation cover the slide at once with a larg e drop of dammar varnish and a cover-glass. The form and size of the ha?min crystals vary. We may obtain from the same blood complete crystals, partly isolated, partly lying crosswise under each other, partly united into stars, along with forms resembling that of a whetstone, and the smallest particles may scarcely exhibit the crystal form. The discovery of hsemin crystals is of great importance for medico-legal pui'poses. It is often thus possible to discover the crystals on portions of clothing, but it is often difficult to furnish proof from small stains, especially on rusty iron, that they have their origin in blood. The instruments- and reagents to be employed in such investigations must be absolutely clean. (e) Hitmatoidin crystals (Fig. 52, p. 129) are found by teazing out old extravasa- tions of blood, which to the naked eye are recognized by their rusty-brown colour. Hffimatoidin crystals occur in apoplectic cysts, in a corpus luteum, etc. (/) Hamoglohin crystals (Fig. 48, p. 119) are produced from human blood (best from the splenic vein) only with difBculty. We sometimes obtain them if we con- vey a drop of the blood of the vein of the spleen to the slide, and after five minutes add a drop of water, cover the whole with a cover-glass and allow it to stand in the light. After some time the crystals may appear on the margin of the pre- paration. 14. Epithelial Cells, Method No. 14, p. 301. Scrape ofi"from the upper surface of the tip of the tongue a little mucus and place it on a slide with a drop of salt solution (p. 260), then put on a cover-glass. Then observe the flat squamous epi- thelium (Fig. 147, 1, p. 301) and the salivary corpuscles (Fig. 143, h, p. 296). Sometimes also one may find a dark mass of fungus threads [Leptolhrix huccalis). Stain under cover-glass with picrocarmine (p. 277), and after adding a little glycerine the preparation may be kept. METHODS OF MAKING HISTOLOGICAL PREPARATIONS. 491 15. Bristle Cells, Method No. 15, p. 302. Cut from the bill of a newly-killed duck or goose the yellowish skin passing over the margin of the mandible and place small portions, 1 to 2 mm. thick and 1 cm. long, into 3 c.cm. of a two per cent. solution of osmic acid along with 3 c.cm. of distilled water. Leave this for twenty-four hours in the dark. Then wash small portions for one hour under the tap and transfer to 20 c.cm. of 90 per cent, alcohol. After six hours cut vertical sections, mount in dammar. Such preparations are better unstained. (See Touch, Vol. II.) 16. Pigment Epithrlixtm of Retina, Method No. 16, p. 302. Open eyeball of sheep or ox in water, allow retina to float off, and on the surface of the choroid behind it the cells will be found. The light spots in the cells are nuclei. They are not readily preserved, but sometimes they keep well in a drop of glycerine. 17. Simple Cylindrical Epithelium, Method No, 17, p. 302. See Method No. 12. 18. Stratified Pavement Epithelium, Method No. 18, p. 303. Remove from the dead body of a cat the larynx, trachea, etc. Place the preparation for a. period of from two to six weeks in from 200 to 400 c.cm. of Miiller's fluid (p. 261), wash under water tap for one hour, harden in 200 c.cm. alcohol of increasing strengths (p. 267). At the end of eight days cut transverse and longiti;dinal sec- tions, through vocal cords, through trachea, and through the thyroid gland ; stain for five minutes in hsematoxylin (p. 262), and mount in dammar. 19. Stratified Ciliated Epithelium, Method No. 19, p. 304. Found in regio respiratoria of nose. Cut small portions, 5 to 10 c. cm. broad, from lower portion of septum narimn, fix and harden in 20 c.cm. of absolute alcohol. For fine sections, use nasal mucous membrane from rabbit. Stain with haematoxylin (p. 262) and mount in dammar. Ciliary motion, p. 321. Kill a frog, lay it on its back, and remove lower jaw, by using scissors ; the roof of the mouth is thus exposed. Cut from the roof of the mouth a small strip of membrane, 5 mm. long, and place it in a few drops of solution of common salt (p. 260) on a slide, and cover with a cover-glass. With a low power a beginner can scarcely perceive anything, unless cun-ents, in which large blood corpuscles swim, direct him to the right spot. Put on, therefore, the high power and search the margins of the preparation. At first the motion of the cilia is still so lively that the observer is unable to distinguish indi- vidual cilia, the entire ciliary margin shows a wavy motion like that of a coriifiehl moved by the wind. After a few minutes this rapid motion abates and the cilia become visible. If the motion is stopped it may be started anew by leading: through one drop of concentrated caustic potash ; the efi'ect is, however, short- lived, so that the eye of the observer must not, during the process of leading through, leave the ocular. The addition of water immediately arrests the ciliary movement. 20. Fat Cells, Method No. 20, p. 305. Take from the axilla of a human subject a small fragment of the reddish yellow gelatinous fat, teaze out with needles on a dry slide, quickly add a drop of salt solution (p. 260), and put on a cover-glass. Thin places show fat cells. Colour under cover-glass (p. 277) with picrocarmine (p. 262), and mount in dilute glycerine. Always examine fat cells in a solution of common salt. 21. Involuntary Muscle, Method No. 21, p. 306. Place a small bit of stomach or intestine of newly killed frog into 20 c.cm. of caustic potash and treat as in Method No. 22 h. 492 APPENDIX I. 22. Striated Muscle, Method No. 22, p. 307. (ti) Trmisvemely striated mmcle fibres of the frog. — Cut with the scissors in the direction of the course of the ■fibres a strip of muscle 1 cm. long out of the adductors of a newly killed frog ; teaze a small part, taken from the inner surface of the strip, in a drop of salt solution (p. 260) ; then add a second larger drop of the same fluid, and lay on a •cover-glass without pressure ; with a low power (50 d. ) observe the cylindrical shape, the various thickness, occasionally even the transverse striation of the isolated muscle fibres ; with high powers (240 d.) notice the distinct transverse striation, sometimes pale nuclei and glittering granules. Very numerous granules in muscle fibres are pathological. In cases in which the muscle fibres are cut transversely, we may perceive the muscle substance squeezed out of the tube of "the sarcolemnia. {h) Of man. — Beautiful transverse striation is seen on human muscles taken from a fresh subject. For preservation, colour under the cover- glass (p. 277) with picrocarmine (p. 262), and after complete colouring, in five minutes, introduce diluted glycerine, and seal up. (c) Sarcole.mma. — Allow a couple of drops of water to flow on preparation for Method No. 25 a. After two to five minutes, observe with low power (50 d.) how the sarcolemma is raised up in the form of transparent vesicles ; in other places, where the torn muscle substance has contracted, the sarcolemma appears as a fine striation. [d) Nuclei. — Take a bit of preparation for Method No. 25 a, and allow a drop of acetic acid to flow on it. Even with low powers the shrivelled or sharply outlined nuclei •appear as dark or spindle-shaped lines, (e) Fibrils. — Place a fresh muscle of a frog in 20 c. cm. of chromic acid "l per cent. (p. 261). After twenty-four hours, teaze in a drop of water, and thus obtain fibres whose ends split up into fibrils. If a permanent preparation is desiderated, place the muscle in water for one hour, then into 20 c. cm. of 33 per cent, alcohol for ten to twenty hours ; or pre- serve it in 70 per cent, alcohol. If the chromic acid is exhausted, protracted immersion for several weeks in alcohol frequently changed will suit. Allow picrocarmine to act on the preparation, and, after complete colouration, replace it by dilute glycerine. (/) Terminations of the muscle fibres. — Place the fresh gastroc- nemius of a frog in 20 c.cm. of concentrated caustic potash. After thirty to sixty minutes the muscle disintegrates on its fibres being pressed slightly with a glass rod. If this does not occur, the potash has been too weak. Transfer a number of fibres into a drop of the same potash on the slide (the fibres cannot be investigated in water or glycerine, for the caustic potash thus diluted immediately destroys the fibres), and cover cautiously with a cover-glass. With low powers, we perceive the terminations of the muscle fibres and numerous glittering nuclei, some of which may be vesicular. 23. Ramified Muscular Fibres, Method No. 23, p. 307. Cut out the tongue from a newly killed frog and place it in 20 c.cm. of pure nitric acid, to which 5 grm. of chloride of potassium are added. Undissolved KCl must still remain at the bottom of the vessel. After fifteen hours, raise cautiously with glass rods the tongue out of the water, and place it in .30 c.cm. of distilled water, which is to be often changed. The tongue may continue in this up to eight days, but it may be operated upon after twenty-four hours. Place it in a small test-tube half filled with water, and shake it for a few minutes ; thereupon the tongue disinte- grates. Now pour the whole into a small dish, and, after one hour or later, place a little of the precipitate formed in a drop of water on to a slide. Teaze with needles so as to isolate fibres. Stain with picrocarmine under the cover-glass and preserve in dilute glycerine. METHODS OF MAKING HISTOLOGICAL PREPARATIONS. 493 24. Striated Mpscle, Method No. 24, p. 311. See Method No. 22 e. 25. Isolated Muscle Fibres, Method No. 25, p. 313. See Method No. 22/. 26. MrscLE Fibre of Heart, Method No. 26, p. 313. Cut out a papillary muscle from human heart, and place in 40 c.cm. of absolute alcohol. After from twenty -four to forty-eight hours, it is suflBciently hard for cutting. Stain with hsematoxylin (p. 262), and mount in glycerine. 27. Gaxglio>-ic Nerve Cells, Method No. 27, p. 314. Teaze out a small portion of the gasserian ganglion of a sheep in a drop of salt solution, and colour with picrocarraine (p. 262). 28. Nerve Cells, Method No. 28, p. 314. Take some grey matter from the fresh spinal cord of an ox and place a portion, 1 to 2 cm. long, in 50 c.cm. of very dilute soliition of chromic acid (5 c.cm. of "5 per cent, solution in 45 c.cm. of dis- tilled water). After changing the fluid often during from three to eight days, the grey matter will be found to be macerated to a soft pulp. Transfer this carefully with a spatula into the undiluted carmine solution (p. 262), and leave it there for from twelve to twenty hours. Then transfer into 50 c.cm. of distilled water, so- as to wash out part of the colour, and, after five minutes, spread it out in a thin layer on a dry slide. The multipolar nerve cells will then be seen, but the pro- cesses are not visible. Allow the layer to dry, cover with dammar, and place a. cover-glass gently over it. 29. Nerve Cells oe Cerebrum akd Cerebellum, Method No. 29, p. 314.. Same process as detailed in Method No. 28. 30. Medullated Nerve Fibres, Method No. 30, p. 315. Remove the sciatic^ nerve of a newly killed frog, cutting out a portion about 1 cm. in length. Teaze a small bit in a drop of a solution of common salt. It is better to teaze on a dry slide. Apply the needle to the lower end of the nerve, so as to pull asunder the fibrils for about half their length, with a drop of salt solution and a cover- glass. The thin film of nerve matter contains numerous isolated nerve fibres. Do- this very quickly (say in fifteen seconds), so that the nerA^e fibres do not dry. 31. Changes of the Maerow Sheath, Method No. 31, p. 315. Let a drop of water flow from the rim of the cover-glass into the preparation. Even in one* minute, the changes shown in Fig. 176, 4, p. 315, may be observed. 32. Axis Cylinder, Method No. 32, p. 315. Teaze when dry (as in No. 30),. and, instead of a solution of common salt, add a drop of absolute alcohol. The discovery of the axis cylinder requires practice. (See Fig. 176, 5, p. 315.) 33. Nerve of Rabbit, Method No. 33, p. 315. Remove the sciatic nerve of » newly killed rabbit, by the following process, without touching it with the fingers. Push a long splinter of wood below the nerve and tie the nerve to the ends of the splinter above and below. Then divide the nerve and lift out the splinter with the nerve attached, and place it in 100 c.cm. of a 1 per cent, solution of chromic acid. Axis Cylinder. After twenty-four hours, the ligatures are cut through ; a small portion of nerve, "5 to 1 cm. long, is cut off, and teazed into fine bundles, not into fibrils. The bundles separate in the solution of chromic acid. After twenty -four hours more, they are transferred into 50 c.cm. of distilled water ; and, after two to tliree hours, they are hardened in alcohol of increasing- strengths (p. 267). It is well to leave the btmdles a longer time, say from one to eight weeks, in 90 per cent, alcohol, because they then stain easily. After com- plete hardening, the bundles are minutely teazed in a drop of picrocarmine, and after completed colouration, which invariably, after the duration of the preceding hardening in alcohol, takes place in from half a day to three days. Preserve in 494 APPENDIX J. ?ici cm. long) for three to six days, in 100 to 150 c.cm. of 0'5 per cent, solution of chromic acid. Make longitudinal sections, stain with ha?matoxylin, preserve in. glycerine or dammar. (As to further details in treating sections of portions of intestinal canal, see Appendix, Vol. II.) 68. Motor Nerve Terminations, Method No. 68, i5. 358. (a) End PlateK, Cut out a thin portion, 1 cm. long, of the muscles of a lizard or the short muscles (intercostal or ocular muscles) of small quadrupeds. Prepare a mixture of chloride of gold and formic acid , boil it and let it cool. Place the bit of muscle in it for one hour in the dark. Transfer the small portions at the end of that tim& into 10 c.cm. of distilled water, ahd after a few minutes into a fresh portion of water to which a few flrops cf formic acid have been added, then expose to daylight (not sunlight) for twenty-four to forty-eight hours. The portions of muscle will now have a dark violet tint. Then transfer them for hardening into 30 c.cm. of alcohol of increasing strengths. After the dai'k violet portions of muscle have lain three to six days in alcohol, teaze the bundles, 2 mm. thick, of muscle fibres in a drop of dilute glycerine, to which a small dr-op of formic acid has been added. On account of the brittleness of the muscle fibres this process may not be successful. A light pressure on the cover-glass is often of advantage. Low powers show nmscle fibres from a delicate rose-red hue up to a deep purple tint, others again of red- violet up to light blue-violet ; in the latter we may notice the end plates most distinctly. In order to find these out, follow the deep black nerve fibres already discernible with low powers. (6) Nuchi of the Motor Plate. Place the METHODS OF MAKING HISTOLOGICAL PREPARATIONS. 501 anterior halves of the muscles of the eye of a freshly killed rabbit in 97 c.cm. of distilled water + 3 c. cm. of acetic acid. After six hours transfer the muscles into distilled water, cut off a flat small piece with the scissors and spread it out on a slide. We may now perceive with the naked eye the raniificatioias of the white looking nerve ; with low power (50 d. ) we notice the anastamoses of the nerve fibres. The blood-vessels are also easily discernible by their transverse nuclei (belonging to the smooth muscle fibres in their walls). The discovery of the end- plates is not easy on account of the great number of sharply outlined nuclei, which belong to the muscles, the intermusciilar connective tissue, etc. Follow a nerve fibre and one may notice that its meduUated sheath suddenly ceases and is apparently lost in a group of nuclei. These are the nuclei of the motor end-plate. m. Ex\d-Plate, Method No. 69, p. 358. See Method No. 68. APPENDIX II. CHEMISTRY OF MUSCLE. The account of the chemical composition of muscle given at p. 360 is founded •chiefly on the researches of Kiihne on the plasma obtained from the muscles' of frogs. Dr. W, D. Halliburton, in an elaborate research, has investigated the chemical composition of the muscles of warm-blooded animals ^ (rabbit, horse), and he has largely extended our knowledge of the subject. He succeeded in obtaining plasma from the muscles of the rabbit by washing out the blood from the vessels by a stream of cold salt solution ( "6 per cent, salt solution at a" C. ), then placing pieces of the muscles in a freezing mixture of ice and salt having a temperature of - 12° C, and then subjecting frozen slices to pressure. A yellowish, somewhat viscid fluid, of a faintly alkaline reaction, was thus obtained, which "set into a solid jelly in the course of from one to two hours ; at a temperature of 40° C. coagulation takes con- .siderably less time, viz. , twenty to thirty minutes : simultaneously an acid reaction is developed." Solutions of neutral salts prevent coagulation of the plasma. Dilution of " salted muscle plasma causes coagulation. Saline extracts of rigid muscle differ from salted muscle plasma in being acid, but resemble it very closely in the way in which myosin can be made to separate from it ; myosin, in fact, undergoes a recoagulation." The separation of the plasma into clot and " salted " muscle serum "does not take place at 0° C. ; it occurs most readily at the tem- perature of the body, and is hastened by the addition of a ferment prepared from muscle in the same way as Schmidt's ferment is prepared from blood." - According to Dr. Halliburton, the proteids of muscle plasma are — 1. Paramyosinogeti, coagulated by heat at 47^ C. 2. Myosinogen, coagulated at 56° C. 3. Myoglobulin, differing from serum globulin in its coagulation temperature (63° C). 4. Albumin, apparently identical with serum albumin a, coagulating at 73° C. ^W. D. Halliburton, M.D., B.Sc, Assist. Professor of Physiology, University ■College, London: "On Muscle Plasma," Journal of Physiology, vol. viii. No. 3. " Halliburton, " On Muscle Plasma," Proceedings of Royal Society, vol. xlii. 502 APPENDIX J J. 5. Mjo-albuu)osc, having the inoperties of deuteio-albuiiiose, aiul identical with, or closely related to, the myosin ferment. The two first bodies form myosin clot and the other three remain in the muscle serum. The three first bodies are globulins, " completelj- precipitated by satura- tion -with magnesium suljihate or sodium chloride, or by dialyzing out the salts from their solutions." Dr. Halliburton has also made the remarkable observation that " when muscle turns acid (by the formation of lactic acid), as it does during ritjor mortis, the pepsin which it contains is enabled to act, and at a suitable temperature (35° to 40" C. ) albumoses and peptones are formed by a process of self- digestion. It is possible that the passing off of nV/or mortis, which is apparently due to the reconversion of myosin into myosinogen, may be the first stage in the self-digestion of muscle." Dr. Halliburton has constructed the following table showing how these pro- teids may be separated from muscle plasma, which he has given me his kind permission to copy — Salted Muscle Plas3l\. Dilute to six times its volume, and expose this to a temperature of 85 C. for one or two honis, It separates into clot and salted muscle serum. Filter. Clot : consists of Myosin. AVash with Avater, redissolve in 5 per cent, magne- sium sulphate solution. Heat to 47^ C. A precipitate is produced. Filter. SaJtcd miisch' serum : contains myoglo- bulin, albumin, and myo-albumose. .Saturate with magnesium sulphate or sodium chloride. A precipitate is pro- duced. Filter. Precipitate : con- Filtrate : contains sists of Pakamyo- M y o s t X o g e X , sixoGEN. which is precipi- tated at 56'^ C. Precipitate: con- sists of Myoglo- bflin. Filtrate : contains- albumin and myo- albumose. Heat to 73" C. A preci- pitate is produced. Filter. Precipit- Filtrat<- : ate : con- contains sists of My'o-Al- ALHrillN. BUMCSE. As regards the muscle-ferment, Dr. Halliburton's conclusions are as follows : — "1. By keeping muscle under alcohol for some months, most of the proteids are coagulated. "Water will, however, extract from the alcoholic precipitate a proteid which has the characters of an albumose. "2. This albumose has the properties of a ferment in caiising the coagulation' of muscle plasma, or it may be that the ferment is in very close combination with, the albumose. " 3. This myosin ferment, as it may be termed, does not hasten the coagulation of blood plasma ; nor does fibrin fennent hasten the coagulation of muscle plasma ; the two are therefore not identical. " 4. The juice expressed from muscle, however, hastens very markedly the- coagulation of salted muscle plasma. This is not due to its containing fibrin CHEMISTRY OF MUSCLE. 503 ferment, but it is due to the proteid substance myosinogen, which enters into the condition of heat coagulum at 56^ C. Fibrin ferment is absent, or only present in exceedingly small quantities. "5. The activity of fibrin ferment is destroyed at 75° to 80° C. ; the activity of myosin ferment is not desti'oyed till the temperature of 100° C. is reached."^ APPENDIX III. LATENT PERIOD OF MUSCLE. Professors Gerald F. Yeo (King's College, London) and Theodore Cash (Aber- deen), after an elaborate research on the muscles both of cold-blooded and of wami- blooded animals, in which thej' made use of the pendulum myograph, have arrived at the following conclusions — - "L Increase in the sti-enfjfh of the ■■stmmlus is accompanied by (a) a steady and gradual shortening of the latent period ; (/3) a sudden prolongation of the actual contraction when a certain degree of stimulation is reached ; (7) an elevation of the altitude of the curve with the early and the final parts of the increase ; (5) and a removal of the sunmiit to a later part of the curve as soon as the elongation of the curve is established. " II. Increase in the iceighf used as a burden for the muscle is accompanied by (a) elongation of the latent period ; (/3) commonly a slight shortening of the dura- tion of the contraction ; (7) depression of the height of the curve ; and (5) no marked change in the position of the summit except in extreme cases. "III. The application of heat causes (a) very marked and continuous shortening of the latent period ; (/3) a gradual and distinct increase in the height of the curve ; and (7) a more rapid arrival at the summit, followed by a sudden fall of the lever, which usually passes considerably below the abscissa. (Extreme warmth has, however, an opposite effect; when above 90° F. , the altitude gets lower and the muscle remains contracted. ) " IV. Cooling causes (a) the latent period rapidly to increase ; (/3) a great increase in the duration of the contraction ; (7) at first a slight elevation in the altitude (extreme cold, however, lowers it) ; (5) the initial part of the curve is flattened, and the summit is delayed until a later period. "V. Gentle activity seems to increase the rate and power of contraction. Very weak interrupted currents have an effect like that produced by gentle beat. If extreme fatigue be induced, (a) the latent period becomes much longer ; (j8) the duration of the contraction is increased ; (7) the height of the curve is consider- ably lessened ; (5) and its summit is moved away from the beginning of the con- traction towards the end of the curve." ' Journal of Physiology, vol. viii. No. 3, p. 182. - Yeo and Cash — On the relation between the active phases of contraction and the latent period of skeletal muscle. Journal of Phy-nology, vol. iv. No. 2. 505 INDEX Abbe's condensers, figures of, 256. Absorption, co-efficient of, of gases, 353 ; of fat by epithelium, 3i!0 ; of fluids by epithelium, 319 ; of gases by epithelium, 319. Accessory appliances to microscopic research, 259, 260 ; for study of muscle, 376-381. Acetic acid series, 57, 161. Acetylene, 47. Acid, acetic, 161, 163 ; butsTric, 163 ; capric, 163 ; caproic, 163 ; caprylic, 163 ; cholalic, 107, 108 ; cryptophanic, 106; formic, 161 ; glycocholic, 106, 107; glycollic, 16i ; hippuric, 104,'l05, 106; ino- .sinic, 106; lactic, 165; leucic, 166; oleic, 16S ; oxalic, 16(i, 167 ; oxalic series, 166 ; oxycaproic, 166; palmitic, 163; propionic, 163 ; stearic, 103; succinic, 167; sulphocyanic, 07; taurocholic, 107; uric, 97, 98, 9;J, 100, 101, 102. Acids, allylic alcoholic series, 167, 168; bile acids, tests for, lOS; glycollic series of, 106, 107 ; non- nitrogenous organic, 57, 161 ; organic nitro- genous, 97, 162; organic, properties of, 46, 48, 49. Aconitine, 89. Actiniohajmatin, 144. Adamkievvicz, reaction of, 64. Adenoid tissue, structure of, 328; how to ex- amine, 496. Adipose tissue cells, 304, 305. Albumin, acid, 75. Albumin, alkali, 75; decomposition of, 16; egg, 73; serum, 72,73; chemical composition of, 81. Albumins, true, 72. Albumin, vegetable, 73. Albumins, 55, 72; derived, 75. Albuminoids, 55 ; crystallizable, 55, 76. Albuminous derivatives, 55, 76; preparation of, 76, 79, Aldehydes, formulas of, 46. Aldehyde, formation of, 48, 49. Alkaloids, 89. Allantoin, 54, 104. AUanturic acid, 88. Alcohols, 57, 146 ; formulae of, 46 ; composition of, 47; diatomic, 4s. Alcohol, ethyl, 47; ethylic, 146 ; propyl, 47 ; oxid- ation of, 47, 48 ; table of, 162 ; monatomic, 48 ; triatomic, 48. Aldehydes, formation of, 49. Amides, 49, 55, 84, 89, 90. Amines, 50, 55. Ammonium salts, in secretions, 40. Amphioxus embryo, figures of sections of, 245. Amyloid matter, 77. Amyloses, 57, 159. Anisotropic substance of muscle fibre, 307. Anemometer, d'Ons-en-Bray's, 383. Animists, 32. Amyloid matter, 55. Aphidein, 144, 145. Aplysiopurpurin, 145. Archenteron, formation of, 245. Area pellucida, changes in the, 247, 248 ; forma- tion of, 246. Aristotle, definition of life, 31, 32. Aromatic compounds, 47. Asparagine, 89. Assinailation, nature of, 294. Atomic constitution of molecules, 50. Atomic theory, 8. Atropine, 89. Ascaris megalocephala, division of egg of, 233; polar bodies of, 229. Axis cylinder, 313, 315 ; how to examine, 493. B Bacillus anthracis, 191, 192 ; subtilis, 192 ; ulna, 192. Baeyeb, on formation of uric acid, 97 ; on indol, 110 ; on protagon, 82. Balance, figure of, 6. Balfocr, on heredity, 237 ; on the muscle ele- ments, 356. Baumank, on phenol, 149. Beadnis, reference to, 39, 40 ; on water in tissues, etc., 36 ; on biliverdin, 131 ; on iron in secre- tions, etc., 42; on nonstriated muscle, 436; table of gases in body fluids, 169; on sulphates in tissues, etc., 43 ; on origin of urea, 86, 87. Bechajip, on alcohol, 146. Beclard, definition of life, 31 ; on production of heat in muscle, 422. Becqukrel's theory of muscle currents, 454, 455. Beneden, Van, on polar bodies, 228, 229 ; on seg- mentation in rabbit ovum, 245, 246. Be.veke, on glycerine, 148. Benzol, 16 ; constitution of, 51. 506 IXDEX. Bernarc's, Claude, method of iireparing glj'co- gen, 159 ; on irritability of muscle, 400, 401. Bkrnstein, on action stream, 458 ; on muscle con- traction, 453; on negative variation, 452. Berzeliu.s, on fermentation, 177. Bezold, Vox, on muscle contraction, 453 ; on negative viiriation, 45:;. Biberine, 89. BiCHAT, definition of life, 31. Bichromate voltaic element, 37.. BiEDERMANx's fluid in muscular contraction, 420, 4-Jl. Bile, acids of, 56, 106, 107, los, lOli ; tests for, 108 ; pigments of, 56, V.W. Bilicyauin, 133. Bilifuscin, 13l'. BilOiumin, 133. Biliprasin, 132. Bilirubin, 130, 131. Biliverdin, 131. Billroth, on production of heat in m\isclc, 423. Biology, general principles of, 13. Biijolar nerve cells, 313. BiscHOFF, on quantity of water in the body, 35. Black pigments, 142. Blaxkexiiorx, on protagon, 82. Blastoderm, 244 ; formation of, 246 ; of hen's ovum, figures of section of, 246. Blastodermic layei-s, formation of, 244-252 ; ves- icle of rabbit, figures of, 251. Blastomeres, 244. Blastopore, formation of, 245. Blood corpuscles, mode of examining, 480 ; struc- ture of, 300 ; crystals, how to examme, 490 ; plaques, how to demonstrate, 489 ; pigments, 56 ; stains, how to examine, 490. Blue pigments of bile, 133. Boeruaave, theory of life, 32. Bogomoloff's test for bile acids, lOS. BoRoaxv, on chemical actions of i)roteids, 60 ; on proteids, 63. Bone, 332-342 ; chemical composition of, 342, 343; development of 336-341 ; how to examine, 497 ; matrix, 324 ; ossification, 337 : structure of, 332, 337 ; lamellaj, how to examine, 498 ; marrow, how to examine, 498. Borelli, theory of life, 32. BoscoviTCH, on atomic theory, 8. BoTTOER-'s test for glucose, 153. Boussole, 442. Bowman, sarcous element of, 309. Bristle cells, how to demonstrate, 491. Brittner, on animal and vegetable proteids, 71. Brownian movement of cells, 296. Browx-SIqi'ard, on muscular irritability, 404 ; on rigor mortis, 432. Brucine, 89. BRrcKE, on muscle substance, 309 ; metliod of preparing glycogen, 160. Budding in cell formation, 297. BuF'KON, on spermatozoa, 214, 215. BuxGE, analysis of soda and potash in enibryo mammals, 40 ; on hippuric acid, 106. BrxsEN's voltaic element, 369, .^70. Burdach, on percentage of fat in body, 150. B urdon-Sandkr.sox, on electric organs of raia batis, 470, 471, 477 ; on polar bodies, 227. Butyric acid, W.\. Cadaveric rigidity, plionoinena of, 432, 433. Caffein, 89. Cagniard de la Tour, on fermentation, 177. Camera lucida, figure of, 259. Canaliculi of bone, 332. Canalis neurentericus, fcirmation of, 245. Cane sugar, 158. Capric acid, 57, 163. Caproic acid, 57, 163. Caprylic acid, 57, 163. Carbohydrates, 151 ; classification of, 151. Carbon compoimds, properties of, 54 ; in organic compounds, 45. Carbonates, in the body, 42. Carbonic acid gas, occurrence of, in body, 170. Carbonic -oxide haemoglobin, 124. Carburetted hydrogen, occuiTcnce of in body, 170. Carnin, 104. Carotin, 139. Carpenter, on vital energy, 20. Cartilage, chemical composition of, 331 ; develop- ment of, 331 : how to examine hvaline, 497 ; structure of, 328, 329, 330, 331 ; white fibro, 330, 331 ; yellow fibro, 330. Casein, 75. Cash, Theodore, on latent period of muscle, 503.. Cells, of adipose tissue, 304, 305; structure of, 299, 317; characters, etc., of, 292-298; coloured blood, 299, 300, 301 ; connective tis.sue. 206, 303, 304 ; division of, 212, 213, 214 ; embryonic, 207 ; epithelial, 301, 302, 303 ; evolution of, 298;. figure of, 903 ; form of, 293, 294 ; general theory of, 292, 293 ; growth of, 298 ; irritability of, 295 ; migrations of, 295 ; movement of, 295 ; muscle, 305-313 ; nerve, 313, 314 ; nutrition of,. 294, 295 ; reproduction of, 296, 297 ; size of. 294 ; structure, details of, 207-212 ; structure, different views of, 206, 207, 20S, 209. Celloidin, use of, in section cutting, 287. Cellular excretion, nature of, 295 ; secretion- nature of, 295. Cellulose, occurrence of, 16, 100, 161. Cerebrin, 55, 82, 83. Cerebrin-pseudo, S3. Chauveau, on fermentation, 182. Chemical composition of bone, 342, 343. Chemical constitution of muscle fibre, 300-363. Chemical forniulw, determination of, 52. Chemical instability, 16. Chemical stimuli in muscular contraction, 419,. 420. Chemical reactions in the living organism, 170. Chemicals, influence of, in fermentation, 194. Chemistry, of the body, 34 ; relation to physi- ology, 3. Chevreil, on nitrogen in body, 168. Chlorocruorin, 143. Chlorophane, spectrum of, 140. Chlorophyll, occurrence of, 23, 142, 143. Cholalic acid, 56, 107, 108. Cholesterin, 146, 147. INDEX. 507 Choletelin, 131, 132. Cholin, 55, 82, S3. Cholohsematin, spectrum analj-sis of, 132. Choloidio acid, 56. Chondi-igen, 55, 79. Chondrin, 79, SO ; chemical composition of, SI. Chondroglucose, SO, 157. Chorda dorsalis, formation of, 248. Chromatin filaments, arrangement of inovum,232. Chromophanes, 140. Chronographs, 385-387. ChronogTaphy, 384-889. Chdbcu, on turacin, 144. Cicatricula, formation of, 240. Ciliary motion, how to examine, 491 ; nature of, 321, 322 ; theories of, 322 ; varieties of, 321. Cinchonine, 89. Cladothrix dichotoma, 189. Clf.rk Maxwell, on matter and motion, 11, 21 ; on the atom, 242, 243. Cochineal, 144. Codeine, 89. Cohesion of connective tissues, 343, 344 ; of muscle, 397, 09S. CoLEM-iN, on temperature in fermentation, 193. Collagen, 55, 79. Colloid matter, 55, 66, 76, 77. Commutator, Pohl's, 378, 379. Compound substances present in the body, 35. Concretions, origin of, 17. Conine, S9. Connective tissue cartilage, how to examine, 497. Connective tissue, ground substance of, 323 ; how to examine, 494, 495; physical properties of, 343-345 ; structure of, 324 ; vital properties of, 346, 347. CoNRADi, on cholesterin, 146. Consistence of connective tissues, 343 ; muscle, 397. Constitution of muscle serum, 361, 362. Contact theory of muscle cun-ents, 456-458. Contractile tissues, 355. Contraction of muscle, modes of exciting, 418- 421 ; period of, 407 ; phases of single, 406-412 ; phenomena of, 405-414. Contraction wave of muscle, propagation of, 412- 414. Costal cartilage, how to examine, 497. Creatin, 56, 94 ; crystals, figure of, 94. Creatinin, 56, 94, 95 ; crystals, figure of, 95 ; test for, 95. Crustaceorubin, 144. Cryptophanic acid, 56, 100. Crystalloids, 66. Crystals, method of increase of, 17. Curare, action of on muscle, 401. Current in a living man, 459. Cuticular formations, 304. Cystin, 56, 97. D DAXiiiLL's voltaic element, 369. Darwin, on pangenesis, 235. Davy, Sir Humphrt, on heat, 11. Death, result of, 30. Decalcification in micros«opic research, 268, 269. Decomposition, in the living organism, 173. Democritus, atomic theory of, S. Density of connective tissues, 343. DepreZj signal of, 389. Deuthyalosome, the, 230. Dextrin, 100. Dialyzer, figure of, 65. Diffusion through organic membranes, 348-353. Direct recording of movement, 389-394. Disassimilation, nature of, 294, 295. Discs, nature of muscle, 307. Discus proligerus, 222. DisQUE, on urobilin, 135. Dissociation, definition of, 173. DiTTMAR, on chemical constitution, 51 ; on fer- mentation, 177. Dobie's line, nature of, 309. DoxDERS, on dissociation, 173; on elasticitj- of muscle, 399. D'ONS-EX-B ray's anemometer, 383. Du Bois-Retmond, discoveries of, 440, 442 ; obser- vations of, on muscle currents, 448, 452 ; mode of electrical action of torpedo, 4S2 ; myo- graph, 390,391; on polarization currents, 479,480; rheocord of, 451 ; theory of muscle currents, 455, 456 ; on currents in living man, 459 ; on electric shocks of fishes, 471, 472 ; on electric .shocks of malapterurus, 474, 475, 476 ; on special electric phenomena of fishes, 477, 478, 479, 480. DuGEs, definition of life, 31. Dumas, on fecundation, 223. DuscH, on fermentation, 179. Dynamical characters of animals, 25, 26, 27; of living things, 20 ; of plants, 24, 25. Dynamometers, 427. E Ebxer, on origin of spermatozoa, 216, 217. Echinochromo, 143. Egg, cleavage of, figure of, 234 ; division of, in sea urchin, 233, 234. Elastic cartilage, 330 ; how to examine, 497. Elastic fibres, 326, 327 ; how to prepare, 494, 495. Elastic intercellular substance, 324. Elasticity of connective tissues, 344, 345, 346 ; of muscle, 398, 399, 400. Elastin, 55, SO; chemical composition of, SI. Elective affinity, definition of, 295. Electric apparatus in study of muscle, 363-381. Electric batteries, 305, 366. Electric fishes, 461, 462 ; general electrical pro- perties of, 471-477; phenomena of, 461-480; special electric phenomena, 477-480. Electric organs, of gymnotus, 465-468 ; malap- terurus electricus, 408-470 ; raia batis, 470, 471 ; torpedo Galvani, 462-405. Electric plates, 364. Electric poles, 364. Electric signals in measuring time, 387, 388 389. Electric stimuli in muscular contraction, 418. Electrical induction, 373, 374. o08 JA'DEX. Electrical iiie:isurements, methods of, 31)8 ; phenomena of nmscle, 436-451) ; resistance, nature of, 3(54, 365, 366, 367. Electricity, discovery of animal, 437 ; definitions of, 363-368 ; effect of upon protoplasm, 2!>0 ; general statement of, 363-368. Electro-capillary theory of muscle currents, 454, '< 455. Electrodes, nature of, 364 ; non-polarizable, 380 ; polarizable of Du IBois-Ueyruoud, 379, 3S0. Electrolytes, nature of, 364. Electrometer, Lipiamanu's capillarj', 443, 444,445. Elements of the bodj', 35. Embedding of tissues, in wax, 279 ; in paraffin, 2V9. Embryo of newt, figures of sections of, -248. Embryology, definition of, 3. Emmerlino, on indol, 110. Enchondrial ossification, 337, 33S, 339. Endogenous cell formation, 297. Endosmometer, 34S. Eiidosmotic equivalent, 348. Energy, conservation of, 10, 11 ; in muscular con- tractions, 28, 29 ; kinetic, lo ; plant versus animal, 27 ; potential, 10 ; relation of its study to physiology, 9 ; vital, Carpenter on, 20; vital, Le Conte on. 21 ; vital, JIayer on, 20 ; vital, older views of, 20. Engelmaxx, disc of in muscle, 309 ; on fermen- tation, 181 ; on muscular contraction, 406 ; on :ionstriated muscle, 435 ; on vital energy, 24. Environment, definition of, 31. Epiblast, formation of, 245, 247 ; structures derived from, 251. Epiplasm, 225. Epithelial cells, 301, 302, 303; how to examine, 490, 491 ; mode of examining, 4S9 ; secreting, figure of, 297. Epithelial layers, varieties of, 302, 303. Epithelium, ciliated, 318-320 ; columnar or pris- matic, 318 ; influence of, in absorption, 319, 320; influence of, in elimination, 319, 320; lammar or stratified, 317 ; physical properties of, 318, 319; physiological properiies of, 317; .simple ciliated, 303 ; simple cylindrical, 303 ; simple, flat or squamous, 317 ; simple pave- ment, 302, 303 ; spheroidal, 318 ; stratified ciliated, 303; stratified cylindrical, 303 ;stratified pavement, 303 ; vital properties of, 319. Ether freezing in section cutting, 282. Ethers, compound, 49, 50. Evolution, of living beings, 20, 30 ; of cells, 298. Ethyleno-lactic acid, 165. EWALD, on Torpedo Galvani, 463. Excitability of muscle, 400. Excretion, cellular, nature of, 295. F«cal pigments, 136. Falk, on physiological action of water, 36. Faraday, relations of magnetism, etc., 11. Fascial, structure of, 327. Fat cells, 304, 305 ; how to examine, 491 ; crystals, figure of, 149 ; in fibrillar tissue, 326 ; origin of, 151. Fatigtie in production of heat in muscle, 423, 424. Fats, constitution of animal, 149, 150; in the body, 57 ; the animal, 149. Fatty compounds, 47 ; nitrogenous matters, 55. Fecundation, figvn-e of, 225 ; true, 230-234. Frdkr, on origin of urea, 87. Fkhi-ISc's test for glucose, 152, 153. Fkltz, on decomposition of urea, 40. Fenestrated membranes, how to demonstrate, 495. Ferment, blood, 185. Fermentation, definition of, 175 ; influence of chemicals in, 194; influence of light in, 193; inflvjence of ozone in, 194 ; influence of i)ressuro in, 194 ; influence of temperature in, 191-193 ; influence of water in, 193; nature of, 175, 17tJ; test for glucose, 153 ; theories of, ISO, 182. Fei-ments, action of, 16 ; amjdolytic, 1S5 ; chemi- cal, actions of, 186-188 ; chemically acting, 186- 188; classification of, 183; hydrolytic, 184; inversive, 185 ; organized, actions of, 185, 186 ; organized, nature of, 188191 ; proteolytic, 184, 185 ; soluble, 55, 76 ; soluble, action of, 183, 184 ; steatolytic, 185; the organised, 185. Fertilization, discoverers of, 223, 224 ; history of, 223, 224. Fibrillar connective tissue, 325, 326. Fibrils, nature of muscle, 307. Fibrin, 55, 74. Fibrinogen, 74. Fibrinoplastin, 74. FiCK, on prodiiction of heat in muscle, 423. Filtration through organic membranes, 347. Fixing fluids, for microscopic research, 266-268 ; general rules for, 266. Fletcher, on albumin, 59. FoL, on polar bodies, 227. Food of animals, 26 ; of plants, sources of, 21. Force, definition of, 9 ; measurement of, 10. Form, organic, 16. Formed connective tissue, 326. Formic acid, 161. Formless connective tissue, 326. Formulse, chemical, determination of, 53 ; deter- mination of, 52. Foster, on spectrum of reduced and oxj-hsemo- globiii, 123. Fkericiis, on oxalic acid, 167. Frey, on hippuric acid, 104. Frog, mode of examining ova of, 489 ; muscles in limb of, 355 ; spermatozoids of, 488. Feitscii, on gymnotus electricus, 46.5-468 ; on malapterurus electricus, 468, 470 ; on torpedo Galvani, 464, 465. G Gaiffe's voltaic element, 371. Galactose, 157. Galkn, on life, 32. • Galvani, on electricity, 437-439. Galvanism, 437. Galvanometer, method of experimenting with, 446-4.54; Thomson's reflecting, 442, 443; Wiede- mann's, 442. Gametes, 225. Gamoee, on alkali albumin, 75 ; analysis of uuclein, 78; on cerebrin, 83; on choiidrin, 80; on collagen, 79; on elastin, 80; on fibrinogen, 74 ; on hasmoglobin, 118 ; on keratin, SO ; on lecithm, S2 ; on legumin, 75 ; un mucin, 78 ; on INDEX. 509 neurin, 83 ; on action of nitrites on blood, 126 ; on paraijlobulin, 74; on peptones, TO; on pro- tagon, 82 ; on the spectrum of blood, 116, 117 ; on spectrum of oxyhemoglobin, 122 ; on solu- bility of proteids, etc., 64. Ganglionic nerve cells, how to examine, 4fi3. Gareod's test for uric acid, 102. Gases of the body, 168 ; absorption of, by fluids, 353, 354 ; absorption of, by membranes, 353, 354 ; effect of, on protoplasm, 291 ; in fluids of body, table of, 16H. Gaskell, on muscular fatigue, 481. Gastrula, formation of, 245. Gat Lussac, on fermentation, 176, ITS. Gelatin, 79 ; chemical composition of, SI ; chem- ical reactions of, 81. Gemmation, nature of, 297. Germ epithelium, 221 ; origin from, 30. Gland cells, nerve endings in, 484 ; secreting, figure of, 298. Globulins, 55, 73. Glucoses, 57, 152. Glucose, estimation of, 153-156; synonyms of, 152 ; tests for, 152, 153. Glycerine, 147, 148; physiological significance, 14S. Glycocholic acid, 106, 107. Glycocin, 90, 91. Glycocholate of sodium crystals, figure of, 107. Glycocholic acid, 56. Glycoffen, 159, 160 ; preparation of, 159, 160 ; tests for, 160. Glycol series of acids, 164. Glycocolle, 55, 90, 91. GlycoUic acid series, 57, 164. Glycin, 90, 91. Gmelin's test for bilirubin, 130. Goitre, 77. GoODSiR, on nature of physiology, 3. Gordon, on polarization, 67, 68. GORTER, De, theory of life, 32. Gorup-Besanez, on magnesium salts in tissues, 41 ; on percentages of fat in tissues and fluids, 150 ; on proteids, 1 ; on oxidation, 171. GoTcn, on electric shocks of electric fishes, 472, 473 ; on electric shocks of malapterunis, 475, 477 ; on electric organs of raia batis, 470, 471 ; on irreciprocity in torpedo, 483 ; on secondary discharges of torpedo, 483. Graafian vesicles, 219, £21 ; figure of, 223 ; struc- ture of, 2:;2. Graham, on colloidal state, 13 ; on dialysis, 65. Graphic method, 3S1-397. Gravimetric estimation of glucose, 156. Grenet's voltaic element, 372. Ground substance of connective tissue, 323. Grove's voltaic element, 370. Growth of cells, 298 ; of living things, 17 ; mode of, 16. GscHEiDLEN, On creatiuin, 95 ; on preparation of hcemoglobin, 119. Guanin, 103. Gymnotus electricus, 465-468. Gun cotton, composition of, 16 ; instability of, 16. H Haas, on albumin, 71. Hakckel, on heredity, 235. Hsematin acid, spectrum of, 127 ; alkaline, spec trum of, 127. Hasmatin, 126, 127. Hsematoidin, 128, 129; crystals, figure of, 129 how to examine, 490. Hsematolin. 128. Haamatoporphyrin, 127, 128 ; acid, spectrum of, 128 ; alkaline, spectrum of, 128. Hajmatoscope, figure of, 113. Hajmin, 129 ; crystals, figure of, 129 ; crystals, how to examine, 490. H?emochromogen, 125. Hasmocj'aniii, occurrence of, 143. Hajmoglobin, 117 ; compounds of, 124, 125 ; con- ditions in which it occurs, 119 ; crvbtals, how to examine, 490 ; occurrence of, 123, 124 ; per- centage composition of, 118 ; preparation of. 118, 119 ; reduced, 119 ; reduced, spectrum of, 123 ; synonyms of, 118. Haller, on irritability of muscle, 400. Hallibcrtox's researches in chemistry of muscle, 501. Hallwachs, on hippuric acid, 106. Haloid derivatives, 49. Ham, on spermatozoa, 214, 215. Hamersten, on glycocholic acid, 107. Hardening in microscopic research, 268. Hartig, on deposition of carbonate of lime, 19. Haversian canals in bone, 333 ; how to examine, 498 ; systems in bone, 333. Heart, how to examine muscles of, 493. Heat, a kind of energy, 12 ; a mode of motion, 11; effect of , on protoplasm, 290 ; measurement of, 12 ; production of by muscle, 421-424 ; re- lations of, to work, 12. Heideshain, on heat production in muscle. 421. 422. Hettzmaxx, on protoplasm, 289, 290. Heller, on indican derivatives, 135. Helmholtz, myograph of, 390 ; on contraction of muscle, 453 ; on energy, 11; on fermentation, 179 ; on induction, 375, 376 ; on negative varia- tion, 452. Helmoxt, Van, theory of life, 32. Hemicollin, 79. Hensen, median disc of, 309. Hereditv, definition of, 30 ; physiological basis- of, 234-244. Hermann's propositions as to currents, 459 :. theory of muscle currents, 456, 457, 458 ; on currents in living man, 459 ; on muscle cur- rents, 457, 458. Hertwig, on polar bodies, 227. Heterodromous currents, definition of, 479. Hippocrates, on life, 32. Hippuric acid, 56, 104, 105, 106 ; crystals, figure- of, 105 ; origin of, 105, 106 ; tests for, 10.5. Histohsematins, 137, 138. Histology, history of, 201-214 ; reference to, 2. Hoffmann's test, 93 ; on fermentation, 179. Hofmeister, on collagen, 79. Holm, on hjematoidin, 129. Homodromous currents, definition of, 479. Hoppe-Seyler, analysis of nuclein, 78 ; on al- bumin, 71 ; on carbonic oxide bajmoglobin, 124-, on chemical ferments, 186 ; on choroiihyll, 23 ; 510 jyoEA-. on cholalic acid, 107, 108 ; oti composition of proteids, 5S ; on constitution of hiemochronio- gen, Vi:> ; on fermcnfcitioii, 1S2, is:i ; on gelatin, 7;) ; on liainiatoporph.vrin, 127 ; on methsenio- globin, 12(5 ; on naphtliylamine, 100 ; on oxida- tion, 171 ; on protagon, 82 ; on proteids, (J-l ; on the spectrum of blood, 110 ; on totronerytbrin, 144. noKMA.N>f, on endosmotic equivalent, 34it. lIowsHir, lacuna; of, 341. Hyaline cartilage, 328, 320 ; matrix of, 323, 324. Hydrobilirubin, 134. Hydrocarbon, derivatives of, 4(1, 47 ; formuhc of. 4(3 ; series of, 40, 47. Hydrochloric acid, in the body, 42. Hydrogen, occin-rence of in body, KiO, 170. Hydroxyl, IG. Hygroplasm, 235. Hypoblast, formation of 24J-247 ; structures derived from, 252. Hypoxanthin, 103. I latro-chemists, 32. latro-mathematicians, 32. Idioplasm, 235. Imbibition, molecular, in tissues, 352. Idio-muscular contraction, 419. Inclination currents in niuscle, 448. Indioan, derivatives of, 135; characters of, 135, 130. Indigo blue, spectrum of, 13(3. Indol, 56, lOCf, 110. Induction coils, 373-37(3 ; electrical, 373, 374. Inductorium of Du Bois-Reymond, 373, 374. Imbibition, capillary, in tissues, 352. Injection in microscopic research, 273. Inorganic constituents of the body, 34 ; com- pounds of the body, 35. Inosite crystals, figure of, 157 ; characters of, 157, 158 ; origin of, 158. Inosinic acid, 5(3, lOii. Intercellular substances, 322, 323, 324. Intestinal muscle fibre, 500. Intussusception, 17. Investigation of fresh objects in microscopic reseai-ch, 270, 277, 278. Involuntary muscle, how to j^repare, 500. Iron in the body, 42. Irritability of cells, 205 ; of muscle, 400-405 ; nature of, 403 ; variations of, 403, 404. Isocholesterin, 147. Isolating fluids for different tissues, 265, 260. Isomerism, definition of, 48. Isotropic substance of muscle fibre, 307. .Jacobt, on fecundation, 223. .Tenkin, on electricity, 373. .Tolly, on endosmatic equivalents, 349. Joule, on heat, 11 ; dynamical equivalent of, 12 ; experiments of, 12. K Karyokinesis, 212, 213^ 297 ; method of demon- strating, 487. Karyosteno.sis, 212, 213 ; nature of, 290, 297. Kkhui.k, on amyloid matter, 77 ; on carbon pro- perties, 45 ; on constitution of benzol, 51 ; oji organic chemistry, 44. Kephalins, 83. Keratin, 55, 80 ; chemical composition of, SI. Ketones, formuhe of, 4(i. Key, l)u Bois-Reymond's friction, 376, 377. Kinoline, 80. Kleix, on temperature in fermentation, 192. Kniriem, on ori^'in of urea, 87. IvoLUEtrn;ii, on fecundation, 223. Kocii, on succinic acid, 107. KoLLiKKR, on heredity, 238; on spcrmatin, 78; on origin of spermatozoa, 215, 216. Kraus, on formation of starch, 23. Krause's membrane, 300. Kroxf.ckek, on genesis of tetanus, 417; on muscular fatigue, 430. Krhkenbero, on biliverdin, 131 ; on lipochromea, 139. KiJHNE, on chromophanes, 140; on hippuric acid, 106 ; on irritability of muscle, 400 ; on motor end plates, 359 ; on indol, 110. KiiLZ, on inosite, 157. I; Lactic acid, 105. Lactose, 158, 1.59. Lasvulose, 157. Lamarck, definition of life, 31. Laxdois, on rapidity of contraction of muscle, 412. Langlev, on vital energy, 24. Lardacein, 77. Latent stimulation, period of, in muscle contrac- tion, 400, 407. Latschinoff, on cholesterin, 147. Laxkester, on peutacrinin, 144. Laurence, definition of life, 31. Lavoisier, on fermentation, 176. Lecithin, 55, 83 ; characters of, 82, 83. Leclanche's voltaic element, 370, 371. Le Coxte, on vital energy, 21. Leedweniioek, on fermentation, 177. Legumin, 75. Length, measurement of, 5. Leucin, 16, 55; characters of, 91, 02; crystals, figure of, 92. Leucippds, atomic theory of, 8. Leucocytes, 299 ; in frog's blood, figure of, 295 ; mode of examining, 489. Leyden, on production of heat in muscle, 423. Lieberkuhn, on albumin, 62. Liebio, on fermentation, 177, 178. LiEBREiCH, on protagon, 82. Life, old theories of, 32 ; theories of, 31 ; pheno- mena of, in living cell, 33. Ligament.s, structure of, 328. Light, influence of, in fermentation, 193 ; polariz- ation of, 66, (37 ; a source of vital energy, 24. Liminal intensity of stimulus, 424. Lipochromes, 139. Liquor sanguinis, 74. Lime salts, quantity in the body, 40 ; physiology of, 41. INDEX. 511 Living things, essential characters of, 31. Liquor folliculi, 222. Locke, John, on heat, 11. LoEw, on chemical actions of protoplasm, GO ; on proteids, 03. Lucre's test for hippuric acid, 105. LuDWiG, on endosmosis, 349. Luteins, 13ft. Lymphoid tissue, how to examine, -tOiJ. M M'Kendrick, on temperature in fermentation, 193. MacMustn, on hiliverdin, 131 ; on choloh»matin 132; on chlorocruorin, 143; on chlorophj'll 143 ; on echinochrome, 143 ; on hsematopor phyrin, 127, 128; on histohaimatins, 137, 13S on myohiematins, 13?, 139 ; on oxy haemoglobin 120, 121, 123 ; on pentacriuin, 144 ; on physio logical spectra, 117 ; on spectrum of hasmatin 127; on spectrum of hsemochromogen, 125 ; on spectrum of indigo blue, 136 ; on spectrum of methremoglobin, 126 ; on spectrum of urabiliu, 135; on stercobilin, 136. Magellan's meteorograph, 383. Magnesium salts in the body, 41. Malapterurus electricus, 46S-470. Maltose, 159. Mannite, 157. Mann itose, 157. Marchand, on hippuric acid, 105. Maret, myograph of, 392, 393 ; on elasticity of muscle, 399 ; on genesis of tetanus, 417 ; on wave of muscular contraction, 412, 413 ; tam- bours of, 394-397. Marie-Davt's voltaic element, 371. Marrow, elements of 334, 335 ; of bone, 333, 334 ; how to examine, 498. Marrow sheath, how to examine, 493. Marshall Ward, on organized ferments, 1S9. Mass, measurement of, 5. Matteucci, discoveries of, 440 ; induced contrac- tion of, 452, 453. Matter, and Energy, permanence of, 4 ; circula- tion of, 16; colloidal condition of, 13; inde- structibility of, 4 ; its condition in living things, 9 ; its importance in vital actions, 9 ; quantita- tive estimation of, 5. ilatrix of bone, 324 ; of hyaline cai'tilage, 323, 324. Mayer, on heat, 12 ; on vital energy, 20. Matow, theory of life, 32. Mechanical stimuli in muscular contraction, 419. Medicus, on constitution of uric acid, 98. Medullated nerve fibres, 315. Medullary, folds, formation of, 248 ; plates, for- mation of, 248 ; ridge, figures of formation of, 247. Mrissner, on creatinin, 95; on hippuric acid, 105, 106; on succinic acid, 167. Melanin, 142. Melsens, on temperature in fermentation, 192. Membrana granulosa, 222. Mbrejkowski, on tetronerythrin, 144. Merkel, on origin of spermatozoa, 216. Mesoblast, development of in amphioxus, 250 ; figures of deveL ipment of 249 ; formation of, 248 ; origin of, 248, 249 ; structures derived from, 251, 252. Metabolic processes in animals, 26. Metabolism, 17; constructive, 22; definition of, 294 ; destructive, 22. Metaplastic type of ossification, 340. Metastasis, definition of, 294. Meteorograph, Magellan's, 383. Methsemoglobin, 125, 126. Methane, 45 ; substitution derivatives of, 45. Metric system, 5. Metronome, in study of muscle, 377. MicellcO, 236. Microscope, the, 252-256 ; description of, 253-257 ; figures of, 254, 255 ; mode of using, 257, 258, 259. Microscopic research, decalcification in, 268, 269 ; methods of, 257-278 ; preparation of sections in, 269, 270; staining in, 270, 271, 272, 273; acces- sory appliances to, 259, 260; dissection of animals for, 264 ; fixing in, 260, 267, 268 ; hard- ening in, 268 ; injection in, 273 ; investigation of fresh objects in, 270, 277, 278 ; isolation in, 205, 206 ; mounting of preparations in, 273, 274, 275, 276 ; nature of material for, 263, 204 ; pre- servation of preparations in, 273, 274, 275, 276, 278; reagents required in, 260, 261, 262, 263. Mierospectroscope, figures of, 114, 115. Micrococcus luteus, 192. Microtomes, 278-287 ; figure of, 283 ; rocking, 285 ; Rutherford's, 279 ; Rivet's, 284 ; Roy's, 282 ; ice freezing, 280, 281. MiEscHER, analysis of nucleln, 78. Migrations of cells, 295. Millon's reaction, 64. Mineral matters present in the body, 37. Mineral matters, table of, in animal solids, 37. Mineral matters, table of, in animal fluids, 37. Mitscherlich, on fermentation, 179. MoHL, Von, on protoi^lasm, 14. Montgomery, on formation of cells, 19. Molecular change, 14 ; instability, 16 ; pheno- mena, occurrence of, in living things, 8. MoLESCfiOTT, on percentage of fat in the body, 150. Moore's test for glucose, 152. MoROCHOwiTZ, on chondrin, 8u. Morphine, 89. Morphology, reference to, 3. MosELEY, on aplysiopurpurin, 145 ; on crus- taceorubin, 144. Motion, ciliary, nature of, 821, 322. Motor end-plates in muscle, 358, 359 ; analogy with electrical organ, 484. Mounting of preparations in microscopic research, 273, 274, 27.5, 276. Movement of cells, 295 ; transmission of, in study of muscle, 394, 397. Mucin, 55, 77, 78 ; chemical composition of, 81 ; chemical reactions of, 81. Mucous connective tissue, 325 ; how to demon- strate, 495. Mulder, on composition of proteids, 58. Mulder-Neubauer, test for glucose, 153. Multipolar nerve cells, 313. MuNK, on creatinin, 95 ; on origin of urea, 87. Murexide test, for uric acid, 101. Muscle, atrophy of, 433, 434, 435. Muscle and tendon, how to study position of, 500; blood of, 357 ; cells, 305-313 ; chemistry of, 501 ; 512 INDEX. coliesion of, 397, 3PS ; coniiectioiiof with tendon, 357 ; consistence of. 3ii7 ; contraction, wiive propagation, 412-il4 ; contraction, remainder of, 412 ; current-', determination of amount of, 449-451 ; currents, direction of, 448 ; duration of contraction of, 412 ; current negative varia- tion of, 452 ; currents, tlieories of, 454-458 ; elasticity of, 398, 399, 400 ; electric apparatus in study of, 3(i3-3Sl ; electrical phenomena of, 430-459 ; ferment, 501, 502 ; fibre, chemical con- stitution of, 300-303; examination of by polarized light, 308 ; formation of, 357 ; gi-owth of, 433, 434, 435 ; how to demonstrate nerve endings in, 500 ; how to demonstrate lamified fibres, 492 ; how to prepai'e for histological purposes, 492 ; inclina- tion currents in, 448 ; involuntary form of, 357, 358 ; involuntary, how to examine, 491 ; irrita- bility of, 4110-405 ; latent period of, 503 ; lymph vessels of, 357; measurement of work done by, 425, 420; metabolism in, 430-432 ; mode of pre- paring for study of structure of, 500 ; motor end-plate of, 358, 359 ; non-striated form of, 357, 35S ; nutritive changes in, 430-432 ; period of contraction of, 407 ; period of relaxation of, 407 ; phenomena of contraction of, 405-414; physical properties of, 397-400; plasma, 300, 301 ; plasma, Halliburton's method of analyzing, 502 ; pro- duction of heat by, 421-424 ; rapidity of contrac- tion of, 412 ; recording of single contraction of, 408, 409; relations of, to nerves, 350-SOO ; serum, constitution of, 301, 302 ; soiind, 427, 428 ; static force of, 42(), 427 ; striated, how to examine, 492 ; structure of, 350-3ii0 ; summarj' of pheno- mena of living, 400, 401 ; telegi'aph, 401 ; ter- mination of nerve in, 358 ; tracings of contrac- tions of, 411 ; work done by, 424-427. JIuscular contraction, amount of, 405, 412, 425 ; dynamical, 29 ; modes of exciting. 418-421 ; observation of, 405, 400 ; fatigue, phenomena of, 428-430 ; fibre, properties of non-striated, 435, 436 ; fibres, figures of, 309, 310, 311, 312 ; smooth, 305 ; transversely striated, 305-313. Jlyeo-protein, 190. Myelins, S3. Myeloplaxes, nature of, 334. Myo-albumose, 502. Myo-epithelial cells, 35C. Myoglobulin, 501. Myograph, the pendulum, 408, 409. Myographs, 390, 391, 392, 393 ; figure of, 28. Myohajmatin, 138, 139. Myosin, 74, 301. Mvosinogen, 501. N Naegeli, on hereditj', 235 ; micellar theory of, 13 ; on schizomj'cetes, 188. Naphthylaraine, 50, 109. Narceine, 89. Narcotine, 89. N.-^ssE, on composition of proteids, 58. Nef.f's interrupter, 373, 374. Negative variation in muscle current, 400, 452. Xen'ckt, on indol, 110. Neox)lastic tyiae of ossification, 340. Xerve cells, 313, 'oU ; how to examine, 493. Xerve endings in muscle, how to prepare, 500. Xerve fibres, 314-317 ; figure of, 315, 310 ; how to examine, 493 ; medullated, definition of, 313 ; how to prepare for histological purposes, 493 ; terminations in muscle, 358 ; muscle prepara- tion, 27, 28, 401 ; nou-medullated, how to pre- pare for histological purposes, 494 ; action of silver nitrate on, 494. Neural canal, formation of, 245, 248. Neurilemma, nature of, 31G. Xcurin, 82, 83. Xewtox, atomic theory of, 8. Nicotine, 89. Nitric-oxide luemoglobin, 124, 125. Nitrogen, presence of in the body, 168. Nitrogenous acids, 50, 97 ; bodies, formation of, 23 ; bodies without oxygen, 56 ; fatty sub- stances, 81 ; substances, 55 ; substances, with- out oxygen, 109. Nitro-glyceriue, composition of, 10 ; instability of, 10. NoBlLi, galvanometer of, 439, 440. NoF-UoRFKEL, thcrmo-electric batteiy, 372. Xon-medullated nerve fibres, 317. Xon-nitrogenous oi-ganic acids, 101. Xon-nitrogenous substances, 57, 140. Non-polarizable electrodes, 380. Non-striated muscle, how to prepare, 500. Notochord, formation of, 248. Nuclear division, diagram of, 228. Nuclei, method of demonstrating, 487. Nuclein, 55, 78. Nutrition of bone. 340 ; of cartilage, 340 ; of con- nective tissue, 340. O Odlinc;, on relations of uric acid, 100. Oersted, discoveries of, 439. Ohm's law in electricity, 365. Olefiant gas and derivatives, 47. Oleic acid, 168. Oleic acid series, 57. Olein, 149. Ontogeny, definition of, 3. OrPLER, on creatinln, 95. Ord, 'William Muller, on the influence of colloids on crystalline form, 17 ; on fonnatiou of bone, etc., 19. Organ current of electrical fishes, 478, 479. Organic compounds of the body, 35, 55 ; com- pounds, classification of, 55 ; composition of, 44 ; constitution of, 50 ; constituents of the body, 43 ; material, formation of, in ijlants, 22. Organisms, life cycles of, 30. Organs, definition of, 292. Ossification, 336-341 ; mode of preparing tissues for study of, 499. Osmosis, causes of, .Sol, 352; in relation to tissues, 347-353. Osteoblasts, nature of, 339. Osteoclasts, nature of, 341. Ova. discoveries of, 219 ; figures of, before and after fecundation, 231 ; origin of, 221, 222 ; female elements, 219 ; mode of examining fresh, 488. Ovaries, structure of, 220, 221 ; examination of, 488 ; figures of transverse section, 220, 221. Ovum, 17 ; development of, 222 ; figure of, 220 ; structure of, 219. Oxalic acid, 100, 107 ; origin of, in the bod}-, 107. Oxalic acid series, 57, 100. Oxaluric acid, 55, 104, 88. Oxidation, in the living organism, 170, 171, 172. INDEX. 513 Oxyhsemoglobin, 120, 121, 122 ; spectruri analysis of, 120, 121, 122, 123. Oxyhsemoglobin crystals, figure of, 119. Oxygen, presence of, in the body, 168. Ozone, influence of, in fermentation, 194. P P.acini, on gymnotus electricus, 46.5, 466, 467 ; on Torpedo Galvani, 462. Palmitic acid, 163. Palmitin, 149. Papaverine, 89. Paracelsus, theory of life, 32. ParaflSn, embedding of tissues, 279 ; infiltration iu section cutting, 282, 283, 284, 285, 286, 287. Paraffins, general formulae of, 46 ; properties of, 46. Paraglobulin, 74. Paralbumin, 55, 76. Paramyosinogen, 501. Paranucleolus, 225. Pasteur, on fermentation, 176, ISO, 181. Pathology, relation to physiology, 3. Pendulum myograph, 408, 409. Pentacrinin, occurrence of, 144. Peptones, 55, 75, 76. Perichondrial ossification, 337, 339, 340. Perichondrium, structure of, 331. Periosteum, structure of, 335, 336. Pettenkofer's test for bile acids, 108. Pfluger, on composition of proteids, 59 ; on heredity, 236 ; on oxidation, 171. Phenol, 16 ; occurrence of, 149. Phosphates in the body, 42. Phosphoglyceric acid, 55, 82. Phylogeny, definition of, 3. Physical properties of connective tissues, 343, 344, 345 ; properties of muscle, 397-400 ; theory of muscle currents, 455, 456. Physics, relation to x)hysiology, 3. Physiological processes in plants, 22. Physiology, definition of, 1 ; means of investigat- ing, 4 ; object of, 4 ; source of its facts, 2. PiCARD, estimation of iron in spleen, 42. Picric acid, composition of, 16 ; test for glucose, 153. PicoT, on physiological action of water, 36. PiCTET, on temperature in fermentation, 192. Pigments, 56, 111 ; of bile, 130; of blood, 56; of the fajces, 136 ; of the tissues, 137 ; of urine, 134 ; physiological significance of, 145, 146 ; spectroscopic detection of, 111, 112. PioTRowsKi's reaction, 64. Piria's test, 93. Plasm cells, 496. Ploesz, on glycerine, 148. Pohl's commutator, 378, 379. Polar body extruding, figure of, 227 ; extrusion of, 225-230 ; extrusion, discoveries of, 226 ; for- mation of, 225-230. Polarimeters, 68, 70. Polarizable electrodes of Du Bois-Reymond, 379 380. Polarization, 66 ; currents in torpedo, 479, 480. Polymerism, definition of, 48. Polymerization, 23. Porret's experiment, 397. Potassium chloride, quantity in the body, 38. Potassium salts in the blood, 39. Potential, electrical, 363. Preparation of organs in research, 263-278 ; of tissues in research, 263-278. Preservation of preparations in microscopic re- search, 273, 274, 275, 276, 278. Pressure, influence of, in fermentation, 194. Prevost, on fecundation, 223. Preyer, on carbonic-oxide hsemoglobin, 124; on hemoglobin, 118; on hsematoidin, 129; on estimation of oxyhsemoglobin, 122 ; on oxy- hEemoglobin, 120, 122. Primary bones, development of, 337-340. Primitive groove, origin of, 248 ; streak, origin of, 248. Products of oxidized bile pigment, 133. Pronucleus, female, 231 ; male, 231. Properties of non-striated muscle, 435, 436. Propionic acid, 163. Protagon, 82 ; formation of processes of, 19 ; pro- perties of, 19. Proteids, 55 ; animal and vegetable, table of, 71 ; living, action of, 63 ; chemical action of, 61 ; chemical, 57, 64; decomposition of, 59; phy- sical characters of, 64 ; physiological characters of, 71 ; quantities of, in tissues and fluids, 71 ; special, 72 ; synthesis of, 61. Proteins, 55, 75. Prothyalosome, 229. Protoplasm, 14, 288-292 ; effects of external agents upon, 290, 291, 292 ; source of vital energy of. 24. Proximate principles, 35 ; characteristics of, in living things, 15 ; definition of, 15. Pseudo-cerebrin, S3. Pyrrol, 56, 111. Quinine, R Rabl, on karyokinesis, 228. Radicles, organic, table of, 162. Raia batis, electrical organ of, 470, 471. Eainey, experiments of, 17; on formation of shells, bone, etc., 17 ; on deposition of car- bonate of lime in presence of a viscid substance, IS ; on formation of shells, bone, etc., IS. Rajewski, on alcohol, 146. Ranvier's cells in tendon, 327 ; nodes of, in nerve, 316 ; on Torpedo Galvani, 464. Ray Lankester, on chlorocruorin, 143; on chloro- phyll, 142, 143. Reactions, chemical, in the living organism, 170. Reagents required in microscopic research, 261, 262, 263. Recording cylinder in measuring time, 384, 385. Reduction, chemical, in the living organism, 172, 173. Reid, on muscular irritability, 403. Relaxation of muscle, period of, 407. Remak, band of, 315. Renson, on origin of spermatozoa, 217. Reproduction of cells, 296, 297. 2 K ol-t INDEX. Residual contractions of muscle, 407; contraction, as a function of muscle, 4'25. Reticular tissue, structure of, 32S. Retina, how to demonstrate pigment cells of, 491. Reymosd, see Du Bois-Reymond. Rheochord of Du Bois-Reymond, 4ol, 45.;. Rheotome, 409. Rhodophane, 141. Rhodopsin, spectrum of, 141. Richardson, on physiological action of water, 3(i. Rigor mortis, phenomena of, 432, 433. RiTTER, on blue pigment of bile, 133 ; on decom- position of urea, 40. Roberts, Sir William, estimation of glucose, 15 J. Rosenthal, on muscular irritability, 402. RuMFORD, on he.at, 11. Rutherford, on electric stimuU, 418; on growth of muscle, 434 ; microtomes of, 279, 280, 281. Saccharimeter, estimation of glucose by, 153, 154, 155 ; figure of, (iS ; Soleil's, (59. Saccharomycetes cerevisiae, 185. Saccharoses, 158. Sachs, on electric shocks of gymnotus, 473, 474. St. George, on origin of sjiermatozoa, 217. Salkowski, on cholesterin, 147; on oriarin of urea, 86, 87 ; on origin of uric acid, 103 ; on urine, 40. Salts, metallic, of organic acids, 49 ; of organic acids in body, 56. Sarcin, 103. Sarcolactic acid, 165. Sarcolemma, nature of, 306. Sarcosin, 56, 97. Sarcous elements of muscle, 307. Savi, on Torpedo Galvani, 462. Sea urchin, division in egg of, 233, 234. Secondary bones, development of, 341. Secreting epithelial cells, figure of, 297 ; gland cells, figure of, 298. Secretion, cellular, nature of, 295 ; in cells, pheno- mena of, 297, 298. Section cutting, celloidin in, 287 ; ether, freezing in, 282 ; paraffin, infiltration in, 2S2, 283, 284, 285, 286, 287. .•Sections, preparation of, 269, 270. Segmentation and development of fowl's egg, 246, 252 ; cavity, f onnation of, 244. Semiglutin, 79. Seminal stains on linen, mode of examining, 488. Sensibility of bone, 346 ; of cartilage, 346 ; of connective tissue, 346. Serial section cutting, 278-287. Sertoli, on origin of spermatozoa, 216, 217. SCHAFER, on cellulose, 160, 161. Schafer, E. a., on muscular fibre, 310. Scherer's test for inosite, 158. ScHiFF, on mechanical stimuli, 419. Schizomycetes, cultivation of, 194-200 ; develop- ment, effects of external influences in, 191, 192, 193, 194 ; figure of, 188 ; multiplication of, 190. Schmidt, on fermentation, 176. Schmiedeberg, on hippurio acid, 106 ; on origin of urea, 87. ScHONBKiN", on oxidation, 171. Schorlemmer, on carbon compounds, 54; on organic compounds, 44. ScHRtEDER, on fermentation, 179. ScHULZE, on cholesterin, 147. Scuoltzes, on origin of urea, 86. ScHUNCK, on indican derivatives, 135. Schutzenbkroer, analysis of yeast by, 177 ; on naphthylamine, 109 ; on proteids, 58. Schwann', on fermentation, 177, 178, 179 ; white substance of, in nerve, 315. Sharpey, fibres of, 336 ; fibres, how to demon- strate, 498. Shells, formation of, 17. Shepard, on hippuric acid, 105, 106. Signal of Deprez, 389. Silver nitrate, action on nerves of, 494. Simony, on bilif uscin, 132. Sinapine, 89. Skatol, 56, 111. Smee's voltaic element, 371. Sodium chloride, quantity of in the body, 38 ; salts of in the blood, 39. Soleil's saccharimeter, 69. Somatopleure, origin of, 249. SoRBY, on aphidein, 144, 145. Spallaszani, on fecundation, 223 ; on oxidation, 171. Spectropolarimeter, figure of, 154. Spectroscope, direct vision, arrangement of prisms in, 114 ; arrangement for detection of pigments by, figure of, 112. Spencer, Herbert, reference to, 21 ; definition of life by, 32. Spermatoblasts, examination of, 488. Spermatin, 55, 78. Spermatogenesis, figures of, 216, 217. Spermatozoa, 217, 218, 219; discoverers of, 214, 215; nature of, 214-219; development of, 215, 216, 217; figures of, 218, 219; influence of on ovum, 223, 224 ; living, examination of, 488. Splanchnopleure, origin of, 249, 250. Spinal cells in connective tissue, 495. Staedeler, on bilihumin, 133 ; on biliprasin, 132 ; hajmatoidin, 129 ; on phenol, 149. Stahl, theory of life by, 32 ; on fermentation, 179. Staining, diffusive, 271 ; fluids, 270, 271, 272, 273 ; in mass, 271, 272 ; in microscopic research, 270, 271, 272, 273 ; of chromatin, 271 ; of nucleus, 270, 271. Starch, 159 ; formation of, 23 ; tests for, 159. Stearic acid, 163. Stearin, 149. Stercobilin, 136, 137 ; spectrum, analysis of, 136, 137. Stereoplasm, 235. Stimulus, contraction as a function of, 424, 425 ; liminal intensity of, 424. Stirling, W., on genesis of tetanus, 417. Stokes, on the spectrum of blood, 116, 117. Stokvis, on product of oxidized bile pigment, 133, 134. INDEX. 515 storage cell, electrical, 373. Strasburger, on heredity, 23T, 238, 239 ; on im- bibition, 13 ; on polar bodies, 225. Strassburg's test for bile acids, 108. Structure, physical of living things, 13. Strychnine, 89. Substantia compacta of bone, 333 ; spongiosa of bone, 332. Succinic acid, 107 ; origin of, 167. Sucroses, 57. Sulphates in the body, 43. Sulphocyanic acid, 56, 97. Sulphuretted hydrogen, 170. Syntonin, 75. Synthesis, in the living organism, 17-1. 175. Tait, on atomic theory, S; on energy, 11 ; on re- cent advances in xjliysical science, 10. Tambours, Marey's, 394-397. Taurin, 56, 96 ; crystals, figure of, 96. Taurocholate of sodium, 107. Taurocholic acid, 56, 107. Telephone in animal electricity, 445, 446. Temperature, influence of, in fermentation, 191, 192, 193. Tendon, how to examine, 496 ; structure of, 326, 327. Tension, in production of heat in muscle, 422. Testis, figure of, section of, 215 ; histological examination of, 4S7, 488. Tetanus, curve of, 415 ; genesis of, 414-418 ; muscular nature of, 414. Tetronerythrin, 144. Thebaine, 89. Theca f olliculi, 222. Theine, 89. Theobromine, 89. Thermal stimuli in muscular contraction, 419. Thermo-electric battery, 372. Thomson, Sir William, galvanometer of, 442 ; vortex atom, hypothesis of, 9. Thudichum, on luteins, 139; on phosphorized bodies, 83 ; on urine pigments, 134 ; on uromelanin, 136 ; on urochrom, 136 ; on crypto- phanio acid, 106. Time, contraction as a function of, 425 ; measure- ment of, in study of muscle, 384, 389 ; measure- ment of, 5. Tissues, complex, definition of, 292 ; origin of, 214-234 ; the physiology of, 201 ; pigments of, 137 ; simple, definition of, 292. Tone-inductorium in tetanus, 417. -Tonicity, muscular, 400. Torpedo Galvani, -162-465; mode of electrical action of, 481 ; secondary discharges from, 483. Transmission of movement in muscle study, 394, 397. Treviranus, definition of life by, 31. Tributyrin, 151. Tricaprin, 151. Tricaproin, 151. Tricaprylin, 151. Trimethylamine, 56, 109. Trimargarin, 151. Triolein, 151. Tripalmitin, 150. Tristearin, 150, 151. Trivalerin, 151. Trommer's test for glucose, 152. TubuU seminiferi, histological examination of, 487 ; figure of, 215, 216. Tunica granidosa, 222. Turacin, 144. Twitch, muscular, nature of, 414. TrNDALL, on temperature in fermentation, 193. TjTian purple, occurrence of, 145. Tyrosin, 16, 55, 93 ; crystals, figure of, 93 ; tests for, 93. U Urate (acid) of ammonia, 101 ; of lime, 101 ; of lithium, 101 ; of potassium, 101 ; of sodium, 101. Urates, 101. Urea, 16, 55, 85 ; constitution of, 84 ; crystals, figure of, 84 ; fermentation of, 85 ; nitrate of, crystals of, figure of, 85 ; occurrence of, 84 ; origin of, 86, 87 ; oxalate of, figure of crystals, 85 ; quantity eliminated of, 84. Uric acid, 97, 98, 99, 100, 101, 102 ; derivatives of, 56 ; occurrence of, 100 ; origin of, 102, 103 ; salts of, 101 ; substances related to, 103 ; tests for, 101, 102. Urine, pigments of, 56, 134. Urobilin, 134, Urochrome, 136. Urohsematin, 136. Uromelanin, 136. Urohasmatoporphyrin, 136. Valektls, on lime salts in tissues, 41. \ks Beseben, on fecundation, 228-230. Van Helmont, on fermentation, 176. Van Mansveldt, on elasticity of muscle, 399. Vapours, effect of, on protoplasm, 291, 292. Variability, definition of, 31. Vater, corpuscles of, 336. VA0QUELIN, on spermatin, 78. Vln'es, physiology of plants, 23 ; on molecular force, 14 ; on polar bodies, 226. Vital action, 16. Vitality, definition of, 33. Vital properties of connective tissues, 346, 347. VitelUn, 73, 74. Vitelline membrane, 246. VoiT, on creatinin, 95 ; on origin of urea, 87. VoLEMANN, on elasticity of muscle, 399. VoLTA, discoveries of 437,-438, 439. Voltaic element, 364, 369-373 ; pUe, discovery of, 439. Volume, measurement of, 7. Volumetric estimation of glucose, 155, 156. Von Helmboltz, see Helmholtz. Von Wittich, on cell movement, 296. W Wagner, on colloid matter, 77. Waller, on muscular fatigue, 430. 51 G INDEX. Waltek, on origin of nrc:i, ST. Water, influence of, in fermentation, 193 ; i>hysio- logical action of, 3() ; proportion of, in the body, tissues, etc., 35, 36. Watts, on polarized light, (i'.i. Wax embedding of tissues, 27'.'. Weber, paradox of, 399 ; on cohesion of muscle, 397 ; on elasticity of muscle, 39S, 390. Weight, measurement of, .5. Weismank, on heredity, 239, 240, 241. Wertheim, on elasticity of tissues, 345. White fibro-cartilage, 330 ; how to examine, 497. WiLLi.\iis, microtome of, 281. Witthaus, on acid urate of lithium, 101; on cholesterin, 147 ; on glycerine, 148 ; on oleic acid, HiS ; on sarcin, 103 ; on taurocholic acid, 107. WoliLEK, on oxalic acid, lii7. Work, definition of, 10 ; done by muscle, 424-427 ; done by muscle, measurement of, 425, 426 ; done in production of heat in muscle, 422, 423 ; measurement of, 10. WouM-MuLLER, analysis of nuclein by, 78. WuNDERLiCH, On production of heat in muscle, 424. WuNDT, on elasticity of muscle, 399. X Xanthin, 103, 104. Xanthophane, spectrum of, 140. Xantho-proteic reaction, 64, 72. y Yeiist, analy^^is of, 177. Yellow fibro-cartilage, how to examine, 497. Yeo, G. F., on latent period of muscle, 503. Young, on the modulus of elasticity, 345. YirsG, on temperature in fermentation, 192. Zaleski, on creatiniu, 95 Zoogloea, 190. END. GLASGOW : PRINTED BV ROBERT MACLEHOSE, UNIVERSITY PRESS. i MM ■mm ■mm. ■ ■ ■■ : ; ■ 1 ,1 1 i( il : -^^a^