!%m •;te' >c 151 m m ~r- ;& m CORNELL UNIVERSITY THE r.osiucll $. Wlttmitv Cibrars THIS BOOK IS THE GIFT OF ^asjoxlJWu^ , ...o#fv6w Saa^sJcoisU. .«&..•&*. .^oauS^... Digitized by Microsoft® Cornell University Library QH 231.F53 Notes on technique. 3 1924 000 297 758 Digitized by Microsoft® This book was digitized by Microsoft Corporation in cooperation with Cornell University Libraries, 2007. You may use and print this copy in limited quantity for your personal purposes, but may not distribute or provide access to it (or modified or partial versions of it) for revenue-generating or other commercial purposes. Digitized by Microsoft® «/A eprinted from the Transactions of the American Microscopical Society, i8q6. NOTES ON TECHNIQUE. PIERRE A. FISH, D. Sc, Cornell University, Ithaca, N. Y. In many of the modern articles, the methods by which certain pathological structures are demonstrated, if mentioned at all, are frequently so meager in the description of import- ant details as to be practically useless to many workers, unless a certain amount of their time is d'evoted to experimentation. A person, who has obtained fairly successful* results with his older methods, is loath to forsake them, especially if his first few attempts with the new are failures. Each investigator may have certain laboratory conveniences ; reagents of the best quality and dyes that have been well tested, all of which will enable him to obtain results much superior to his less fortunate colleague. It is difficult, therefore, to work suc- cessfully unless details are carefully attended to, and the reasons for the various steps understood. The methods fol- lowing have been well tested, and have been attended with uniformly good results, which in some cases, it is belieyed, would have ended in failure with the older methods. FIXATION. The fixation of pathological tissues, with strong alcohol for histological study, is very commonly employed for the double purpose of killing at once any microorganism that may be present and at the same time to preserve the structure of the part. With many tissues this caused a too rapid withdrawal of the contained water or lymph, so that the specimen becomes hard and gives unsatisfactory results when it comes to the cutting process. Some experiments with different reagents, upon known pathological material, were of service in formulating a mix- Digitized by Microsoft® 2 PIERRE A. FISH : ture, which obviated the defects of strong alcohol when used alone. This mixture, while quickly killing the bacteria, also preserves most faithfully the histological structure. Various solutions of formalin, including the undiluted, were employed, and gave good results, particularly the presentation of the bacteria, after the usual staining methods. The tissues were more or less swollen by the weaker solutions, in marked con- trast to the contraction caused by alcohol. Various combi- nations of formalin with alcohol were also tried, and that which seemed to be most completely satisfactory for quick penetration and convenience, bacteriologically and histologi- cally, was as follows : 95 per cent, alcohol ioo parts. Commercial formalin (40 per cent, formic aldehyde) . 10 parts. Pieces of tissue, ^ centimeter square, are well fixed in from twelve to twenty-four hours, after which it is well to leave for a few hours in 95 per cent, alcohol before clarifying for the paraffin bath. Specimens, transferred directly from the fixing mixture, have been clarified in chloroform or cedar oil, but it requires a longer time. The addition of the formalin is advantageous, because in a way it brings about a state of equilibrium. The alcohol alone shrinks the tissue, while on the other hand formalin swells it, so that in this respect the one reacts against the other. ADHESION TO THE SLIDE. After the infiltration and imbedding of the tissue in paraf- fin, the question of the treatment of the sections is one of some importance. If they are to be carried through a series of reagents in watch glasses, and not placed upon the slide until they are mounted, the sections must necessarily be rather thick, in order to withstand the manipulation. Very much thinner sections, if adherent to the slide, and conse- quently supported by it, can be carried through the different steps of the process without injury, and show the structural elements to much better advantage. Digitized by Microsoft® NOTES ON TECHNIQUE. 3 The albumen or collodion adhesive, usually employed for this purpose, however, possesses the disadvantage of taking the aniline colors used in bacteriology, sufficiently to disfigure the preparations. If a clean slide be coated with a thin film of glycerine and then rubbed very nearly dry with a cloth or the hand, and a drop or two of 35 per cent, alcohol be placed upon it, the section, if curled, will tend to flatten itself when placed on the alcohol. If the slide now be placed in a thermostat for a few hours, at a temperature near the melting point of paraffin, the heat will cause any wrinkles or irregu- larities of the section to disappear ; the alcohol slowly evaporates and when the slide is thoroughly dry the albumen molecules of the tissue adhere quite firmly to the slide, as noted by Gaule. After this the slide may be heated gently over a flame until the paraffin begins to melt. If any mois- ture remains the section will be quite likely to loosen during the latter stages. Thick sections do not adhere so firmly as thin ones. The slides may then be immersed in a jar of tur- pentine or any solvent of paraffin and carried through the various grades of alcohol to water. A shorter method, in which there is as firm adhesion of the section to the slide, is to bring the slide in contact with aniline oil for a few minutes after the treatment with the tur- pentine, absorbing the superfluous turpentine with filter paper. The aniline oil is also removed by means of filter paper. The section is then thoroughly washed in distilled water which removes the oil, and the tissue is then stained and washed in water. If aniline stains are used, a hurried rinsing is sufficient. Drain or absorb the water and again apply the aniline oil. Besides clearing the section the oil tends to remove the aniline stain and care must be exercised in not letting this process go to far. Displace the aniline oil with xylol and mount in balsam. The color ought not to fade if the aniline oil has been thoroughly removed. With certain stains, or combinations of them, the aniline oil may not succeed in preserving the sharp definition of the color. Under such conditions the section, after staining, Digitized by Microsoft® 4 NOTES ON TECHNIQUE. may be treated directly with absolute alcohol to dehydrate and remove any superfluous stain. Some aniline dyes are not as soluble in absolute alcohol as in the weaker grades. Clear in xylol and mount in balsam. The use of aniline oil in the treatment of the sections will be recognised as having been recommended by Weigert for bacterial purposes. It likewise gives most excellent results in ordinary histological work and is a saving of time and- material. MOUNTING. Many valuable specimens are ruined for the want of suf- ficient precaution in the preparation of the balsam. In its commercial state it contains many volatile principles and traces of acids, which, in the course of time, act upon the specimen and diminishes or entirely removes the color. All this may be lessened, if the balsam be heated sufficiently to drive off the volatile constituents, or more thoroughly obvi- ated if a little potassium carbonate or mild alkali be added to neutralise the acid just before the balsam is heated. When the balsam becomes hard it can be broken into flakes and stored. When wanted for use dissolve in xylol to the desired consistency and filter through absorbent cotton. Specimens stained with the Biondi-Ehrlich mixture (which fades so ■easily) have at the end of a year shown no signs of losing their pristine clearness. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® «y vi?. ;:f*£-a*S' w£ft