324 



METHODS AND FORMULAS 



DS 12.20 



or if handled with undesirable reagents in 

 any stage of the proceedings. It is there- 

 fore recommended that the following de- 

 scription be followed rather closely, for it 

 can be adapted almost without variation 

 to any other heavily muscularized tissue 

 which it is desired to stain. 



As this preparation is intended to show 

 only the gross histological elements pres- 

 ent, it is unnecessary to specify the man- 

 ner in which the rat should l)e killed; but, 

 the sacrificed animal may be used also for 

 the staining of taste buds in the posterior 

 lobe of the tongue by the method de- 

 scribed in some detail in Chapter 23, 

 "Demonstration of the nerve ending in 

 taste buds by the method of Bielschowsky 

 1914," in which case the rat had better be 

 killed by a blow on the head rather than 

 by an anesthetic. 



The tongue may be easily removed by 

 severing the articulation of the lower jaw 

 and by removing this together with the 

 adherent tongue which may be detached 

 with a short scalpel or cartilage knife. A 

 portion of the tongue approximately 5 

 mm. in length is now cut off and placed 

 in a large volume of the selected fixative. 



Though opinions vary widely as to the 

 most desirable fixative to use for muscular- 

 ized tissues, it may be said at once that 

 no alcoholic solution and no solution con- 

 taining picric acid or osmic acid can be 

 recommended. The author's choice would 

 be for the cupric-acetic-phenol formula of 

 Behrens 1898 (Chapter 18 F 4000.0010), 

 which he has employed most successfully 

 on a variety of heavily muscularized tis- 

 sues. This formula would also provide an 

 excellent prior-mordanting for the stain- 

 ing techniques which follow. Whatever 

 formula is selected, however, a large 

 volume (about 100 cc. for the piece of rat 

 tongue described) should be employed 

 and permitted to act for no longer than is 

 necessary to secure the complete impreg- 

 nation of the tissues. When the piece has 

 been successfully fixed it must be washed 

 overnight in running water and then de- 

 hydrated. The process of dehydrating, 

 clearing, and embedding is the point at 

 which most n^usculurized tissues become 

 unmanageable. Nothing, of covn-se, can 

 counteract the effect of improper fixation, 



but even with good fixation the utmost 

 attention must be paid to the selection of 

 dehydrating agent and clearing agent, and 

 to the temperature at which the embed- 

 ding takes place. It has been the experi- 

 ence of the writer that the newer substi- 

 tutes for alcohol in dehydrating tend to 

 harden or render brittle muscular tissue 

 to a greater extent tlian does the more old- 

 fasliioned method of using ethanol. There 

 is little choice in the matter of clearing 

 prior to embedding, for it has been found 

 by numerous workers that benzene has a 

 less hardening effect on muscular tissue 

 than have other agents. 



Unless it is desired to cut very thin sec- 

 tions, a wax of no higher melting point 

 than 52°C. should be emploj^ed. It may be 

 stated categorically that should the tem- 

 perature be permitted to rise above 56°C., 

 it would be better to throw the prepara- 

 tion away than to waste time endeavoring 

 to section it. Paraffin sections are now cut 

 from the block by the standard method, 

 then flattened and attached to a slide by 

 either gelatin or egg albumen. It is recom- 

 mended that as soon as the sections are 

 flattened, they should be pressed to the 

 slide with a piece of wet filter paper, rolled 

 into position with a rubber roller, and 

 dried with the maximum possible speed. 



As soon as they are dried, the sections 

 are deparaffinized l)}^ the usual techniques 

 and taken down to distilled water, where 

 they may remain until one is ready to 

 stain them. Coelestin blue B as the nuclear 

 stain is selected in this instance because 

 the contrast of muscularized tissues is 

 better brought out with the aid of a picro- 

 contrast than by any other method. These 

 picro-contrasts are, however, so acid that 

 hematoxylin-stained nuclei are often de- 

 colorized in the course of counterstaining. 

 Any of the oxazine formulas (DS 11.41 

 above) may be chosen, the writer's prefer- 

 ence being for the first (DS 11.41 Anony- 

 mous 193G). The sohition presents no 

 difficulties of pi'eparation and need not be 

 rejected if it shows a shght precipitate at 

 the bottom. The sections are passed di- 

 rectly into it from the distilled water and 

 allowed to remain until an examination 

 under the low power of the microscope 

 shows the nuclei to be clearly and deeply 



