DS 21.16-DS 21.20 DYE STAINS OF SPECIAL APPLICATION 



395 



21.16 Unna 1910 safranin-anilin-blue-tannin Ehrlich, Krause, et al. 1910, 247 



REAGENTS REQUIRED: A. 1% safranin; B. water 50, anilin l)lue 0.5, water 50, tannin 15 

 method: [sections of ale. fixed material] —* A, 10 mins. — > water, thorough wash — > B, 15 



mins. — » water, rinse —> abs. ale., till color clouds cease -^ balsam, via xylene 

 RECOMMENDED FOR: demonstration of collagen fibers in alcohol-fixed material. 

 result: collagen, blue. 



21.16 Verocay 1908 test. 1948 Romeis Romeis 1948, 351 



reagents required: A. \% chromic acid; B. DS 11.122 Delafield 1885 

 method: [paraffin sections] —> water —* yl, 24 hrs., 46°C. — * wash — > B, 



wash — > counterstain, if desired -^ balsam, via u.sual reagents 

 recommended for: collagen fibers. 



21.2 Special Stains for Nervous Tissues 



1-2 hrs. 



It has become so customary today for 

 all nervous tissues to be stained by one 

 of the metal-staining techniques that it is 

 a surprise to many histologists to learn 

 that excellent methods exist for dye- 

 staining these structures besides those of 

 the classic "Weigert" methods given in 

 division 21.212 l)elow. These stains for 

 nervous tissues are divided into those 

 destined to stain the neuroglia, which, 

 though they cannot, of course, properly 

 be called nervous tissues, are so closely 

 associated with them that the staining 

 technique must be kept in the same place. 



21.20 TYPICAL EXAMPLES 



Preparation of a transverse section of 



the brain of a frog using the stain 



of Bethe 1896 



The method of Bethe (DS 21.211 Bethe 

 1896) for the preparation of class-teaching 

 material or demonstration slides from the 

 brains of the lower vertebrates is one of 

 the best arguments against rejecting a 

 technique for the mere reason that it is 

 obsolete. The process is rapid and simple, 

 yet it yields as good images as can be ob- 

 tained by many of the metal-staining 

 techniques, which would involve weeks of 

 work, and which are capricious and un- 

 rehable in the extreme. The technique de- 

 pends essentially upon the fact that solu- 

 tions of methylene blue are differentially 

 absorbed, in a living animal, into the nerve 

 cells and processes of the brain. The dye is 

 fixed in this position with the aid of am- 

 monium picrate, used also as a fixative of 

 the tissues, and finally precipitated in 

 place with the aid of either sodium or 

 ammonium molybdate. A frog is an easy 



animal on which to experiment, not only 

 because it is readily available, but also 

 because of the simplicity with which the 

 brain may be removed from the cranium. 



It is necessary to start wdth a saturated 

 solution of methylene blue in'any standard 

 physiological saline solution, but it need 

 not be sterile since the animal is going to 

 be sacrificed shortly after the application. 

 It is simpler and pleasanter to work on an 

 anesthetized frog, and though any anes- 

 thetic may be used for this purpose, the 

 writer's choice for all amphibian anes- 

 thetization is tricaine methanosulfonate, 

 more frequently known under its trade 

 designation of M2.2.2. Tliis should be pre- 

 pared as a 0.2% solution in physiological 

 saline, and the frog placed in about a liter 

 of the solution. The frog will become non- 

 receptive to stimuli after about ten min- 

 utes and is unlikely to die even upon an 

 exposure of some hours. 



As soon as the frog is thoroughly anes- 

 thetized, fill a 5-milliliter hypodermic 

 syringe with the saturated solution of 

 methylene blue in physiological saline, 

 and inject about 1 milhhter into the ab- 

 dominal cavity. Take care not to damage 

 the internal structures. It is simplest to 

 pick up a fold of the frog's skin with the 

 fingers and to insert the hypodermic 

 syringe into the length of the fold, parallel 

 to the main axis of the body. The point of 

 the syringe should be watched carefully at 

 the beginning of the injection to make 

 sure that it has not merely slipped under 

 the skin, but has actually penetrated 

 through the muscular layers into the 

 coelom. The needle is then withdrawn and 

 the frog returned to its anesthetic saline. 

 After three or four minutes a slight blue 



