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IX.— Two Valuable Methods of 

 Staining in Bulk and Counter- Staining. 



By E. J. Sheppard. 



Read April 17, 1918. 



A METHOD which will materially cut short a lengthy process such 

 as Heidenhain's iron-htematoxyliu staining, and, at the same time, 

 give as good results, should be of value to either histologist or 

 cytologist ; either of the following methods here described does 

 this, and for the first I claim some measure of originality. 



My work has been confined chiefly to animal and insect tissues 

 for the study of spermatogenesis, but there is reason to believe that 

 the methods are equally applicable to vegetable tissues. 



1. Details of the actual staining must be prefaced by particulars 

 of a fixation method which, in my opinion, excels that of fixing 

 with riemming's chromo-osmic-acetic mixture. 



The formula* for the fixative (a modification of Bouin's) is as 

 follows : — 



Picric acid, saturated sol. in water . 75 c.cm. 

 Formol 40 p.c. . , . . 25 „ 



Acetic acid glacial . . . . 5 ,, 

 Chromic acid . . . -. . I'Sgrms. . 

 Urea ...... 2-0 ,, 



And should be used warm (38° C) in the case of tissues from warm- 

 blooded animals, and cold, or at an ordinary room temperature, for 

 cold-blooded animals such as the frog, etc., the time being regulated 

 according to the size of the tissue. Roughly the rate of penetration 

 at 38° C. upon an object, such as the testis of a rat, is about 

 one- eighth inch per quarter hour, and when used cold, or at room 

 temperature, slightly slower. Tissues usually float for some time, 

 and complete penetration has not taken place until they sink. 



When fixation is complete remove the objects and wash in 

 repeated changes of 70 p.c. alcohol, until no further yellow colora- 

 tion of the spirit (due to the picric acid) takes place. This usually 

 takes a day or two. When ready transfer to methylated spirit full 

 strength, and allow to remain for forty-eight hours. This com- 

 pletes the hardening process. 



Graduate back to distilled water by stages such as 70 p.c, 



* R.M.S. Journal, June, 1917, page 347, under " Improved Technique for 

 showing Details of Dividing Cells.'' 



