390 Transactions of Hie Society. 



XIII. — On the Douhle and Treble Staining of Animal Tissues for 

 Microscopical Investigations; with a Note on Cleaning Thin 

 Cover-glasses. By Heneage Gibbes, M.B., F.K.M.S. 



(Read 10th March, 1880.) 



While engaged last year in an examination into the structure of 

 the Vertebrate spermatozoon, I tried the effect of a large number of 

 staining agents, and succeeded at last in staining the head and 

 body of the spermatozoon of Trito7i cristatus different colours, 

 showing thereby a different chemical reaction. This led me to try 

 these stains on sections of animal tissues, and the specimens under 

 the Microscopes will show with what results. It may be interest- 

 ino- to give a few details of the different processes I have used, 

 in the hope that some who have more leisure than myself may 

 work out the subject thoroughly. 



The first double stain to be mentioned is the well-known 

 picrocarmine and logwood, which gives very good results in 

 sections of skin and other parts. 1 have also found it answer 

 better than any other stain in an investigation into the develop- 

 ment of spermatozoa, in which I am at present engaged with 

 Dr. Klein. 



There is one point which greatly facilitates a good result with 

 this process— that is, after staining the sections in picrocarmine to 

 place them in plain water acidulated with a few drops of acetic or 

 picric acid for an hour before staining with logwood ; they take the 

 second stain better and do not fade afterwards. 



The next process consists of — 



1. A solution of carmine and borax. 



2. A mixture of hydrochloric acid and absolute alcohol. 



3. A solution of indigo-carmine. 



The carmine solution is prepared by mixing 



Carmine Sss 



Borax 5U 



Aqua 



^lY 



o 



and pouring off the clear fluid. It must not be filtered. Three 

 or four drops of this solution are placed in a watch-glass, and the 

 sections immersed for a few minutes ; they are then removed to a 

 mixture of hydrochloric acid and absolute alcohol, one part of 

 the acid to twenty parts of alcohol, and allowed to remain there 

 until they take on a bright rose colour; this happens in a few 

 seconds. They must then be washed in methylated spirit several 

 times to remove the acid, when they will be ready for staining with 

 the indigo-carmine, which is prepared in the following manner : — 

 A saturated solution of indigo-carmine is made in distilled 



