STAINING ENTIRE OBJECTS 491 



until they are thoroughly penetrated (for days if necessary). They 

 are then put into alcohol of 70 per cent, acidified with from four to 

 six drops of hydrochloric acid for every 100 c.c. of the alcohol. The 

 acid alcohol at once begins to remove the excess of colour from the 

 objects, which may be seen to give it off in rosy clouds. They 

 remain in it until the colour no longer comes away freely and they 

 have exchanged their primitive opaque red coloration for a brilliant 

 transparent coloration. This may require days (the acid alcohol 

 should be changed frequently). 



The staining is now complete, and the objects arc washed in pure 

 neutral alcohol, cleared and mounted in balsam or any other desired 

 medium. The result is a brilliant nuclear stain, quite permanent. 

 The process must not be used for objects containing calcareous 

 elements that it is desired to preserve. 



For delicate objects, and for very impermeable objects, it may be 

 well to increase the proportion of 70 per cent, alcohol in the 

 solution ; the proportion of alcohol may be brought up to about 

 50 per cent., but should not exceed 60 per cent, in any case. 



This process is an example of what is known as regressive or 

 indirect staining ; the objects are first overstained in the carmine 

 solution, and the excess of stain is then removed to the required 

 degree in the acid alcohol. 



If, as is frequently the case, especially in studies on the 

 Arthropoda, a still more highly alcoholisecl stain be desired, Mayers 

 alcoholic cochineal may be tried. Cochineal in coarse powder is 

 macerated for several days in 70 per cent, alcohol. For each 

 gramme of the cochineal there is required 8 to 10 c.c. of alcohol. 

 Stir frequently. Filter, and the solution is ready for staining. 



The objects to be stained must previously be well imbibed with 

 70 per cent, alcohol. They may remain for almost any length of 

 time in the staining bath. After staining they are washed in 

 70 per cent, alcohol, which is frequently changed until it takes up no 

 more colour from the objects. Overstating seldom happens : it 

 may be corrected by means of 70 per cent, alcohol containing 1 per 

 <3ent. of acetic acid or T ^ per cent, of hydrochloric acid. 



Small objects or thin sections are stained in a few minutes ; large 

 objects require hcurs or days ; a nuclear stain, either red or blue, 

 according to the chemical composition of the tissues stained. It 

 does not succeed with all objects. The best stains are obtained with 

 objects that have been prepared with chromic or picric acid 

 combinations, or with absolute alcohol. Osmic acid preparations 

 stain very weakly unless they have been previously bleached. All 

 acids should be carefully washed out of the objects before staining. 

 The stain is permanent in oil of cloves and balsam. 



Kleinenberg 1 s Alcoholic, Hftimatoxylin, once very much vised, is 

 highly irrational and very inconstant in its composition and its 

 effects, and is now with reason generally abandoned. 



Nuclear Stains for Sections. Any of the foregoing stains may 

 of course be used for sections if desired. But in many cases other 

 stains are indicated, as being more powerful, or more precise, or of 

 a richer selectivity. 



