Stains and Staining 85 



soluble in water or alcohol but is readily soluble in strong alkalis (e.g., 

 ammonium hydroxide) and slightly soluble in weak alkalis (e.g., borax) 

 or weak acids (e.g., acetic or propionic). Carmine staining solutions are 

 no longer used for sections but are widely employed as indirect stains for 

 wholemounts or as direct stains for squash preparations. 



Indirect Carmine Staining. Both of the carmine formulas given below 

 can be used as direct stains if they are diluted several hundred times 

 with an alum solution. It is not safe to dilute them with distilled water, 

 for there is a risk of the carmine precipitating out, particularly within 

 the cavities of whole animals, from which it is almost impossible to re- 

 move it subsequently. The best known of all the carmine nuclear stains is : 



Grenacher's Alcoholic Borax Carmine: 



Staining solution 



Boil together 10 g of borax and 8 g of carmine in 250 ml of water for 

 about 30 min. Cool overnight and filter. Evaporate the filtrate to dryness 

 and then store this powder, labeled as "Borax Carmine Powder." This 

 powder may be dissolved in any strength alcohol from 30 per cent to 

 70 per cent; in all cases a saturated solution should be employed. This 

 is the original method of making up the solution and is undoubtedly 

 the best. 



For those, however, who do not wish to take the time for the later 

 preparation, a working solution may be made directly in the following 

 manner: 



Boil 2 g of borax and 1.5 g of carmine with 50 ml of water for 30 min. 

 Cool and then add 50 ml of 70 per cent alcohol. This solution should be 

 filtered after two or three days. 



Method of Using the Solution Prepared from the Dry Stock Powder Is 

 as Follows: 



1. Accumulate the objects in whatever percentage of alcohol is con- 

 venient. 



2. Make up a saturated solution of the dry powder in alcohol from 10 

 to 20 per cent stronger than that in which the objects are accumulated. 



3. Transfer the objects to stain until they have become a deep red 

 color. This will take from 5 min for an individual protozoan to overnight 

 for a medium-sized flatworm. 



4. Transfer the objects to 0.1 per cent hydrochloric acid in alcohol of 

 the same strength as that used for making up the stain. Let them remain 

 in this solution until they become pink and translucent. 



5. Dehydrate and mount the objects in the usual manner. This stain is 

 not only good, used in the manner described, for staining wholemounts 



