Stains and Staining 87 



Note: The orcein is dissolved in the dilute acid, the concentration of 

 which is varied according to the material to be stained. Fifty per cent 

 acetic acid is commonly used by plant cytologists: 70 per cent acetic 

 acid is preferred for Drosophila. 



Method for Squashes 



1. Place freshly dissected living material in a drop of stain. 



2. Leave 3 to 10 min. 



3. Apply coverslip and squash to shatter cells and display chromosomes. 



SYNTHETIC NUCLEAR STAINS 



Of the four stains given below, only celestine blue B and safranin are 

 usually considered to be true nuclear stains, the other stains having been 

 developed for staining bacteria. However, they are all considered together 

 since the bacterial stains often can be used to demonstrate nuclei in 

 material in which conventional stains break down. For example, when one 

 is staining a section of a frog larva or egg, which is heavily loaded with 

 yolk, one has great difficulty in endeavoring to use hematoxylin for the 

 reason that some of the albuminous yolk particles pick up this stain. 

 Either carbolmagenta or crystal violet, however, will demonstrate the 

 nuclei clearly without being picked up by the yolk. 



Celestine Blue B. This is much the most satisfactory synthetic nuclear 

 stain for sections since a stable staining solution is now available. Previous 

 staining solutions, including that given in the last edition of this book, 

 were both unstable and erratic in their action and have given this dye a 

 bad name, but the following method is foolproof. It is the only direct 

 nuclear stain available for sections. 



Gray's Celestine Blue B: 



Dissolve 2.5 g iron alum in 100 ml water with 14 ml glycerin. Place 

 1.0 g celestine blue B in a beaker. Tilt the beaker and tap to accumulate 

 the dye in one place. Pour on 0.5 ml concentrated sulfuric acid and mix 

 with a glass rod. When effervescence has ceased, the dye will be in the 

 form of a friable mass. Break up this mass coarsely and pour on, with 

 constant stirring, the iron alum solution heated to 50° C. Cool to room 

 temperature and adjust to pH 0.8 with concentrated sulfuric acid. 



Note: Both the method of compounding and the pH are critical. At 

 pH 0.6 there is no staining. At pH 1.0, nuclei are well stained but the 

 cytoplasm picks up some stain. At pH 1.2 the cytoplasm is rather densely 

 stained. Those who lack a pH meter and a magnetic stirrer, which is the 

 only really satisfactory combination for adjusting the pH, should add 1.5 

 ml concentrated sulfuric acid to the cooled solution. After thorough mix- 



