STAINS AND STAINING 21 



3. Transfer the object to the stain and leave it until examination shows the 

 nuclei or internal structures to have been stained a fairly dark red, while 

 the other portions are stained only pink. This will take about six hours with 

 a fern prothallium in the dilution described or about three weeks for a 

 liver fluke at the low concentration. 



4. Wash the object in running water until all alum has been removed and 

 then mount in the ordinary manner. 



Synthetic Nuclear Stains 



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

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

 oped 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 diffi- 

 culty 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. 



The stain given below is an excellent blue nuclear stain which the author 

 much prefers to hematoxylin because it is impossible to overstain in it. 



Celestin Blue B: 

 Staining solution 



Boil together for 5 minutes 0.5 Gm. celestin blue B and 2.5 Gm. iron 

 alum in 100 ml. of water. Cool, filter, and then add to this mixture 14 ml. 

 of glycerin and 2 ml. of strong sulfuric acid. 

 STAIN IS USED ONLY ON SECTIONS IN THE FOLLOWING MANNER: 



1. Accumulate the sections in water. 



2. Transfer them to the staining solution until the nuclei are sufficiently 

 stained. This takes anywhere from five minutes to an hour or two, accord- 

 ing to the fixative used and the manner in which the sections have been 

 handled previously. 



3. Wash sections in water for a few minutes. 



It will be seen that this is one of the simplest of all the nuclear staining 

 methods, but it differs from most in that it cannot be used to stain the xylem 

 in plant tissues. 



Safranin nuclear staining in English speaking countries is usually con- 

 fined to botanical specimens, though its use in European countries is wide- 

 spread for histological purposes. Safranin is not easy to use and is a very slow 

 stain. Undoubtedly the best method is: 



