Stains and Staining 93 



removed in absolute alcohol, so that differentiation must be watched very 

 carefully. The dyes used are extremely sensitive to alkali, so that the slides, 

 if they are to be kept for some time, should be mounted in a very acid 

 medium. A procedure that has been recommended is to keep a saturated 

 solution of salicylic acid in xylene and to dip each coverslip in this before 

 applying it to the finished preparation. This is much less trouble than and 

 just as effective as making up a special salicylic acid balsam. 



Another complex contrast stain, in which the nuclei first must be stained 

 with hematoxylin, is: 



Patay's Triple Stain: 



First staining solution 



1% Ponceau 2R 

 Differentiating and mordanting solution 



1% phosphomolybdic acid 

 Second staining solution 



0.5% light green in 90% alcohol 



Method of Use 



1. Stain the sections in Delafield's hematoxylin (see page 83) and dif- 

 ferentiate until not only the nuclei but also the cartilage remains blue. 

 Wash the sections in alkaline tap water until they are blue and then wash 

 thoroughly in distilled water to get rid of all the alkali. 



2. Put the sections in the first stain for 2 min. 



3. Rinse them briefly in water. 



4. Place them in the differentiating and mordanting solution for 2 min. 



5. Rinse sections briefly in water. 



6. Transfer them to 95% alcohol until dehydrated. 



7. Transfer each section individually to the second staining solution for 

 30 sec. 



8. Rinse each slide individually in absolute alcohol until no more color 

 comes away. 



9. Transfer the slides to balsam and mount in the usual manner. 



This is one of the most brilliant of all the triple stains, and a successful 

 preparation shows the nuclei in blue-black and the cartilage in clear blue. 

 Other connective tissues are green, white fibrous connective tissue being 

 light green, and bone is a most brilliant green in contrast to the orange 

 muscle and blue cartilage. Red blood cells are yellow and most nerve 

 tissues are colored a neutral gray. This stain is little more trouble to apply 

 than Mallory's, but the results are both more brilliant and more permanent. 



Many complex stains rely on the fact that a mixture of eosin and meth- 

 ylene blue solutions gives rise to a precipitate which, though insoluble 

 in water, is soluble in methyl alcohol. These stains are used mainly for 



