364 Special Procedures I (chap. 22) 



solutions: 



Davidson's fixative: 



95% ethyl alcohol 30.0 ml. 



formalin 20.0 ml. 



glacial acetic acid 10.0 ml. 



distilled water 30.0 ml. 



Buffered thionin: 



A. thionin, C.I. 52000, saturated solution in 50% alcohol. Filter. 



B. sodium acetate 9.714 gm. 



sodium barbiturate 14.714 gm. 



distilled water, CO2 free 500.0 ml. 



C. hydrochloric acid, sp. gr. 1.19 8.50 ml. 



distilled water 991.5 ml. 



Working solution (pH 5.7): 



thionin solution (A) 40.0 ml. 



buffer solution (B) 28.0 ml. 



hydrochloric acid (C) 32.0 ml. 



procedure: 



1. Deparaffinize and hydrate slides to water. 



2. Hydrolyze in 5N HCl (page 408), 20-25 °C: 20 minutes. 



3. Rinse thoroughly in distilled water, several changes; no acid 

 should be carried over into thionin. 



4. Stain in buffered thionin: 15-60 minutes. 



5. Rinse in distilled water, and 50% alcohol. 



6. Rinse in 70% alcohol luitil clouds of stain cease to appear. 



7. Dehydrate, clear, and mount. 



results: 



sex chromatin — deep blue-violet 



nuclear chromatin — lightly colored 



cytoplasm — unstained 



fibrin and related structures will show metachromasia 



comments: 



The Feulgen technique may be used for sex chromatin body 

 identification, but the above method is simpler and quicker. The 

 principle is the same in both methods: the sex chromatin body 

 differentiates from nonspecific nuclear chromatin because of a higher 

 content of DNA, which takes longer to extract. If the basophil shell 

 of the nucleolus is not visible, no attempt at sexing should be made 



