Rickettsia and Virus Staining 325 



Stock Giemsa stain: 



Giemsa stain 1.0 gm. 



glycerol 66.0 ml,. 



Mix and place in oven 2 hours, GO^C. Add 



methyl alcohol, absolute 66.0 ml. 



Working solution A : 



distilled water 100.0 ml. 



glacial acetic acid 1 drop 



Jenner stock solution 20.0 ml. 



Working solution B: 



distilled water 100.0 ml. 



glacial acetic acid 1 drop 



Giemsa stock solution 5.0 ml. 



procedure: 



1. Deparaffinize and hydrate slides to water. 



2. Stain in solution A, 37°C: 15 minutes. 



3. Transfer directly to solution B, 37°C: 30-60 minutes. 



4. Dehydrate quickly, 2 changes absolute alcohol. 



5. Clear and motint. 



results: 



rickettsiae — blue to purplish blue 



Castaneda's Method (gradwohl, 1956) 



fixation: a general fixative, but Regaud recommended. 



solutions: 



Buffer solutions: 

 Solution A : 



di-basic sodium phosphate, Na2HP04- I2H4O 23.86 gm. 



distilled water 1000.0 ml. 



Solution B: 



monobasic sodium phosphate, anhydrous, NaHo 



PO4 11.34 gm. 



disdlled water 1000.0 ml. 



Working solution: 



solution A 88.0 ml. 



solution B 12.0 ml. 



formalin 0.2 ml. 



