SECTION TWO 



CRESYL FAST VIOLET 



For the demonstration of Cryptococciis Neoformans in 



polarized light 



Solutions required: 



A. Sodium acetate, i% aqueous 



B. Cresyl fast violet . . . . . , o-i gm. 



Acetic acid, i% aqueous . . 30 ml. 



Solution A . . . . . . 20-5 ml. 



Distilled water . . . . . . 67-5 ml. 



Technique: 



1. Fix pieces of tissue in 10% formalin and embed in paraffin 

 wax in the usual manner. 



2. Fix sections to slides, then dewax with xylol. 



3. Take sections into distilled water through the usual descend- 

 ing grades of alcohol. 



4. Stain in solution B (buffered cresyl fast violet, pH 3-5) for 

 fifteen to thirty minutes. 



5. Differentiate in 95% alcohol, controlling by microscopic 

 examination while the section is still wet, until the background is 

 clear. 



6. Dehydrate quickly in three changes of absolute alcohol. 



7. Clear in xylol or toluene. 



8. Mount in D.P.X. or Clearmount. 



9. Examine in polarized light. 



Results: 



The spherical cytoplasm of the fungi appears as a pink luminous 

 centre, surrounded by brilliantly birefringent spinous radiations 

 of the capsule. 



Reference: Klatzo, I. (1958). 



Notes : 



(a) The author states that in his experience the intensity of 

 birefringence of the organism in sections stained by routine 

 histological and special fungal techniques is frequently too low to 

 be of any value in diagnostic problems, but with the buffered 

 cresyl fast violet technique, the appearance of the organisms is 

 so distinct and characteristic that even single isolated cells can 

 be identified easily under the low power. 



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