SECTION TWO 



C. Crystal violet 2% aqueous. 



D. Solution B . . . . • • 3 ml- 

 Solution C . . . . • • 27 ml. 



This mixture should be prepared immediately 

 before use. 



E. Gram's iodine. 



F. Aniline oil . . . . . . . . i volume 



Xylol . . . . . . . . I volume 



Technique: 



1. Fix material in absolute alcohol, Carnoy or alcohol-formalin 

 and embed in paraffin wax. 



2. Fix sections to slides; de-wax and pass through descending 

 grades of alcohol down to distilled water in the usual way. 



3. Stain in the lithium carmine solution for two to five minutes. 



4. Wash thoroughly in distilled water. 



5. Immerse in Solution D for five to ten minutes. 



6. Rinse in distilled water; drain well and blot carefully. 



7. Cover with Gram's iodine solution and allow the stain to act 

 for five to ten minutes. 



8. Pour off the excess iodine solution and blot carefully with 

 filter paper. 



9. Differentiate with the aniline xylol solution until no more 

 purple coloration comes out. 



10. Drain, and blot carefully. 



1 1 . Rinse with several changes of xylol. 



12. Mount in balsam or in cristalite. 



Results: 



Fibrin and Gram-positive organisms are blue to blue-black 

 while nuclei are red. 



References : 



Cowdry, E. V. (1952). 

 Mallory, F. B. (1938). 

 Weigert, C. (1887). 



