SECTION TWO 



3. Pour off excess and blot, without washing. 



4. Flood with Gram's iodine and allow the stain to act for three 

 minutes. 



5. Destain with the acetic acid alcohol (Solution C above) 

 until no more colour comes away, and the sections assume a dirty 

 straw colour. 



6. Rinse quickly in distilled water. 



7. Stain in neutral red-fast green diluted one part with three 

 parts of distilled water, for five minutes. 



8. Wash quickly with distilled water. 



9. Decolorize with the acetic alcohol solution until no more 

 red stain comes out. 



10. Rinse quickly in absolute alcohol. 



1 1 . Clear in xylol and mount. 



Results: 



Nuclei are stained red, while cytoplasm is light green. Gram- 

 positive bacteria, dark blue. Gram-negative bacteria, pink. 

 Erythrocytes, green. 



J. Path. & Bad., 59, 357-8, Ollett, W. S. (1947). 



NILE BLUE SULPHATE 

 For demonstrating fatty acids and neutral fats 



Solution required: 



To 100 ml. saturated aqueous Nile Blue sulphate 

 add 0'5 ml. cone, sulphuric acid; then boil under a 

 reflux condenser for two hours ; allow to cool ; then 

 use as follows : 



Technique: 



1. Fix small pieces of tissue in 10% formalin. 



2. Frozen sections are stained for about ten minutes to half an 

 hour at 37° C. ; or overnight at room temperature. 



171 



