STAINING, PRACTICAL AND THEORETICAL 



NEUTRAL RED - FAST GREEN 



For staining both Gram-positive and Gram-negative 



bacteria in sections 



Solutions required: 



A. Aniline crystal violet. 



B. Gram's iodine. 



C. Absolute alcohol . . . . 98 ml. 

 Glacial acetic acid . . . . 2 ml. 



D. Twort's stain, modified (neutral red-fast 



green) 



Technique: 



1. Fix tissue in 5% formal-saline, dehydrate, clear; embed in 

 paraffin wax. Cut sections 3// in thickness. 



2. Stain in aniline crystal violet for three to five minutes. 



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. 



11. 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. 



Reference: Ollett, W. S. (1947). 



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