MEDICAL AND BIOLOGICAL STAINING TECHNIQUES 



4. Wash with absolute alcohol until no more colour comes out 

 of the film ; then wash with distilled water and drain. 



5. Counterstain with Jensen's neutral red (o-i gm. neutral red, 

 0-2 ml. 1% acetic acid, 100 ml. distilled water) for thirty to sixty 

 seconds. 



6. Wash with distilled water; drain, blot and dry in air. 



Note: Ziehl Neelsen's carbol fuchsin diluted i : 15 with dis- 

 tilled water may be used (staining time, twenty to thirty seconds) 

 for routine work. For demonstrating gonococcus and other intra- 

 cellular Gram-negative bacteria, however, neutral red should be 

 used. 



GRAM'S STAIN 

 (Kopelofif and Berman modification) 



For bacterial smears 



Solutions required: 



A. Crystal violet 1% aqueous. 



B. Sodium bicarbonate 5% aqueous. 



C. Potass, iodide . . . . . . 2 gm. 



Iodine . . . . . . . . i gm. 



Distilled water . . . . . . 89 ml. 



N/i NaOH . . . . . . 10 ml. 



D. Equal volumes of acetone and ether. 



E. Safranin 2% aqueous. 



Technique: 



1. Thin, unfixed, air-dried smears are stained for five to ten 

 minutes in a mixture consisting of 3 ml. Solution A, and o-8 ml. 

 of Solution B. 



2. Rinse with distilled water then stain with Solution C for two 

 to five minutes. 



3. Wash with tap water and drain and blot water from the sur- 

 face of the film, but do not allow the films to dry. 



370 



