SECTION TWO 



Staining Procedure 



Solutions required: 



A. Ethyl alcohol (95% or absolute) i volume 

 Ether . . . . . . . . i volume 



B. Acetic acid 1% aqueous 



C. Acridine orange (Michrome No. 



87) . . . . . . . . 2 gm. 



Distilled water . , . . . . 100 ml. 



*D. Disodium hydrogen phosphate 



(Na2HP04) 9-465 gm. 



Distilled water . . . . . . to i litre 



*E. Potassium dihydrogen phosphate 



(KH2PO4) 9-072 gm. 



Distilled water . . . . . . to i litre 



*F. M/15 phosphate buffer pH 6-o . . 



Solution D . . . . • . 50 ml. 



Solution E . . . . . . 300 ml. 



ATo/e; The original author (F. D. BertalanfFy, i960) directs that solution 

 F be checked with a pH meter to pH 60. I would suggest, however, 

 that in some laboratories it might be preferable that the M/15 phosphate 

 buffer should readily and simply be prepared as follows: 



G. 5 Michrome buffer tablets pH 6-o 



Distilled water . . . . . . 500 ml. 



Crush the tablets between the folds of a piece of 

 clean paper with the thumb or with a coin, then 

 dissolve the powder by shaking with the water. 

 This not only obviates the preparation of solutions 

 D, E, and F, but also the necessity for checking the 

 pH with a pH meter. 



H. Solution C 



97 



