102 STAINING METHODS 



This solution is at its best some four or five days 

 after manufacture ; it keeps for about a couple of weeks, 

 but must be re-centrifugalised each time, before use. 



(b) Stain. 



Alum, saturated aqueous solution 25 c.c. 



Gentian violet, saturated alcoholic solution . . 5 c.c. 



Filter. 



This stain must be freshly prepared. 



METHOD. The cultivations employed should be 

 smear agar cultures, twelve to eighteen hours old if 

 incubated at 37 C., twenty-four to thirty hours if 

 incubated at 22 C. 



1. Remove a very small quantity of the growth by 

 means of the platinum spatula. 



2. Emulsify it with a few cubic centimetres of dis- 

 tilled water in a watch-glass, by gently moving the 

 spatula to and fro in the water. Do not rub up the 

 growth on the side of the watch-glass. Some workers 

 prefer to use tap water, others employ normal saline 

 solution, but distilled water gives the best emulsion. 



3. Spread a thin film of the emulsion on a newly 

 flamed cover-slip, using no force, but rather leading 

 the drop over the cover-slip with the platinum loop. 



4. Allow the film to dry in the air, properly protected 

 from falling dust. 



5. Fix by passing thrice through the Bunsen flame, 

 holding the cover-slip whilst- doing so by one corner 

 between the finger and thumb. 



6. Pour on the film as much of the mordant as the 

 cover-glass will hold. Grasp the cover-slip with the 

 forceps and hold it, high above the flame, until steam 

 rises. Allow the steaming mordant to remain in con- 

 tact with the film two minutes. 



7. Wash well in water and dry carefully. 



8. Pour on the film as much of the stain as the cover- 

 glass will hold. Steam over the flame as before for 

 two minutes. 



