Staining 191 



fortunately worked out the amounts required for some 

 species, and of the more important ones the following 

 solutions of B and C must be added to 16 c.c. of solution 

 A to attain the desired effect: 



Cholera spirillum -1 drop of solution C $ 



Typhoid fever 1 c.c. of solution B 



Bacillus subtilis 28-30 drops of solution B 



Bacillus of malignant edema .36 or 37 drops of solution B 



Part of the success of the staining depends upon having 

 the bacteria thinly spread upon the glass, and as free from 

 albuminous and gelatinous materials as possible. The 

 cover-glass must be cleaned most painstakingly; too much 

 heating in fixing must be avoided. After using and washing 

 off the mordant, the preparation should be dried before the 

 application of the anilin-water-fuchsin solution. 



Pitfield's Method. Pitfield* has devised a single solution, at 

 once mordant and stain. It is made in two parts, which are 

 filtered and mixed: 



(A)- 



Saturated aqueous solution of alum 10 c.c. 



Saturated alcoholic solution of gentian violet . . 1 " 



(B)- 



Tannic acid " 1 gram 



Distilled water 10 c.c. 



The solutions should be made with cold water, and im- 

 mediately after mixing the stain is ready for use. The 

 cover-slip is carefully cleaned, the grease being burned off 

 in a flame. After it has cooled, the bacteria are spread 

 upon it, well diluted with water. After drying thoroughly 

 in the air, the stain is gradually poured on and by gentle 

 heating brought almost to a boil; the slip covered with the 

 hot stain is laid aside for a minute, then washed in water 

 and mounted. 



Smith's Modification of Pitfield' s Method.* A boiling saturated 

 solution of bichlorid of mercury is poured into a bottle in which 

 crystals of alum have been placed in quantity more than sufficient 

 to saturate the fluid. The bottle is shaken and allowed to cool; 

 10 c.c. of this solution are added to the same volume of freshly 

 prepared tannic acid solution and 5 c.c. of carbol fuchsin added. 

 Mix and filter. The filtrate, which is the mordant, is caught 



* "Medical News," Sept. 7, 1895. 



t ''British Medical Journal," 1901, i, p. 205. 



