Staining 169 



A specimen thus treated may or may not show the flagella. 

 If not, before proceeding further it is necessary to study 

 the chemic products of the micro-organism in culture 

 media. If by its growth the organism elaborates alkalies, 

 from i drop to i c.c. of solution C in 16 c.c. must be added 

 to the mordant A, and the staining repeated. It may be 

 necessary to stain again and again until the proper amount 

 is determined by the successful demonstration of the fla- 

 gella. On the other hand if the organism by its growth 

 produces acid, solution B must be added, drop by drop, 

 and numerous stained specimens examined to see with 

 what addition of alkali the flagella will appear. Loffler 

 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 simple and 

 excellent method of staining flagella, a single solution at 

 once mordant and stain being employed. 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 

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



