10 ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 
SECTION 3: INDIRECT STAINING METHODS 
EXERCISE 12 
INDIA INK PREPARATION 
A useful method for examining bacteria under the microscope is to suspend 
them in India ink. When this method is used, the bacteria remain unstained in 
a dark background. By the use of India ink it is possible to examine any or- 
ganism unstained. It has the advantage that the organisms do not shrink or 
change their form. 
Procedure 
Place a drop of India ink on a microscopic slide. Mix with this a small 
amount of the culture to be examined. Spread the preparation to a thin uniform 
film and allow to dry. Using the oil immersion objective, examine the prepara- 
tion and make drawings. 
EXERCISE 13 
NIGROSINE PREPARATION 
As it is difficult to obtain an India ink preparation which is satisfactory 
_nigrosine is recommended as a better substance for indirect or negative staining. 
A one per cent aqueous solution of nigrosine is used. 
Procedure 
Place a small drop of the nigrosine solution on a slide, mix with it a small 
loopful of a broth culture and spread well. Allow to dry before examination 
under the microscope. Make drawings. 
Note 
Nigrosine may also be used to demonstrate motility in bacteria. For this 
purpose, add a very small amount of the nigrosine solution to a hanging drop. 
EXERCISE 14 
CONGO RED PREPARATION 
Make a slide preparation as in Exercise 13, using a saturated aqueous so- 
lution of congo red instead of nigrosine. When the preparation has dried, im- 
merse the slide in a 95 per cent alcoholic solution containing one per cent hydro- 
chloric acid. The bacteria will be unstained on a deep blue background. By use 
of the oil immersion, study and make drawings. 
