198 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
D. Intra-vitam Staining.— Nakanishi^ has applied the method of 
intra-vitam staining to the study of spores and granules in living 
bacterial cells. The method consists essentially in emulsifying a 
small amount of bacterial growth in normal salt solution containing 
sufficient aqueous methylene blue to impart a distinct blue color to 
the solution. The preparation is viewed as in the hanging-drop slide. 
The organisms absorb sufficient dye to impart to them a faint color, 
and granules within their bodies frequently stain with moderate 
intensity. The development of spores from presporogenic granules 
may be studied by this method. 
II. Staining of Bacteria.— A. Chemistry of Stains.^— The stains of 
value for coloring bacteria are almost exclusively anilin dyes which 
contain one or, more commonly, several benzene rings. Their color- 
4 ing properties have been shown to depend upon two distinct radicals; 
double-bonded atoms as C = C, C = O, C = N, N = N, known as 
chromophoric groups, and auxochromic groups, which impart to or 
intensify the color. Of the chromogenic groups, NH2 and —OH 
are the more important. The latter form salts which may be either 
basic or acidic in character. Bacteria usually stain best with basic 
dyes, as do nuclei of higher plant and animal cells.'^ 
The chemistry of the staining process itself is a matter of discussion. 
It was formerly held that the cell protoplasm united chemically with 
the stain as an acid unites with a basic salt, but later investigations, 
particularly those of Michaelis,^ are not in harmony with this view. 
It is probable that the physical state of the cell membrane as well as 
the composition of the cytoplasm plays a part in the staining process. 
B. Preparation of Stains.— Stains are conveniently kept in stock as 
saturated aqueous or alcoholic (96 per cent) solutions. The solubility 
of stains in water and in alcohol respectively varies, but, as a rule, the 
solubility in alcohol is greater than that in water. Saturated solutions 
of anilin dyes are unsuited for the staining of microorganisms, but they 
are more stable than diluted solutions provided they are kept in 
tightly-stoppered bottles away from the light. Dilutions of saturated 
solutions are prepared as they are needed for current use. 
C. Technique of Staining Bacteria. — 1. Preparation of a film of bac- 
teria for staining: A drop of a culture of bacteria from a fluid medium 
—as broth — is removed with a platinum loop and spread upon a clean 
cover-glass or glass slide. Bacteria from a solid medium are emulsified 
in a small drop of water on the slide. '^ 
1 Miinchen. med. Wchnschr., 1900, 47, 187; Centralbl. f. Bakteriol., 1901, 30, 97, 145, 
193, 225. 
- For a brief and comprehensive discussion of the chemistry of stains, see Fairbrother: 
The Industrial Chemist, 1926, p. 101. 
3 An excellent brief account of the principles of staining will be found in Bayliss's 
Principles of General Physiology, p. 70, Longmans Green & Co., 1924, 4th ed. 
^ Einfilhrung in die Farbstoffchemie, 1902, Berlin. 
s It is essential that the emulsion shall be but faintly opalescent when viewed by 
reflected light; a distinct clouding indicates that too many organisms have been added, 
in which event the preparation will be found to be unsatisfactory. 
