178 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 



C. Dark Field Illumination and Ultramicroscopic Examination. For 

 the study of very minute particles in suspension, the ultramicroscope 

 of Siedentoff and Zsigmondy 1 has been used, but the dark-ground 

 illumination apparatus of Reichert, 2 a much simpler device, readily 

 adjusted to any microscope, has largely supplanted it for bacterial 

 examinations. With the Reichert apparatus the flagella of bacteria 

 and other structures of low-refractive index may be observed. Tre- 

 ponema pallidum in fresh smears from lesions is readily seen with the 

 dark ground illuminating apparatus. 



D. Intra Vitam Staining. Nakanishi 3 has applied the method of 

 intra vitam staining to thie study of spores and granules in living bac- 

 terial cells. The method consists essentially in emulsifying a small 

 amount of bacterial growth in normal salt solution containing suffi- 

 cient 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 inten- 

 sity. The development of spores from pre-sporogenic 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- 

 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 chromophoric groups, NH 2 and OH are 

 the more important. The latter form salts which may be either basic 

 or acid 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, 4 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 play a part in the staining process. 



B. Preparation of Stains. Stains prepared by Griibler or Merck 

 are commonly used for the staining of bacteria. They are conveniently 



1 Zeit. f. wissenschaftl. Mikroskopie, 1909, xxvi, 391. 



2 Munchen. med. Wchnschr., 1906, 2351; Hyg. Rund., 1907, No. 18; Cent. f. Bakt. 

 Orig., 1909, li, 14. 



3 Miinchen. med. Wchnschr., 1900, No. 6; Cent. f. Bakt., 1901, xxx, 97, 145, 193, 225. 



4 Einfiihrung in die Farbstoffchemie, 1902, Berlin. 



