METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 183 



gentian violet, leaving them unstained. In the unstained condition 

 the organisms were colored with the second or counter stain. Subse- 

 quent investigation has largely discredited this view. It has been 

 shown by Kruse 1 that the cytoplasm of Gram-positive bacteria is 

 more resistant to autolysis, to the action of trypsin, and to solution in 

 dilute KOH than that of Gram-negative organisms, probably because 

 the cytoplasm of the former is less permeable to these various reagents 

 than is that of the latter. Eisenberg, 2 and Guerbet, Mayer and Schaef- 

 fer 3 have advanced the hypothesis that Gram-positiveness is due to 

 the lipoidal content of the cell membrane (ectoplasm) and specifically 

 to unsaturated fatty acids and phosphatids. The addition of iodin, 

 according to this theory, through the formation of alcohol-insoluble 

 combinations with the lipoids in the ectoplasm, renders the cell wall 

 impermeable to alcohol and thus prevents removal of the dye which 

 has already penetrated into the cell contents. 

 Preparation of Stain: 



Solution A Saturated aqueous solution of anilin 4 90 c.c. 



Saturated alcoholic solution of gentian violet . . 10 c.c. or, 



Five per cent, aqueous solution of carbolic acid . . 90 c.c. 



Saturated alcoholic solution of gentian violet . . 10 c.c. 



The above solutions are unstable, but retain their tinctorial value 

 for two or three days if they are kept stoppered. 



Solution B 5 Distilled water 300 c.c. 



Potassium iodide 2 grams 



Iodin crystals 1 gram 



Solution C Bismarck brown, saturated aqueous solution ... 10 c.c. 

 Distilled water 90 c.c. 



Procedure. (a) Prepare and fix film of bacteria in the usual manner. 

 (6) Flood with anilin-oil gentian violet (or carbolic gentian violet) 

 and stain five minutes. 



(c) Pour off excess of stain and flood with iodin solution. 



(d) Decolorize with 96 per cent, alcohol until no more stain can be 

 removed. 



(e) Wash thoroughly in water. 



(/) Counterstain with Bismarck 1 brown 6 13r two minutes. 

 (g) Wash in water, dry, and mount. 



1 Miinchen. med. Wchnschr., 1910, p. 685. 



2 Cent. f. Bakt., 1909, xlix, 465; 1910, li, 115; liii, 481, 551; Ivi, 183. 



3 Compt. rend., Soc. biol., Ixviii, 353. 



4 Three c.c. of anilin oil are shaken for several minutes in 100 c.c. of distilled water. 

 The solution is filtered through filter paper to remove the undissolved anilin. 



5 This iodin solution is variously known as Gram's iodin solution or Lugol's solution. 



6 Dilute aqueous fuchsin, 1 to 10, may be used in place of Bismarck brown. 



