180 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 



Fixed films of bacteria are stained from one to five minutes with 

 this stain, or the films are flooded with the stain and heated until 

 steam rises (not boiled) for one to three minutes. It is difficult to 

 overstain with Loffler's methylene blue unless evaporation takes 

 place to such a degree that the stain dries on the slide. The stain is 

 washed off with water, dried, and mounted. 



E. Stains for Special Structures of the Bacterial Cell. 1. Spores. 

 (a) Flood fixed film of bacteria with carbol-fuchsin 1 and steam (not 

 boil) for five minutes. 



(6) Wash thoroughly in running water. 



(c) Decolorize with 1 per cent, sulphuric acid until excess stain is 

 removed. 



(d) Wash thoroughly in running water. 



(e) Flood with saturated aqueous solution methylene blue (or 

 Loffler's alkaline methylene blue) and allow to stain one minute. 



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

 Spores stain red, vegetative cells blue. 



Holler's Spore Stain: 2 (a) Suspend the fixed film of bacteria in 

 chloroform for two minutes. 

 (6) Wash with water. 



(c) Flood with 5 per cent, chromic acid solution for two minutes. 



(d) Wash thoroughly in running water. 



(e) Flood with carbol-fuchsin and steam for five minutes. 

 (/) Wash thoroughly in water. 



(g) Decolorize with 1 per cent, sulphuric acid until excess stain is 

 removed. 



(h) Wash thoroughly in water. 



(i) Flood with Loffler's alkaline methylene blue and allow to stain 

 one minute. 



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



Spores stain red, vegetative cells blue. 



2. Capsule Stains. Welch Method* (a) Fixed films of bacteria are 

 flooded with glacial acetic acid for a few seconds. 



(6) The acid is poured off and the preparation is washed two or 

 three times with anilin oil gentian violet, then flooded with the stain, 

 which is allowed to act for three to five minutes. 



(c) Wash with 2 or 3 per cent, aqueous solution of sodium chloride. 



(d) Mount in salt solution and examine. 

 Capsules faint purple, bacterial body deep purple. 



1 Saturated alcoholic solution of basic fuchsin, 10 c.c.; 5 per cent, aqueous phenol 

 solution, 90 c.c. 



2 Cent. f. Bakt., 1891, x, 273. 3 Johns Hopkins Hosp. Bull., 1892, 128. 



