METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 181 



Hiss's Method. 1 (a) Place a drop of sterile blood serum upon a 

 slide and emulsify bacteria in it. 

 (6) Dry in the air and fix by heat. 



(c) Flood smear with 5 per cent, solution 2 of gentian violet or 

 fuchsin; steam for thirty seconds. 



(d) Remove excess of stain by washing in a 20 per cent, solution 

 of copper sulphate. 



(e) Dry with filter paper. Mount and examine. 



Capsule faint pink or purple; body of organism deep red or purple. 



Rosenow Method. 3 (a) Prepare the smear on perfectly clean cover- 

 glass. 



(6) When smear is nearly dry, cover with 10 per cent, aqueous 

 solution of tannic acid for twenty seconds. 



(c) Wash with water; remove moisture with filter paper. 



(d) Flood with anilin-oil gentian violet and steam gently for thirty 

 to sixty seconds. 



(e) Wash thoroughly in water. 



(/) Cover with Gram-iodin solution, one minute. 

 (g) Decolorize with 96 per cent, alcohol. 



(h) Stain one or two minutes with a saturated (60 per cent.) alco- 

 holic solution of Griibler's eosin. 



(i) Wash in water; dry and mount in balsam. 

 Capsules pink; bacteria blue. 

 3. Polar Bodies. Neisser Stain. 4 

 Preparation of Stain. 



Solution A Methylene blue 1 gram 



Ninety-six per cent, alcohol 20 c.c. 



Glacial acetic acid 50 c.c. 



Distilled water 950 c.c. 



Solution B Bismarck brown 1 gram 



Distilled water 500 c.c. 



(a) The air-dried film, fixed by heat, is flooded with solution A for 

 three to five seconds. 

 (6) Wash with water. 



(c) Flood with solution B for five seconds. 



(d) Wash with water, dry, and mount. 

 Polar bodies stain blue; bacterial cells brown. 



1 Jour. Exp. Med., 1905, vi, 338. 



2 Saturated alcoholic solution of the dye, 5 c.c. ; distilled water, 95 c.c. 



3 Jour. Infect. Dis., 1911, ix, 1. 



* Ztschr. f. Hyg., 1897, xxiv, 443. 



