128 LABORATORY GUIDE IN BACTERIOLOGY 



1. Routine study. — Observe the colonies formed on 

 agar plates. Also prepare gelatin plates and study the 

 colonies. 



2. Special study. — Make an "impression prepara 

 tion" (Klatschpraparat) from a surface colony on a 

 gelatin plate. 



Method — 



a) Clean and flame a cover slip. 



b) Place on a colony of suitable size, and gently 

 press down, taking care not to press so hard as to 

 disturb the characteristic shape of the colony. 



c) Lift the cover shp with the forceps. 



d) Dry, fix, and stain with methylene blue or by 

 Gram's method. 



e) Examine under low and high power (dry lens). 



3. Special study. — Staining of spores. 

 MoUer's method: 



a) Prepare several (five or six) films from agar 

 or potato cultures of B. anthracis (or B. subtilis). 

 6) Place in chloroform for 2 minutes. 



c) After drying in the air, cover with a 5 per cent 

 solution of chromic acid for 2 minutes. 



d) Wash in water. 



e) Cover with carbol f uchsin and heat for 5 minutes 

 on a water bath, or over a small flame. 



/) Decolorize with i per cent sulphuric acid for 25- 

 30 seconds. 



g) Wash in water. 



h) Counterstain with methylene blue for 10-15 sec- 

 onds without heat. 



i) Wash, dry, and mount in balsam. 



Note. — ^The body of the cell should appear blue; the spore, 

 red. 



