40 General Bacteriology. 



B. Differential stain (Hauser's method). 



a. Make cover-glass preparation of a spore-bearing culture, fix and stain with hot 

 carbol-fuchsin until spores are thoroughly colored. This must be determined by mount- 

 ing in water and examining under microscope. 



b. Cautiously decolorize with acetic acid, 5%, until stain is removed from the vege- 

 tative portion only. This to be determined as above. 



c. Wash in water and counter- stain with methylen blue. 



d. Examine. Crimson spores will be seen in blue bacilli. 

 REFERENCES. Other methods, see A. 164-167. 



SPECIAL DIRECTIONS. Stain by each method spores in cultures of B. subtilis or 

 other spore-bearing organisms. 



EXERCISE XXXIV. STUDY OF ENDOSPORES. 



a. Make cultures on peptoneless agar, or agar to which a few drops of calcium 

 hydrate has been added, of the following organisms and incubate at 28 or 38 C. depend- 

 ing upon the organisms : 



Bacillus subtilis (EHRENB.) COHN. 



Bacterium anthracis (KOCH) MIG. 



Bacillus amylobacter VAN TIEGHEM (or any clostridium form). 



Bacillus tetani NICOLAIER (or any "drumstick" bacillus). 



Pseudomonas erythrosporus (CORN) MIG. 



b. When the cultures are 48 hours old mount films without staining, examine and 

 note: 



1) Form. 



2) Size. 



3) Color. 



4) Power to refract light. 



5) Relation to mother-cell. 



(1) Median or central. 



(2) Intermediate. 



(3) Terminal or polar. 



(4) Clostridium form. 



(5) Drumstick form. 



c. Make drawings. 



READ: J. H. 26; L. 60; L. & N. 76; M. & R. 6; N. 46; P. 46; P. B. C. 15; 



S. 114. 



EXERCISE XXXV. FLAQELLA STAIN (Bunge). 



GENERAL DIRECTIONS. 



a. Make an agar streak of the organism to be stained. 



b. After 18 to 24 hours, by means of the platinum needle, remove a portion of the 

 growth (being careful to avoid the culture medium) to a large drop of tap water on a 

 perfectly clean cover-glass (XV.) and allow to stand 5 minutes rather than spread, as 

 there is less danger of breaking off the flagella. 



c. Spread carefully 2 or 3 loopfulsof this drop on each of several clean cover-glasses 

 and dry at room temperature. 



d. Fix by passing the cover- glass through the top of the flame while it is held in the 

 hand, not in the forceps, as over heating will injure the preparation. 



