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 afew drops of calcium 
hydrate has been added, of the following organisms and incubate at 28° or 88° C. depend- 
ing upon the organisms: 
Bacillus subtilis (EHRENB.) COHN. 
Bacterium anthracis (Koca) Mie. 
Bacillus amylobacter Van TyecHEM (or any clostridium form). 
Bacillus tetani Nicouater. (or any ‘‘drumstick’’ bacillus). 
Pseudomonas erythrosporus (Conn) Mia. 
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. 
Reap: J. H. 26; L. 60; L.& N.76; M. &R. 6; N. 46; P. 46; P. B.C. 15; 
8. 114. 
EXERCISE XXXV. FLAGELLA STAIN (Bunge). 
GENERAL DIRECTIONS. 
a. Make an agar streak of the organism to be stained. 
db. 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. 
ec. Spread carefully 2 or 3 loopfuls of 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. 
