STAINING SPORES AND FLAGELLA 59 
89. Work for this exercise. Examine and carefully describe 
the cultures of B. subtilis made in Exercise XIV. omit 
Make a hanging-drop preparation from the bouillon and one 
from the agar culture and examine them microscopically. 
Describe the bacilli and observe carefully the appearance of the 
spores both within and without the organisms. 
Make a cover-glass preparation from each culture and stain) 147, 
with alkaline methylene blue. Examine carefully and note et Spor 
appearance of spores which remain unstained. Make a draw- 
ing of a few of the bacteria containing spores. 
Make a few (about 3) cover-glass preparations and stain 
them for spores. 
Clean about 10 cover glasses after the special method for 
flagella staining (§4). Make about 5 films on these from the 
water suspension prepared’ at the last exercise and stain for 
flagella. Use Johnston and Mack’s modified method, with 
_ Loeffler’s mordant and stain. The films should not be heated 
either before or during the staining process, but if it does not 
succeed, ’Loeffler’s process may be tried. 
90. A method for staining spores. Make a cover-glass 
preparation, dry, and. flame as already described. Take the 
preparation by the edge with the fine forceps, cover the film 
surface with carbol fuchsin, and hold the preparation over the 
+ gas flame until steam is given off; then remove it for a few 
seconds and heat again. Repéat the heating three or four 
times. After the stain has acted for from 3 to 5 minutes, rinse 
the preparation in water and decolorize it by immersing it in 
a watch glass containing about 3 cc. of 1% solution of sul- 
phuric acid or 95% alcohol. After about 4 minute remove 
the preparation and rinse it thoroughly in water. If it is not 
decolorized, repeat the bleaching process. This removes the 
coloring matter from the bodies of the bacteria, but leaves 
it in the spores. After thoroughly washing the preparation, 
counterstain it with a saturated aqueous solution of methylene 
blue for about 30 seconds, rinse in water, and examine. The 
