ANTHRAX GROUP 99 



b) 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 thoroughly in water. 



e) Cover with carbol-fuchsin and heat for 5 minutes 

 over the water-bath at 100°, or over a small flame, sim- 

 mering gently all the while. 



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

 30 seconds. 



g) Wash thoroughly in water. 



h) Mount in water, and examine under the micro- 

 scope to see if spores are cherry-red and the protoplasm 

 colorless or faintly pink. 



i) Counterstain with methylene-blue for 10-15 

 seconds without heat. 



f) Wash, examine in water, and then mount in balsam. 



NoTE.= — The body of the cell should appear blue; the spore, 

 ired. 



4. Special study C. — Demonstration of filament- 

 formation. 



a) Spread a loopful of a broth-culture of B. anthracis 

 or B. subtilis on a clean cover-glass. 



b) Dry and fix in the flame. 



c) Cover with strong acetic acid (80 per cent.) for 

 5-10 seconds. 



d) Wash in water. 



e) Stain with gentian-violet. 



f) Examine in water, dry, and mount in balsam. 

 AVhat is the object of applying acetic acid ? 



5) Special study D. — Inoculate a guinea-pig subcu- 

 taneously with 0.2 c.c. of a 24-hour-old broth-culture of 

 B. anthracis, or insert a loopful of a 24-hour-old agar- 



