THE PREPARATION OF TUBERCULIN 303 



2. Incubate one tube of each at 20 C., one at 37 C., 

 and one at 42 C. (Study and record the effect of these 

 temperatures upon the growth and spore formation of the 

 organism.) 



3. Make cover-glass preparations and stain with meth- 

 ylen blue, fuchsin and Gram's stain. Stain for spores 

 (Anjeszky's method). 



4. Transfer a small quantity of the agar culture to 

 4 or 5 c.c. of sterile physiological salt solution and 

 inject 0.25 c.c. subcutaneously into a guinea pig. Make 

 daily observations and an autopsy of the animal at 

 death. 



5. Make cultures on agar slants, and smear prepara- 

 tions from the blood, liver, spleen and kidney after the 

 autopsy. 



6. Fix the smears in the flame, stain with methylen blue 

 or fuchsin. After twenty-four and forty-eight hours ex- 

 amine the cultures microscopically. 



7. State your results and conclusions in full. 



REFERENCES 



MARSHALL: Microbiology, pp. 469, 476, 559, 561, 599-604. 

 JORDAN: General Bacteriology, 4th Ed. (1914), pp. 223-236. 

 BESSON: Practical Bacteriology, Microbiology and Serum Therapy, 



transl. by Hutchens (1913), pp. 517-535. 

 KOLMER: Infection, Immunity and Specific Therapy (1915), pp. 653- 



654. 

 ZINSSER: Infection and Resistance (1914), pp. 15, 18, 53, 64, 296. 



EXERCISE 4. THE PREPARATION OF TUBERCULIN 



Apparatus. Two 500 c.c. Erlenmeyer flasks; glycerin- 

 ated veal broth; evaporating dish; 0.5% phenol salt solu- 

 tion; Berkefeld filter; heavy filter paper; 20 c.c. homeo- 

 pathic vials; sealing wax. 



Culture. Bact. tuberculosis (specially adapted for 

 tuberculin) . 



Method, 1. Place about 200 c.c. of glycerinated veal 



