46 General Bacteriology. 
b. Thoroughly mix, solidify gelatin in ice water and then inoculate (stab) each 
tube with the organism to be studied, making a control culture in a tube of neutral 
gelatin. 
c. Incubate at 18° C. and note the effect of the chemicals on the rate, amount and 
character of the growth. 
REFERENCES. L. & N. 87; McF..46. 
SPECIAL DirEcTIONS. Use B. subtilis and B. coli. 
EXERCISE XXXIX. EFFECT OF CONCENTRATION OF MEDIA ON GROWTH. 
a. Pour about 2 ec. of ‘‘condensed milk’’ into each of two sterile test-tubes,. dilute 
one with five times the volume of sterile water. 
b. Inoculate both with a pure culture of B. subtilis and incubate at 28° C. 
Explain changes which occur. ; 
-¢. Test extract of beef or syrup in the same way. 
EXERCISE XL. EFFECT OF TEMPERATURE VARIATIONS ON RATE OF GROWTH. 
GENERAL DIRECTIONS. 
a. Make four agar streak cultures of organism to be studied. 
b. Incubate them at the following temperatures: Ice chest (7° C.), room (20° C.), 
low incubator (28° C.), blood heat (38° C.). 
c. By frequent observations as to luxuriance of growth, determine the optimum 
temperature of growth for each. 
REFERENCES. F. 73; L. & N. 98. 
SpeciaL Directions. Use B. campestris and B. coli. 
EXERCISE XLI. DETERMINATION OF THERMAL DEATH POINT. 
GENERAL DIRECTIONS. 
a. Make a bouillon culture of the organism to be tested. 
b. 48 hours later heat a large water-bath to 45° C. Place in this, in close proximity 
to a thermometer, a test-tube (16 mm. in diam.) containing exactly 10 cc. of standard 
bouillon. (Reaction +1.5.) i 
c. After 15 minutes exposure at this temperature remove the cotton plug from the 
tube, inoculate the broth with three loopfuls (standard size, XII) of the culture prepared 
above (a.), and carefully mix by slightly agitating the tube, without removing it from the 
bath. ; 
d. After a further exposure of 10 minutes remove the tube from the bath and place 
it in a vessel of ice cold water to cool. Then incubate at a temperature favorable to the 
development of the organism under observation. 
e. In the same manner expose the organism to the following temperatures: 50°, 55°, 
60°, and 65° C. 
f. In all cases incubate at least a week and take as the thermal death point the low- 
est temperature at which growth fails to appear. (In more accurate work the tempera- 
ture should be determined within 2° C.). 
REFERENCES. P. B. C. 32. 
Specian Directions. Use B. coli or B. typhosus. 
