68 GENERAL BACTERIOLOGY 



b. Inoculate both with a pure culture of B. subtilis and incubate 

 at 28° C. Explain changes which occur. 



c. Test extract of beef or syrup in the same way. 



EXERCISE 40. EFFECT OF TEMPEEATUBE 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, deter- 

 mine the optimtim temperature of growth for each. 



Eeperences. F. 73 ; L. & N. 44 ; McF. 44. 



Special Directions. Use a mesophilic bacterium as B. coli and 

 a psychrophilie organism as Ps. violacea. 



exercise 41. 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, 5 test-tubes (16 mm. in 

 diam.) containing exactly 10 cc. of standard bouillon. (Reaction 

 + 1.5.) 



c. After 15 minutes exposure at this temperature remove the 

 cotton plug from one of the tubes, inoculate the broth with three 

 loopfuls (standard size, 12) of the' 48 hour old culture (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 immediately in a vessel of ice cold water to 

 cool. Then incubate at a temperature favorable to the development 

 of the organism under observation. 



e. Raise the temperature of the bath 5 degrees, i. c, to 50° 'C, 

 inoculate another tube. Keep it at 50° for 10 minutes, remove, cool 

 and incubate. 



/. In the same manner expose the organism to the following 

 temperatures : 55°, 60°, and 65° C. for a period of 10 minutes each. 



g. In all cases incubate at least a week and take as the thermal 

 death point the lowest temperature at which growth fails to appear. 



