REACTION OF MEDIUM 305 



2. Pipette 5 c.c. sterile normal saline into the culture 

 tube and emulsify the entire surface growth in it. Ex- 

 amine microscopically to determine the presence of 

 spores in large numbers. 



3. Spread thin even films on twelve sterile cover- 

 slips and place each cover-slip in a separate sterile 

 capsule. 



4. Expose each capsule in turn for ten minutes to a 

 different fixed temperature, varying 5 C., between 

 100 C. and 160 C. 



5. Complete the examination as for vegetative forms. 



III. Reaction of Medium. 



(A) Range. 



1. Prepare a bouillon culture of the organism and in- 

 cubate, under optimum conditions as to temperature 

 and atmosphere, for twenty-four hours. 



2. Pipette o.i c.c. of the cultivation into a ster- 

 ile capsule; add 9.9 c.c. sterile bouillon and mix 

 thoroughly. 



3. Prepare a series of tubes of nutrient bouillon of 

 varying reactions, from +25 to 30 (vide page 155), 

 viz.: +25, +20, +15, +10, +5, neutral, -5, -10, 



-15. - 20 ~ 2 5> -3- 



4. Inoculate each of the bouillon tubes with o.i c.c. 



of the diluted cultivation by means of a sterile gradu- 

 ated pipette and incubate under optimum conditions. 



5. Observe the cultures at half -hourly intervals 

 from the third to the twelfth hours. Note the reaction 

 of the tube or tubes in which growth is first visible 

 macroscopically (probably optimum reaction) . 



6. Continue the incubation until the completion, if 

 necessary, of seven days. Note the extremes of acidity 

 and alkalinity in which macroscopical growth has 

 developed (Range of reaction) . 



7. Control the result of these observations. 



(B) Optimum Reaction. The optimum reaction has 



20 



