SYNCHRONOUS GROWTH FOR SPORE PRODUCTION 11 



Description of the test flask. A two-liter Krlenme)er flask was fitted with a 

 side arm which was covered with a rubber cap before autoclaving. Samples 

 could be removed from the flask with a hypodermic syringe. City gas was 

 passed aseptically through the flask immediately after inoculation until all 

 oxygen was removed both from the medium and from the space above the 

 medium. A rubber stopper with an inlet and outlet tube was used in the 

 mouth of the flask with a suitable water trap to insure anaerobic conditions. 

 A magnetic stirring rod placed in the flask before sterilization provided a 

 means for constant stirring of the medium in the flask during growth of 

 the anaerobe. All media were used immediately after preparation. 

 Test media. The original test medium [the medium used in the test flask] 

 was composed of the following: 



[1] Trypticase 1.5% 



Sodium Chloride 0.5% 



K.HPO4 M/90 



Dextrose 0.2 % 



Initial pH 7.0 to 7.2 



The modified tris test medium was composed of the following: 



[2] Trypticase 1.5% 



Sodium Chloride 0.5% 



K0HPO4 M/30 



Tris buffer M/80 



Dextrose 0.2 % 



Initial pH 7.5 to 8.0 



Counting of the spores. Percentage of spores was determined by staining 

 with crystal violet; the spores remain unstained while the sporangia, the 

 germinated spores, and the vegetative ceUs absorb the dye. 



Results 



When the original test medium was used, sporulation occurred usually 

 within five and one-half to six and one-half hours. Table I summarizes the 

 results obtained. 



It is obvious that not all cells were producing spores at the same time. This 

 was thought to be due to one of two things : possibly the culture was not 

 in synchronous cell division or the medium itself was not satisfactory for 

 complete simultaneous sporulation. Many investigators have obtained syn- 

 chronous growth of bacteria by lowering the temperature of a culture for 

 a period of time and then allowing the culture to incubate at the normal 

 growth temperature. We used this technique for Clostridium roseum. Just 

 prior to the inoculation of the test flask with the active culture, the inoc- 



