BIOLOGY OF CLOSTRIDIUM WELCHII 399 



In no case was there more than a degree difference in duplicate 

 samples. Control tubes were always run at 37° and 80° for 

 fifteen minutes to determine whether the organism was alive 

 before heating and to ascertain the presence of spores. All 

 cultures used showed the characteristic "stormy fermentation' 7 

 of milk previous to the test. The temperatures used to deter- 

 mine the thermal death point of vegetative forms were 56°, 

 57°, 58°, 59°, 60°, 61°, 62°, 63° and 80°. 



3. Thermal death point of spores. The determination of the 

 thermal death point of Clostridium Welchii in the spore stage 

 was determined in a manner similar to that described above using 

 the same pure cultures grown for eight hours by inoculating 

 0.5 cc. of the "steaming" culture into a neutral medium of pep- 

 tone-egg and growing at 37.5° for one week. At the end of the 

 seven days' incubation, the resistance of the spores to high 

 temperatures was determined. The cultures to be tested were 

 subjected to temperatures from 85° to 101°, raising the temper- 

 ature one degree at a time. All the spore cultures survived 86° 

 and some survived 100° for fifteen minutes and thirty minutes. 

 The tubes containing the inoculated material were cooled im- 

 mediately after the fifteen minute exposure at the desired temper- 

 ature and incubated at 37° until definite results were obtained. 

 In the majority of cases twenty-four hours' incubation was suffi- 

 cient for the organism to germinate, but in some cases germination 

 did not occur until after four or five days in the incubator. 

 When doubtful results were obtained, 0.5 cc. of the culture 

 containing spores was inoculated instead of the five loopfuls to 

 make sure that there were sufficient spores present. The use 

 of 0.5 cc. was however resorted to only in exceptional cases, for 

 where negative results were obtained with five loopfuls, the same 

 result was found with the larger quantity. 



4. Discussion of results, a. Vegetative forms. The thermal 

 death point of vegetative forms of Clostridium Welchii varies 

 according to the source from which the cultures are isolated and 

 the duration and conditions of growth previous to the test. 

 Cultures isolated from market milk survive 56° and die at 61°, 

 although cultures immediately after isolation die at 59°. The 



