398 J. RUSSELL ESTY 



the fecal sources were tested as soon as possible after they were 

 isolated in pure culture. For the determination of the thermal 

 death point, tests were made on 75 cultures from market milk, 

 54 cultures from human feces, 18 from cow feces and 18 from 

 horse feces. The thermal death point of the spores in cow and 

 horse feces was not determined. 



2. Thermal death point of vegetative forms. Tubes containing 

 10 cc. of glucose-liver broth were inoculated with two-tenths 

 cc. of the pure cultures and grown for eight, twenty-four and 

 forty-eight hours at 37.5°. Cultures were also grown in sterile 

 milk. The best and most consistent results were obtained by 

 growing at 37.5° for eight hours. A double water-bath was used 

 to keep the temperature uniform. This bath was heated to the 

 desired temperature and a sufficient supply of tubes each con- 

 taining 10 cc. of sterile glucose-liver broth was placed in the 

 inner bath. The temperature of the bath was recorded in a 

 control tube by a thermometer placed in the broth which was 

 not allowed to vary more than half a degree in either direction. 

 The water in the bath was agitated by means of a stirring rod 

 throughout the heating. After fifteen minutes exposure at the 

 desired temperature, the broth was inoculated with five loopfuls 

 of the eight-hour broth culture to be tested by simply removing 

 the cotton plug, without removing the tube from the bath. No 

 attempt was made to determine the numbers per cubic centimeter 

 before and after the heat exposure, either of the vegetative or 

 the spore forms. The amount was kept as uniform as possible 

 using the same size loop and uniform 1 cc. pipettes. The inocu- 

 lated tubes were exposed for fifteen minutes at this temperature 

 and were then transferred at once to a vessel of cold water in 

 order to cool them quickly and prevent further action of the 

 heat upon the bacteria. When cold they were placed in the 

 incubator at 37.5° and kept under observation long enough to 

 ascertain whether growth occurred. Clostridium Welchii in the 

 vegetative stage showed evidence of growth in twelve to forty- 

 eight hours if the heat had not killed the organism. Duplicate 

 tubes were inoculated to check the determination and in prac- 

 tically all cases the same thermal death point was obtained. 



