100 



Scientific Proceedings (118). 



the same time, with absolute protection against any possibility 

 of contamination after their contents had been subjected to the 

 heating process. 



After a number of trials we have adopted the following method 

 of procedure in our experiments, and, although it cannot be 

 adapted to the investigation of all the problems which present 

 themselves in thermal death time experiments, it has proved to 

 be satisfactory for the problems under immediate investigation. 



Soft glass tubes, iox 150 mm., are used in the experiments. 

 Three cubic centimeters of 1 per cent, glucose peptic digest liver 

 broth, adjusted so that the final Ph is between 7.3 and 7.5, are 

 placed in each tube and covered with a thin layer of oil to prevent 

 evaporation. The medium is sterilized at fifteen pounds pressure 

 for thirty minutes. 



Immediately before they are to be inoculated the tubes of 

 broth are exposed to live steam for twenty minutes to expel the 

 air. A known number of spores is added to each tube in yi c.c. 

 of the medium in which they have grown, the number of spores 

 in the suspension being determined by actual count in a counting 

 chamber. The tubes are then sealed in an oxygen flame and are 

 ready for heating. 



Although it has been found by actual counts that there is no 

 appreciable change in the number of spores within five hours 

 after they have been placed in the tubes, not more than ninety 

 minutes are allowed to elapse after the tubes are inoculated before 

 they are submitted to the heat. 



The spores are heated by immersing the sealed tubes in racks 

 into oil which is maintained constant at the required temperature 

 and vigorously agitated. At the end of the required time of 

 heating the tubes are removed from the oil, placed in deep pans 

 of cold water to cool and immediately labelled. They are then 

 incubated at 37. 5 0 C. 



The appearance of growth in the sealed tubes is characteristic 

 and easily detected. Incubation is continued for at least ten 

 days after growth is recognized in each instance to allow time 

 for the formation of toxin, after which the tubes are opened under 

 sterile precautions, deep agar, broth and meat mediums are in- 

 oculated for the purpose of observing the cultural characteristics 



