296 DETERMINATIONS OF THERMAL DEATH-TIME 



Esty and Meyer' report germination of subcultures prepared from heated suspensions 

 after an incubation of 378 days at 37° C. The cultures produced a virulent toxin and 

 presented typical morphological and biological characteristics. They also' observed 

 that subcultures prepared from diluted unheated or slightly heated spore suspensions 

 (80° C. for I hour or 100° C. for 2 minutes) exhibited the phenomenon of retarded 

 germination. Similar findings have been recorded by Burke.^ In view of this, the 

 dormancy of unheated and heated suspensions must be determined for each organism. 



Thermophilic organisms and many aerobic types^ germinate readily even when 

 only a few viable cells are present. In fact, germination of heated spores of thermo- 

 philic organisms occurs from 48 to 120 hours when cultures are incubated under 

 optimum conditions. 



It has previously been suggested that another reason for irregularities and vari- 

 able results may be unsatisfactory technique. The method by Bigelow and Esty^ has 

 been used with minor modifications generally by research workers of this country. 

 This method deserves wider recognition and, therefore, is briefly described. 



A definite amount of a uniform, preferably strained or filtered, suspension is 

 inoculated into a series of sterile glass tubes (hard or soft) of standard size, approxi- 

 mately 7-mm. inside diameter with i-mm. thickness of wall. The inoculated tubes are 

 sealed in an oxygen flame, preheated in boiling water and then completely immersed 

 in an electrically heated oil bath so adjusted and controlled as to maintain a constant 

 uniform temperature throughout. The preheating and an initial rise in the tempera- 

 ture of the bath of from 2° to 3° during the first 2 minutes' exposure of the tubes 

 reduces the lag period to approximately 3 minutes. The bath is so operated that at 

 the end of the lag period the temperature reaches the desired degree and thereafter 

 remains constant. 



At definite periods tubes are removed, cooled immediately in a bath of ice water 

 and the entire content subcultured in a favorable medium, if not already in suitable 

 material, and incubated to determine survival. 



Weisss modified the foregoing procedure by using 6"Xf" test tubes, in which he 

 found that a lag of about 30 minutes raised the inside of the tubes from room tempera- 

 ture to 99° C. (with a bath at 100° C), of 15 minutes to 105° C. and 8 minutes to 

 120° C. It is evident that in the tests reported by Weiss, 3 minutes at 120° C, the 

 spores were destroyed during the period of lag when the temperature inside the tube 

 was still rising and long before the bath temperature was attained. He also modified 

 the method of subculturing: A loopful of the heated material was inoculated into 

 meat infusion glucose agar tubes, which were finely layered with paraffin oil and 

 incubated at 37.5° C. 



Magoon** used thin-walled capillary tubes and introduced them into the culture 

 tubes for sterility tests. 



' Esty, J. R., and Meyer, K. F.: loc. cit. 



' Burke, G. S.: J. Infect. Dis., 33, 274. 1923. 



3 Bigelow, W. D., and Esty, J. R.: loc. cii.; Esty, J. R., and Williams, C. C: ibid., 34, 516. 1924. 



" Bigelow, W. D., and Esty, J. R.: loc. cit. 



5 Weiss, H.: loc. cit. 'Magoon, C. A.: loc. cit. 



