J. RUSSELL ESTY 



297 



Dickson and Burke' adopted the so-called "sealed-tube technique" to exclude 

 all possibility of contamination and to obtain accurate information as to the inci- 

 dence of "skips" (bacterial growth occurring in tubes which have been heated for a 

 longer period than those which frequently remain sterile). The majority of their final 

 tests on the heated resistance of CI. hotulinum spores were made in sealed tubes 

 heated and incubated in i per cent glucose peptic digest broth (pH 7.0-7.4) covered 

 by a thin layer of mineral oil. For comparison a smaller series was run in glucose 

 broth without oil, glucose agar, and brain medium. Heating records show a lag period 

 of from 2^ to 4 minutes at each temperature. 



With this technique, Dickson^ et al. demonstrated "skips" in all the media and 

 at all the temperatures at which the spores were heated. 



Using the Bigelow and Esty^ method to determine the spore resistance of numer- 

 ous strains of the common aerobic soil and water organisms, facultative and obligate 

 thermophiles, CI. botidimmi, and allied anaerobes, "skips" as noted by Dickson 

 were frequently observed by the writer. Cultures heated for periods up to and includ- 

 ing a certain time showed growth uniformly when incubated in a favorable environ- 

 ment, but for longer periods the results were irregular. These observations indicated 

 that spore suspensions in a single-tube series occasionally contained viable organisms 

 that had been heated for a considerable time in excess of those in which uniform 

 growth had been observed. To explain these irregularities and to determine more ac- 

 curately the actual resistance of organisms to moist heat, Esty and Williams^ modified 

 the original method by subjecting a large number of tubes, from twenty-five to thirty, 

 instead of one or two, each containing the same suspension, to various temperatures. 

 This procedure conclusively showed that these "skips" are due to the variable re- 

 sistance of individual spores in any suspension. They occurred in cultures containing 

 different numbers of spores and to some extent in all media. Table IV^ gives typical 

 results of tests on twenty-five to thirty tubes each containing the same spore suspen- 

 sion, all heated alike, as compared to those obtained when single tubes were treated. 

 It will be seen that spores in a phosphate solution pH 5.1 in single tubes were viable 

 for 18 minutes at 110° C. (230° F.), while even after 12 minutes, one of twenty-six 

 tubes failed to show growth. In other words, 96.4 per cent of the tubes showed viable 

 organisms after 12 minutes and 64.3 per cent after 15 minutes; and although a single 

 tube indicated complete destruction in 20 minutes, yet 39.4 per cent of the tubes gave 

 growth by the multiple method. 



Chart II illustrates graphically the results obtained with phosphate solutions, 

 pH 7.09 and 6.2, corn juice 6.1 and pea juice 5.74, as given in Table IV. A straight 

 line is drawn as nearly as possible through the experimentally determined points. 

 These graphs are suggestive of logarithmic relations similar to those found in the 

 mortaHty curves of bacteria generally and in thermal death-time relations as pointed 



' Dickson, E. C, and Burke, G. S.: Proc. Soc. Exper. Biol. &° Med., 19, 99. 1921. 



= Dickson, E. C, Burke, G. S., Beck, D., and Johnston, J.: loc. cit. 



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



" Esty, J. R., and Williams, C. C: loc. cit. 



sEsty, J. R., and Williams, C. C.: /. Infect. Dis., 34, 518. 1924. 



