J. RUSSELL ESTY 291 



which would scarcely be differentiated from granular forms of vegetative bacilli have 

 a direct bearing on this aspect of the problem. Furthermore, spores generated in dif- 

 ferent flasks of the same medium, inoculated with equal amounts of the same stock 

 culture, and incubated for the same period show striking differences in heat resistance. 

 Although the most resistant strains of CI. bokilinum behave fairly consistently, yet 

 in the most suitable medium they may exhibit a low degree of heat resistance. No 

 explanation can be offered for this peculiar behavior. These differences are independ- 

 ent of the final reaction of the culture fluid within the range of pH 6-8, the numbers 

 and the structure of the spores, as demonstrated by Burke's staining reaction.' 

 However, a well-buffered medium which furnishes abundant food material for "pro- 

 gressive growth" produces spores of average or low resistance. Kronig and PauP have 

 noted that the same food does not always induce the production of spores of equal 

 resistance. 



Von Hibler^ found that the spores from a single species were very constant in heat 

 resistance if they were not too young or too old or had not been exposed too long to incubator 

 temperatures or in an acid medium. Weiss'* and Esty and Meyer^ found young spores to be 

 the most heat resistant. Dickson et al.^ report that the age of botulinum spores which have 

 been kept at room temperature in the culture medium in which they developed does not 

 materially influence the resistance after they reach an equilibrium. They found that the 

 22-day-old spores were considerably less resistant than those in a culture 66 days, but that be- 

 tween 66 and 315 days the resistance was practically constant. Magoon,^ working with spores 

 of B. mycoidcs grown on sand moistened with broth, concluded that the degree of resistance is 

 influenced by age, temperature, humidity, and possibly other factors. He states that the 

 maximum resistance to heat develops under conditions of moderate temperature and hu- 

 midity and is probably reached by the time the spores are 60 days old. Furthermore, the 

 change in resistance took place slowly when spores were dry and cold, while a low tempera- 

 ture accompanied by high humidity resulted in the development of resistant spores. 



Esty and Meyer* observed that the dehydration of moist spore suspensions of CI. 

 botulinum lowered the heat resistance, although this remained constant over a period of 347 

 days. In fact, perfectly dry spores were preserved equally well at 37° C, room temperature, 

 or 20°C. indefinitely, and as such were well suited for comparative studies. 



Convincing experimental evidence indicates that some of the variations in heat resistance 

 are due to the chemical composition of the solutions in which the organisms are heated, pH 

 and other so-called "extrinsic" factors. For instance, in some cases vegetative forms and 

 spores were heated in the medium in which they were produced, in others a portion of the 

 suspension was transferred to fresh culture media or to different solutions, while in others 

 the organisms were washed and then resuspended in the test solution. Striking variations 

 have been noted in the resistance of spores heated in different solutions, and Table H^ 

 illustrates the results obtained with peptone solutions as compared to a phosphate mixture. 



In view of these and numerous other observations, the spore material should be concen- 

 trated by centrifuging and the sediment washed and resuspended in a well-buffered solution, 

 preferably a phosphate mixture of a neutral reaction for comparative heat-resistance tests. 



' Burke, G. S.: J. Infect. Dis., 32, 6, 433. 1923. 



2 Kronig, B., and Paul, T.: Ztschr.f. Hyg. u. Infektionskrankh., 25, i. 1897. 



3 von Hibler, E.: loc. cit. 6 Dickson, E. C, et al.: loc. cit. 

 " Weiss, H.: loc. cit. 7 Magoon, C. A.: loc. cit. 



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



