124 THE ROYAL SOCIETY OF CANADA 



one-quarter of an hour too frequently gave false results, since for a 

 considerable part of the time of exposure the spores might be sub- 

 jected to a temperature below that recorded in the immediately sur- 

 rounding fluid, when the temperature was taken not within the test 

 tubes, but in that surrounding fluid. It therefore appeared that this 

 source of error would be eliminated, and a more thorough test afforded, 

 if the period of exposure were extended from fifteen minutes to an 

 hour. Thus unless otherwise stated, our results represent the exposure 

 of the spores in suspension to a given heat for a period of one hour. 

 At the end of sixty minutes the tubes were taken out of the bath, im- 

 mersed in cold water, and surface inoculations made upon Lemco 

 peptone broth agar, the original tubes being employed now as controls 

 to demonstrate the complete destruction — or otherwise — of all the 

 spores. 



THE WATER-BATH. 



Studying the earlier literature, it is impossible not to realize that 

 the methods employed by most observers, whether to test the thermal 

 death-point of bacteria, or to determine the heat resistance of en- 

 dospores, have been very imperfect. In the course of these observa- 

 tions an endeavour has been made to eliminate as far as possible the 

 imperfections. 



1. As already indicated, if a glass tube containing fluid be im- 

 mersed in a water-bath, it requires many minutes before the contents 

 of that tube attain the same temperature as that of the surrounding 

 fluid. Therefore, to determine accurately the temperature to which a 

 suspension of spores is exposed, it is essential that the thermom.eter be 

 immersed not in the surrounding fluid, but actually within the sus- 

 pension itself, in other words, for accurate work the amount of a sus- 

 pension of spores employed must be sufficiently large to permit the 

 placing in it of a thermometer or, what comes to the same thing, a 

 control test tube must be employed in which the thermometer is 

 immersed. This must be of same size and contain same amount of 

 fluid as the test tubes that are employed to hold the suspensions of 

 spores. 



2. The amount of loss of heat from the free surface of a water- 

 bath is much greater than, we think, is ordinarily imagined. At 

 temperatures between 60°C. and 100°C. it was found that this loss 

 was so great that employing a large water-bath of two jackets we 

 repeatedly found it impossible in the dry, winter air of Montreal to 

 maintain the water in the inner receptacle within three or four degrees 

 of that in the outer. When using a very large surface, as, for example, 

 that of the routine "serum inspissator," with which we made our 

 first experiments, the water in the outer compartment might be boiling 



