20 MASS. EXPERIMENT STATION BULLETIN 183. 



four hours, but these were spores in the center of the drop of water, or 

 directly in the agar, which seemed to give them some protection. There 

 was no germination whatever after thirty-six hours. 



The first method more nearly approximates natural conditions, but 

 under any conditions we may safely draw the conclusion from these 

 experiments that all spores are killed by freezing during thirty-six hours. 



Thermal Death Point of Spores. 

 Investigation of this point was undertaken with a view to the possi- 

 bility of sterilization by heat. Thermal death point is defined as the 

 lowest temperature at which an organism is killed by an exposure for ten 

 minutes. Since tliis point might be different for spores than for mycelium, 

 each was tried separately. 



Method. — Spores from a young culture immersed in a drop of water were placed 

 in a thin pipette tube, sealed at one end and covered with a rubber cap at the 

 other. The tubes were then dropped into vessels of water kept at the desired 

 temperature. Each vessel was supplied with a thermometer, and could be iieated 

 by a Bunsen burner when necessary. After ten minutes the tubes were removed, 

 the sealed end filed off, and the spores forced out through it on to a glass slide 

 by pressing the rubber cap at the other end. The slides were then put in moist 

 chambers as previously described in germination tests. These were kept at ordi- 

 nary laboratory temperatures. Temperatures at intervals of 1°, from 40° to 55°, 

 were tried. All tests were made in duplicate several times. 



Up to and including 46° the spores did not seem to be affected by ten- 

 minute exposures. Above this the percentage remaining alive declined 

 very rapidly to the absolute thermal death point of 49°, At this tem- 

 perature none ever germinated. 



It was also found that spores can be killed at lower temperatures than 

 49° by exposing them for longer periods. In some previous experiments it 

 had been determined that they are killed by an exposure to 37.5° for 

 twenty-four hours. At 42° they are killed in two hours. To determine 

 the efTect of varying the period of exposure at a given temperature, 40° 

 was selected as a standard, and spores exposed (in drops of water on 

 slides in Petri dishes) during periods differing by intervals of one hour. 

 They were then brought back to room temperature and tested as above. 

 The results of this series are given in Table II. 



