GERMICIDAL ACTION OF FREEZING TEMPERATURES 427 



tested over a long period of time without relation to the quanti- 

 tative aspect, while we have studied chiefly its influence over 

 very short periods and have consistently made quantitative 

 studies.. 



In order to eliminate crystallization and possible mechanical 

 crushing of bacteria during the freezing of the medium, the 

 freezing point was depressed by the addition of a non-electrolytic 

 substance, grape sugar. From the formula given in Harper's 

 Scientific Memoirs for the lowering of the freezing point of water 

 in degrees Centigrade, produced by dissolving a gram mole- 

 cule of a given substance in a liter of water, it was possible to 



TABLE 4 



To depress the freezing point to — 6°C.. . 

 To depress the freezing point to -4°C.. . 

 To depress the freezing point to — 3°C.. . 

 To depress the freezing point to -2°C.. . 

 To depress the freezing point to — 1.5°C. 

 To depress the freezing point to -0.5°C. 



GLUCOSE 



grams 



56.2 

 37.5 

 28.1 

 18.7 

 14.0 

 4.7 



calculate the amount of glucose (CeHiaOs) which must be added 

 to a liter of water to depress its freezing point by any desired 

 increment. The table above gives the amount of glucose 

 required to depress the freezing point to a certain degree as 

 worked out by the above formula. 



The several sugar solutions were made with chemically pure 

 glucose and sterilized in streaming steam on three successive 

 days. The pure cultures of B. coU—six strains in all were used 

 —were frequently transferred and a twenty-four hour old cul- 

 ture in standard bouillon was always used in the tests. In all 

 of the experiments sterile tap water was used as a control, and 

 for comparison. The culture was inoculated into the tubes of 

 water and solutions to be tested, the initial numbers determined 

 by plating from each tube in several dilutions, always using 

 duplicate plates, and then the tubes were immersed in an ice- 



