EXPERIMENT STATION BULLETINS, 305 



varied with the cultures under study, as will be noted later in the detailed 

 work. In making them, 1 c. cm. was the smallest measurement employed, 

 because it was found that anything smaller would not give uniform results, 

 although the pipettes were supposed to be accurately graduated to .1 and 

 1-100 c. cm. The flasks containing the solutions for dilution were 375 c. cm. 

 Erlenmeyer flasks, in which was placed 99 c. cm of the suspending diluent. 

 From the last dilution of a twenty-four hour culture, 1 c. cm. was trans- 

 ferred to the milk flasks which were also 375 c. cm. Erlenmeyer flasks, con- 

 taining 100 c, cm. of litmus milk* The cultures consisted of the associate 

 micro-organism and the lactic micro-organism together with the combined 

 culture. The milk flasks inoculated may be designated thus, as 



la, in which 1 c. cm. of the associate culture la, diluted as above and 

 recorded under this particular combination, is added to one milk 

 flask containing 100 c. cm. of litmus milk, 



Ij, in which 1 c. cm. of lactic culture, Ij, diluted as above and recorded 

 under this particular combination, is added to one milk flask con- 

 taining 100 c. cm. of litmus milk. 



la + Ij, in which 1 c, cm. of the associated culture, la, and 1 c. cm. of 

 the lactic culture, Ij, each, diluted as above and recorded under this 

 particular combination, is added to one milk flask containing 100 

 c. cm. of litmus milk. 



This gives one milk flask culture of each micro-organism, la and Ij as 

 checks against the combination la + Ij. These checks grew under iden- 

 tical conditions as the combination. The combination, therefore, differs 

 only in having been inoculated with the same number of micro-organisms, 

 la, as were placed in the milk flask culture la, and the same number of 

 micro-organisms, Ij, as were placed in the milk flask culture Ij. The num- 

 ber of micro-organisms introduced into each was controlled by plating. 

 The three milk flask cultures la, Ij, la + Ij, were placed at 21° C. and main- 

 tained at this temperature until la + Ij or Ij loppered. From time to time 

 ordinary and whey agar plates were made to determine the number of micro- 

 organisms present in each culture. Note was made of any visible changes 

 and the acidityf determined. The records of the individual combinations 

 will illustrate with what results this was accomplished, 



SAMPLE I. 



This sample of milk was secured from a ten gallon can, placed in the milk- 

 room of the College stable and was taken as soon as the milkers had filled 

 the can. The milk was received in sterile Erlenmeyer flasks and conveyed 

 to the laboratory, where it was immediately plated and tested for acidity. 



The flask of milk was then placed in a temperature room, at a temperature 

 of 21° C. Plates were made from the flask of milk from time to time, as 

 indicated later, and the milk tested for acidity at the time of each plating, 



♦Wherever milk was used for cultures in which combinations were to be made, such milk was in- 

 variably of the same lot, containing the same amount of litmus and sterilized in an identical manner- 

 In other words, its treatment was identical in every instance. See article on "Extended Studies of 

 the Associative Action of Bacteria in the Souring of Milk," in which "The Significance of Different 

 Milks and Their Relation to Germ Development." is treated. Cent. f. Bakt. II. Bd. XV. p. 400. 



tThe matter of measuring the acidity of milk cultures has already been referred to by the senior 

 author in his articles on "Aeration of Milk." Cent. f. Bakt. II, Bd. IX, p. 313. "Extended Studies of 

 the Associative Action of Bacteria in the Souring of Milk." Cent. f. Bakt. II, Bd. XV, p. 400. 



39 



