EXPERIMENT STATION BULLETINS. 459 



micro-organism B, some whey-agar was made out of a milk-culture of B which 

 had grown for forty-eight hours. The casein was precipitated by rennet, the 

 whey was clarified and made into agar. When this agar was planted with 

 micro-organism A and the culture compared with the growth upon whey-agar 

 made from fresh milk, in twenty-four hours time at 37V_'° C, there was so 

 much difference in the prolificacy of growth manifested between the two whey- 

 agars in fayor of the agar made from the milk-culture of micro-organism B 

 that it was plainly evident the products of micro-organism B found their way 

 into the agar. The stability of these products through repeated heating is 

 only another instance of what has been repeatedly demonstrated in case of 

 many other products of bacteria. 



In an effort to secure definite data upon the influence of the products re- 

 sulting from the growth of micro-organism B in milk, a quantitative determina- 

 tion was resorted to. Eight flasks, 500 c. cm. in capacity, were employed as 

 containers. Into each was placed 100 c. cm. of milk of the same lot, to which 

 litmus had been added. These were heated at 100° C. for three consecutive 

 days. For the purpose of reference we shall designate these flasks as 1, 2, 3, 4, 5, 

 6, 7, and 8. When sterilization was completed, flasks 5, 6, 7, and 8 were inocu- 

 lated each with a definite and the same quantity of micro-organism B. These 

 flasks were placed at 23° C. for forty-eight hours, at the end of which time 

 visible milk changes, characteristic of micro-organism B, were noted. They 

 were then heated again at 100° C. for three consecutive days. Bouillon tubes 

 inoculated from these flasks gave no signs of growth after several days. At 

 this stage, therefore, we have four flasks, 1. 2, 3, and 4 in which no special 

 germ had been permitted to grow, and four flasks, 5, 6, 7, and 8 in which micro- 

 organism B had been allowed to develop for forty-eight hours at 23° C. 



Each of these flasks, 1. 2, 3, 4. 5, 6, 7, and 8, was inoculated synchronously 

 with 1-2000 c. cm. of a fresh milk-culture of micro-organism A. An estimatedl 

 determination made the number of organisms 49,800 for each flask. In handling 

 this amount of culture, to obtain accuracy and uniformity, high dilutions in 

 physiological salt solutions were employed. Our work was accurate, for the 

 eight plates employed scarcely deviated over one or two colonies from the 

 average. The further results of our work also bear us out in the correctness 

 of our manipulations. 



All the flasks after inoculation as above were placed at a temperature of 23" 

 C. Observations were made every twenty-four hours. 



Observations at the end of twenty-four hours. 



The litmus in the milk in flasks 1, 2, 3, and 4 was slightly reddened 

 only. There was no apparent change in milk. In flasks 5, 6, 7, and 8 

 the litmus was wholly reduced except a very thin layer on the imme- 

 diate surface. The milk was a firm lopper with whey separated. 



Observations at the end of forty-eight hours. • 



Litmus in milk in flasks 1, 2, 3, and 4 redder than twenty-four hours 

 previous. Milk still unchanged to naked eye. Flask cultures 5, 6, 7, 

 and 8 remained as the day previous. Growth was checked and oxygen 

 had permeated milk to half its depth, reddening the litmus. 



Observations at the end of seventy-two hours. 



Milk in flasks 1, 2, 3. and 4 was beginning to lopper. A thin layer 

 of loppered milk had formed on bottom of flasks but surface milto 

 was still fluid. Litmus almost wholly reduced. Flask cultures 5, 6, 

 7, and 8 remained same as previously; the litmus had become red 

 throughout. 



Upon reviewing the above observations, it should be emphasized that flasks 

 1, 2. 3, and 4 developed together as nearly identically as it is possible: a study 

 of the acidities will also establish this; hence it follows that these four flaska 

 may be considered as dem.onstrating the unity of action. Flasks 5, 6, 7, and 

 8 may also be treated in the same manner. The difference in time of loppering 

 between these two sets is about forty-eight hours. Flasks 5, G, 7, and 8 were thor- 

 oughly loppered and the whey separated in twenty-four hours, while flasks 

 1, 2, 3, and 4 did not begin to lopper till seventy-two hours. Flasks 1, 2, 3, and 



