SPOKOGENES TEST. 9 



the number of stormy reactions and the dilution the number of 

 spores in the manure was estimated. Assuming that the manure 

 as it enters milk contains only one- fourth as much moisture as sam- 

 ples of moist feces, and multiplying, therefore, the average spore 

 content by four, it may be assumed from the results that the manure 

 as it enters the milk contains approximately 1,200 spores per gram. 

 When this average figure is compared with the 10,000 obtained by 

 Weinzirl it is apparent that there must be an extremely variable 

 number of spores of B. enteritldis sporogenes in cow feces. Even 

 if it were possible to assume that these spores could gain entrance 

 into milk from cow manure only (without definite proof this as- 

 sumption can not safely be made) their variable number in this 

 material would decidedly interfere with the accuracy of the test. 

 In the light of these facts it is probably possible to explain the dis- 

 crepancies that exist between the test and the manurial content of 

 the milk. 



Spores of the organism under consideration are known to be 

 widely distributed in nature. They are found in waters and soils, 

 and, as is shown in Table 2, they are present in considerable numbers 

 in silage, grain, and mixed feeds. How many spores may gain 

 entrance into milk from these sources is of course problematical, 

 although on the whole it seems probable that they are introduced in 

 the greatest numbers through manure. 



It seems apparent from the results of other investigations and of 

 our experiments that if the sporogenes test is to be of any definite 

 value its greatest possibility lies in the relation which the test bears 

 to the general conditions of production. 



ATTEMPTS TO IMPROVE THE CHARACTERISTIC STORMY 



REACTION. 



Before going further with the sporogenes test it was considered 

 desirable (1) to reduce if possible the number of doubtful reactions 

 caused by peptonizing faculative anaerobes and (2) to hasten and 

 make more dominant the growth of B. enteritidis sporogenes by add- 

 ing to milk certain substances likely to promote more rapid growth 

 of the organism. 



To eliminate peptonization, attempts were made to utilize the selec- 

 tive action of gentian violet. Churchman (3) has shown that gen- 

 tian violet in a dilution of 1 to 100,000 of medium prevented the 

 growth of B. subtUis while it permitted the growth of B. welchii. 

 Dilutions of 1 to 50,000 and 1 to 100,000 were tried, the necessary 

 amount of a stock solution of 1 to 1,000 being added previous to heat- 

 ing the milk tubes. It was found that the proportions of 1 to 50,000 

 25154°— 21 2 



