ERADICATION OF PULLORUM DISEASE 1931-32 7 



Contrary to the report of Stafseth and Thorp (86) no change was observed 

 in the turbidity. There was a shght change noticed in the pH, however, begin- 

 ning at the eighth week when the pH of the 8.4 antigen had decreased shghtly. 

 By the twelfth week, the alkahnity had decreased so that the pH was 8.2, and 

 at the last test (fifteenth week) it was 8.0-8.2. The pH 7.2 antigen began to show 

 a decrease in alkalinity about the twelfth week and had a pH of 7.0 by the fif- 

 teenth week. Comparing the results on a numerical basis as in the case of the 

 concentrated antigen, the antigen of pH 7.2 raised to 8.4 at the time of the test 

 was the most sensitive in 14 of the 25 times it was tested; in 5 tests it was more 

 sensitive than one of the other antigens and in 3 it was identical with one other 

 antigen; and in only 3 tests was it the least sensitive. The almost parallel ten- 

 dency of the three to fluctuate is plainly shown in Graph 1. The summary of the 

 values from which the data for the making of the graph was derived is as follows : 



pH 7.2 



antigen 



Number of tests 25 



Number of teats most sensitive 4 



Number of tests more sensitive than one other antigen 7 



Number of tests least sensitive 11 



Number of tests identical with one other 3 



The type of agglutination in the tests showed no appreciable difference dur- 

 ing the course of the experiment. Cloudy reactions occurred in the pH 7.2 antigen 

 in a few instances. The pH 7.2 antigen seemed slightly less sensitive on the 

 whole and the pH 8.4 antigen not quite as sensitive as that to which sodium 

 hydroxide was added at the time of the test. However, the results suggest that 

 a diluted antigen with pH 8.4 loses very little, if any, of its antigenic value dur- 

 ing a period of fifteen weeks. 



Effect of Frequent Transfers and Low Temperature on the Cultures 



In view of the fact that very frequent transfer to fresh medium and storage 

 at refrigerator temperature may cause changes in the behavior and morphology 

 of many bacteria, ^S. ptillorum was treated in this way to observe whether any 

 marked changes resulted and whether the antigens produced from the cultures 

 were in any way less satisfactory for agglutination tests. Speaking of 5. ptillorum 

 for agglutinable antigen, Jones (53) states that freshly isolated or even second 

 or third generation cultures give better results than those under cultivation for 

 a longer time; but Tittsler (88) after the work had been in progress much longer 

 was unable to estabUsh any correlation between "agglutinability and the length 

 of time strains had been carried in stock on artificial media." 



Casman, Valley, and Rettger (17) in some detailed work found that antigens 

 prepared from cultures grown at different temperatures (37°, 34°, 30°, 24°, 

 20°, and 16° C.) and for different periods of time (1, 2, 3, 5, 6, 9^, and 10 weeks) 

 revealed no agglutinable dift"erences. Antigens of equal value were also prepared 

 from cultures transferred twice a week for a period of 10 weeks and incubated 

 at 37°, 30°, and 16° C. 



The temperature chosen for the observations at this laboratory was approx- 

 imately 8° C, and the interval between transfers to fresh medium was one week. 

 A set of cultures held at a temperature approximately 22 ° C. and transferred to 

 fresh medium once a month served as a control. Three strains of 5. pullorum, 

 designated as Nos. 17, 19, and 20, were studied. The strains were all isolated 

 by Dr. Rettger of Yale University, strain No. 17 from a baby chick in 1916, 

 strain No. 19 from a hen in 1911, and strain No. 20 from a baby chick in 1917. 

 Three agar slant subcultures were made from each strain and incubated 48 

 hours at 37 ° C. The cultures were distributed as follows: one of each strain placed 



