4 CONTROL SERIES No. 63 



decrease in agglutinability or become less stable and that fresh antigens are 

 usually more satisfactory than those that have been kept more than 2 weeks. 

 Doyle (27) found that, in antigens from cultures being studied, storage up to 

 36 days had no influence on the titer. Biely (5), using the rapid serum method, 

 found perfect agreement in 171 tests between an antigen 3 years and 7 months 

 old and an antigen 1 day old. Jones (531 found that diluted test fluid might be 

 kept for "several months in a refrigerator." 



For the study of the efi'ect of age on concentrated antigen, a quantity of 

 antigen was prepared from three strains, Nos. 10, 11, and 20, of 5. pullorum, 

 which had been successfully used in the production of antigen for a large number 

 of tests. Strain No. 11 was isolated from the ovary of a hen and Nos. 10 and 20 

 were isolated by Dr. Rettger of Yale University from baby chicks in 1916 and 

 1917, respectively. This stock antigen which was about 20x tube No. 1 of the 

 McFarland nephelometer standard was stored at 8° C. A portion was diluted 

 and tested when it was prepared. Further tests were made at biweekly intervals, 

 with three exceptions when 4 weeks elapsed between tests, for the 583 days over 

 which the investigation extended. On the second and succeeding tests a fresh 

 antigen composed of the same strains was prepared and tested in the same 

 manner as the stored antigen. Both antigens were adjusted to a turbidity be- 

 tween 0.75 and 1.0 on the McFarland nephelometer scale and a pH of 8.4. Fresh 

 sera were used for each test. Samples were taken from each of 10 birds known 

 to be reactors and 5 non-reactors. Comparative tests were made 37 times with 

 a total of 555 sera (365 positive, 185 negative, and 5 which were cloudy and 

 recorded as unsatisfactory). 



Table 1 gives the results of the tests in numerically computed values and an 

 analysis of the positive and negative reactions and relative sensitivity. Judged 

 by this standard, there appears to be little choice between the two antigens 

 and no evidence of inferiority in the stored antigen as it grows older. In 18 of 

 the tests the stored antigen had a sUghtly higher value against 19 times when 

 the freshly prepared had a higher value. The variations observed at each test 

 might readily be ex-plained in part as due to differences in individual sera. It 

 may be possible that a difference in the contents of any sera, aside from agglu- 

 tinins, might cause a variation in the reaction with even the most satisfactory 

 antigen. Fiu-thermore, such factors as technique of testing and interpretation 

 of reactions might influence the variations that have been observed. The macro- 

 scopic observation of the tests showed no difference in the type of agglutination. 

 The reactions with the one antigen were as typical and distinct as with the other. 

 No progressive change in the stored antigen affecting its efficiency as an agglu- 

 tination test fluid occurred. The turbidity of the stock antigen decreased from 

 20x to 15x tube No. 1 of the nephelometer during the duration of the ex-periment. 



Effect of Age on Dilute Antigen 



The effect of storage on dilute antigen was investigated in two trials: the 

 first, less extensive, over a period of 5 weeks, and the second extending over 15 

 weeks. A report of the first is omitted because the results were similar to those 

 of the second. In many laboratories sodium hydroxide is added to dilute antigen 

 to eliminate, as far as possible, "cloudy reactions" and has been reported as a 

 valuable agent in this respect, first by Mathews (62) and later by Stafseth and 

 Thorp (86), Casman, Valley, and Rettger (17), Bleecker and Schilling (6, 7), 

 and the Connecticut (Storrs) Agricultural Experiment Station (19). Dilute 

 antigens with and without the excess sodiimi hydroxide were tested. 



The three strains of S. pullorum used for this experimental antigen were the 

 same as those used in the work with concentrated antigen. The stock antigen 



