ERADICATION OF PULLORUM DISEASE 1931-32 3 



The Technique Used in Making the Antigen 



The method of making antigen has changed since the first application of the 

 test by Jones (52\ as reported in 1912. In his early work he used an antigen 

 prepared by incubating the culture for 72 hours and used a saline solution con- 

 taining 0.6 per cent sodium chloride and 0.5 per cent phenol for washing off the 

 growth, and then inspissated the washings for 23^ hours at 60° C; but later (53) 

 he stated that the heated antigen was less satisfactory and also changed the sal- 

 ine solution to 0.88 per cent sodium chloride. Gage, Paige, and Hyland (36) 

 incubated inoculated agar slants one to two days and shook the washings for 

 one-half hour in a mechanical shaking machine before the suspension was filtered 

 through cotton. This, with the omission of the shaking, was essentially the 

 method used by Rettger, Kirkpatrick, and Jones (76). Gwatkin (40) incubated 

 the inoculated agar for four days, standardized the turbidity by Gates' method, 

 and used phenol as a preservative. Brunett (10) incubated his cultures for 24 

 hours. 



Antigens for this investigation were prepared in the same way throughout, 

 and the technique of making the agglutination tests and the interpretation of 

 the results were identical. Strains of Salmonella pullorum chosen for antigen 

 were checked for purity by microscopic examination of stained smears and 

 inoculation into broth media containing 1 per cent of the carbohydrates, dextrose, 

 lactose, dulcite (or maltose), and sucrose. The soHd medium used was meat 

 extract agar containing 0.3 per cent meat extract, 1 per cent peptone, and 1.5 

 per cent agar. Kolle flasks were inoculated with the pure cultures, incubated for 

 72 hours at 37° C., and the growth washed off with a salt solution containing 

 0.85 per cent sodium chloride and 0.5 per cent phenol. The washings were fil- 

 tered through cotton in a funnel and combined. The diluent for the test fluid 

 was physiological saline solution containing 0.25 per cent phenol. The turbidity 

 and pH of the antigens were standardized as required. Agglutination tests were 

 set up in dilutions of 1:10 and higher, suificient to detect the titer of the servun. 

 In a few cases in the earlier tests not enough tubes were used for a few sera where 

 the titer exceeded 20,480. Tests were incubated for 24 hours at 37° C. and an 

 additional 20 to 24 hours at room temperature. Reactions were recorded as 

 follows, and given a corresponding numerical value for the purpose of compara- 

 tive study: 



Recorded Numerical 

 Reaction Value 



Complete agglutination 4 4 



Incomplete agglutination 3 3 



Partial agglutination 2 2 



Slight agglutination 1 1 



No agglutination 



Effect of Age on the Quality of Concentrated Antigen 



A number of workers studying antigen or using it in routine tests have ex- 

 pressed opinions concerning the length of time antigen may be held without 

 deterioration. Gage, Paige, and Hyland (36) reported in 1914 that "test fluid 

 properly preserved on ice will keep in a very active state for more than two 

 months"; and later, in 1925, Brunett (10) stated that a "quantity of antigen can 

 be prepared and kept for a period of months when stored in a cool place in an 

 uncontaminated condition." Gwatkin (40) agreed that "antigen was found to 

 stand up well and could be kept for months in the ice chest." Mailman (60) 

 found that S. pullorum antigen could be kept for 12 months at approximately 

 10° C, but Stafseth and Thorp (86) differ widely, stating that an antigen may 



