GERM-FREE FILTRATES AS ANTIGENS IN THE COMPLE- 

 MENT-FIXATION TEST 



By William S. Gochenour 

 Veterinary Inspector, Pathological Division, Bureau of Animal Industry, United States 



Department of Agriculture 



In the production of germ-free blackleg filtrates, to insure uniformly 

 good results it is of prime importance to check or control properly each 

 lot of culture flasks, so as to know definitely that the blackleg organism 

 alone has been growing therein. After the culture flasks have been 

 inoculated and have been incubated for from six to nine days it is quite 

 simple to remove one or more cubic centimeters of the culture and test 

 it for the presence or absence of aerobic organisms. The detection of for- 

 eign anaerobes, however, should any be present, is not at all a simple 

 procedure. Moreover, it would be quite impracticable to resort to the 

 complicated process of anaerobic plating or fishing in the search of 

 contaminating anaerobic microorganisms as a routine procedure with 

 each lot of culture flasks. For this reason serological studies were 

 made with pure germ-free blackleg filtrates to ascertain whether they 

 would act as antigens in the complement-fixation test and, if so, what 

 range of specificity could be obtained therewith, using the results as an 

 index to what a satisfactorily produced product should possess. 



Accordingly, blackleg filtrates were prepared, and a horse was repeat- 

 edly injected with them at intervals extending over a period of approxi- 

 mately three months. Blood serum drawn from this animal constituted 

 the positive or immune serum. Antigenic titrations were then made, 

 using 0.2 cc. of positive and 0.2 cc. of negative (normal horse) serum; 

 and grading amounts of the germ-free filtrate were added as the antigen. 

 The titration given in Table I will exemplify the character of reaction 

 that has been obtained. 



When the filtrate is concentrated over sulphuric acid in vacuo to 

 one-half or one-third its original volume, the antigenic unit and the 

 anticomplementary dose are reduced in the same ratio. 



So far as the writer is able to learn by search through the literature, 

 the use of a germ-free filtrate as an antigen in the complement-fixation 

 test is an entirely new phenomenon; and it promises to serve a very 

 important role in the separation and differentiation of the spore-bearing 

 anaerobes. With this purpose in mind it is contemplated to parallel this 

 reaction with the other pathogenic spore-bearing anaerobes as Bacillus 

 edemaiiens, vibrion septique, B. tetanus, B. hotulinus, etc. ; and evidence 

 of the feasibility of doing this is shown in the tests already made with 



Journal of Agricultural Research, Vol. XIX. No. lo 



Washington, D. C. Aug. id, 1920 



nu Key No. A-51 



(SI3) 



