Problems in the Use of Emulsifying Agents with Water-insoluble Substrates.

and a loss of weight. The pooled sera contained 0.68 mg of antibody N per ml as determined by the quantitative precipitin test of Heidelberger and Kendall (J. Exptl. Med. 50:809, 1929). Standard curves for both the gel diffusion test and the precipitin test were prepared with the purified toxin and antitoxin. Agar diffusion plates were incubated at 30 C in a glass-walled water bath. The movement of the precipitate band was measured at about 24-hr intervals, using a cathetometer. A single band of precipitate was formed in the interaction of these two reagents. For quantitative precipitin tests, the preparations were incubated at 37 C for 4 hr and then at 4 C for 4 days. Washed precipitates were analyzed for total N by the micro-Kjeldahl technique. Tests of the supernatant serum obtained after centrifugation and removal of the precipitate never showed the simultaneous presence of both antigen and antibody. The comparative tests were performed on a number of filtrates of S. aureus cultures grown in various media at 37 C for 18 to 24 hr. The cultures were centrifuged and the clarified fluid was sterilized by filtration through an ultrafine sintered-glass filter. Undiluted filtrate or filtrate diluted 1:5 with the buffered saline was tested by the Oudin test. In the quantitative precipitin tests, 0.5-ml amounts of undiluted or diluted filtrate (1:5) were mixed with 0.5 ml of antitoxin. Representative results are presented in Table 1. Analysis of the data by the t test showed no significant difference between the results obtained with the two tests at the 95% level. These tests indicated that excellent correlation existed between the results obtained by the gel diffusion and the quantitative precipitin tests.