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The biological and immunological activities of Staphylococcal enterotoxin B are stable in pH 2.0 approximately 11.0, and also resistant to proteolytic enzyme (trypsin, pepsin) digestion for 3 approximate 4 hours. Therefore, the toxin could be purified with trypsin digest and then absorbed on kaolin and Kieselgel, followed by eluting the different pH buffer solutions. Further purifications was chromatographied on CM-Sephadex column.

A method for rapidly identifying and quantitating small amount of staphylococcal enterotoxin B was developed on capillary tube single diffusion test. The high sensitivity of method has the advantages to save antiserum and incubation period. Some factors such as concentration of antigen and antiserum, incubation temperature, buffer systems, and position of capillary tube all might affect the test accuracy. If all those affecting factors were concerned, the toxin would probably be standardized. The most accessible quantitative estimation was accomplished by means of (1) plotting a precipitation curve from the different concentrations of standardized staphylococcal enterotoxin B; (2) the sample to be test was diluted by serial two-fold method with the same buffer system; (3) select suitable 3--5 dilutions to perform capillary tube diffusion test, taking the values of precipitation bands as abcissa and sample dilutions as longitudinal axis, rendered the precipitation curve of sample exquisitely paralled to that of the standard; (4) calibrate the figure values of sample exactly on the precipitation curve, and reading the toxin content of specimen to be tested from standard curve.

Enterotoxigenic strain of Staphylococcus aureus (ATCC 14458) was grown under various conditions with constant shaking to determine the requirements for maximum toxin production. It was evident that 3% tryptic soy broth, 3% NZ-Amine NAK + 3% casein hydrolysate, 3% NZ-Amine NAK + 1% yeast extract, and 3% NZ-Amine NAK + 1% yeast extract + 0.2% glucose are most available toxin production media. But concentration of glucose could strictly triggered the enterotoxin producing efficiency. When glucose concentration was less than 0.5%, although with higher yield, the toxin production was delayed for certain period of time. However, if glucose concentration was up to more than 0.5%, the enterotoxin production was almost inhibited. Some metabolites of glucose to elucidate the inhibitory effect have also investigated. Our results indicated that glycerol and citric acid inhibited the toxin production directly, while the inhibitory effect of lactic acid and acetic acid were due to those acidic metabolites, decreased the pH value of media, and adversely suppressed the bacterial growth.