The sac culture method in combination with 6% NZ-Amine A plus 1% yeast extract was found to be the optimal condition for staphylococcal enterotoxins A (SEA) and D (SED) production. This growth condition was tested for the production of unidentified staphylococcal enterotoxins (SEs). Twenty-one Staphylococcus aureus strains that previously showed an emetic response in monkeys but were negative for any of the identified SEs when cultured by the membrane-over-agar method were grown by the sac culture method. All 21 strains produced at least one known SE. One strain produced only enterotoxin C (SEC). Nine strains produced only SED. One strain produced both SEA and SED. Four produced both SEC and SED. One produced SEA, enterotoxin B (SEB), and SED. Two produced SEA, SEC, and SED. Three produced SEA, SEB, SEC, and SED. One of these strains, FRI-569, that produced only SED at a low level (10 ng/ml) was selected to confirm the production of an unidentified SE by the monkey feeding test. Emesis was induced in five out of six monkeys. The total amount of SED in the supernatant fluids fed to monkeys was only 0.5 μg, or 2.5% of the 50% emetic dose (20 μg). Production of an unidentified SE by strain FRI-569 was therefore confirmed. This optimal condition can be used to produce increased amounts of unidentified SEs for purification.

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