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A simple homogeneous enzyme immune assay was developed for detection of staphylococcal enterotoxin B by a conjugate of beta-amylase coupled with the enterotoxin.

One percent added sugar (sucrose, maltose, lactose, glucose and glucose plus fructose) to casein hydrolysate (NAK) medium with or without starch or low methoxyl pectin did not significantly affect enterotoxin C production but did significantly decrease the pH at 12 and 24 h. The increased viscosity of colloidally thickened media resulted in decreased aeration in shaken cultures and decreased quantities of enterotoxin C. Sugars tested did not differ practically in their effects on the parameters of multiplying Staphylococcus aureus 361 and 265-1 which were measured. Addition of 1% of glucose, sucrose, or maltose prevented the decreased growth rate observed when strain 265-1 was grown in 20% O 2 + 80% N 2 instead of air.

From comparisons of 4% N-Z Amine NAK made with distilled water, naturally hard water, and synthetic salt solutions, it appeared that magnesium and, to a lesser extent, iron were limiting factors in the production of staphylococcal enterotoxins B and C but not A. Maximum enterotoxin production with NAK medium was achieved by the addition of 5 mg of Mg2/ per liter (for a total of 9 mg of Mg2+ per liter) and 0.5 mg of Fe2+ per liter. Higher levels of magnesium were not inhibitory. Supplementing NAK with commonly used complex components, which added Mg2+ above the 9-mg/liter level, did not result in maximum yields of enterotoxin. Variability in the ability of different lots of NAK to support enterotoxin production may be minimized by supplementing NAK medium with magnesium and iron.

Larger amounts of enterotoxin were produced when Staphylococcus aureus S-6 was grown under still (nonshaken) conditions in a medium that was a paste or gel than were produced in a liquid dispersion with the same colloidal ingredient or in control basal broth (4% NZ Amine-NAK containing 50 mug of thiamine per 100 ml and 1 mg of niacin per 100 ml). Four colloidal ingredients were used which had been previously demonstrated to not support enterotoxin production in buffer. The effect of the type of dispersion occurred earlier than that of the colloidal ingredient, but interactions were found. This effect was not observed when the cells were grown with aeration (shaken). Four other strains of S. aureus followed a similar pattern for enterotoxins A, B, and C, although liquid and paste with cornstarch and carrageenan were the only media compared to the control broth. Enterotoxins A and B were produced earlier by S. aureus S-6, and much greater quantities of enterotoxins were produced for all strains when incubated shaken.