Rapid, sensitive methods for enumeration and enterotoxin assay for Staphylococcus aureus Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/bn999981f

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  • A selective, differential plating medium for the enumeration of coagulase-positive staphylococci was developed. The ingredients of this medium were analysed for their effect on normal, heat treated and frozen staphylococcal cells. The nutrient base consisted of 10 gm Tryptone, 5 gm Beef Extract, 1 gm Yeast Extract and 17 gm Agar, per liter. Selectivity of this medium was obtained by incorporating 5 gm lithium chloride, 10 gm glycine and 0.1 gm of potassium tellurite, per liter. Pyruvate at 5 gm/liter was added for recovery of stressed cells. The medium was made differential for coagulase-positive staphylococci by the addition of five percent sterile pig plasma and 1 mg/ml of bovine fibrinogen. Colonies of coagulase-positive staphylococci were surrounded by opaque fibrin halos. The new medium designated Tryptone Pyruvate Plasma Fibrinogen Agar (TPPF) gave 30 percent higher counts than those obtained from two non-selective media, Plate Count Agar and Tryptic Soy Agar. Recovery of heat treated staphylococci was 360 times higher than counts from Vogel-Johnsons Agar and 70 times higher than Tryptic Soy Broth containing 10 percent NaCl. The TPPF medium has the following advantages over staphylococcus medium in current use: highly selective for staphylococci, a quantitative pour plate method, coagulase-positive counts in 24 hours and the highest recovery from heated or cold injured cells of any staphylococcus medium tested. The slide electroimrnunodiffusion (EID) technique was applied to staphylococcus enterotoxin detection. The variables associated with this method were independently analysed to determine optimum conditions. These conditions were found to be: a supporting medium of 1.2 percent Noble's agar, an electrolyte consisting of 0.082N barbituate buffer at pH 8.4, 40μg /ml Antitoxin A or 20 μg/m1 Antitoxin B incorporated into gel and electrophoresis for 30 minutes at 150 volts, with the temperature maintained at 30° C. The sensitivity of the EID method was 0.40 μg/m1 of enterotoxin B. The Brinkman equipment used was capable of electrophoresis of 27 quantitative enterotoxin tests simultaneously in two hours, at a cost of 40 cents per sample.
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