Examination of Foods for Staphylococcus aureus - DB Server Test ...

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lysostaphin sensitivity, coagulase and thermonuclease production, and glucose and mannitol fermentation were conducted on 100 enterotoxigenic and 51 nonenterotoxigenic strains of S. aureus (3). In all cases, the reactions of enterotoxigenic and nonenterotoxigenic strains varied by 12% or less. This research indicates that none of these tests can be relied upon to differentiate toxic and nontoxic staphylococci. Experiment: Direct Plate Count Method This method is suitable for the analysis of foods in which more than 100 S. aureus cells/g may be expected. It conforms to the method in ref. 1. A. Materials Tuesday, October 15: Plating on Baird Parker Medium plates, incubating plates at 35 o C for 48 hours, plating on 3M Petrifilm Rapid S. aureus Count Plates, incubating plates at 35 o C for 24 hours 1. Food sample 2. Sterile spatulas: 1 per team 3. Scale 4. Sterile stomacher bags: 1 per team 5. Butterfield's phosphate-buffered dilution water: One 225-ml flask per team 6. Butterfield's phosphate-buffered dilution water: Four 90-ml bottles per team 7. Sterile 10-ml pipettes 8. Sterile 1-ml tips (blue) 9. Sterile 0.2-ml tips (yellow) 10. Baird Parker Medium plates: 15 plates per team 11. 3M Petrifilm Rapid S. aureus Count Plates: 5 plates per team Wednesday, October 16: Transferring 3M Petrifilm Rapid S. aureus Count Plates to a 62 o C incubator, incubating plates 1-4 hours, placing reactive disks, incubating plates with reactive disks at 35 or 37 o C for 1-3 hours, counting and recording colonies 1. Sterile forceps: 1 per team 2. 62 o C incubator 3. Colony counter Thursday, October 17: Counting and recording colonies, transferring suspect S. aureus colonies into BHI broth, inoculating agar slants of TSA, incubating BHI culture suspension at 35 o C for 18-24 hours, incubating agar slants of TSA at room temperature for 18-24 hours 1. Colony counters 2. Brain Heart Infusion (BHI) Broth: 0.2-0.4 ml per tubes; 3 tubes per team 3. Trypticase Soy Agar (TSA) slants: 3 slants per team 2
Tuesday, October 22: Catalase test, adding coagulase plasma to the BHI culture and incubating at 35oC and examining periodically for clot formation, Gram stain (from TSA slants), inoculating carbohydrate fermentation medium containing glucose (0.5%), inoculating carbohydrate fermentation medium containing mannitol (0.5%), testing for lysostaphin sensitivity, testing for thermostable nuclease production 1. 3% Hydrogen peroxide 2. Reconstituted coagulase plasma(rabbit) with EDTA: 1.5 ml per team 3. Gram stain materials 4. Carbohydrate fermentation medium containing glucose (0.5%): 5 tubes per team 5. Positive and negative controls (bacterial cultures that ferment and do not ferment glucose): One set per bench 6. Carbohydrate fermentation medium containing mannitol (0.5%): 5 tubes per team 7. Positive and negative controls (bacterial cultures that ferment and do not ferment mannitol): One set per bench 8. Sterile paraffin oil: Enough to cover 100 tubes 9. Phosphate-saline buffer: 0.2 ml per tube; 3 tubes per team 10. Phosphate-saline buffer: 0.1 ml per tube; 3 tubes per team 11. Lysostaphin (50 µg/ml) dissolved in 0.02 M phosphate-saline buffer containing 1% NaCl: 0.3 ml per team 12. DNase Test Agar with Methyl Green plates: 1 plate per team Thursday, October 24: Read the results of carbohydrate fermentation tests. No materials needed. B. Preparation of sample Using aseptic technique, weigh 25 g of sample into sterile stomacher bag. Add 225 ml Butterfield's phosphate-buffered dilution water (1:10 dilution) and blend for 1 min at high speed. Using the 1:10 dilution, make serial dilutions of sample for enumeration of S. aureus. C. Isolation and enumeration of S. aureus 1. For each dilution to be plated, aseptically transfer 1 ml sample suspension to 3 plates of Baird-Parker agar, distributing 1 ml of inoculum equitably to 3 plates (e.g., 0.4 ml, 0.3 ml, and 0.3 ml). Spread inoculum over surface of agar plate, using sterile bent glass streaking rod. Retain plates in upright position until inoculum is absorbed by agar (about 10 min on properly dried plates). If inoculum is not readily adsorbed, place plates upright in incubator for about 1 h. Invert plates and incubate 45-48 h at 35°C. Select plates containing 20-200 colonies, unless only plates at lower dilutions (>200 colonies) have colonies with typical appearance of S. aureus. Colonies of S. aureus are circular, smooth, convex, moist, 2-3 mm in diameter on uncrowded plates, gray to jet-black, frequently with light-colored (off-white) margin, surrounded by opaque zone and frequently with an outer clear zone; colonies have buttery to gummy consistency when touched with inoculating needle. Occasionally from various foods and dairy products, nonlipolytic strains of similar appearance may be encountered, except that surrounding opaque and clear zones are absent. Strains isolated from frozen or desiccated foods that have been 3
Page 1: Experiment 12: Examination of Foods
Page 5 and 6: Dilution 10 -3 Dilution 10 -4 Dilut
Page 7 and 8: Colony 3 5. Thermostable nuclease p
Page 9 and 10: 2. AOAC INTERNATIONAL. 1995. Offici

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