AOAC Official Method 2001.05 Petrifilm TM Rapid S
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3062E2C585EA4946B6687B592FC5866E |
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0.02 |
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2 |
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日期： |
2024-7-30 |
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17.5.07,AOAC Official Method 2001.05,PetrifilmTM Rapid S. aureus Count Plate Method for,the Rapid Enumeration of Staphylococcus aureus,in Selected Foods,First Action 2001,(Applicable to the enumeration of confirmed Staphylococcus,aureus organisms in pasta filled with beef and cheese; frozen hash,browns; cooked chicken patty; egg custard; frozen ground raw pork;,and instant nonfat dried milk.),Safety note: Autoclave materials after use.,See Table 2001.05 for results of the interlaboratory study supporting,acceptance of the method.,A. Principle,Method uses bacterial culture plates of dry media and,cold-water-soluble gel. Undiluted or diluted test portions are added,to plates at 1.0 mL/plate. Pressure, when applied to plastic spreader,placed on overlay film, spreads the product evenly over a 30 cm2,growth area. Gelling agent is allowed to solidify, and plates are incubated,at 35 ± 1°Cfor24±2h. The plate is incubated at a high temperature,(62 ± 2°C) to deactivate heat labile deoxyribonuclease; then an,indicator disk which detects the heat stable enzyme TNase is placed,between the top and bottom films of the plate. Plates with disks are,incubated at 35 ± 1°C for 1–3 h to identify TNase-positive staphylococci.,B. Apparatus and Reagents,(a) Petrifilm Rapid S. aureus (RSA) plates.—Plates, available,from 3M Microbiology Products (St. Paul, MN 55144, USA) contain,modified Baird-Parker nutrients and a cold-water-soluble gelling,agent.,(b) Petrifilm TNase Reactive disk.—Disks (3M Microbiology,Products) contain DNA, Toluidine Blue-O, and a tetrazolium indicator.,(c) Plastic spreader.—With handle (3M Microbiology Products).,(d) Pipets.—Calibrated 1.0 and 10.0 mL serological pipets with,0.1 mL graduations. (Calibrated electronic pipettor may be used to,deliver 1.0 mL.),(e) Colony counter.—Standard apparatus, Quebec Model, available,from many suppliers, or one providing equivalent magnification,and visibility.,(f) Sterile 1M NaOH.—Dissolve 40 g NaOH in water and dilute,to 1 L. Autoclave 15 min at 121°C.,(g) Dilution water.—(1) Stock solution.—Dissolve 34 g,KH2PO4 in 500 mL water, adjust to pH 7.2 with 1M NaOH (ca,175 mL), and dilute to 1L. (2) Buffered water for dilutions.—Dilute,1.25 mL stock solution to 1 L with boiled and cooled H2O. Autoclave,15 min at 121°C.,(h) Blender.—Use high-speed blender (16 000–18 000 rpm) with,sterile jar.,(i) Incubators.—Maintaining 35 ± 1°C and 62 ± 2°C.,C. Preparation of Test Suspensions,Use balance with capacity of 2 kg and sensitivity of 0.1 g to aseptically,weigh 50 g unthawed test portion into blender jar, B(h). Add,450 mL dilution water, B(g)(2), and blend at 16 000–18 000 rpm for,2 min to homogenize. If entire test sample is <50 g, weigh a portion,of the test sample and add dilution water to make 1:10 dilution. As,required, adjust pH of diluted test sample to 6.5–7.5 with NaOH,B(f), (ca 0.1 mL/g test portion).Donot use buffered peptone water or,diluents containing citrate or thiosulfate. Prepare all decimal dilutions,with 90 mL dilution water plus 10 mL from previous dilution.,Do not use pipets to deliver <10% of their total volume. For example,to deliver 1 mL, do not use pipet > 10 mL; to deliver 0.1 mL, do,not use pipet > 1 mL. Mix all dilutions by shaking 25′ through 30 cm,arc in 7 s.,D. Analysis,Place dry Petrifilm RSA plate, B(a), on flat surface. Lift top film,and inoculate 1mL test suspension onto center of bottom film. Carefully,roll top film down onto inoculum. Distribute test suspension,over 30 cm2 growth area with downward pressure on handle of plastic,spreader, B(c). Leave plate undisturbed to permit gelling agent to,solidify. Incubate plates for 24 ± 2 h at 35 ± 1°C. In incubator, place,plates in horizontal position or in a Petrifilm plate rack, clear side up,in stacks not exceeding 20 units. After incubation, place plates in 62,± 2°C incubator for 1 h in stacks not exceeding 10 units. Open,Petrifilm plates and place Petrifilm TNase reactive disk, B(b), into,each well formed by the foam dam, being careful not to trap air bubbles,below disks. Disks are coated with substrate on both sides;,therefore, orientation in relation to Petrifilm plate does not matter.,Lower top film onto disk being careful not to trap air bubbles between,film and disk. To ensure uniform contact between disk and,medium, apply gentle pressure across reactive disk area. Slide a bent,rod across the plate to complete contact between disk and gel by,pushing the air bubbles out past edge of disk. Incubate Petrifilm,plates with their disks at 35 ± 1°C. After 1 h, remove Petrifilm plates,from incubator. Using a colony counter, B(e), count and record,“confirmed” colonies with……
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