AOAC Official Method 962
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6873DD68ECAA40EEA580E910B900CF44 |
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0.01 |
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1 |
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日期： |
2024-7-30 |
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33.2.60,AOAC Official Method 962.14?,Beta-Lactam Antibiotics in Milk,Bacillus subtilis,Qualitative Field Disk Assay,First Action 1962,Final Action 1965,Surplus 1999,(Sensitive to ca 0.05 unit/mL.),See also 982.16 (see 23.1.15), 979.14 (see 23.1.16).,A. Culture Media,(a) Agar medium A.—For carrying test organism and for performing,assay. See 957.23A(a) (see 5.3.01).,(b) Agar medium G.—For preparing test suspension. See,957.23A(g) (see 5.3.01).,B. Reagents,(a) Stock penicillin solution.—Accurately weigh, in atmosphere,of ￡50% relative humidity, ca 10 mg USP potassium (or sodium),Penicillin G Reference Standard. Dissolve in enough pH 6 buffer,957.23B(f) (see 5.3.01), to give concentration of exactly,100 units/mL. Store in dark at ca 5°C ￡2 days.,(b) Control disks.—Dilute stock penicillin solution with antibiotic-,free, whole, homogenized milk to final concentrates of 0.05 and,0.1 unit/mL. Use S&S 740E 1,4 in. (6.4 mm) disks or prepare 1,4 in.,(6.4mm)round, white disks fromS&S470W or 470 paper, of equivalent,absorption performance qualities and purity. Dip disks into,standard penicillin-containing milks and shake off excess. Dry on,rust-proof wire screening under fan. Stored in refrigerator in,tightly-stoppered vial with desiccant, disks are usable 1 month.,C. Stock Culture of Test Organism,Maintain Bacillus subtilis on agar medium A, transferring to fresh,slant monthly. Inoculate fresh slant of agar medium A with test organism,and incubate 16–24 h at 37°C. Wash culture from slant with,2–3 mL sterile 0.9% NaCl solution (w/v) with aid of sterile glass,beads onto surface of Roux bottle containing 300 mL agar medium,G. Incubate 5 days at 37°C. Wash resulting growth from surface with,aid of the beads and 50 mL sterile 0.9% NaCl solution into sterile,centrifuge tube, centrifuge, and decant supernate. Reconstitute sediment,with ca 70mLsterile 0.9% NaCl solution and heat-shock spore,suspension by heating 30 min at 70°C. Store in glass-stoppered flask,in refrigerator. Suspension is stable several months.,With each new suspension, determine amount to be used as follows:,Prepare plates as directed below with varying amounts of,inoculum, i.e., 0.2, 0.5, 1.0, and 2.0 mL/100 mL medium. Refrigerate,3–5 days. Place control disks of 0.05 and 0.1 unit/mL on plates,and incubate as in test. Use as concentration of inoculum that concentration,showing best response, considering both sensitivity and,discernibility of zones of inhibition.,D. Preparation of Plates,Melt agar medium A, cool to 55–60°C, and add 0.2–2.0 mL,spore suspension, C, to each 100 mL agar medium A. Mix well.,Add 10 mL inoculated agar to each 20 ′100 mm glass or plastic,Petri dish. Distribute agar evenly, cover with porcelain covers,glazed only on outside, and let harden on level surface. Store in refrigerator,33 but ￡5 days. Remove each dish from refrigerator as,needed and use within 15 min.,E. Assay,With forceps place 0.05 and 0.1 unit/mL control disks on each plate.,Thoroughly shake each milk test sample to disperse fat evenly.,Using forceps, dip blank disk completely into test sample, withdraw,and shake off excess milk by vigorously waving in air several times.,Place disk on surface of agar and touch gently with tip of forceps to,assure proper contact. Do not touch so heavily that milk is squeezed,out of disk. Place disks so that they are 320mmapart, measured center,to center, to avoid overlapping of zones. Flame tips of forceps to,avoid carryover from contaminated test portions. Place control and,test sample disks on plate within few min of one another.,Incubate plates 2.5–3 h at 37°C, and examine for zones of inhibition.,Hold plates at various angles to light (either electric bulb or,daylight) to determine optimum conditions for observation. Zones,around control disks indicate sensitivity of test.,To determine if zone of inhibition is due to penicillin, add 0.05mL,(ca 1 drop) penicillinase concentrate (BBL or Difco penicillinase,have been found satisfactory) to 5 mL aliquot milk test sample and,shake well. Prepare 3 disks from this treated test portion and from,untreated test portion. Place all disks on same plate, incubate as before,and observe. Zone around untreated test portion disks but no,zone around treated test portion disks is positive test for penicillin.,Zone of inhibition around both treated and untreated test portion,disks indicates antibacterial activity other than penicillin.,F. Quantitative Overnight Method,Use method for penicillin in feed, 967.41 (see 5.3.20), preparing,standard solutions for standard curve with antibiotic-free, whole, homogenized,milk instead ofpH6 buffer. Cylinders must be used for applicat……
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