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AOAC Official 967.40 Lincomycin in Feeds Microbiological First Action 1967_A

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AOAC Official 967.40 Lincomycin in Feeds Microbiological First Action 1967_AOAC.rar,AOAC Official 967.40 Lincomycin in Feeds Microbiological First Action 1967_A

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5.3.11AOAC Official Method 967.40Lincomycin in FeedsMicrobiological MethodFirst Action 1967Method I(Applicable to feeds containing 3.63 g/907 kg)A. Standard Solutions(a) Lincomycin stock solution.Accurately weigh ca 40 mgUSP LincomycinHCl Reference Standard and dissolve in enoughpH 8 buffer, 957.23B(b) (see 5.3.01), to give concentration of ex-actly 100 g lincomycin base/mL. Store 30 days at 210C.(b) Standard response line.Dilute aliquots stock solution, (a),with enough pH 8 phosphate buffer, 957.23B(b) (see 5.3.01), to ob-tain concentrations of 0.2, 0.4, 0.8, 1.6, and 3.2 g lincomycinbase/mL. Reference concentration is 0.8 g/mL.B. Plates(a) Base layer.Add 10 mL melted agar medium E 957.23A(e)to sterile Petri dishes, distribute evenly, and let harden on perfectlylevel surface.(b) Seed layer.Before assay, determine by preparation of trialplates optimum concentration of organism suspension of M. luteususually 0.020.05% of suspension prepared as in 957.23D(e)(1)(see5.3.01)or0.21%asin957.23D(e)(2)(see5.3.01)tobeaddedto agar medium J, 957.23A(j) to obtain zones of inhibition of ade-quatesize(16 mm10%with0.8g/mL)andsharpness.Forassay,add appropriate amount of organism suspension to agar medium Jpreviously melted and cooled to 48C. Mix thoroughly and add4.0 mL to each plate containing base layer.C. Assay SolutionObtain and prepare test sample as in 965.16 (see 5.1.01) and950.02 (see 5.1.02). Accurately weigh ca 10 g ground test portionand transfer to 250 mL glass-stoppered, round-bottom flask, add20 mL H2O, and shake 10 min on wrist-action shaker. Add 50 mL0.1M HClmethanol (1 + 4) and shake 10 min. Filter throughWhatmanNo.4paper,using42mmBchnerand500mLflask.Re-peatextractiontwice,using50mLHClmethanoleachtime.DonotaddmoreH2O.Alternatively,conductextractionsin250mLcentri-fuge bottle and centrifuge to clarify.Transfer combined filtrates to 500 mL round-bottom flask andevaporate to 1520 mL, using rotary evaporator. Do not heat 60?C.Transfer aqueous extract to 125 mL separator. Rinse flask succes-sively with 10 mL Skellysolve B, 951.01A(o) (see 3.3.04), 78 mLphosphate buffer pH8, 957.23B(b) (see 5.3.01), and 10 mLSkellysolve B. Add all rinsings to separator, shake, and let separate.Drainaqueousphase,extractSkellysolveBtwicewith78mLbufferpH8, and adjust combined extracts to pH 8.0 with dilute NaOH solu-tion. Adjust volume with pH 8 buffer to 0.61.0 g lincomycinbase/mL.D. AssayUsing lincomycin standard response line and assay solution, pro-ceed as in 957.23EF (se