AOAC Official Method 960
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7FFF4B05BA7A4BE500D270C1D906CE6D |
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0.08 |
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3 |
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日期： |
2016-2-28 |
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45.2.07,AOAC Of fi cial Method 960.47,Amino Acids,in Vi ta min Prep a ra tions,Mi cro bi o log i cal Methods,First Ac tion 1960,(Ap pli ca ble only to ma te ri als con tain ing free forms of amino,ac ids in ab sence of ap pre cia ble amount of pro tein.),A. Stock So lu tions for Basal Me dia,(Store all so lu tions un der to lu ene; in ad di tion, store vi ta min and,amino acid so lu tions in dark at ca 10°C.),(a) Amino acid so lu tion.—Pre pare stock so lu tion of each amino,acid as in Ta ble 960.47A. Use amino ac ids of high est pu rity,avail able. (DL form may be pre ferred since it is less likely to be,con tam i nated with other amino ac ids.) If DL form is used, dou ble,weight given in ta ble. Dis solve amino acid with heat, if nec es sary,and di lute to in di cated vol ume with H2O. Where spe cial sol vents are,in di cated, dis solve amino acid in vol ume ini tial sol vent spec i fied,and di lute to fi nal vol ume with H2O.,(b) Ad e nine–gua nine–ura cil so lu tion.—See 960.46A(b) (see,45.2.01).,(c) Salt so lu tion A.—Dis solve 50 g an hy drous K2HPO4 and 50 g,an hy drous KH2PO4 in H2O, di lute to 500 mL, and add 5 drops HCl.,(d) Salt so lu tion B.—Dis solve 20 g MgSO4×7H2O, 1 g NaCl, 1 g,FeSO4×7H2O, and 1 g MnSO4×H2O in H2O, di lute to 500 mL, and add,5 drops HCl.,(e) Vi ta min so lu tion I.—Dis solve 25 mg ri bo fla vin, 25 mg,thi a mine×HCl, 50 mg ni a cin, and 0.15 mg bi o tin in 0.02M,CH3COOH to make 1 L.,(f) Vi ta min so lu tion II.—Dis solve 50 mg pyridoxal, 50 mg,pyridoxamine×HCl, 25 mg cal cium pantothenate, 5 mg,p-ami no ben zoic acid, and 0.25 mg fo lic acid in 25% al co hol to make,1 L.,B. Basal Me dium,Com plete as say me dium is given in Ta ble 960.47B. In pre par ing,me dium for given as say, omit par tic u lar amino acid to be as sayed,from me dium. Con cen tra tion of all in gre di ents in basal me dium is,twice what it will be in as say tube af ter ad di tion of test or stan dard,so lu tion and H2O. Ta ble lists amounts of in gre di ents re quired for 50,100, 150, and 200 tubes. Pre pare fresh for each as say.,To com bined so lu tions add glu cose and CH3COONa with mix ing.,When so lu tion is com plete, ad just to pH 6.8 and di lute to vol ume.,(Note: Amounts of glu cose and CH3COONa de pend upon or gan ism,used in as say.),C. Culture and Suspension Media,(a) Agar cul ture me dium.—See 960.46B(b) (see 45.2.01).,(Avail able from Difco Lab o ra tories as Lactobacilli Agar AOAC,Cat. No. 290010.),(b) Liq uid cul ture me dium.—Di lute mea sured vol ume of,com plete amino acid basal me dium, B, with equal vol ume H2O. Add,10 mL por tions to test tubes, cover to pre vent con tam i na tion (or,screw-cap tubes may be used), ster il ize 15 min in au to clave at,121°–123°C, and cool tubes as rap idly as prac ti ca ble to keep color,for ma tion at min i mum. Store in dark at ca 10°C.,(c) Liq uid sus pen sion me dium.—Di lute mea sured vol ume,ap pro pri ate basal me dium (with out amino acid be ing as sayed), B,with equal vol ume H2O. Add 10 mL por tions to test tubes, cover to,pre vent con tam i na tion, ster il ize 15 min at 121°–123°C, and cool as,rap idly as prac ti ca ble to keep color for ma tion at min i mum. Store in,dark at ca 10°C.,Table 960.47A. Amino acid stock so lu tions,Amino acid CAS Registry No. Weight, g Ini tial solvent, mL Fi nal volume, mL,Final concentration,mg/mL,(a) L-Alanine 56-41-7 5.000 900 H2O 1000 5,(b) L-Arginine×HCl 7048-02-4 12.095 400 H2O 500 20,(c) L-Asparagine 70-47-3 5.000 400 H2O 500 10,(d) L-As par tic acid 56-84-8 10.000 100 1M NaOH 1000 10,(e) L-Cysteine×HCl 52-89-1 5.000 100 2M HCl 1000 5,(f) L-Cystine 56-89-3 5.000 100 2M HCl 1000 5,(g) L-Glutamic acid 56-86-0 10.000 100 1M NaOH 1000 10,(h) Glycine 56-40-6 10.000 400 H2O 500 20,(i) L-Histidine×HCl 7048-02-4 12.351 400 H2O 500 20,(j) L-Hydroxyproline 51-35-4 5.000 150 H2O 250 20,(k) L-Isoleucine 73-32-5 5.000 900 H2O 1000 5,(l) L-Leucine 61-90-5 10.000 900 H2O 1000 10,(m) L-Lysine×HCl 657-27-2 6.248 400 H2O 500 10,(n) L-Methionine 63-68-3 5.000 900 H2O 1000 5,(o) L-Norleucine 327-57-1 5.000 100 2M HCl 1000 5,(p) L-Phenylalanine 63-91-2 5.000 900 H2O 1000 5,(q) L-Proline 147-85-3 5.000 150 H2O 250 20,(r) L-Serine 56-45-1 5.000 900 H2O 1000 5,(s) L-Threonine 72-19-5 5.000 900 H2O 1000 5,(t) L-Tryptophan 73-22-3 5.000 50 1M NaOH 500 10,(u) L-Tyrosine 60-18-4 5.000 50 1M NaOH 500 10,(v) L-Valine 72-18-4 5.000 900 H2O 1000 5,2006 AOAC INTERNATIONAL,D. Stock Cul tures of Test Or gan isms,Pre pare and test cul tures as in 960.46C (see 45.2.01). Use,fol low ing or gan isms:,(a) Strep to coc cus faecalis (faecium).—ATCC No. 9790. For use,in as say of isoleucine, leucine, threonine,……
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