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    Aoac 960.09

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    Chapter 6, p. 10 AOAC Ormcut Merio0s oF ANALYSs (2000) 6.3.03 AOAC Official Method 960.09 Germicidal and Detergent Sanitizing Action of Disinfectants First Action 1960 Final Action (Suitable for determining minimum concentration of chemical that can be permitted for use in sanitizing precleaned, nonporous food contact surfaces. Minimum recommended starting concentration is ‘2-4 this concentration. Test also determines maximum water hard- ‘ess for claimed concentrations. As contol, check accuracy of hhard-water tolerance esults with pure C,alky! dimethyl benzyl am- ‘monium chloride at 700 and 900 ppm (g/mL) hardness, and pure Cyealkyl dimethyl benzyl ammonium chloride (Cetalkonium Chlo- ‘de}, at 400 and 550 q.g/mL) ppmhardness, expressed as CaCO},) A. Reagents (@) Culture media 1) Nurient agar —Boil3 geet extract, 5 epentone (rom Difco No. 0118 or equivalent special rads must noxbe used), and IS gsalt-sfeagarin 1 HyO. Donotuse remixed, dehyated media, Tube, and astclave 20 min a 121°C. Use for daily transfer of test culture. (2) Nutrient agar Prepare as above butuse 30g agar. Use for growing test calturesin French square bot- tes @) Nutrient agar (AOAC) —See 9S5.11AC0) see 6.19). Use forpreparng stock culture slants. (b) Subculture media—1) Use tryptone glucose exact agar (@ifco No. 0002), adding 25 mL. stock neutralizer, (€JL. (2) Trypone glucose extract agar (Dio). (©) Neutralizer stock slution—Mix 40 ¢ Lecithin (Aleolec Granules, American Lecithin, PO Box 1908, Danbury, CT 06813, USA (25-50 kg containers oly} or Advanced Lecithin Products PO Box 677, Danbury. CT 0680S, USA), 280 mi. poysorbate 80, and 1.25 ml. phosphate afer): ise with HO | Landadjst to pH 72. Dispense in 100 ml. porions and aoc 20 min at nrc. (@) Neutralize blanks —For we with 200 ppm quaternary am- menium compound, Mix 100 ml neutralizer stock sohtion, (©) 25 mi. 0.25M phosphate bute stock solution) and 1675. H,0. Dispense 9 mi portions into 20% 150 mm tubes. Autoclave 20min nrc. (©) Phosphate bufer stock soluion —0.25M. Dissolve 340 g KH,PO in 500ml 0 adjust to pH 72 with IM NaOH, and ate wl (©, Phosphate buffer dition water —Add | 25 mL.0.25M phos- phate buffer stock solution, (¢), to 1 L H,O and dispense in 99 mL. portions. Autoclave 20 min at 121°C. (g) Test organisms.—Use Escherichia coli ATCC No. 11229 of Staphylococcus aureus ATCC 6538. Incubate 24 and 48 h,respec- tively. Maintain tock cultures on nutrient agar (AOAC), (a)(3), at refrigerator temperature B. Resistance to Phenol of Test Cultures Determine resistance to phenol at least every 3 months by 9S8.11 (see 6.1.01), Resistance of E. coli should be equivalent o that speci fed forS.ryphi in 985.11D (see 6.1.01) and that for S. aureus equiv- lent o that specified for this organism in 958.12 (se 61.02}; also, use procedures under 991.48A(b) (se 6.2.03) for S. aureus. ©. Apparatus (a) Glassware—250 mL. wide-mouth Erlenmeyers: 100 ml ‘graduate: Mohr, serological, and/or bacteriological (APH specifi- cation) pipets; 20 x 150 mm test tubes. Sterilize at 180°C in ot air oven 22h (b) Petr dishes —Sterile. (©) French square botles—175 mL, lint glass. (@) Water bath —Controlled at 25°C. . Preparation of Culture Suspension From stock culture inoculate tube of nutrient agar A, A(a)(), and make 23 consecutive daly transfers (<30), incubating transfers 20-24 h at 35-37°C. Do not use transfers 330 days. If only I daily twansfer has been missed, no special procedures are required; if 2 daily transfers ae missed, repeat with 3 daily transfers. Prepare 175 ml. French square eulture bottles containing 20 ml. nutrient agar B, A(a)2), autoclave 20 min at 121°C, andlet solidify tn botle in horizontal postion. Inoculate culture bottles by wash: zrowth from slant with 5 mL phosphate buffer dilution H,0, A(D, into 9 mi phosphate buffer dilution H,O, and adding 2 mL of this suspension to each culture bole, ing back and fort wo dis tribute suspension; then drain excess liquid. Incubate 18-24 h at '35-37°C, agar side down. Remove culture from agar surface of 4 or ‘more bottles, using 3 mL. phosphate buffer dilution H,O and glass beads in each bottle to suspend grow. Filter suspension through ‘Whatman No.2 paper prewet with 1 mL sterile phosphate buffer. and collect in sterile ube. (To hasten filtration rub paper gently with sterile policeman.) Standardize suspension to give average of 10 « 10” organisms/l_ by dilution with sterile phosphate bufer dilution 1,0. ACD. ‘Table 960.08A Percent light transmission at various wavelengths corresponding to bacterial concentrations ‘6 Light transmission with fits, Am ‘Average bacterial 70 420 490 530 560 580 50 count. 70 40 60 60 60 79 80 13010 80 60 790 79 70 80 80 ns 90 60 80 80 8 90 100 102 100 790 90 90 90 110 110 8 no 80 100 10.0 100 120 130 1 130 20 120 120 s20 130 150. er ‘© 3000 AOAC INTERNATIONAL ‘AOAC Orci. Memcos oF ANALYsis (2000) Disnezcrirs ‘Chapter 6.11 ‘Table 960.098 Preparation of 8aSOs suspensions ‘corresponding to bacterial concentrations ‘2% Bel, 1%H,SO, Average bacterial Standard No._ solution, mi._(w) solution, mL countiml. 1 40 96.0 50x10 2 50 95.0 75 3 60 ono 8s ‘ 70 93.0 100 5 80 20 120 6 100 900 135 z 120 880 180. If Lumetron colorimeter is used, dilute suspension in sterile Lumen tube to give % T according to Table 960.09. McFarland nephelometer and BaSO, standards are used, select, 7 tubes of same id as that containing test culture suspension. Place 10 mL of each suspension of BaSO,, prepared as indicated in Ta- ble 960.098, in each tube and seal tube. Standardize suspension to correspond to No. 4 standard. , Synthetic Hard Water wn Prepare Solution I by dissolving 31.74 g MgCl, (or equivalent of Inydrates) and 73.99 g CaCl; in boiled distilled H,O and diluting to 1. Prepare Solution by dissolving 36.03 g NaHCO, inboiled dis- tiled H,0 and diluting to IL. Solution 1 may be heat sterilized: So: lion 2 must be sterilized by filtration. Place required amount Solurion In sterile IL flask and add 2600 ml. sterile distilled H,0; then add 4 mL. Solution 2 and dilute to 1 L with sterile distilled H,0, Each mL. Solution 1 wil give a water equivalent to ea 100 ppm of hardness calculated as CaCO, by formula: ‘Total hardness as ppm g/ml.) CeCO, = 2.495 x ppm g/m.) Ca 4+ 4.115 « ppm (ugimL) Me pH of all test waters $2000 ppm (g/mL) hardness should be 7.6-8.0. Check prepared synthetic waters chemically forhardness at time of tests, using following method or other methods described in APHA, Standard Methods for the Examination of Water and Wastewater 20th Ed, 1998. F. Hardness Method (a) EDTA standard solution.—Dissolve 4.0 § [NaH,EDTA-2H,0 and 0.10 g MgCl 6H,0 in 800 mL H,O and ad- just by subsequent dilution so that 1 mL. of solution is equivalent to 1 mg CaCO when titrated as in (€). Check EDTA solution after preparation or, if commecially purchased, against CaCO, standard at least every 2 months (b) Calcium standard solution —1 mL. = 1 mg CaCO,, Weigh 1.00 g CaCO, dried ovemight or longer at 105°C, into $00 mL Erlenmeyer and add dilute HCI through funnel until CaCO, is dis- solved. Add 200 mL. H,O, boil to expel COs, and cool. Add few drops methyl red indicator and adjust color to intermediate orange with dilute NOH or HClasrequired. Transfer quantitatively to VL. volumetric ask and dilute to volume. (©) Determination.—Dilute 5-25 mL test sample (depending on hhardness) to 50 mL. with H,0 in Erlenmeyer or casserole. Add ml. buffer solution (67.5 g NHCl and S70mL.NH,OH diluted'o 1 Lwith 1,0), | mL inhibitor (5.0 g NaS 9H,0 or 3.7 gNa,S-SH,0 dissolved in 100 mL H,0), and one or 2 drops indicator solution (0.5 g Chrome Black Tin 100 mL. 60-80% alcohol). Titrate wth EDT standard so- lution slowly, string continuously, until last reddish tinge disappears from solution, adding lst few drops at 3-5 s intervals Hardness as mg CaCOL = (an standard solution « 1000y/m. test sample G. Preparation of Test Samples Use composition declared or determined as guide to test sample \weight required for volume sterile H,O used to prepare 20 000 ppra (g/mL) solution. From his stock dilution, transfer | mL into99 mL. Of the water 10 be used in test 10 give concentration of 200 pra (g/mL) - In making transfer. ill 1 mL pipet and drain back into stock solution; then refill 1 correct for adsorption on glass. After mixing, discard I mL to provide 99 ml. ofthe test water in . 1H. Operating Technique Measure 99 mL water tobe used in est, containing bactericide at concentration to be tested, into chemically clean, sterile, 250 ml. \wide-mouth Erienmeyer and place in constant temperature bath un- Uilitreaches 25°C, or 220 min. Prepare duplicate flasks for each ger- mice to be tested. Also prepare similar flask containing 99 ml. sterile phosphate buffer dilution H,0, A(P), as “inital numbers” control ‘Add 1 ml-cultre suspension to each test Mask as follows: Whit! ‘ask, stoppin just before suspension is added, creating enough re- sidual motion of liquid .o prevent pooling of suspension at point of ‘contact with est water. Add suspension midway between center and ‘edge of surface with ip of pipet slightly immersed in test solution, ‘Avoid touching pipet o neck o side of flask during addition. Trans- fer | mL portions of this exposed culture to neutralizer blanks ex- actly 30 and 60s after addition of suspension. Mix well immediately after transfer. FFor “numbers control” transfer, add 1 ml culture suspension to 99 mL. sterile phosphate dilution H,0 in same manner. In case of ‘numbers contol plants need be made only immediately after adding and mixing thoroughly 530 s. (It is advantageous to use milk pipets to add culture and withdraw test samples.) Plate from neutralizer tube to agar, using subculture medium ‘A(by(J) for quaternary ammonium compounds and A(b)(2) with ‘numbers control. Where 0.1 mL portions ae planted, use mL pipet sraduated in 01 mL intervals. For dilutions to give countable paces, tse phosphate buffer dilution H,O. A(D. For numbers contol. use following dilution procedure: Transfer | mL. exposed culture (1 ml. culture suspension transfered to 99 ml. phosphate bufer dilution 1,0 in HO bath) 1099 mL phosphate buffer dilution H;0, A(D, (di lugion 1). Shake thoroughly and transfer | mL. dilution Ito 99 mL. phosphate buffer dilution H,0, A(N, (dilution 2). Shake thoroughly and transfer 1 mi. dilution ?t099 mL phosphate buffer dilution H,O. (dilution 3). Shake thoroughly and transfer four 1 ml. and four (0.1 mL aliquots from diluion 3 to individual sterile Petr dishes, Fortest samples, use following dilution procedure: Transfer 1 mL. ‘exposed culture into9 ml neutralizer, A(). Shake and transfer four {ImL and four 0.1 mL aliquots to individual sterile Petri dishes, For numbers control, use subculture medium A(b)2); forests with qus- ‘© 200 AOAC INTERNATIONAL Disnrecranrs ‘Chapter 6, p. 12 ‘AOAC Orci Mer#o08 oF Ansys (2000) temary ammonium compounds, use medium A(b)1). Cool agar to solidify, and then inver and incubate 48h at 35°C before counting. 1. Results ‘Tobe considered valid, results must meet standard effectiveness: 99.9999 reduction in count of numberof organisms within 308. Re- port results according to actual count and percent reduction over ‘numbers control. Counts on numbers control for germicide test mix- ture should fll between 75 and 125 « 10‘hmL for percent reductions tobe considered valid. J. Steritty Controls (2) Newralizer—Plate 1 mL. from previously unopened tube. (b) Water—Plate | mL. from each ype of water use. (©) Sterile distilled water—Plate 1 mL. After counting plates, confirm that surviving organisms are Ecol by transfer to brilliant sgreen bile broth fermentation tubes or lactose broth and EMB agar; confirm S. aureus by microscopic examination, References: Am J. Public Health 38, 1405(1948). J. Milk Food Technol. 19, 183(1956) Fed. Regist. 21, 7020(1956) JAOAC 41, $41(1958): $6, 308(1973), 6.3.04 4 AOAC Official Method 961.02 * Germicidal Spray Products ‘as Disinfectants First Action 1961 Final Action 1964 (Suitable for determining effectiveness of sprays and pressurized spray products as spot disinfectants for contaminated surfaces.) A. Reagents Use culture media and reagents specified in 991.47A(a) and (f) (see 62.02); 991.48A(a) (see 62.03); and 991.49A(a) and (b) (see 62.05). Use as test organisms Trichophyton mentagrophytes ATCC No. 9533, prepared as in 988.17D (see 63.02), 10 which has been added 0.02 ml octyl-phenoxy-polyethoxy-ethanol (Triton x100, Un- {on Carbide Corp.) 10 mL suspension to facilitate spreading, Salmo- nella choleraesuis ATCC No. 10708, maintained as in 991.47A(b) (see 62.02), Staphylococcus aureus ATCC No. 6538, maintained as in 991.48A(b) (see 62.03), and Pseudomonas aeruginosa ATCC No, 15442, maintained as in 991.49A() (see 6:2.05).Inevbate all bac~ terial cultures for 48 h, except pseudomonas. 8. Apparatus Use apparatus specified in 991.47B(a), (b), (e), (a), and (0) (see 62.02), and in addition: (2) Capillary pipets—0.1 mL, graduated to deliver 0.01 mi. Sterilize in air oven 2h at 180°C () Microscope slides—Noncorrosve, 25 x25 mm (1% 1 in.) oF 18 x 36 mm glass slide. Sterilze by placing individual slides in Petri dish matted with 2 pieces 9m ter paper (Whatman No.2, ot equivalent) in air oven 2 hat 180°C. (©) Bacteriological culture ubes.—Pyrex, 32 x200 mm (Bellco Gass, Ine., PO Box B, Vineland, NJ 08360, USA). (@) Metal forceps.—Sharp points, straight, 115 ram long, ‘©2000 AOAC INTERNATIONAL ©. Operating Technique Thoroughly shake 48 h nutrient broth cultures of S. choleraesuis and S. aureus and let settle 10 min. For P. aeruginosa, follow preparation of culture under 991.49A(c) (see 6.2.05). With sterile capillary pipet or sterile 4.0 mm loop, transfer 0.01 ml culture onto I sqin. sterile test slide in Petri dish and immediately spread uniformly over entire area. Cover dish immediately and repeat operation until 12 slides have been pre- pared for each organism. (Use 2 slides as control.) Dry al slides 3040 min at 37°C. Spray 10 slides for specified time and distance. If no time or distance specified, use 10 s at 1 ft. (30 em). Hold each slide 10 min, drain off excess liquid, and transfer slide to individual 32 x 200 mm tube containing 20 mL appropriate subeulture me- dium, 988.11A(@) (see 6.1.01), with flamed forceps. Shake cul- ture thoroughly. If broth appears cloudy after 30 min, make subculture to fresh individual tubes of subculture broth, Transfer 2 unsprayed slides, as viability controls, vo individual subculture tubes in same manner. Tncubate all tubes used for primary and secondary transfers 48hat| 37°C. Readas + (growth) or -(no growth), Killing of test organisms in 10 of 10 trials i presumptive evidence of disinfecting action For procedures tobe followed in assuring standard cultures, for S. choleraesuis, see 991ATA(D) (see 6.2.02) for S. aureus, 991.48A(b) (see 6.2.03); for P. aeruginosa, see 991.49A\6) (see62.05).For Tmentagrophytes ee958.17A and D (see. 3.02) I there is reason to believe that lack of growth in subtransfer tubes is due to bacteriostass, inoculate all incubated subculture tubes with loop needle inoculation of respective test culture and reincubate. Growth of these inocula eliminates bacteriostasis as cause of lack of growth. If there is question as to possibility of contamination as source of growth in subculture tubes, make {gram stains and/or subculture for identification, according 0 re- spective test culture If fungicidal activity as well as germicidal activity is involved, lise test suspension of T. mentagrophytes spores, 955.17D (see 6.3.02), and prepare 12 slides, using 0.01 ml. standard spore suspension, spraying and subculturing exactly as above. Make subcultures in glucose broth, 955.17B (see 6.3.02). incubating 7 days at 25-30°C, References: JAOAC 44, 422(1961); $0, 763(1967). Soap Chem. Spec. 382), 69(1962); 61, 400(1978). 6.3.05 AOAC Official Method 966.04 Sporicidal Activity of Disinfectants First Action 1966 Final Action 1967 (Suitable for determining sporicidal activity of liquid and ‘gaseous chemicals. Applicable to germicides for determining presence or absence of sporicidal activity against specified pore-forming bacteria in various situations and potential effi- cacy as sterilizing agent.) A. Reagents (2) Culture media —(1) Soil extract nutrient broth Extract 1b (454 g) garden soil in | LH,O, filter several times through S&S No. $88 pape, and dilute to volume (pH should be 25.2). Add 5 g |AOAC Orricit MeTH008 oF AnALYss (2000) Disnrecras Chapter, p. 13 beef extract DifeoNo. 0126, § g NaCI and 10g peptone [Anatone, 985.11A(a) (see 6.1.01)} Boil 20 min, dilute to volume, adjust with IM NaOH to pH 69, and filter trough pape. Dispense in 10 mL. portions into 25 x 150 mm tubes, and autoclave 20 min at 121°C. Use this broth to propagate test culture of Bact. 2) Nutrient «agar—See 985.11A(6) (see 6.1.01), Use slants ofthis medium to maintain stock culture of Bacil (3) Modified lid thioglycolae medium USP XX.—Prepar asin 9S8.11A(B)2) (see 6.1.01), excep suid 20 ml. IM NaOH to each L before dispensing for strization. Use this medium to subculture spores exposed to 25M HCI. For spores exposed to unknown germicides se fui thoglycolat me- dium, 985.11A(@)2) (see 6.1.00. (4) Soil exractegg-meat me- dim —Add 15 g Bacio egg-meat medium dehydrated (Difco No. 042-17) 025 x 150-mm tube: then add 15 mL garden sil ex- tract, (1), and sterilize 20 min at 121°C. Use this medium to propa- gate test cultures of Clostrida and maintain stock cultures of species ofthis genus (0) Testorganisms.—Use Bacillus subtilis, ATCC No. 19659,0r Clostridium sporogenes, ATCC No. 3584, for routine evaluation. Methodisalso applicable forusewithsher spore forming species (©) Didwe hydrochloric acid —2.5M. Use to determine resis- tance of dried spores. Standardize and adjust to 25M asin 936.158 (see 41.06). B. Apparatus (@) Glassware —Bacterological culture thes. flared, 25 x 150:mm: 100 mL las-stoppered cylinders graduated in 1 mi divi- sions: 65 mm id funnels: supply of 15x 110mm Petr dishes mated with? sheets 9em S&S No. 597.or Whatman No.2 filter paper. Ster ize al glassware and matted Petr dishes 2hinair oven at 180°C. () Water bath —See 955.11B() (Se 61.1) (©) Racks.—See 985.11B() (see 6.101 (@) Transfer loop, hook —See 988.14%B(6) and (f (see 62.01) Forceps, see 961.028(8 (see 63.08). (©) Tissue grinder —Thomas Scientific, No. 3431-E20, size B, or equivalent. (©) Sure oop carrier —From spool of size 3 surgical stk su- ture (3, 6.0 metric, silk black braided SA-9G, USP, Ethiocon, Ine., Rte 22, Sommerville, NI 08876, USA), pre- pare standard loops by wrapping the silk around ordinary pen- cil 3 times, slipping coil so formed off end of pencil. and holding it firmly with thumb and index finger of left hand while passing another piece of suture through coi, knotting, and ying securely. Then sear off end of coil and knotted suture to within 2mm. This should provide overall length of ea 65 mn of suture in 2-loop coil that can be conveniently handled in ordi- nary aseptic transfer procedure Extract Toapsin groups f 100-200 in Soxhlet extraction appar tus using CHCl, for 248, Aird 12-18 hat room temperature in hood. Pace 100 oops in 100 mL 0.5M HCC for 10 min or uni all Joops are completely submerged in solution. Decant, and rinse re- peatedly with distilled H,O for 15 min. Check rinse H,O for ab- sence of HCL using litmas paper. ir-dry onfilterpaper mats under ambient conditions or in incubator. () Cylinder carvers —"Penicyliners.”porelain, 81 mmod, (61 mmid, 10 | mm ong. (Available from ALSIMAG Tech Ce- ramic, Laurens, SC, USA, Cat. No.LE15819.) Senize 2hin 180°C air oven. Wash used Penicylinders with ton X-100 and ise with, 0 4 times. ©. Operating Technique Grow all Bacil in soil extract nutrient broth and all Cosridia in soil extract~meal-egg medium. Make monthly transfer ofB. subtilis stock culture on Nutrient Agar. Clostridia do not require periodic transfer. Inoculate 3 tubes, using one loop stock culture, and incu- bate 72h at 37°C. Pace supply of suture loops and cylinder carers inseparate Petri dishes matted with filter paper and sterilize 20 min at 121°C, Use new loops for each test. Penicylinders must be free from chips or eracks. Filter C.sporogenes through funnel containing 25% S em sq piece of moist cotton or glass wool into sterile 25 x 150 mm test tubes, using same funnel In preparing B. subtilis ‘culture, pour tube of 72 hcultureinto tissue grinder and macerate to break up pellicle. Filter through sterile funnel containing ‘moist cotton or glass wool into sterile 25 x 150 mm tube, repeat- ing operation for other 2 tubes. Place 10 sterile suture loops or Penicylindersinto each of 3 tubes containing 10 mL filtrate from 72h culture of Cl. sporogenes, agitate, and let stand 10-15 min. Using this technique, contaminate 35 loops or cylinders. Place ‘contaminated suture loops and/or cylinders into Pets dish mat- ted with 2 layers of filter paper. Drain. Proceed similarly for B. subtilis. Place the 35 suture loops or cylinders contaminated with C.sporogenes or B. subtilis in vactum desiccator containing CaCl, and draw vacuum of 69 cm (27 in) Hg for 20 min. Dry 24 h under vacuum. (Spores dred and held under these conditions willretainre- sistance 27 days.) ‘Transfer 10 mL 2.5M HCI. A(e), into sterile 25 x 150 mm tbe Place tube in 20°C constant temperature H,O bath and let come to temperature. Rapidly transfer dried, contaminated loop or cylinder carriers to acd tube. Transfer remaining dred, contaminated suture loop or eylindercariersto tube of thioglycolate subculture medium, ‘A(a)(3), as viability control. After, 5, 10,and20 min, withdraw in-

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