AOAC965_12.pdf

6.3.06AOAC Official Method 965.12Tuberculocidal Activityof DisinfectantsFirst Action 1965Final Action 1967Revised First Action 1988(Suitablefordeterminingmaximumtuberculocidaldilutionofdisin-fectants used on inanimate surfaces. This method has not been vali-dated for glutaraldehyde-based products.)I. Presumptive in vitro Screening TestUsingMycobacterium smegmatisA. Reagents(a) Testorganism.Mycobacteriumsmegmatis(PRDNo.1;avail-able from Microbiology Laboratory, U.S. Environmental ProtectionAgency, Biological and Economic Analysis Division, Bldg. 306,BARC-East, Beltsville, MD 20705, USA). Maintain on nutrient agarslantsbymonthlytransfers.Incubatenewstocktransfer2daysat37C;then store at 25C. From stock culture inoculate tubes ofProskauerBeckbroth,(b)(1),incubate48hinslantingposition,carry30 days, using 48 h transfers, and use these 48 h cultures to start testcultures. Inoculate one or 2 tubes of ProskauerBeck broth. Incubate67 days at 37C. Incubate tubes 48 h in slanting position to providemaximumsurfaceaerationandtheninuprightposition45days.Add1.5 mL sterile 2.0% BactoGelatin solution and homogenize culturewithsterilizedglasstissuegrinder,966.04B(e)(see6.3.05).Adjustto20%Tat650nmwithsterileProskauerBeckbrothforuseintesting.(b) Culturemedia.(1)ModifiedProskauerBeckbroth.Dis-solve 2.5 g KH2PO4, 5.0 g asparagine, 0.6 g MgSO47H2O, 2.5 gmagnesium citrate, 20.0 mL glycerol, 0.0046 g FeCl3, and 0.001 gZnSO47H2Oin1LH2O.AdjusttopH7.27.4with1NNaOH.Filterthroughpaper,place10mLportionsinseparate20150mmtubes,and sterilize 20 min at 121C. Use for propagating 48 h test startercultures and 67 day test cultures. (2) Subculture media.Use (1)withadditionofsuitableneutralizingagentssuchaspurifiedlecithin(Azolectin) or sodium thioglycolate, where necessary. (3) Nutrientagar.Prepareasin955.11A(c)(see6.1.01).Usetomaintainstockculture. (4) Sterile distilled water.See991.47A(f) (see 6.2.02).B. Apparatus(a) Glassware, Petri dishes, water bath, transfer loops,andwirehook.See991.47B(a)(c), (e), and (f) (see 6.2.02).(b) Carriers.See966.04B(g) (see 6.3.05).C. Operating TechniqueTransfer 20 sterile Penicylinder carriers, using flamed nichromewirehook,into20mL67dayhomogenizedstandardizedbrothcul-ture,A(a),insterile25150mmtesttube.After15mincontact,re-move cylinders and place on end in vertical position in sterile Petridish matted with filter paper, 991.47B(b) (see 6.2.02). Cover andplaceinincubatorat37Candletdry20minbut60min.Thiswillprovide dried test carriers in groups of 20 in individual Petri dishes.Witheachgroupof20carriers,add1driedcylinderat30sintervalsto each of 20 tubes containing 10 mL dilution of germicide to betested(at20CinH2Obath).Flamelipsoftestandsubculturetubes.Immediately after placing carrier in test tube, swirl tube 3 times be-fore placing it back in H2O bath. (Thus, by time 20 tubes have beenseeded, 9 min and 30 s have elapsed, leaving 30 s interval prior tosubculturing series at 10 min exposure for each carrier. The 30 s in-terval between transfers allows adequate time for flaming and cool-ingtransferhookandmakingtransferinmannersoastodrainallex-cess chemical from carrier.) Transfer carrier to 10 mL subculturemedia, A(b)(2). Shake all subculture tubes thoroughly and incubate12 days at 37C. Report results as + (growth) or (no growth).Where there is reason to suspect that results may be affected bybacteriostaticactionofantibacterialchemicalcarriedoverinsubcul-ture tubes, use suitable neutralizer in subculture media.Make 30 carrier exposures at each of 3 relatively widely spaceddilutions of germicide under test between no response and total re-sponsedilutionlevels.Calculatepercentofcarriersonwhichorgan-ismiskilledateachdilution.Usinglogpercentprobitpaper(3cyclelogarithmic normal No. 32.376, Codex Book Co., Inc., 74 Broad-way,Norwood,MA02062,USA),locatepercentkillpointsondilu-tion lines employed (log scale). Draw best fitting straight linethroughthese3pointsandextendtointercept99%killline.Readdi-lution line (log scale) at point of intercept. This is presumed 95%confidence end point for product. (Do not use presumptive test or-ganism for checking validity of this presumptive end point.)II. Confirmative in vitro Testfor Determining Tuberculocidal ActivityFirst Action 1965D. Reagents(a) Culture media.(1) Modified ProskauerBeck me-dium.PrepareasinA(b)(1),andinaddition,place20mLportionsin 25 150 mm tubes. Use 10 mL portions for daily transfers of testcultures and 20 mL portions for subculturing porcelain cylinders.(2) Middlebrook 7H9 Broth Difco A. Dissolve 4.7 g in 900 mLH2Ocontaining2mLglyceroland15.0gagar.Heattobptodissolvecompletely. Distribute in 180 mL portions and autoclave 15 min at121C. To each 180 mL sterile medium at 45C, add 20 mLMiddlebrookADCEnrichment(DifcoNo.0713)underasepticcon-ditionsanddistributein10mLportionsinsterile20150mmtubes.Slant. Use to maintain test culture. (3) Middlebrook 7H9 BrothDifco B.(No. 0713.) Dissolve 4.7 g in 900 mL H2O containing2mLglyceroland1.0gagar.Heattobptodissolvecompletely.Dis-tribu