1、Designation: E 1153 03Standard Test Method forEfficacy of Sanitizers Recommended for Inanimate Non-Food Contact Surfaces1This standard is issued under the fixed designation E 1153; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, th
2、e year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method is used to evaluate the antimicrobialefficacy of sanitizers on precleaned inanimate, nonpo
3、rous,non-food contact surfaces.1.2 This test method may also be used to evaluate theantimicrobial efficacy of one-step cleaner/sanitizer formula-tions recommended for use on lightly soiled, inanimate,nonporous, non-food contact surfaces.1.3 It is the responsibility of the investigator to determinewh
4、ether Good Laboratory Practices (GLP) is required and tofollow them where appropriate (see section 40 CFR, 160) or asrevised.1.4 This standard may involve hazardous materials, chemi-cals and microorganisms and should be performed only bypersons who have had formal microbiological training. Thisstand
5、ard does not purport to address all of the safety concerns,if any, associated with its use. It is the responsibility of the userof this standard to establish appropriate safety and healthpractices and determine the applicability of regulatory limita-tions prior to use.2. Referenced Documents2.1 ASTM
6、 Standards:2D 1193 Specification for Reagent WaterE 1054 Practices For Evaluating Inactivators of Antimicro-bial Agents Used in Disinfectant, Sanitizer, Antiseptic, orPreserved Products2.2 Federal Standard:40 CFR, Part 160, Good Laboratory Practice Standards33. Significance and Use3.1 This test meth
7、od shall be used to determine if a chemicalhas application as a non-food contact sanitizer or as a one-stepcleaner/sanitizer.4. Apparatus4.1 BalanceA balance with a platform to accommodate a100-mL volumetric flask. This balance should be sensitive to0.01 g.4.2 Nonporous Test Surfaces, pre-cleaned.4.
8、2.1 Borosilicate Glass Squares, 25 by 25 by 2 mm slides,nonchipped.4.2.2 Glazed Glass or Stainless Steel, of appropriate type,approximately same size as in 4.2.1.4.3 Glass Culture Tubes, recommended sizes: 18 to 20 by150 mm and 25 by 150 mm without lip.4.4 Culture Tube Closures, appropriate sized no
9、ntoxic clo-sures.4.5 Pipets or Dispensing Syringes, (or both), appropriatelycalibrated and sterile.4.6 Bacteriological Transfer Loop, 4 mm inside diameterloop of platinum or platinum alloy wire or sterile, disposableplastic loops of approximate size.4.7 Flasks or Containers:4.7.1 Appropriate sizes w
10、ith closures for preparation ofculture medium and sterile distilled water.4.7.2 Volumetric, 100 and 1000 mL, sterile.4.8 Petri dishes, recommended sizes: 50 by 9 mm plastic,and 100 by 15 mm, glass and plastic; sterile.4.9 Jars, ointment jars, 2 oz (60 mL) with nontoxic lids,sterile.4.10 Graduated Cy
11、linders, recommended sizes; 100 and500 mL.4.11 Flaming ApparatusA bunsen burner or other appro-priate heat sterilizer.4.12 MixerA “vortex” mixer is recommended.4.13 TimerA reliable stopwatch or laboratory timer ca-pable of measuring elapsed time in seconds and minutes.4.14 pH MeterA reliable pH mete
12、r to determine pH ofculture media.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 10, 2003. Published July 2003. Originallyappro
13、ved 1987. Last previous edition approved in 1994 as E 1153 94.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website
14、.3Available from the Superintendent of Documents, U.S. Government PrintingOffice, Washington, DC 20402.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.4.15 Desiccator, recommended size: 200 mm inside diam-eter with approximately 125-
15、mm chamber depth from insideplate to cover flange, glass.4.16 Incubator, capable of maintaining temperature of 37 62C.4.17 Sterilizer, steam sterilizer and hot air oven (180 6 2Cfor 2 h).4.18 Colony CounterAny one of several types may beused, for example Quebec.4.19 Membrane Filters, of 0.22 m pore
16、size.4.20 Filter Assembly, autoclavable.4.21 Forceps.5. Reagents and Materials5.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents shall conform to the specifications of the Commit-tee on Analytical Reagents of the Ameri
17、can Chemical Society,where such specifications are available.4Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.5.2 Water for Dilution of Product Under Test:5.2.1 Water, steril
18、e, deionized or distilled, equivalent to orbetter than Type 3, see Specification D 1193.5.2.2 Association of Offcial Analytical Chemists (AOAC)Synthetic Hard Water:5(c)5.2.2.1 Solution 1Dissolve 31.74 g magnesium chloride(MgCl2) (or equivalent of hydrates) and 73.99 g calciumchloride (CaCl2) in boil
19、ed distilled water and dilute to 1 L.Sterilize by autoclaving.5.2.2.2 Solution 2Dissolve 56.03 g sodium bicarbonate(NaHCO3) in boiled distilled water and dilute to 1 L. Sterilizeby membrane filtration.5.2.2.3 Place the desired amount of Solution 1 in a sterile1-L flask and add approximately 600 mL s
20、terile distilled water;then add 4 mL of Solution 2 and dilute to exactly 1 L withsterile distilled water. Each millilitre of Solution 1 will give awater equivalent to 100 ppm of hardness calculated as calciumcarbonate (CaCO3) by the following equation:Total hardness as ppm CaCO3(1)5 2.495 3 ppm Ca#
21、1 4.115 3 ppm Mg#5.2.3 The pH of synthetic hard water should be from 7.6 to8.2.5.2.4 The synthetic water to be used for the testing shouldbe analyzed chemically for hardness at the time of test.Analysis may be performed by the method described infootnote 6(c) or by commercial available kit.5.2.5 All
22、 water used for preparation of test solutions shall besterile.5.3 Sanitizing SolutionsFreshly prepared solutions ofsanitizers shall be used in all tests.5.4 Neutralizing SolutionsSolutions appropriate to inacti-vate sanitizing solutions shall be used in accordance withPractices E 1054.5.5 Culture Me
23、dia:55.5.1 Nutrient Broth.(5(a)5.5.2 Nutrient Agar.(5(b)5.6 Soil, Bovine Serum, aseptically derived and maintained.6. Preparation of Apparatus6.1 Constant Humidity Chamber (Desiccator):6.1.1 At least one day prior to use, fill the lower portion ofa large size desiccator with about 500 mL of glycerin
24、 solutionhaving a refractive index of 1.4529 at 25C (approximately86.5 % glycerin in distilled water will provide this refractiveindex). This will provide a constant 40 to 41 % relativehumidity at 37 6 2C in which the inoculated nonporoussquare surfaces will be dried prior to treatment with thesanit
25、izer. Replace the porcelain floor plate of the desiccator andstore at 37 6 2C to allow to come to equilibrium.6.2 Test Squares:6.2.1 Test squares shall be dipped in 70 to 95 % ethyl orisopropyl alcohol, rinsed with distilled water, and air driedbefore sterilization.6.2.2 Place test squares into a la
26、rge, glass petri dish andsterilize in a hot air oven for2hat180C.6.2.3 After sterilization, place each square into separate 50by 9 mm sterile plastic petri dishes using sterile technique.7. Test Organisms7.1 Klebsiella (K.) pneumoniae American Type CultureCollection (ATCC) 4352 and Staphylococcus (S
27、.) aureusATCC 6538.7.2 Maintenance of Test OrganismsMaintain stock cul-tures of K. pneumoniae and S. aureus on nutrient agar. Incubate2 days at 37 6 2C, then refrigerate at 5 to 7C. Transferculture every 3 days. Stock cultures used for inoculation shouldnot be more than five passages removed from th
28、e ATCCcultures (USP XXIII).6Information on long term culturemaintenance and storage is found in “Manual of Methods forGeneral Bacteriology”7and “ATCC Catalogue of Bacteria andBacteriophages”.88. Preparation of Inocula8.1 K. pneumoniae and S. aureusK. pneumoniae and S.aureus are grown in nutrient bro
29、th. From stock cultures, (nomore than 30 days old), inoculate tubes containing 10 mL ofappropriate broth, and incubate for 24 h at 37 6 2C. Using a4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlis
30、ted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,MD.5“Official Methods of Analysis of the Association of Official Analyti
31、calChemists,”Association of OfficialAnalytical Chemists, Washington, DC, Chapter 6:Disinfectants, 15th ed., 1990.(a) Page 133, Section 955.11 A. (a).(b) Page 133, Section 955.11 A. (c).(c) Page 139140, Section 960.09A.6Sterility Tests (71), United States Pharmacopeia (USP) XXII.7Manual of Methods fo
32、r General Bacteriology, 1981, P. Gerhardt (ed. in chief)ASM Microbiology, Washington, DC.8Associated Concentrates, Inc., 32-60 61st St., Woodside, NY 11377.E11530324 mm inside diameter transfer loop, transfer a loopfull of theculture into fresh broth. Make three consecutive daily transfersprior to u
33、se as an inoculum. The final transfer is incubated for48 h, and this culture is used for the test.8.2 Inocula for Testing Sanitizers for Use on PrecleanedSurfacesThoroughly mix 48 h culture of test organism on“vortex” mixer, then allow the culture to settle for 15 min.Decant upper two thirds of this
34、 suspension and use this as theinoculum for testing sanitizers for use on precleaned surfaces.8.3 Inocula for Testing Formulations as One-Step Cleaner/Sanitizers or Sanitizers for Use on Lightly Soiled SurfacesThoroughly mix 48 h culture of test organism on “vortex”mixer, then allow the culture to s
35、ettle for 15 min. Decant uppertwo thirds of this suspension and add bovine serum (forexample, 19 mL of a 48 h bacterial culture and 1 mL bovineserum). Use this suspension now containing bovine serum at5 % concentration as the inoculum for testing one-step cleaner/sanitizers or sanitizers for use on
36、lightly soiled surfaces.9. Preparation of Test Solutions9.1 Prepare the sanitizer in accordance with the manufac-turers recommended dilution. Dilutions for the test may bemade in sterile distilled water or in AOAC formula synthetichard water of any hardness desired (see 5.2).9.2 For each organism to
37、 be tested prepare 100 mL aliquotsof the test solution.10. Preparation of Neutralizer Solutions10.1 Quarternary and Phenolic Solutions:10.1.1 Phosphate Buffer Stock Solution (0.25 M)Dissolve34.0 g of potassium phosphate, monobasic (KH2PO4)in500mL distilled water; adjust the pH to 7.2 with 1N NaOH an
38、ddilute to 1 L.10.1.2 Phosphate Buffer Dilution WaterAdd 1.25 mL of0.25 M phosphate buffer stock solution to 1 L water anddispense in 99 mL portions. Autoclave for 20 min at 121C.10.1.3 Neutralizer StockMix 40.0 g Azolectin,8280 mLpolysorbate 80, and 1.25 mL phosphate stock solution buffer(see 10.1.
39、1). Adjust to pH 7.2 with 1N NaOH. Dilute to 1 Lwith distilled water. Dispense in suitable portions and sterilizefor 20 min at 121C.10.1.4 Neutralizer SolutionMix 62.5 mL of neutralizerstock (see 10.1.3), 6.25 mL of phosphate buffer stock solution(see 10.1.1), and 381.25 mL of distilled water. Dispe
40、nse 20 mLportions into 25 by 150 mm culture tubes and sterilize for 20min at 121C.10.2 Halogen SanitizersNeuralizer Solutions, Dissolve0.31 g of sodium thiosulfate and 0.30 mL of Triton X-100 in500 mL of distilled water. Dispense 20 mL portions into 25 by150 mm culture tubes and sterilize for 20 min
41、 at 121C.10.3 Other Sanitizing AgentsUse appropriate neutralizers(see Practices E 1054).11. Procedures11.1 Inoculation of Test Squares:11.1.1 Inoculate each sterile glass or other nonporous sur-face (see 6.2.3) squares with exactly 0.02 mL of 48 h culture.Spread the inoculum to within 3 mm of the ed
42、ges of the square.Prepare appropriate number of test squares, depending uponthe test and its parameters.11.1.2 Number each plate used in the order in which thesquares are inoculated. Place all plates containing the inocu-lated squares in the 37C constant humidity desiccator. Set thelids of the plate
43、s slightly ajar and close the desiccator lid.Allow the squares to remain at this temperature and humidityfor exactly 35 min.NOTE 1Caution: Be very careful to remove the desiccator lid onlylong enough to place the 50 by 9 mm plates on the porcelain floor plateand set their lids ajar.11.2 Inoculum Cou
44、nt:11.2.1 Plate the appropriate dilutions of K. pneumoniae andS. aureus using nutrient agar. Incubate the organisms for 48 hat 37 6 2C. Count the colonies to determine the number oforganisms per mL of culture and present at the start of the test.Cultures used for further testing must be kept at 5 to
45、 7C for nomore than 8 h.11.2.2 Report inoculum count for the test organisms.11.3 Sanitizer or Cleaner/Sanitizer Treatment of InoculatedTest Squares:11.3.1 Transfer five squares to five sterile 2 oz (60 mL)ointment jars using sterile forceps. Be sure to resterilize theforceps between each transfer. (
46、Dip in 70 to 95 % ethyl orisopropyl alcohol and burn off). Mark each jar with a numbercorresponding to that on the plate from which the square wastaken.11.3.2 At zero time on the timer, cover inoculated squareNo. 1 (the first one inoculated) with exactly 5 mL of the testsolution using a sterile 5 mL
47、 pipette. At exactly 1 min, coversquare No. 2 with 5 mL of the test solution. Treat square No.3 in a like manner at 2 min, square No. 4 at 3 min, and squareNo. 5 at 4 min.11.3.3 At exactly 5 min on the timer, add 20 mL ofappropriate neutralizer solution into jar No. 1 and rotate the jarvigorously on
48、 an even plane for approximately 50 rotations tosuspend the surviving organisms. At 6 min, add 20 mL ofneutralizer into jar No. 2 and rotate as in No. 1. Continueaddition of neutralizer to jars No. 3, No. 4 and No. 5 at 1 minintervals, and rotate each in turn.11.3.4 Within 30 min after the addition
49、of the neutralizer tothe test sanitizer or cleaner/sanitizer, plate in duplicate 1.0 and0.1 mL of the neutralizer solution from each of the five jarsusing standard pour plate techniques. Use nutrient agar for bothorganism types. Incubate for 48 h, K. pneumoniae and S.aureus at 37 6 2C. Count the number of colonies on theplates.11.4 Inoculation of Control SquaresAllow the refriger-ated cultures to come to ambient temperature. Prepare threeglass squares for each organism type as in 11.1.1 and 11.1.2.11.5 Treatment of Inoculated Control Squ
