ASTM E1054-2002 Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents《评价抗菌剂活力失效的标准试验方法》.pdf

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1、Designation: E 1054 02Standard Test Methods forEvaluation of Inactivators of Antimicrobial Agents1This standard is issued under the fixed designation E 1054; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision

2、. 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 These methods are used to determine the effectivenessof procedures and agents for inactivating (neutralizing,quenching) the microb

3、iocidal properties of antimicrobialagents and to ensure that no components of the neutralizingprocedures and agents, themselves, exert an inhibitory effect onmicroorganisms targeted for recovery.NOTE 1Knowledge of microbiological and statistical techniques isrequired for these procedures. These meth

4、ods are not applicable to testingwith viruses (see Test Method E 1482).1.2 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine th

5、e applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E 645 Test Method for Effectiveness of Microbicides Usedin Cooling SystemsE 1115 Test Method for Evaluation of Surgical Hand ScrubFormulationsE 1482 Test Method for Neutralization of Virucidal Agentsin

6、 Virucidal Effectiveness Evaluations3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 antimicrobial agenta test formulation, chemicalcompound, or product designed to prevent the growth ofmicrobes either by inhibiting growth or destroying the mi-crobe.3.1.2 antimicrobial effectiv

7、eness evaluationa determina-tion of microbiocidal properties of an antimicrobial agent bymethods, such as Test Methods E 645 and E 1115.3.1.3 CFU/mLcolony-forming units of a microorganismper millilitre of fluid.3.1.4 neutralizera procedure or chemical agent used toinactivate, neutralize, or quench t

8、he microbiocidal properties ofan antimicrobial agent.3.1.5 neutralizer effectivenessa neutralizers ability toinactivate, neutralize, or quench the microbiocidal properties ofan antimicrobial agent.3.1.6 neutralizer toxicityany inhibitory effects a neutral-izer may have on the survival of a microbial

9、 population.3.1.7 test material controlan evaluation of the activity ofan test material in reducing a known population of microor-ganisms.3.1.8 test organism viabilitythe population or viability ofa challenge microorganism used in a neutralization assay.4. Summary of Test MethodsNOTE 2The neutraliza

10、tion test method selected must be identical tothe method used in the antimicrobial effectiveness evaluation.4.1 Neutralization Assay with Recovery on Solid MediumNeutralization assay for antimicrobial effectiveness tests thatrecover and quantify microorganism populations on solid(agar) media. This m

11、ethod is appropriate for antimicrobialagents that can be chemically inactivated or diluted to sub-inhibitory levels.4.2 Neutralization Assay with Recovery in LiquidMediumNeutralization assay for antimicrobial effectivenesstests that recover surviving microorganism populations inliquid media for a gr

12、owth/no growth determination. Thismethod is appropriate for antimicrobial agents that can bechemically inactivated or diluted to sub-inhibitory levels.4.3 Neutralization Assay with Recovery by MembraneFiltrationNeutralization assay for antimicrobial effective-ness tests that recover and quantify mic

13、roorganism populationsby using membrane filtration. This method is appropriate forantimicrobial agents that cannot be chemically inactivated ordiluted to sub-inhibitory levels. This method should not beused when difficulties are incurred during the filtration process.1These test methods are under th

14、e jurisdiction of ASTM Committee E35 onPesticides and are the direct responsibility of Subcommittee E35.15 on Antimicro-bial Agents.Current edition approved May 10, 2002. Published August 2002. Originallypublished as E 1054 85. Last previous edition E 1054 91.2For referenced ASTM standards, visit th

15、e 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.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,

16、United States.5. Significance and Use5.1 The effectiveness of antimicrobial agents such as disin-fectants, sanitizers and antiseptics are measured by their abilityto kill microorganisms at or for a specified contact time.Accurate determination of antimicrobial effectiveness thereforerequires efficie

17、nt and effective inactivation (neutralization) ofthe antimicrobial agent. Inefficient or incomplete neutralizationwill permit killing or inactivation of microorganisms to con-tinue beyond the experimental exposure time, resulting in anover-estimation of antimicrobial activity.5.2 The neutralization

18、methods commonly used in antimi-crobial effectiveness evaluations are chemical inactivation,dilution and filtration. All critical parameters, for example,media, microorganism(s), equipment, and temperature of solu-tions, of the antimicrobial effectiveness evaluation must bemimicked when evaluating a

19、 neutralization procedure to beused in the antimicrobial effectiveness evaluation.5.3 The evaluation must include at least three replications(five replications in Section 9) so that a statistical analysis canbe performed with the recovery data. The number of replicatesused in the evaluation depends

20、on the statistical significancerequired for the expected results, the variability encountered inthe evaluation, and the relative efficacy of the neutralizationprocedure.5.4 A limitation of these evaluation procedures is that theyuse microorganisms that have not been exposed to an antimi-crobial. Und

21、er experimental conditions, cells exposed to neu-tralization procedures are likely to be damaged to differentdegrees by the antimicrobial agent. Sublethal injury may be afactor in recovery and the role of the neutralization procedurein recovery of injured organisms should be examined.NOTE 3Ideally,

22、all microorganisms used in the antimicrobial effec-tiveness evaluation should be tested in the neutralization assay. However,“representative” organisms may be selected for testing, as judged appro-priate by the investigator. The investigator is cautioned that failure toidentify neutralizer efficacy

23、and toxicity for all microorganisms couldresult in exaggerated microbial reductions in an antimicrobial effective-ness evaluation. Also, for studies involving multiple antimicrobial prod-ucts and a sample containing multiple species of microorganisms (forexample, skin flora), a single neutralizing p

24、rocedure and/or combinationof agents suitable for the multiple products must be used for testing.6. Apparatus6.1 Standard bacteriological devices and equipment shouldbe used for performance of the neutralization assay.6.2 Colony CounterAny of several types may be used; forexample, Quebec colony coun

25、ters and similar devices, orautomated, computerized plater/counter systems.6.3 IncubatorAny incubator capable of maintaining anappropriate temperature for growth of the microorganism maybe used.6.4 SterilizerAny steam sterilizer capable of producingthe conditions of sterilization.6.5 Timer (stopwatc

26、h)One that displays hours, minutes,and seconds.6.6 Vortex Mixer or equivalent.6.7 Membrane Filter UnitsAny sterilizable unit that per-mits filtration of microorganisms for enumeration. The mem-brane filter unit should be suitable for testing the antimicrobialagent and recovery of the microorganisms.

27、7. Reagents and Materials7.1 Phosphate Buffered Saline Dilution WaterPBS (seeTest Method E 645).7.1.1 Phosphate Buffer Solution, StockDissolve 34.0 g ofpotassium dihydrogen phosphate (KH2PO4) in 500 mL ofwater. Adjust pH to 7.2 6 0.2 with 0.1 N NaOH or 0.1 N HCland bring to 1000 mL with deionized wa

28、ter.7.1.2 Phosphate Buffer Saline Dilution WaterAdd 1.25mL of stock phosphate buffer solution and 8.75 g of NaCl to avolumetric flask, fill with deionized water to the 1000 mLmark, and mix. Final pH should be 7.2 6 0.2. Sterilize byfiltration or autoclave.7.2 Because the types of materials and reage

29、nts required forvarious antimicrobial effectiveness evaluations are so diverse,it is impractical to list them in this method. The specificmaterials and reagents to be used in the antimicrobial effec-tiveness evaluation, however, should be tested in the neutral-ization assay to confirm that the antim

30、icrobial agent is beingneutralized in a particular evaluation.7.3 Table 1 provides a partial list of materials that have beenemployed by researchers to inactivate the microbiocidal prop-erties of various antimicrobial agents. This list is provided as aguide for selecting neutralizers. A neutralizati

31、on assay shouldbe performed to determine a selected neutralizers effective-ness.8. Neutralization Assay with Recovery on Solid Medium(Fig. 1)8.1 At least three replicates are required for these proce-dures. The number of replicates used in the evaluation dependson the statistical significance requir

32、ed for the expected results,the variability encountered in the evaluation, and the relativeefficacy of the neutralization procedure.8.2 All tests must be performed in a timely manner so thatreplication of the test organism does not occur.8.3 Test ANeutralizer Effectiveness:8.3.1 Add a volume of prod

33、uct, or solution containingproduct, to neutralizer that will result in the same dilution ratioto be used in the antimicrobial effectiveness evaluation. If theantimicrobial effectiveness evaluation will employ the use ofcarriers, use instead a carrier containing an amount of productrepresentative of

34、that to be used in the test.NOTE 4The dilution ratio of product to neutralizer can be manipu-lated to determine the dilution at which adequate neutralization of theproduct will occur, particularly when testing products not readily neutral-ized by chemical means.NOTE 5The sequence of product-into-neu

35、tralizer, followed by thechallenge microorganism, allows the neutralizing action to take place. Ifthe microorganism is introduced into the neutralizing solution prior toadding the product, there is possibility of the product acting on themicroorganism there by reducing the population and disqualifyi

36、ng theneutralizer.8.3.2 Within5sofexecution of 8.3.1, inoculate the product/neutralizer mixture with a volume of the challenge microor-ganism suspension so that the resulting suspension contains 30to 100 CFU/mL of the microorganism.E1054022NOTE 6The challenge inoculum should be prepared in the same

37、manner used in the antimicrobial effectiveness evaluation. The volume ofTABLE 1 Processes Applied for Neutralization of Certain Antimicrobial AgentAAntimicrobial Agent Neutralizers/InactivatorsAlcoholsIsopropanol, Phenoxyethanol Polysorbate 80, dilution to sub-inhibitory levelsAldehydes2-Bromo-2-nit

38、roproprane-1, 3-diol (bronopol) Serum, cysteine, thiosulfate, thioglycolate, metabisulfiteFormaldehyde Sodium sulfite, ammonia, histamineGlutaraldehyde Dilution to sub-inhibitory levels, sodium bisulfite, sodium sulfite,glycine, cystine, cysteineChlorallytriazaazoniaadamantane (Dowicil 200) Dilution

39、 to sub-inhibitory levelsDimethyloldimethyl hydantoin (Glydant) Dilution to sub-inhibitory levelsBiguanides and Bis-biquanidesChlorhexidine Lecithin/polysorbate 80, sodium oleatePolyhexamethylene biguanide HCL (Cosmocil CQ) Polysorbate 80/lecithinPhenolicsPhenylphenol, Chloroxylenol, Cresols, Chloro

40、cresols, Phenol Nonionic surfactants, polysorbate 80, and/or dilution to sub-inhibitory levelsBis-PhenolsTriclosan 10 % polysorbate 80/lecithin, and dilution to sub-inhibitory levelsHexachlorophene 10 % polysorbate 80/lecithin, and dilution to sub-inhibitory levelsQuaternary Ammonium CompoundsCetrim

41、ide, Benzalkonium and Benzethonium Chloride Lecithin/polysorbate, suramin sodium, organic material, 0.5 %polysorbate 80, cyclodextrinsMercurials Sulfhydryl compounds, thioglycolic acid, thiosulfate, bisulfite,ammonium sulfiteOrganic AcidsBenzoic, Propionic, Sorbic Nonionic surfactants, dilution to s

42、ub-inhibitory levels, pH 7 ofaboveHalogensHypochlorite Thiosulfate and/or dilution to sub-inhibitory levelsIodine Thiosulfate, polysorbate 80, skim milkBromine Thiosulfate and/or dilution to sub-inhibitory levelsEDTA Mg+2or Ca+2ionsImidazolidinyl urea Dilution to sub-inhibitory levelsMethyl-, and me

43、thylcholoroisothiazolinone (Kathon) Amines, sulfites, mercaptans, sodium bisulfite, heparinParabensMethyl-, ethyl-, propyl-, butyl-parahydroxybenzoate Lecithin, filtration, dilution to sub-inhibitory levels, polysorbatesurfactants, 1 % polysorbate 80 or 20Hydrogen Peroxide CatalasePeroxyacetic Acid

44、Sodium ThiosulfateASutton, S. V. W., “Neutralizer Evaluations as Control Experiments for Antimicrobial Effectiveness Tests,” Ch. 3 in Handbook of Disinfectants and Antiseptics,Marcel-Dekker, NY, 1996, p. 300.FIG. 1 Testing Schema for Neutralization Assay with Recovery on Solid MediumE1054023the chal

45、lenge inoculum should be kept to a minimum so it does not causesignificant dilution of the product/neutralizer mixture.8.3.3 Within 1 min of execution of 8.3.2, enumerate theproduct/neutralizer/microorganism suspension by quantitativepour or spread plates, in duplicate, using appropriate platingmedi

46、um. If neutralizers are to be incorporated in the platingmedium for the antimicrobial effectiveness evaluation, use thissame medium for plating the suspension.8.3.4 Allow the product/neutralizer/microorganism suspen-sion to stand for the longest exposure period representative ofthat to be used in th

47、e antimicrobial effectiveness evaluation.For example, if the product/neutralizer/microorganism fromthe antimicrobial effectiveness evaluation will be plated within30 min, then the longest exposure period for the neutralizationassay is 30 min.8.3.5 After the hold-time, enumerate the product/neutraliz

48、er/microorganism suspension by quantitative pour orspread plates, in duplicate, using an appropriate plating me-dium. If neutralizers are to be incorporated in the platingmedium for the antimicrobial effectiveness evaluation, use thissame medium for plating the suspension.NOTE 7The duration of the h

49、old time must not be such that replica-tion of the test organism introduces a variable.8.3.6 Repeat this procedure (8.3.1-8.3.5) an additional twotimes, for a total of three replicates.8.3.7 Incubate the plates under the same conditions as thoseto be used in the antimicrobial effectiveness evaluation.8.4 Test BNeutralizer Toxicity:8.4.1 Add a volume of PBS or other appropriate bufferingagent to neutralizer that will result in the same dilution ratio asthat used in Test A (see 8.3.1).8.4.2 Within5sofexecution of 8.4.1, inoculate the PBS/neutralizer mixt