ASTM E1054-2008 374 Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents《评估抗菌剂灭活的标准试验方法》.pdf

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1、Designation: E 1054 08Standard 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 test methods are used to determine the effective-ness of procedures and agents for inactivating (neutralizing,quenching) the

3、 microbicidal properties of antimicrobial agents,and to ensure that no components of the neutralizing proce-dures and agents, themselves, exert an inhibitory effect onmicroorganisms targeted for recovery.NOTE 1Knowledge of microbiological and statistical techniques isrequired for these procedures. T

4、hese methods are not suitable when testingthe virucidal activity of microbicides (see Test Method E 1482).1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 This standard does not purport to address all of thesafety concern

5、s, 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 the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E 645 Test Method for Efficacy of Microbic

6、ides Used inCooling Water SystemsE 1115 Test Method for Evaluation of Surgical Hand ScrubFormulationsE 1482 Test Method for Neutralization of Virucidal Agentsin Virucidal Efficacy Evaluations3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 antimicrobial, adjdescribes an agent t

7、hat kills orinactivates microorganisms or suppresses their growth orreproduction.3.1.2 antimicrobial effectiveness evaluation, adj/na deter-mination of microbicidal properties of an antimicrobial agentby methods, such as Test Methods E 645 and E 1115.3.1.3 CFU/mL (abbrev.)colony-forming units of a m

8、icro-organism per millilitre of fluid.3.1.4 neutralization, na physical or chemical procedurethat inactivates or quenches the microbicidal properties of anantimicrobial agent.3.1.5 neutralizer effectiveness, adj/nability of a neutral-ization procedure to inactivate or quench the microbicidalproperti

9、es of an antimicrobial agent.3.1.6 neutralizer toxicity, adj/nany inhibitory effects aneutralization procedure may have on the survival of a micro-bial population.3.1.7 test material control, adj/nan evaluation of theactivity of a test material in reducing a known population ofmicroorganisms.3.1.8 t

10、est organism viability, adj/nthe population of achallenge microorganism used in a neutralization assay.3.1.9 viability, nthe ability of a challenge microorganismto form colonies or grow on a nutrient medium.3.1.9.1 DiscussionIn the context of these test methods,“viability” is understood to be synony

11、mous with culturability.4. Summary of Test MethodsNOTE 2The neutralization test method selected must be consistentwith the methods of testing used in the antimicrobial effectivenessevaluation.4.1 Neutralization Assay with Recovery on Semi-solidMediumNeutralization assay for antimicrobial effectivene

12、sstests that recover and quantify microbial populations on solid(agar) media. This method is appropriate for antimicrobialagents that are chemically inactivated or diluted to sub-inhibitory levels and performed entirely in vitro or including anin vivo component to verify neutralization of an antimic

13、robialformulation sampled from the skin of a human volunteer.4.2 Neutralization Assay with Recovery in LiquidMediumNeutralization assay for antimicrobial effectivenesstests that recover surviving microbial populations in liquidmedia for a growth/no growth determination. This method isappropriate for

14、 antimicrobial agents that are chemically inac-tivated or diluted to sub-inhibitory levels.1These test methods are under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and are the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition

15、approved April 1, 2008. Published May 2008. Originallyapproved in 1985. Last previous edition approved in 2002 as E 1054 02.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, r

16、efer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.4.3 Neutralization Assay with Recovery by MembraneFiltrationNeutralization assay for antimicrobial effective-ness tests th

17、at recover and quantify microbial populations byusing membrane filtration. This method is appropriate forantimicrobial agents that cannot be chemically inactivated ordiluted to sub-inhibitory levels, as well as for those that can be.5. Significance and Use5.1 The effectiveness of antimicrobial agent

18、s incorporatedinto disinfectants, sanitizers, and antiseptics is measured bytheir ability to kill microorganisms within a specified contacttime. Hence, accurate determination of antimicrobial effective-ness requires complete and immediate inactivation (neutraliza-tion) of the antimicrobial agent. In

19、efficient or incompleteneutralization will permit killing or inactivation of microorgan-isms to continue beyond the experimental exposure time,resulting in an overestimation of antimicrobial activity.5.2 The neutralization methods commonly used in antimi-crobial effectiveness evaluations are chemica

20、l inactivation,dilution, and filtration. All critical parameters of an antimicro-bial effectiveness evaluationfor example, media, equipment,microorganism(s), and temperature of solutionsmust beduplicated when evaluating a neutralization procedure to beused.5.3 The neutralization evaluation must incl

21、ude at least threereplications (five replications in Section 9) so that a statisticalanalysis of the recovery data can be performed. The number ofreplicates used in the evaluation depends on the statisticalsignificance required for the expected results, the variabilityencountered in the data, and th

22、e relative effectiveness of theneutralization procedure.5.4 A limitation of these evaluation procedures is that theyuse microorganisms that have not been exposed to an antimi-crobial agent. Under experimental conditions, cells exposed toneutralization procedures are likely to be damaged to different

23、degrees 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. Thismethod is not intended to assess injured organism recovery.NOTE 3Ideally, all microorganisms used in the antimicrobial

24、 effec-tiveness evaluation should be tested in the neutralization assay. However,representative organisms may be selected for testing, as judged appropri-ate by the investigator. The investigator is cautioned that failure to identifyneutralizer efficacy and toxicity for all microorganisms could resu

25、lt inexaggerated microbial reductions in an antimicrobial effectiveness evalu-ation. Also, for a study testing multiple antimicrobial formulations, and inwhich samples will contain multiple species of microorganisms (forexample, skin flora) that are exposed to the formulations, a singleprocedure and

26、/or combination of agents suitable for neutralizing theantimicrobial activities of the multiple formulations must be used fortesting.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

27、used; forexample, Quebec colony counters and similar devices, orautomated, computerized plater/counter systems.6.3 IncubatorAny incubator capable of maintaining anappropriate temperature for growth of the test microorganismmay be used.6.4 SterilizerAny steam sterilizer capable of producingthe condit

28、ions of sterilization.6.5 Timer (stopwatch)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 must be chemically compatible with theantimicrob

29、ial agent and appropriate to efficient recovery of thetest microorganisms.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.

30、2 6 0.2 with 0.1 N NaOH or 0.1 N HCland bring to 1000 mL with deionized water.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 adjusted to 7.2 6

31、 0.2, ifnecessary. Sterilize by filtration or autoclave.7.2 Because the types of materials and reagents 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-t

32、iveness evaluation, however, should be tested in the neutral-ization assay to confirm that the antimicrobial agent is beingneutralized in a particular evaluation.7.3 Table 1 provides a list of materials employed byresearchers to inactivate the microbicidal properties of variousantimicrobial agents.

33、This list is provided as a guide forselecting neutralizers and is not exhaustive. A neutralizationassay must be performed to determine a selected neutralizerseffectiveness.8. Neutralization Assay with Recovery on Semi-solidMedium (Fig. 1)8.1 The number of replicates necessary in the evaluationdepend

34、s on the statistical significance required for the expectedresults, the variability encountered in the data, and the relativeeffectiveness of the neutralization procedure. At least threereplicates are required for these procedures.8.2 All tests must be performed in a timely manner so thatsignificant

35、 proliferation of the test organism does not occur.8.3 Test ANeutralization Effectiveness:8.3.1 Inoculate the neutralizer with a volume of the chal-lenge microbial suspension to result in a suspension thatcontains 30 to 100 CFU/mL of the microorganism.NOTE 4The challenge inoculum should be prepared

36、in the samemanner to be used in the antimicrobial effectiveness evaluation. Thevolume of the challenge inoculum should be kept to a minimum so that itdoes not cause significant dilution.8.3.2 Add a volume of product, or solution containingproduct, to the neutralizer/microbial suspension that will re

37、sultE1054082in the same dilution ratio used in the antimicrobial effective-ness evaluation. If the antimicrobial effectiveness evaluationwill employ the use of carriers, use instead a carrier bearing anamount of product used in the effectiveness evaluation.NOTE 5The dilution ratio of product to neut

38、ralizer 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.8.3.3 Within 1 min of execution of 8.3.2, transfer aliquots ofthe product/neutralizer/microbial suspension to

39、 pour or spreadplates, in duplicate, using an appropriate semi-solid medium. Ifneutralizers are incorporated in the plating medium for theantimicrobial effectiveness evaluation, use this same mediumfor plating the suspension.8.3.4 Allow the product/neutralizer/microbial suspension tostand for the lo

40、ngest exposure period representative of thatTABLE 1 Processes Applied for Neutralization of Certain Antimicrobial AgentAAntimicrobial Agent Neutralizers/InactivatorsAlcoholsIsopropanol, Phenoxyethanol Polysorbate 80, dilution to sub-inhibitory levelsAldehydes2-Bromo-2-nitropropane-1, 3-diol (Bronopo

41、l) Serum, cysteine, thiosulfate, thioglycolate, metabisulfiteFormaldehyde Sodium sulfite, ammonia, histamineGlutaraldehyde Dilution to sub-inhibitory levels, sodium bisulfite, sodium sulfite, glycine, cystine, cysteineN-(3-Chloroallyl)hexaminium Chloride (Dowicide Q) Dilution to sub-inhibitory level

42、sDimethylol-5, 5-dimethylhydantoin (Glydant) Dilution to sub-inhibitory levelsBiguanides and Bis-biquanidesChlorhexidine Lecithin/polysorbate 80, sodium oleatePolyhexamethylene biguanide HCL (Cosmocil CQ) Polysorbate 80/lecithinPhenolicsPhenylphenol, Chloroxylenol, Cresols, Chlorocresols,PhenolNonio

43、nic 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 CompoundsCetrimide, Benzalkonium and

44、 Benzethonium Chloride Lecithin/polysorbate, suramin sodium, organic material, 0.5 % polysorbate 80, cyclodextrinsMercurials and Silver Sulfhydryl compounds, thioglycolic acid, thiosulfate, bisulfite, ammonium sulfiteOrganic AcidsBenzoic, Propionic, Sorbic Nonionic surfactants, dilution to sub-inhib

45、itory levels, pH 7 of aboveHalogensHypochlorite 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 dimethylc

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

47、m 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.Test A Test B Test C Test DNeutralizer Effectiveness Neutralizer Toxicity Test Organism Viability

48、 Test Material Control30-100 CFU/mL 30-100 CFU/mLTest Organism Test Organism30-100 CFU/mL 30-100 CFU/mLNeutralizer Neutralizer Test Organism Test OrganismProduct PBS PBS ProductPlate Count Plate Count Plate Count Plate CountHold Hold Hold HoldPlate Count Plate Count Plate Count Plate CountFIG. 1 Tes

49、ting Schema for Neutralization Assay with Recovery on Semi-solid MediumE1054083used in the antimicrobial effectiveness evaluation. For ex-ample, if the product/neutralizer/microorganism from the anti-microbial effectiveness evaluation will be plated within 30min, then the longest exposure period for the neutralizationassay is 30 min.8.3.5 After the hold-time, transfer aliquots of the product/neutralizer/microbial suspension to pour or spread plates, induplicate, using an appropriate semi-solid medium. If neutral-izers are incorporated in the plat