ASTM E1054-2008(2013) 4375 Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents《评定抗菌剂灭活剂的标准试验方法》.pdf

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1、Designation: E1054 08 (Reapproved 2013)Standard Test Methods forEvaluation of Inactivators of Antimicrobial Agents1This standard is issued under the fixed designation E1054; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year

2、of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () 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

3、,quenching) the 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 thes

4、e procedures. These methods are not suitable when testingthe virucidal activity of microbicides (see Test Method E1482).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 the

5、safety 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 the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E645 Test Method for Efficac

6、y of Microbicides Used inCooling Water SystemsE1115 Test Method for Evaluation of Surgical Hand ScrubFormulationsE1482 Practice for Use of Gel Filtration Columns for Cyto-toxicity Reduction and Neutralization3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 antimicrobial, adjdes

7、cribes an agent that 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 E645 and E1115.3.1.3 CFU/mL (abbrev.)colony-formi

8、ng units of a micro-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 micro

9、bicidalproperties 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 ofmicroor

10、ganisms.3.1.8 test 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 understo

11、od to be synonymous 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 antimicrob

12、ial effectivenesstests 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 neutralizatio

13、n of an antimicrobialformulation sampled from the skin of a human volunteer.4.2 Neutralization Assay with Recovery in Liquid MediumNeutralization assay for antimicrobial effectiveness tests thatrecover surviving microbial populations in liquid media for agrowth/no growth determination. This method i

14、s appropriatefor antimicrobial agents that are chemically inactivated ordiluted to sub-inhibitory levels.1These test methods are under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and are the directresponsibility of Subcommittee E35.15 on Antimi

15、crobial Agents.Current edition approved April 1, 2013. Published April 2013. Originallyapproved in 1985. Last previous edition approved in 2008 as E1054 08. DOI:10.1520/E1054-08R13.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.or

16、g. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States14.3 Neutralization Assay with Recovery by MembraneFiltrationNeutra

17、lization assay for antimicrobial effectivenesstests that recover and quantify microbial populations by usingmembrane filtration. This method is appropriate for antimicro-bial agents that cannot be chemically inactivated or diluted tosub-inhibitory levels, as well as for those that can be.5. Signific

18、ance and Use5.1 The effectiveness of antimicrobial agents 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 inact

19、ivation (neutraliza-tion) of the antimicrobial agent. Inefficient 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

20、 in antimi-crobial effectiveness evaluations are chemical 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 procedu

21、re to beused.5.3 The neutralization evaluation must include 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

22、 results, the variabilityencountered in the data, and the 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 toneutral

23、ization 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. Thismethod is not intended to assess injured organism recovery.NOT

24、E 3Ideally, 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 appropri-ate by the investigator. The investigator is cautioned that failure to identifyneutralizer

25、 efficacy and toxicity for all microorganisms could result 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) tha

26、t are exposed to the formulations, a singleprocedure and/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 neutralizat

27、ion assay.6.2 Colony CounterAny of several types may be 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 Steri

28、lizerAny steam sterilizer capable of producingthe conditions 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 filt

29、er unit must be chemically compatible with theantimicrobial agent and appropriate to efficient recovery of thetest microorganisms.7. Reagents and Materials7.1 Phosphate Buffered Saline Dilution WaterPBS (seeTest Method E645).7.1.1 Phosphate Buffer Solution, StockDissolve 34.0 g ofpotassium dihydroge

30、n 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 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 100

31、0 mLmark, and mix. Final pH should be adjusted to 7.2 6 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 specificmateria

32、ls and reagents to be used in the antimicrobial effec-tiveness 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 m

33、icrobicidal properties of variousantimicrobial agents. 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

34、 number of replicates necessary in the evaluationdepends 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

35、must be performed in a timely manner so thatsignificant 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 microor

36、ganism.NOTE 4The challenge inoculum should be prepared in the samemanner to be used in the antimicrobial effectiveness evaluation. Thevolume of the challenge inoculum should be kept to a minimum so that itE1054 08 (2013)2does not cause significant dilution.8.3.2 Add a volume of product, or solution

37、containingproduct, to the neutralizer/microbial suspension that will resultin 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 effec

38、tiveness evaluation.NOTE 5The dilution ratio of product to neutralizer can be manipulatedto determine the dilution at which adequate neutralization of the productwill occur, particularly when testing products not readily neutralized bychemical means.8.3.3 Within 1 min of execution of 8.3.2, transfer

39、 aliquots ofthe product/neutralizer/microbial suspension to 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

40、product/neutralizer/microbial suspension tostand for the longest exposure period representative of thatused in the antimicrobial effectiveness evaluation. Forexample, if the product/neutralizer/microorganism from theTABLE 1 Processes Applied for Neutralization of Certain Antimicrobial AgentAAntimicr

41、obial Agent Neutralizers/InactivatorsAlcoholsIsopropanol, Phenoxyethanol Polysorbate 80, dilution to sub-inhibitory levelsAldehydes2-Bromo-2-nitropropane-1, 3-diol (Bronopol) Serum, cysteine, thiosulfate, thioglycolate, metabisulfiteFormaldehyde Sodium sulfite, ammonia, histamineGlutaraldehyde Dilut

42、ion to sub-inhibitory levels, sodium bisulfite, sodium sulfite, glycine, cystine, cysteineN-(3-Chloroallyl)hexaminium Chloride (Dowicide Q) Dilution to sub-inhibitory levelsDimethylol-5, 5-dimethylhydantoin (Glydant) Dilution to sub-inhibitory levelsBiguanides and Bis-biquanidesChlorhexidine Lecithi

43、n/polysorbate 80, sodium oleatePolyhexamethylene biguanide HCL (Cosmocil CQ) Polysorbate 80/lecithinPhenolicsPhenylphenol, Chloroxylenol, Cresols, Chlorocresols,PhenolNonionic surfactants, polysorbate 80, and/or dilution to sub-inhibitory levelsBis-PhenolsTriclosan 10 % polysorbate 80/lecithin, and

44、dilution to sub-inhibitory levelsHexachlorophene 10 % polysorbate 80/lecithin, and dilution to sub-inhibitory levelsQuaternary Ammonium CompoundsCetrimide, Benzalkonium and Benzethonium Chloride Lecithin/polysorbate, suramin sodium, organic material, 0.5 % polysorbate 80, cyclodextrinsMercurials and

45、 Silver Sulfhydryl compounds, thioglycolic acid, thiosulfate, bisulfite, ammonium sulfiteOrganic AcidsBenzoic, Propionic, Sorbic Nonionic surfactants, dilution to sub-inhibitory levels, pH 7 of aboveHalogensHypochlorite Thiosulfate and/or dilution to sub-inhibitory levelsIodine Thiosulfate, polysorb

46、ate 80, skim milkBromine Thiosulfate and/or dilution to sub-inhibitory levelsEDTA Mg+2or Ca+2ionsImidazolidinyl urea Dilution to sub-inhibitory levelsMethyl-, and dimethylchloroisothiazolinone (Kathon) Amines, sulfites, mercaptans, sodium bisulfite, heparinParabensMethyl-, ethyl-, propyl-, butyl-par

47、ahydroxybenzoate Lecithin, filtration, dilution to sub-inhibitory levels, polysorbate surfactants, 1 % polysorbate 80 or 20Hydrogen Peroxide CatalasePeroxyacetic Acid Sodium thiosulfateASutton, S. V. W., “Neutralizer Evaluations as Control Experiments for Antimicrobial Effectiveness Tests,” Ch. 3 in

48、 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 Test Material Control30-100 CFU/mL 30-100 CFU/mLTest Organism Test Organism30-100 CFU/mL 30-100 CFU/mLNeutralizer Neutralizer T

49、est Organism Test Organism Product PBS PBS ProductPlate Count Plate Count Plate Count Plate CountHold Hold Hold Hold Plate Count Plate Count Plate Count Plate CountFIG. 1 Testing Schema for Neutralization Assay with Recovery on Semi-solid MediumE1054 08 (2013)3antimicrobial 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 approp