1、Designation: E2315 03 (Reapproved 2008)E2315 16Standard Guide forAssessment of Antimicrobial Activity Using a Time-KillProcedure1This standard is issued under the fixed designation E2315; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revis
2、ion, the year 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 This guide covers examples an example of a basic method to measurethat measures the changes ofin a po
3、pulation of aerobicmicroorganisms within a specified sampling time when tested against antimicrobial test materials in vitro.are present. Severaloptions for organism selection and growth, inoculum preparation, sampling times and temperatures are provided. When the basictechnique is performed as a sp
4、ecific test method, it is critical when evaluating the results to ensure that such that the abovementioned variables have been standardized.Antimicrobial activity of specific materials, as measured by this technique, may varysignificantly depending on variables selected. It is important to understan
5、d the limitations of in vitro tests, especially comparisonsof results from tests performed underwith different circumstances.parameters. As an example, test results of microorganismsrequiring growth supplements,supplements or special incubation conditions,conditions may not be directly comparable to
6、 morerobust organisms under the conditions of a single procedure.organisms evaluated without those stated conditions.1.2 Knowledge of microbiological techniques is required for this test.procedure.1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are inc
7、luded in this standard.1.4 This standard may involve hazardous materials, operations and equipment. This standard does not purport to address allof the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriatesafety and health pr
8、actices and determine the applicability of regulatory requirements prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE1054 Test Methods for Evaluation of Inactivators of Antimicrobial AgentsE2783 Test Method for Assessment of Antimicrobial Activity for Wate
9、r Miscible Compounds Using a Time-Kill Procedure3. Terminology3.1 Definitions:3.1.1 inoculum suspension, nthe initial suspension of test organism used to inoculate the test materialmaterial. This may alsobe known as the organism inoculum (see 8.28.3).3.1.2 microbial population, nthe microbial count
10、(cfu/mL) in the final volume of test material (see 9.4). This may also beknown as the “initial population” or “numbers control.”“numbers control.” The measurement may be taken at time zero which maybe termed “Initial Population.” Alternatively, the measurement may be taken at each exposure time or t
11、he longest exposure timeused during testing to simulate the test procedure which may be termed “Final Population.”3.1.3 neutralization, na process which results in the inactivation or quenching of the antimicrobial the process for inactivatingor quenching the activity of a test material. This may be
12、 achieved through dilution of the test material(s) or with the usephysicalmeans (for example, filtration, dilution) and/or the addition of chemical agents, called neutralizers, to reduce or quench theantimicrobial activity.neutralizers.1 This guide is under the jurisdiction of ASTM Committee E35 on
13、Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2008Jan. 15, 2016. Published May 2008March 2016. Originally approved in 2003. Last previous edition approved in 20032008 asE2315
14、 03.E2315 03(2008). DOI: 10.1520/E2315-03R08.10.1520/E2315-16.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website
15、.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior ed
16、itions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.4 neutralizer, na procedure or chemical age
17、nt used to inactivate, neutralize, or quench the microbiocidalmicrobicidalproperties of an antimicrobial agent.3.1.5 total test volume, nthe volume of test material plus the volume of inoculum suspension.4. Summary of a Basic Test Method4.1 The test material or a dilution of the test material is bro
18、ught into contact with a known population of microorganisms fora specified period of time at a specified temperature. TheAn appropriate and specified neutralization technique is applied to quenchthe antimicrobial activity of the test material is quenched at specified sampling intervals (for example,
19、 30 s, 60 s, or any rangecovering several minutes or hours) with an appropriate neutralization technique. The test material is neutralized at the samplingtime hours), and the surviving microorganisms are enumerated. The percent orand/or log10 reduction, or both, from either an initialmicrobial popul
20、ation, or test blank is calculated. reduction is calculated by comparison with the microbial population.5. Significance and Use5.1 This procedure may be used to assess the in vitro reduction of a microbial population of test organisms after exposure toa test material.6. Apparatus6.1 Sterile Vials or
21、 Test Tubes, or equivalent.6.2 Timer (Stop-clock),(Stop-clock) one that displays minutes and seconds.6.3 Shaking Water Bath or Bath, Controlled Temperature Chamber, or equivalent capable of maintaining test system at thespecified exposure temperature 6 2C.62C.6.4 Colony Counter, any of several manua
22、l or automated types may be used.6.5 Incubator, any incubator capable of maintaining a specified temperature 62C may be used.6.6 Sterilizer, any suitable steam sterilizer capable of producing the conditions of sterilization.6.7 Vortex Mixer, Magnetic Stirrer, or equivalent.6.8 Spiral Plating System,
23、 (optional).6.9 Sterile Bacteriological Pipettes, for viscous test materials, positive displacement pipettes or syringes may be necessary.6.10 Water Dilution Bottles, any sterilizable container having appropriate capacity and tight closures may be used.6.11 Sterile Cotton Applicator Swabs.7. Reagent
24、s and Materials7.1 Dilution Fluid or Diluent, Diluentsterile water, 0.65 % 0.9 % (w/v) saline, sterile Butterfields buffered phosphatediluent,3 or equivalent.7.2 Broth Growth Medium, Mediumsoybean-casein digest broth,broth or equivalent and other liquid media appropriate tosupportsupporting growth o
25、f the test organism(s), with appropriate neutralizers, if required (see 3.1).7.3 Solid Growth and Plating Medium, Mediumsoybean-casein digest agar,agar4 or equivalent, and other equivalent solidmedia appropriate to support growth of the test organism(s), with appropriate neutralizers, if required (s
26、ee 3.1.3 and 3.1.4).7.4 Sterile Deionized Water, or equivalent (Specification D1193, Type III).8. Test Methods Organism Preparation8.1 The test organism selected may be representative of the microbial flora encountered under the conditions of use of a testmaterial or may be standardized strains.8.2
27、Test Organisms: Organism Preparation8.1.1 The test organisms selected may be representative of the microbial flora encountered under the conditions of use, or mayrepresent standardized strains. The organism should be capable of providing reproducible results under specific test condition-s.Transfer
28、culture(s) from stock twice (once every 18 to 24 h or as appropriate for the test organism) into appropriate growthmedium to maintain the organism in growth phase. The second transfer may be made into a volume of growth medium that willprovide a microbial suspension sufficient to conduct testing and
29、 controls. Consider that additional volume may be needed to permittesting of multiple samples or time points.3 Horowitz, W., Ed., Offcial Methods of Analysis of the AOAC, 17th Ed.18th Ed., ch. 17, p. 4, sec. 17.2.01,A(m),Association of OfficialAnalytical Chemists, Washington,DC, 2000; (As cited in,
30、Butterfields Phosphate Buffer, Journal of the Association of Official Analytical Chemists. Vol 22, No. 635, 1939.)1939.4 U.S. Pharmacopoeia, 24th Revision,Pharmacopeia, 38NF33, The United States PharmacopoeiaPharmacopeial Convention, Inc. Rockville, MD, 2000.E2315 1628.2.1 Organism PreparationTransf
31、er culture(s) from stock twice (once every 18 to 24 h or as appropriate for the testorganism) into appropriate growth media. The second transfer Alternatively, the transfers may be made into a volume of growthmedium to produce sufficient microbial suspension to inoculate. Volumes used should permit
32、testing of multiple samples or timepoints.onto agar plates or slants, and the inoculum suspension prepared by washing the organism from the slant with an appropriatebroth or diluent.8.1.2.1 Alternatively, the transfers may be made onto agar plates or slants and the inoculum suspension may be prepare
33、d bywashing the organism from the slant with an appropriate broth or diluent.NOTE 1Reports in the published literature have noted differences in microbial kill or antimicrobial resistance susceptibility as a result of cellprotection in broth or as a result of washing cells. different propagation met
34、hods. It is recommended that tests be conducted with either all cells preparedin broth dilutions or with all cells prepared by washing.using a consistent procedure for organism propagation.8.3 Inoculum Suspension Preparation and Determination of the Microbial Population or Numbers Control:Population
35、:58.3.1 To prepare inoculum suspension directly from broth growth medium, a dilution in sterile broth (diluent is same as that usedfor broth growth medium) may be performed to achieve the desired concentration.8.3.2 To prepare inoculum suspension directly from broth, a dilution in sterile broth (the
36、 same as that used for growth medium)in dilute broth, an up to 1:10 dilution of the suspension into Butterfields buffered phosphate diluent or equivalent may beperformed to reduce the concentration of the microorganisms to the appropriate level.growth medium.8.2.1.1 To prepare inoculum suspension in
37、 dilute broth, a 1:10 dilution of the suspension into Butterfields buffered phosphatediluent or equivalent may be performed to reduce the concentration of the growth medium.8.2.1.2 Inoculum suspensions grown from broth may be diluted to appropriate concentration or they may be centrifuged andreconst
38、ituted in Butterfields buffered phosphate diluent, broth, saline, or equivalent, to the appropriate concentration.8.3.3 Inoculum suspensions in broth may be diluted to achieve the desired concentration or they may be centrifuged andreconstituted in Butterfields buffered phosphate diluent, broth, sal
39、ine, or equivalent, to achieve the desired concentration.8.3.4 To prepare the inoculum suspension from an agar plate or slant, wash microbial growth or transfer the growth asepticallyusing a sterile swab from the agar surface with Butterfields buffered phosphate diluent, saline, or equivalent.NOTE 2
40、Because certain antimicrobials (for example, alcohol and iodine) are sensitive to organic material and may have activity reduced by even theslightest organic load, washed inoculum suspensions, whether established initially in broth or from solid media, may be used.NOTE 2Antimicrobials sensitive to o
41、rganic material (for example, alcohol and iodine) may have reduced activity by even the slightest organic loadand therefore thoroughly washed inoculum suspensions only, whether grown initially in broth or from solid media, should be used.8.3.5 The inoculum suspension should be prepared to achieve a
42、minimum population concentration of 106 cfu/mL microbialpopulation (see 9.4). Results of tests where the initial microbial populations differ from the test population by greater than 2log10should be interpreted with care because of the exponential nature of the populations. The final inoculum suspen
43、sion should be wellmixed well-mixed prior to additiontransfer to test materialsmaterial (see 9.5).8.3.6 The inoculum suspension should be enumeratedplated in duplicate by standard microbiological procedures at the initiationand completion of testing. Appropriate dilutions are should be prepared and
44、enumerated by standard microbiological procedures(Spread(spread- or pour plating, pour-plating, microbial filtration, or spiral plating). spiral-plating). The initial and final counttiterof the inoculum should be within 60.5 log10 for a valid test. This step may be omitted where Microbial Population
45、 enumerationis conducted.8.2.4.1 To perform the population quantitation of the control blank, a volume of inoculum suspension equivalent to thatinoculated into the test material is added to a dilution blank containing the same volume as used for the test material. The initialand final count of the p
46、opulation in the blank must be within 60.5 log10 for a valid test.8.2.5 Incubate plates at the specified temperature 62C for 24 to 48 h or as appropriate for the test organism(s).8.2.6 Count colonies and record raw data as cfu/plate to determine surviving organisms.Average duplicate plates (2 plates
47、 fromeach replicate dilution) and multiply by the dilution factor to calculate (cfu/mL) microbial population of both the control blank andtest system.8.4 To perform the Microbial Population (3.1.2) quantitation, a volume of inoculum suspension equivalent to that inoculatedinto the test material is a
48、dded to a dilution blank containing the same volume as used for the test material. The Initial Populationand Final Population counts must be within 60.5 log10 for a valid test.8.4.1 Incubate plates at the specified temperature 62C for 24 to 48 h or as appropriate for a test organism.8.4.2 Count colo
49、nies and record raw data as cfu/plate to determine the number of surviving organisms. Average duplicate platecounts (2 plates from each dilution) and multiply by the dilution factor to calculate cfu/mL of inoculum.9. Basic Procedure9.1 Select the test concentrations of the test material. The concentrations selected may reflect the anticipated concentration ofthe test material during use. material to be tested. Each concentration is tested at least in duplicate. Each recovery sample is platedin duplicate. See Fig. 1.5 Brown, M. R. W., Gilbert P., Micro
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