1、Designation: E 2315 03 (Reapproved 2008)Standard Guide forAssessment of Antimicrobial Activity Using a Time-KillProcedure1This standard is issued under the fixed designation E 2315; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, t
2、he 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 guide covers examples of a basic method tomeasure the changes of a population of aerobic microorgan-i
3、sms within a specified sampling time when tested againstantimicrobial test materials in vitro. Several options for organ-ism selection and growth, inoculum preparation, samplingtimes and temperatures are provided. When the basic techniqueis performed as a specific test method it is critical wheneval
4、uating the results to ensure that such variables have beenstandardized. Antimicrobial activity of specific materials, asmeasured by this technique, may vary significantly dependingon variables selected. It is important to understand the limita-tions of in vitro tests, especially comparisons of resul
5、ts fromtests performed under different circumstances. As an example,test results of microorganisms requiring growth supplements,or special incubation conditions, may not be directly compa-rable to more robust organisms under the conditions of a singleprocedure.1.2 Knowledge of microbiological techni
6、ques is requiredfor this test.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard may involve hazardous materials, opera-tions and equipment. This standard does not purport to addressall of the safety concerns,
7、 if any, associated with its use. It isthe responsibility of the user of this standard to establishappropriate safety and health practices and determine theapplicability of regulatory requirements prior to use.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterE 1054 Te
8、st Methods for Evaluation of Inactivators ofAntimicrobial Agents3. Terminology3.1 Definitions:3.1.1 inoculum suspension, nthe initial suspension of testorganism used to inoculate the test material This may also beknown as the organism inoculum (see 8.2).3.1.2 microbial population, nthe microbial cou
9、nt (cfu/mL) in the final volume of test material (see 9.4). This may alsobe known as the “initial population” or “numbers control.”3.1.3 neutralization, na process which results in theinactivation or quenching of the antimicrobial activity of a testmaterial. This may be achieved through dilution of
10、the testmaterial(s) or with the use of chemical agents, called neutral-izers, to reduce or quench the antimicrobial activity.3.1.4 neutralizer, na procedure or chemical agent used toinactivate, neutralize, or quench the microbiocidal properties ofan antimicrobial agent.3.1.5 total test volume, nthe
11、volume of test material plusthe volume of inoculum suspension.4. Summary of a Basic Test Method4.1 The test material or a dilution of the test material isbrought into contact with a known population of microorgan-isms for a specified period of time at a specified temperature.The activity of the test
12、 material is quenched at specifiedsampling intervals (for example, 30 s, 60 s, or any rangecovering several minutes or hours) with an appropriate neu-tralization technique. The test material is neutralized at thesampling time and the surviving microorganisms enumerated.The percent or log10reduction,
13、 or both, from either an initialmicrobial population, or test blank is calculated.5. Significance and Use5.1 This procedure may be used to assess the in vitroreduction of a microbial population of test organisms afterexposure to a test material.6. Apparatus6.1 Sterile Vials or Test Tubes, or equival
14、ent.1This guide is under the jurisdiction ofASTM Committee E35 on Pesticides andAlternative Control Agents and is the direct responsibility of Subcommittee E35.15on Antimicrobial Agents.Current edition approved April 1, 2008. Published May 2008. Originallyapproved in 2003. Last previous edition appr
15、oved in 2003 as E 2315 03.2For 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.1Copyright ASTM International, 100
16、Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6.2 Timer (Stop-clock), one that displays minutes and sec-onds.6.3 Shaking Water Bath or Controlled Temperature Cham-ber, or equivalent capable of maintaining test system at thespecified exposure temperature 6 2C.6.4 Col
17、ony Counter, any of several manual or automatedtypes may be used.6.5 Incubator, any incubator capable of maintaining aspecified temperature 62C may be used.6.6 Sterilizer, any suitable steam sterilizer capable of pro-ducing the conditions of sterilization.6.7 Vortex Mixer, Magnetic Stirrer, or equiv
18、alent.6.8 Spiral Plating System, (optional).6.9 Sterile Bacteriological Pipettes, for viscous test materi-als, positive displacement pipettes or syringes may be neces-sary.6.10 Water Dilution Bottles, any sterilizable container hav-ing appropriate capacity and tight closures may be used.7. Reagents
19、and Materials7.1 Dilution Fluid or Diluent, sterile water, 0.65 % saline,sterile Butterfields buffered phosphate diluent3or equivalent.7.2 Broth Growth Medium, soybean-casein digest broth, orequivalent and other liquid media appropriate to supportgrowth of the test organism(s), with appropriate neut
20、ralizers, ifrequired (see 3.1).7.3 Solid Growth and Plating Medium, soybean-casein di-gest agar,4or equivalent, and other solid media appropriate tosupport growth of the test organism(s), with appropriateneutralizers, if required (see 3.1.3 and 3.1.4).7.4 Sterile Deionized Water, or equivalent (Spec
21、ificationD 1193, Type III).8. Test Methods8.1 Test Organisms:8.1.1 The test organisms selected may be representative ofthe microbial flora encountered under the conditions of use, ormay represent standardized strains. The organism should becapable of providing reproducible results under specific tes
22、tconditions.8.1.2 Organism PreparationTransfer culture(s) fromstock twice (once every 18 to 24 h or as appropriate for the testorganism) into appropriate growth media. The second transfermay be made into a volume of growth medium to producesufficient microbial suspension to inoculate. Volumes usedsh
23、ould permit testing of multiple samples or time points.8.1.2.1 Alternatively, the transfers may be made onto agarplates or slants and the inoculum suspension may be preparedby washing the organism from the slant with an appropriatebroth or diluent.NOTE 1Reports in the published literature have noted
24、 differences inmicrobial kill or antimicrobial resistance as a result of cell protection inbroth or as a result of washing cells. It is recommended that tests beconducted with either all cells prepared in broth dilutions or with all cellsprepared by washing.8.2 Inoculum Suspension Preparation and De
25、termination ofthe Microbial Population or Numbers Control:58.2.1 To prepare inoculum suspension directly from broth, adilution in sterile broth (the same as that used for growthmedium) may be performed to reduce the concentration of themicroorganisms to the appropriate level.8.2.1.1 To prepare inocu
26、lum suspension in dilute broth, a1:10 dilution of the suspension into Butterfields bufferedphosphate diluent or equivalent may be performed to reducethe concentration of the growth medium.8.2.1.2 Inoculum suspensions grown from broth may bediluted to appropriate concentration or they may be centrifu
27、gedand reconstituted in Butterfields buffered phosphate diluent,broth, saline, or equivalent, to the appropriate concentration.8.2.2 To prepare the inoculum suspension from an agar plateor slant, wash microbial growth from the agar surface withButterfields buffered phosphate diluent, saline, or equi
28、valent.NOTE 2Antimicrobials sensitive to organic material (for example,alcohol and iodine) may have reduced activity by even the slightestorganic load and therefore thoroughly washed inoculum suspensions only,whether grown initially in broth or from solid media, should be used.8.2.3 The inoculum sus
29、pension should be prepared toachieve a minimum of 106cfu/mL microbial population (see9.4). Results of tests where the initial microbial populationsdiffer from the test population by greater than 2log10should beinterpreted with care because of the exponential nature of thepopulations. The final inocu
30、lum suspension should be wellmixed prior to addition to test materials (see 9.5).8.2.4 The inoculum suspension should be enumerated induplicate by standard microbiological procedures at the initia-tion and completion of testing. Appropriate dilutions areprepared and enumerated by standard microbiolo
31、gical proce-dures (Spread or pour plating, microbial filtration, or spiralplating). The initial and final count of the inoculum should bewithin 60.5 log10for a valid test.8.2.4.1 To perform the population quantitation of the controlblank, a volume of inoculum suspension equivalent to thatinoculated
32、into the test material is added to a dilution blankcontaining the same volume as used for the test material. Theinitial and final count of the population in the blank must bewithin 60.5 log10for a valid test.8.2.5 Incubate plates at the specified temperature 62C for24 to 48 h or as appropriate for t
33、he test organism(s).8.2.6 Count colonies and record raw data as cfu/plate todetermine surviving organisms. Average duplicate plates (2plates from each replicate dilution) and multiply by the dilutionfactor to calculate (cfu/mL) microbial population of both thecontrol blank and test system.3Horowitz,
34、 W., Ed., Offcial Methods of Analysis of the AOAC, 17th Ed., ch. 17,p. 4, sec. 17.2.01, A(m), Association of Official Analytical Chemists, Washington,DC, 2000; (As cited in, Butterfields Phosphate Buffer, Journal of theAssociation ofOfficial Analytical Chemists. Vol 22, No. 635, 1939.)4U.S. Pharmaco
35、poeia, 24th Revision, The United States Pharmacopoeia Con-vention, Inc. Rockville, MD, 2000.5Brown, M. R. W., Gilbert P., Microbiological Quality Assurance: A GuideTowards Relevance and Reproducibility of Inocula, CRC Press, New York, NY,1995.E 2315 03 (2008)29. Basic Procedure9.1 Select the test co
36、ncentrations of the test material. Theconcentrations selected may reflect the anticipated concentra-tion of the test material during use. Each concentration is testedat least in duplicate. Each recovery sample is plated induplicate.9.2 Prepare each test concentration at least in duplicate.Dilutions
37、should be prepared using sterile distilled water.Dilution using other materials, such as saline or a buffer maybe appropriate if test material is typically diluted that wayunder conditions of use or for informational purposes. Ensurethat the test material is completely dispersed. Some testmaterials
38、may require gentle heating before they becomecompletely dispersed. Allow the solutions to equilibrate to 256 2C.NOTE 3Additional test temperatures may be considered based on theintended use of the test material (for example, 22 6 2C room tempera-ture; 30 6 2C temperature of human skin; and 38 6 2C t
39、emperature of“warm” water). A solid test material(s) may require warming to andholding at 45 6 2C to disperse the test material and maintain uniformityduring testing. Under no circumstances should a test temperature bechosen where the temperature effects alone cause microorganism death.9.3 For selec
40、tion of contact times, a minimum time periodshould be selected based on the ability to reproducibly conductthe test sampling in this short time frame (for example, 15, 30,or 60 s). Other time points may be selected based on theintended use of the test material, or over a period of time inorder to co
41、nstruct a kinetic kill model.Additional contact timesmay be selected at the discretion of the investigator.9.4 To minimize buffer interference and reduction of anti-microbial activity, the volume of inoculum suspension shouldbe kept less than or equal to 5 % of the total volume of the testvolume. Th
42、e microbial population or numbers control shouldcontain a minimum of 106cfu/mL test material.9.5 Start mixing the test sample, add the appropriate inocu-lum suspension to the sample or control blank and maintainmixing to disperse the inoculum suspension. The inoculumsuspension should be uniformly mi
43、xed. Maintaining uniformmixing throughout the test is crucial for test repeatability.Where applicable mix carefully to minimize foam formation.The formation of foam may cause anomalous results.NOTE 4Methods of mixing include use of a vortex, stomacher,syringe, positive displacement pipette, constant
44、 velocity mix plate or othermethods that rapidly disperse inoculum into the test material, whilemaintaining uniform mixing of the test system. Viscous materials maycause difficulty in mixing sample from the sample tube.9.6 At predetermined time intervals remove an aliquot,typically 1 mL, from the sa
45、mple/inoculum container and makeappropriate dilutions in sterile Butterfields buffered phosphatediluent or equivalent containing appropriate neutralizers, ifneeded (see 3.1.3 and 3.1.4). See Fig. 1.9.7 Recover viable organisms from appropriate dilutions byculturing in duplicate (see 7.3) by use of s
46、pread- or pour-plating, microbial filtration, spiral plating, or other validmicrobial recovery methods. See Fig. 1.9.8 Incubate plates at the specified temperature 62C for 24to 48 h or as appropriate for each organism selected. Incubationtime should allow for growth of surviving organisms, withoutov
47、ergrowth of colonies, making enumeration difficult.9.9 To determine surviving organisms, count colonies andrecord raw data as cfu/plate. Average duplicate plates (2 platesfrom each replicate dilution) and multiply by the dilution factorto arrive at cfu/mL test suspension. This averaged count shouldt
48、hen be converted into log10.9.10 Formula for Calculating Microbial log10Reduction:Step 1: Transform the measured initial microbial popula-tion of the control blank (see 8.2.4.1) into the log10scale.Step 2: If more than one tube was used for determining themeasured initial microbial population, trans
49、form each mea-sured value to the log10scale first, then calculate the mean andvariance associated with the use of multiple tubes.Step 3: Transform the measured surviving microbial popu-lation (see 9.9) into the log10scale.Step 4: If more than one tube was used for determining thesurviving microbial population, transform each measured valueto the log10scale first, then calculate the mean and variance forassociated with the use of multiple tubes.Step 5: Calculate the log reduction.Log10reduction LR!5mean log10measured initial microbial population!2mean log10
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