1、Designation: E 2315 03Standard 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, the year of last re
2、vision. 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-isms within a speci
3、fied 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 whenevaluating the results
4、 to ensure that such variables have beenstandardized. Antimicrobial activity of specific materials, asmeasured by this technique, may vary significantly on vari-ables selected. It is important to understand the limitations of invitro tests, especially comparisons of results from tests per-formed und
5、er different circumstances. As an example, testresults of microorganisms requiring growth supplements, orspecial incubation conditions, may not be directly comparableto more robust organisms under the conditions of a singleprocedure.1.2 Knowledge of microbiological techniques is requiredfor this tes
6、t.1.3 The values stated in SI units are to be regarded asstandard.1.4 This standard may involve hazardous materials, opera-tions and equipment. This standard does not purport to addressall of the safety concerns, if any, associated with its use. It isthe responsibility of the user of this standard t
7、o establishappropriate safety and health practices and determine theapplicability of regulatory requirements prior to use.2. Referenced Documents2.1 ASTM Standards:D 1193 Specification for Reagent WaterE 1054 Practices for Evaluating Inactivators of Antimicro-bial Agents Used in Disinfectant, Saniti
8、zer, Antiseptic orPreserved Products3. 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 count (cfu/mL) in the final vol
9、ume 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 the testmaterial(s) or with
10、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 volume of test material plus
11、the 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 material is quenched at spe
12、cifiedsampling 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, or both, from either an ini
13、tialmicrobial 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 equivalent.6.2 Timer (Stop-clock),
14、one that displays minutes and sec-onds.1This guide is under the jurisdiction of ASTM Committee E35 on Pesticides andAlternative Control Agents and is the direct responsibility of Subcommittee E35.15on Antimicrobial Agents.Current edition approved Oct. 1, 2003. Published November 2003.1Copyright ASTM
15、 International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.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 Colony Counter, any of several manual or autom
16、atedtypes may be used.6.5 Incubator, any incubator capable of maintaining aspecified temperature 6 2C 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 equivalent.6.8 Spiral Plating System, (optional
17、).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 and Materials7.1 Dilution Fluid or Diluent
18、, sterile water, 0.65 % saline,sterile Butterfields buffered phosphate diluent2or 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 neutralizers, ifrequired (see 3.1).7.3 Solid G
19、rowth and Plating Medium, soybean-casein di-gest agar,3or 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 (SpecificationD 1193, Type III).8. Test Methods
20、8.1 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 testconditions.8.1.2 Organism PreparationTran
21、sfer 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 usedshould permit testing of multiple samples or
22、 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 differences inmicrobial kill or antimicro
23、bial 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 Determination ofthe Microbial Population or
24、Numbers Control:48.2.1 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 inoculum suspension in dilute broth, a1:10 dilu
25、tion 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 centrifugedand reconstituted in Butterfields buffe
26、red 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 equivalent.NOTE 2Antimicrobials sensitive to o
27、rganic 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 suspension should be prepared toachieve a min
28、imum 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 inoculum suspension should be wellmixed prior t
29、o 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 microbiological proce-dures (Spread or pour plating,
30、 microbial filtration, or spiralplating). The initial and final count of the inoculum should bewithin 6 0.5 log10for a valid test.8.2.4.1 To perform the population quantitation of the controlblank, a volume of inoculum suspension equivalent to thatinoculated into the test material is added to a dilu
31、tion blankcontaining the same volume as used for the test material. Theinitial and final count of the population in the blank must bewithin 6 0.5 log10for a valid test.8.2.5 Incubate plates at the specified temperature 6 2C for24 to 48 h or as appropriate for the test organism(s).8.2.6 Count colonie
32、s 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.2Horowitz, W., Ed., Offcial Methods of Analysis o
33、f 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 the Association ofOfficial Analytical Chemists. Vol 22, No. 635, 1939.)3U.S. Pharmacopoeia, 24th Revision, The United State
34、s Pharmacopoeia Con-vention, Inc. Rockville, MD, 2000.4Brown, M. R. W., Gilbert P., Microbiological Quality Assurance: A GuideTowards Relevance and Reproducibility of Inocula, CRC Press, New York, NY,1995.E23150329. Basic Procedure9.1 Select the test concentrations of the test material. Theconcentra
35、tions 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 should be prepared using sterile distilled wate
36、r.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 may require gentle heating before they becomeco
37、mpletely 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 temperature of“warm” water). A solid test materi
38、al(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 selection of contact times, a minimum time periodsho
39、uld 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 construct a kinetic kill model. Additional contac
40、t 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. The microbial population or numbers control shou
41、ldcontain 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 mixed. Maintaining uniformmixing throughout the
42、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 velocity mix plate or othermethods that rapid
43、ly 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 sample/inoculum container and makeappropriate di
44、lutions 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 spread- or pour-plating, microbial filtration,
45、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, withoutovergrowth of colonies, making enumeration diffi
46、cult.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 shouldthen be converted into log10.9.10 Formula for C
47、alculating 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, transform each mea-sured value to the log10scale fi
48、rst, 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 th
49、e 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 log10surviving microbial population!Step 6: Calculate the standard error of the meanStandard error 5square root variance of the log10measured initial microbial population/number of replicates!1variance of the log10surviving microbial population/number of replicates!#NOTE 5The above calculations for standard error assess only thewith-in experiment va
