1、Designation: E2783 10Standard Test Method forAssessment of Antimicrobial Activity for Water MiscibleCompounds Using a Time-Kill Procedure1This standard is issued under the fixed designation E2783; the number immediately following the designation indicates the year oforiginal adoption or, in the case
2、 of revision, 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 test method measures the changes of a populationof aerobic and anaerobic microorganisms
3、 within a specificsampling time when tested against antimicrobial test materialsin vitro. The organisms used are standardized as to growthrequirements and inoculum preparation and must grow underthe conditions of the test. The primary purpose of this testmethod is to provide a set of standardized co
4、nditions and testorganisms to facilitate comparative assessments of antimicro-bial materials miscible in aqueous systems.1.2 This test method allows the option of using a test samplesize of 10 mL or 100 mL.1.3 Knowledge of microbiological techniques is requiredfor this procedure.1.4 Aseptic techniqu
5、e should be practiced at all times.1.5 In this test method, SI units are used for all applications,except for distance in which case inches are used and SI unitsfollow in parentheses.1.6 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is there
6、sponsibility 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:2E691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test
7、 MethodE1054 Test Methods for Evaluation of Inactivators of An-timicrobial AgentsE2315 Guide for Assessment of Antimicrobial ActivityUsing a Time-Kill Procedure3. Terminology3.1 Definitions:3.1.1 antimicrobial, ndescribes an agent that kills orinactivates microorganisms or suppresses their growth or
8、reproduction.3.1.2 drug, narticles intended for use in the diagnosis,cure, mitigation, treatment, or prevention of disease in man orother animals. Drugs are intended to affect the structure or anyfunction of the body of man or other animals.3.1.3 initial microbial population, nbacterial count (CFU/m
9、L) in the final volume of test material. Also known as initialbacterial population, numbers control or control.3.1.4 inoculum, nthe viable microorganisms used to con-taminate a sample, device or surface, often expressed as tonumber and type.3.1.5 microbiocide, na physical or chemical agent thatkills
10、 microorganisms.3.1.6 neutralization, nthe process for inactivating orquenching the activity of a microbiocide. Often achievedthrough chemical or physical means (for example, filtration ordilution).3.1.7 room temperature, ntemperature in the range of 20to 30C (68 to 85F).3.1.8 test material, na form
11、ulation which incorporatesantimicrobial ingredient(s). Also known as test formulation.3.1.9 total test volume, nthe volume of test material plusthe volume of inoculum suspension.4. Summary of Test Method4.1 A dilution/aliquot of the test material is brought intocontact with a known population of tes
12、t organisms for specifiedperiods of time, at a specified temperature. The activity of thetest material is quenched at specified sampling intervals1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommit
13、tee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2010. Published November 2010. DOI:10.1520/E278310.2For 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
14、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.(example 15, 30, and 60 s, or any range covering severalminutes or hours) with an appropriate neutralizing technique.The test m
15、aterial is neutralized at the sampling time and thesurviving microorganisms enumerated. The percent and log10reduction, from an initial microbial population is calculated.5. Significance and Use5.1 This procedure may be used to assess the in vitroreduction of a microbial population of test organisms
16、 afterexposure to a test material.6. Apparatus6.1 Adjustable or Fixed Volume PipetCapable of dispens-ing 0.1 mL and 1.0 mL.6.2 Anaerobic Jar or IncubatorAny incubator or appara-tus in an incubator that creates an environment having a levelof oxygen that does not support the growth of oxygen-requirin
17、gmicroorganisms. Required only for organisms that need ananaerobic environment to grow.6.3 BalanceAny suitable laboratory balance with a mini-mum readability of 0.01 g.6.4 Beakers and Magnetic Stir BarsFor 100 mL samplesize. A 250 mL beaker containing a 51 by 8 6 2 mm magneticstir bar. The beaker an
18、d stir bar should be sterile.6.5 Colony CounterAny of several types may be used.6.6 IncubatorAny incubator capable of maintaining asuitable temperature 62C may be used.6.7 Laboratory CentrifugeAny centrifuge capable of pro-ducing 3200 r/min (1520 RCF).6.8 Magnetic Stirring PlateAny rotor powered mag
19、neticstirrer.6.9 Positive Displacement Pipet1.0 mL capacity. Re-quired for viscous test materials.6.10 SterilizerAny suitable steam sterilizer capable ofproducing the conditions of sterility.6.11 Sterile ContainerAny container of sufficient size todilute test material into.6.12 Test Tubes with CapSt
20、erile. Alternate sample con-tainer to (7.5) for 10 mL sample size. Size of test tube shouldallow the thorough mixing of samples.6.13 Timer (Stop Clock)One that displays minutes andseconds.6.14 Vortex MixerAny suitable vortex mixer capable ofmixing test material and diluents.6.15 WaterbathAny waterba
21、th capable of maintainingsuitable temperature.7. Reagents and Materials7.1 Bacteriological LoopsAny type of disposable sterileor sterilizable bacteriological loop is suitable.7.2 Bacteriological PipetsSterile. 1.1, 2.0, 5.0, 10.0 mLcapacity.NOTE 1Pre-sterilized/disposable bacteriological pipets are
22、availablefrom most local laboratory supply houses.7.3 Broth Growth MediumSterile soybean-casein digestbroth (tryptic soy broth) or other broth media appropriate tosupport growth of the test organisms.7.4 Dilution Fluid or DiluentSterile Butterfields bufferedphosphate diluent3or other suitable diluen
23、t with appropriatelyvalidated neutralizers. Perform Test Method E1054 to deter-mine what diluent or neutralizers are required. Volume is 9.0mL after sterilization.7.5 Flip Top Centrifuge TubesSterile. For 10 mL samplesize. Minimum of 50 mL capacity.NOTE 2Pre-sterilized/ disposable flip-top centrifug
24、e tubes are avail-able from most laboratory supply houses.7.6 Petri Dishes100 by 15 mm. Required to plate samplesand control.NOTE 3Pre-sterilized/disposable plastic petri dishes are availablefrom most local laboratory supply houses.7.7 Physiological SalineSterile. Used to prepare inocu-lum.7.8 Pipet
25、 TipsSterile. 0.1 and 1.0 mL capacity.7.9 Positive Displacement Pipet TipsSterile. 1.0 mL ca-pacity.7.10 Solid Growth and Plating MediumSterile soybean-casein digest agar (tryptic soy agar) or other solid mediaappropriate to support growth of the test organism(s) withappropriately validated neutrali
26、zers. Perform Test MethodE1054 to determine what neutralizers are required. Should betempered to between 40 to 50C if using a pour plate method.7.11 WaterSterile deionized or distilled water.8. Hazards8.1 All test organisms listed for use in this method fall underthe Biosafety level 1 or 2 categorie
27、s and should be handled inaccordance with CDC and NIH guidelines.49. Calibration and Standardization9.1 Ensure that all equipment used in this test method hasbeen calibrated and standardized as required for that piece ofequipment.10. Test Organisms10.1 The following list of organisms may be used in
28、thisprocedure. This list is not all inclusive and other organismsmay be used. Organisms selected may be representative of themicrobial flora encountered under the conditions of use, or mayrepresent standardized strains. The organism should be capableof providing reproducible results under the test c
29、onditions.10.2 The organisms listed shall be maintained as specifiedby ATCC or other validated methods.10.2.1 Acinetobacter species.10.2.2 Candida albicans ATCC 10231.10.2.3 Enterobacter species.10.2.4 Enterococcus faecalis ATCC 29212.10.2.5 Enterococcus faecium.10.2.6 Escherichia coli ATCC 8739, 11
30、229, or 25922.3Horowitz, W., (Ed.), Offcial Methods of Analysis of the AOAC, 17th Ed., Sec.6.3.03 A.(f), Chapter 6, 2000, p.10. Official Methods of Analysis of AOACInternational, Gaithersburg, MD.4Richmond, J. Y. and McKinney, R. W. (eds.), 1999, Biosafety in Microbio-logical and Biomedical Laborato
31、ries, 4th ed., Washington DC, U.S. GovernmentPrinting Office.E2783 10210.2.7 Klebsiella pneumoniae ATCC 10031 or 51504.10.2.8 Micrococcus luteus ATCC 7468.10.2.9 Pseudomonas aeruginosa ATCC 9027, 15442, or27853.10.2.10 Proteus mirabilis ATCC 4675 or 7002.10.2.11 Salmonella enterica ATCC 10708.10.2.1
32、2 Serratia marcescens ATCC 14756.10.2.13 Shigella species.10.2.14 Staphylococcus aureus ATCC 6538, 29213, 33591,or 33592.10.2.15 Staphylococcus epidermidis ATCC 12228.10.2.16 Staphylococcus haemolyticus.10.2.17 Staphylococcus hominis.10.2.18 Staphylococcus saprophyticus.10.2.19 Streptococcus pyogene
33、s.10.2.20 Streptococcus pneumoniae.11. Preparation of Organism11.1 All organisms used in the test method shall be preparedusing a validated method. The same method shall be used toprepare all organisms for the test.11.2 A minimum starting inoculum level of 1.0 3 108CFU/mL should be used for the test
34、.11.3 Other starting inoculum levels may be used dependingon the organisms growth potential.11.4 All organisms shall be no more than five passagesremoved from the original source.11.5 The inoculum shall be used within4hofpreparation.11.6 If a validated method is not available, the followinginstructi
35、ons shall be used to prepare the inoculum.11.7 Inoculum Preparation Directly from Agar:11.7.1 The stock culture, frozen or lypholized should be atleast one 24 h broth growth media (7.3) transfer from theoriginal source.11.7.2 Inoculate a sufficient number of solid growth mediaslants or plates (7.10)
36、.11.7.3 Incubate at appropriate temperature and environmentfor the organism.11.7.4 Wash each slant by adding 5 to 10 mL of physiologi-cal saline (7.7) to each slant.11.7.5 Using bacteriological loop (7.1), gently scrape or rubsurface of agar to remove growth.11.7.6 Using a sterile pipet, collect the
37、 washings in a 50 mLcentrifuge tube (7.5). Repeat for all slants, adding washing tothe centrifuge tube.11.7.7 Centrifuge at conditions appropriate to sediment theculture completely.11.7.8 Decant or pipet supernatant and re-suspend organismpellet in 20 mL physiological saline (7.7).11.7.9 Centrifuge
38、at conditions appropriate to sediment theculture completely.11.7.10 Decant or pipet supernatant and re-suspend organ-ism in an amount of physiological saline (7.7) sufficient toachieve a minimum final suspension of 1.0 3 108CFU/mL.Use McFarland Barium Sulfate Standard #2, turbidimetry,optical densit
39、y, or other technique that correlates to an aerobicplate count.11.8 Inoculum Prepared Directly from Broth:11.8.1 The stock culture, frozen, or lypholized should be atleast one 24 h liquid growth media (7.3) transfer from theoriginal source.11.8.2 Incubate at appropriate temperature and environmentfo
40、r the organism.11.8.3 Centrifuge at conditions appropriate to sediment theculture completely.11.8.4 Decant or pipet supernatant and re-suspend organismpellet in 20 mL physiological saline (7.7).11.8.5 Centrifuge at conditions appropriate to sediment theculture completely.11.8.6 Decant or pipet super
41、natant and re-suspend organismin an amount of physiological saline (7.7) sufficient to achievea minimum final suspension of 1.0 3 108CFU/mL. UseMcFarland Barium Sulfate Standard #2, turbidimetry, opticaldensity, or other technique that correlates to an aerobic platecount.NOTE 4Antimicrobials sensiti
42、ve to organic material (for examplealcohol and iodine) may have reduced activity by even the slightestorganic load and therefore only use thoroughly washed inoculum suspen-sions, whether initially grown in broth or from solid media.12. Test Conditions12.1 Test should be performed at room temperature
43、.12.2 Test materials and diluents should be at room tempera-ture.12.3 Test materials may require a lower or higher tempera-ture than room temperature (for example, solids that requirewarming to and be held at 45C, or test material may only bestable at a certain temperature). If this is a requirement
44、, allsteps of the test should be run at that temperature.12.4 This test method allows the use of either a 10 or 100mL sample size. The test shall be run using the same samplesize for all test materials.13. Test (Contact) Times13.1 For selection of contact times, a minimum time periodshould be select
45、ed based on the ability to reproduce the testsampling in this short time frame (for example 15, 30 and 60s).13.2 Time points should reflect the intended use of the testmaterial.13.3 Times may be chosen to construct a kinetic kill model.14. Test Material Concentration14.1 Select the test concentratio
46、ns of the test material. Theconcentrations selected may reflect the anticipated concentra-tion of the test material during use. Each concentration is testedin triplicate.14.2 If test material is to be diluted, dilute using sterilewater (7.11). Dilution using other materials, such as sterilesaline or
47、 sterile buffer may be appropriate if test material istypically diluted that way under conditions of use. Dilute alltest material needed at once. Use sterile container (6.11). Mixthoroughly. Test material can be mixed using a sterile magneticstir bar and a magnetic stir plate.E2783 10315. Inoculum (
48、Start) Count15.1 To be done at the start and end of the test phase. Thestart and end counts must be within 60.5 log10for test to bevalid.15.2 Prepare serial ten-fold dilutions of the inoculum pre-pared in Step 11 using 9.0 mL dilution blanks (7.4).15.3 Enumerate, in duplicate, using standard plating
49、 tech-niques. Ensure that the proper dilutions are plated in order toobtain plate(s) that are within a countable range.15.4 Incubate at the appropriate temperature, time, andenvironment for the test organism.15.5 Count colonies using standard plate count rules andrecord raw data as CFU/plate. Average duplicate plates andmultiply by the dilution factor to calculate CFU/mL.NOTE 5Inoculum suspension should be thoroughly mixed prior toeach instance of use.16. 100 mL Sample Size Test Procedure16.1 The control and all replicates of the test
