1、Designation: E2783 11 (Reapproved 2016)Standard 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 adopti
2、on or, in the case 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 anaero
3、bic microorganisms 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
4、of standardized conditions 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.
5、4 Aseptic technique 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 it
6、s 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:2E691 Practice for Conducting an Interlaboratory Study toDetermine the P
7、recision of a Test MethodE1054 Test Methods for Evaluation of Inactivators of Anti-microbial Agents3. Terminology3.1 Definitions:3.1.1 antimicrobial, ndescribes an agent that kills orinactivates microorganisms or suppresses their growth orreproduction.3.1.2 drug, narticles intended for use in the di
8、agnosis,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/mL) in the final volume of test material. Also known as initia
9、lbacterial 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 microorganisms.3.1.6 neutralization, nthe process for inacti
10、vating 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 formulation which incorporatesantimicrobial ingredient(s). Also k
11、nown 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 test organisms for specifiedperiods of time, at a specified temp
12、erature. The activity of thetest material is quenched at specified sampling intervals(example 15, 30, and 60 s, or any range covering severalminutes or hours) with an appropriate neutralizing technique.The test material is neutralized at the sampling time and thesurviving microorganisms enumerated.
13、The percent and log10reduction, from an initial microbial population is calculated.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edi
14、tion approved April 1, 2016. Published May 2016. Originallyapproved in 2010. Last previous edition approved in 2011 as E2783 11. DOI:10.1520/E278311R16.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMSta
15、ndards 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 States15. Significance and Use5.1 This procedure may be used to assess the in vitroreduction of a micr
16、obial population of test organisms 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 sup
17、port the growth of oxygen-requiringmicroorganisms. 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
18、mm magneticstir bar. The beaker and 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 S
19、tirring PlateAny rotor powered magneticstirrer.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 materi
20、al into.6.12 Test Tubes with CapSterile. 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
21、diluents.6.15 WaterbathAny waterbath 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/disp
22、osable bacteriological pipets are 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 bufferedphosphat
23、e diluent3or other suitable diluent 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-sterili
24、zed/ disposable flip-top centrifuge 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. U
25、sed to prepare inocu-lum.7.8 Pipet 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) wi
26、thappropriately validated neutralizers. 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 underth
27、e Biosafety level 1 or 2 categories 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
28、 list of organisms may be used in 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 rep
29、roducible results under the test conditions.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.3Ho
30、rowitz, 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 Microbiologi-cal and Biomedical Laboratories, 4th ed., Wa
31、shington DC, U.S. GovernmentPrinting Office.E2783 11 (2016)210.2.6 Escherichia coli ATCC 8739, 11229, or 25922.10.2.7 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
32、.11 Salmonella enterica ATCC 10708.10.2.12 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 sa
33、prophyticus.10.2.19 Streptococcus pyogenes.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.
34、0 108CFU/mL should be used for the test.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 i
35、s not available, the followinginstructions 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 so
36、lid growth mediaslants or plates (7.10).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
37、.7.6 Using a sterile pipet, collect the 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 physio
38、logical saline (7.7).11.7.9 Centrifuge 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 108CFU/mL. UseMcFarland Barium Sulfate Sta
39、ndard #2, turbidimetry, opticaldensity, or other technique that correlates to an aerobic platecount.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 appr
40、opriate temperature and environmentfor 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 co
41、mpletely.11.8.6 Decant or pipet supernatant and re-suspend organismin an amount of physiological saline (7.7) sufficient to achievea minimum final suspension of 1.0 108CFU/mL. UseMcFarland Barium Sulfate Standard #2, turbidimetry, opticaldensity, or other technique that correlates to an aerobic plat
42、ecount.NOTE 4Antimicrobials sensitive to organic material (for examplealcohol and iodine) may have reduced activity by even the slightestorganic load and therefore only use thoroughly washed inoculumsuspensions, whether initially grown in broth or from solid media.12. Test Conditions12.1 Test should
43、 be performed at room temperature.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 tempe
44、rature). If this is a requirement, 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 mi
45、nimum time periodshould be selected 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 Concentration
46、14.1 Select the test concentrations 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 ma
47、terials, such as sterilesaline or sterile buffer may be appropriate if test material isE2783 11 (2016)3typically 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
48、and a magnetic stir plate.15. Inoculum (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 Enumera
49、te, in duplicate, using standard plating 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 Procedure
