ASTM E2111-2005 Standard Quantitative Carrier Test Method to Evaluate the Bactericidal Fungicidal Mycobactericidal and Sporicidal Potencies of Liquid Chemical Microbicides《评估液体化学杀虫.pdf

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1、Designation: E 2111 05Standard Quantitative Carrier Test Method toEvaluate the Bactericidal, Fungicidal, Mycobactericidal, andSporicidal Potencies of Liquid Chemical Microbicides1This standard is issued under the fixed designation E 2111; the number immediately following the designation indicates th

2、e year oforiginal adoption or, in the case of revision, the 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.INTRODUCTIONThe need for better tests to assess the microbici

3、dal activity of chemicals was recognized (1)2andseveral simpler and quantitative test methods have been developed for working with a wide variety ofmicroorganisms (2). The test method described here uses glass vials as carriers; the same basic set ofmaterials and procedures can be used to test the p

4、otency of liquid microbicides against vegetativebacteria, fungi, mycobacteria, and bacterial spores. However, the test method is not appropriate for usewith viruses because of the relatively high levels of eluate dilutions required and the need formembrane filtration. Further evaluation of products

5、under more stringent test conditions may benecessary for their registration. Performance standards for the categories of products to be tested andthe specific types of organism(s) to be used may also vary depending on the regulatory agency.1. Scope1.1 This test method is designed for use in product

6、devel-opment and for the generation of product potency data. Thistest method permits the loading of each carrier with a knownvolume of the test organism. The incorporation of controls canalso determine the initial load of colony forming units (CFU)of organisms on the test carriers and any loss in CF

7、U after themandatory drying of the inoculum.1.2 This test method is designed to have survivors and alsoto be used with a performance standard. The surviving micro-organisms on each test carrier are compared to the mean of noless than three control carriers to determine if the performancestandard has

8、 been met. To allow proper statistical evaluation ofresults, the size of the test inoculum should be sufficiently largeto take into account both the performance standard and theexperimental variation in the results. For example, if anarbitrary performance standard of 6-log10reduction in theviability

9、 titer of the test organism is used, and an inoculum sizeof 107CFU, then theoretically a maximum of ten survivors percarrier is permitted; however, because of experimental vari-ability, the exact target may need to be higher than 106CFU/carrier, thus fewer survivors would be permitted.1.3 This test

10、method should be performed by persons withtraining in microbiology and in facilities designed andequipped for work with infectious agents at the appropriatebiosafety level (3).1.4 In this test method, SI units are used for all applications,except for distance, in which case inches are used and SI un

11、itsfollow.1.5 It is the responsibility of the investigator to determinewhether Good Laboratory Practice Regulations (GLPs) arerequired and to follow them where appropriate (40 CFR, Part160 for EPA submissions and 21 CFR, Part 58 for FDAsubmissions).1.6 This standard does not purport to address all o

12、f thesafety concerns, if any, associated with its 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:3D 1129 Terminology Rela

13、ting to WaterD 1193 Specification for Reagent Grade WaterE 1054 Practices for Evaluating Inactivators of Antimicro-bial Agents Used in Disinfectant, Sanitizer, Antiseptic, orPreserved Products1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agent

14、s and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Nov. 1, 2005. Published December 2005. Originallyapproved in 2000. Last previous edition approved in 2000 as E 2111 00.2The boldface numbers in parentheses refer to the list of references at the

15、 end ofthis standard.3For 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 Barr

16、Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.2.2 CFR Standards:40 CFR, Part 160421 CFR, Part 5843. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 carrier, ninanimate surface or object inoculatedwith the test organism.3.1.2 eluate, neluent, which con

17、tains the recovered organ-ism(s).3.1.3 eluent, nany solution that is harmless to the testorganism(s) and that is added to a carrier to recover theorganism(s) in or on it.3.1.4 neutralization, nprocess to quench the antimicrobialactivity of a test formulation. This process may be achieved bydilution

18、of the organism/test formulation mixture and/or byadding to it one or more chemical neutralizers. (Refer toPractices E 1054 in 2.1 for further details3.1.4.1 DiscussionThis process may be achieved by dilu-tion of the organism/test formulation mixture or by adding to itone or more chemical neutralize

19、rs, or both.3.1.5 soil load, nsolution of one or more organic, orinorganic substances, or both, added to the suspension of thetest organism to simulate the presence of body secretions,excretions, or other extraneous substances.3.1.6 test formulation, nformulation that incorporates an-timicrobial ing

20、redients.3.1.7 test organism, napplied inoculum of an organismthat has characteristics that allows it to be readily identified. Italso may be referred to as a surrogate or a marker organism.4. Summary of Test Method4.1 This is a fully quantitative carrier test method suitablefor assessing the potenc

21、y of chemicals against vegetativebacteria, fungi, mycobacteria, as well as bacterial spores. It isdesigned primarily for testing formulations to be used on hardenvironmental surfaces and medical devices. This test methoduses the flat inside bottom surface of glass vials as the carrier.Each vial rece

22、ives 10 L of the test organism with or withouta soil load. The contamination of the inside surface of thecarrier with microaerosols is avoided by the use of glassinserts. The inoculum is dried and exposed to 1 mL of the testmicrobicide for the desired contact time at the recommendedtemperature; cont

23、rol carriers receive 1 mL of normal salineinstead. At the end of the contact time, 9 mL of an eluentwithout or with a neutralizer, is added to the vial to dilute/neutralize the microbicide and any inoculum adhering to thecarrier surface is recovered using a magnetic stir bar with athreaded surface.

24、The eluate is passed through a membranefilter, the carrier vial is then rinsed several times with eluent/diluent and the rinses are also passed through the same filter.The total rinse volume is no less than 100 mL. Control and testeluates requiring dilution to get countable colonies are firstsubject

25、ed to a series of tenfold dilutions and the material fromsuitable dilutions is passed separately through membranefilters. Each filter is placed on the agar surface of an appropri-ate recovery medium in a 100-mm diameter petri plate. Theplates are held for the required period at the desired incubatio

26、ntemperature, colonies counted, and log10reductions in theviability titer of the test organism calculated.NOTE 1Do not soak the magnetic stir bars in ethanol or other solventsfor decontamination as this may damage the sealant on them.5. Significance and Use5.1 This test method is fully quantitative

27、and it also avoidsany loss of viable organisms through wash off. This makes itpossible to produce statistically valid data using many fewertest and control carriers than other quantitative methods basedon most probable numbers (MPN).5.2 The design of the carriers makes it possible to place intoeach

28、a precisely measured volume of the test suspension. Theuse of the threaded stir bars allows for efficient recovery of theinoculum even after its exposure for several hours to strongfixatives such as glutaraldehyde.5.3 The membrane filtration step allows processing of theentire eluate from the test c

29、arriers and therefore the capture andsubsequent detection of even low numbers of viable organismsthat may be present.5.4 This test can be performed with or without a soil load todetermine the effect of such loading on microbicide perfor-mance. The soil load developed for this test is a mixture ofthr

30、ee types of proteins (high molecular weight proteins, lowmolecular weight peptides, and mucous material) to representthe body secretions, excretions, or other extraneous substancesthat chemical microbicides may encounter under field condi-tions. It is suitable for working with the various test organ

31、ismsincluded here. The components of the soil load are readilyavailable and subject to much less variability than animal sera.5.5 Since the quality of tap water varies considerably bothgeographically and temporally, this test method incorporatesthe use of water with a specified and documented level

32、ofhardness to prepare use-dilutions of test products. The U.S.Environmental Protection Agencys Scientific Advisory Panel(SAP) on Germicide Test Methodology has recommended theuse of water with a standard hardness of 400 ppm as CaCO3.6. General Equipment and Labware6.1 Laminar Flow CabinetA Class II

33、(Type A) biologicalsafety cabinet for this work. The procedures for the propermaintenance and use of such cabinets are given in Ref 3.6.2 IncubatorAn ordinary incubator and an anaerobicincubator. If only one ordinary incubator is available, itstemperature will require adjustment depending on the typ

34、e oforganism under test.6.3 SterilizerAny steam sterilizer suitable for processingculture media, reagents and labware is acceptable. The steamsupplied to the sterilizer must be free from additives toxic tothe test organisms.6.4 Filter Sterilization System for Media and ReagentsAmembrane or cartridge

35、 filtration system (0.22-m pore diam-eter) is required for sterilizing heat-sensitive solutions.4Available from U.S. Government Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.E21110526.5 Membrane Filtration System for Capture of the TestOrgan

36、ismsSterile 47-mm diameter membrane filters (0.22-or 0.45-m pore diameter) and glass, metal, or plastic holdersfor such filters are required.6.6 Environmental Chamber/IncubatorTo hold the carri-ers at the desired test temperature.6.7 FreezersA freezer at 20 6 2C is required for thestorage of media a

37、nd additives. A second freezer at 70C orlower is required to store the stocks of test organisms.6.8 RefrigeratorA refrigerator at 4 6 2C for storage ofmedia, plates, and reagents.6.9 TimerAny stopwatch that can be read in minutes andseconds.6.10 Hot Air OvenAn oven at 60C to dry and sterileclean gla

38、ssware.6.11 Magnetic Stir Plate and Stir BarsLarge enough for a5-L beaker or Erlenmeyer flask for preparing culture media orother solutions.6.12 Positive Displacement PipetteA pipette and pipettetips that accurately can dispense 10-L volumes for inoculationof carriers.6.13 Air Displacement PipettesE

39、ppendorf or equivalent,100 to 1000 L with disposable tips.6.14 Orbital ShakerFor shaking the broth cultures ofbacteria during their incubation.6.15 Sterile Dispenser10 mL, for dispensing diluent/eluent.6.16 GlasswareOne-liter flasks with a side-arm and ap-propriate tubing to capture the filtrates fr

40、om 47-mm diametermembrane filters; 250-mL Erlenmeyer flasks for culture media;FIG. 1 Components of a Carrier for the Quantitative Carrier TestE2111053100 mL and 5 L beakers, reusable or disposable glass pipettescapable of handling 10-, 5-, and 1-mL volumes; and 25-mL testtubes with caps.6.17 Vacuum

41、SourceA vacuum pump, access to an in-house vacuum line or a water faucet vacuum apparatusrequired to pull the samples through the membrane filters.6.18 Sterile Disposable Plastic Petri Dishes, 100 by 15 mm.6.19 Forceps, straight or curved, with smooth tips to handlemembrane filters.6.20 Flat-Bottome

42、d Glass Vials, 20 mL, with regular andseptate caps (Fig. 1A). Flat-bottomed glass vials may bemanufactured such that the bottom of the vials is completelyflat with no ridges.5,66.21 Vials, wide-mouth, glass, 25 mL, for use as dilutionvials.6.22 Desiccator, recommended size is 25 cm wide by 20 cmdeep

43、, with an active desiccant for drying the inocula on thecarriers.6.23 Stir Bars with Threaded TFE-Fluorocarbon-CoatedSurface, to dislodge inoculum from the carriers surface. Stirbars may be manufactured according to Fig. 1B.76.24 Magnet, strong enough to hold the threaded stir bar inplace in the gla

44、ss carrier while the liquid is being poured out ofit for membrane filtration.6.25 Aluminum Foil, to wrap items to be sterilized.6.26 Vortex Mixer, to vortex the eluate and rinsing fluid inthe carrier to ensure efficient recovery of the test organism(s).6.27 Glass Inserts, to be placed inside the gla

45、ss carriersduring inoculation with the test organism. Such inserts havebeen found to eliminate the deposition of microaerosols on theinside walls of the carriers. Glass inserts may be manufacturedaccording to Fig. 1C.6,86.28 Centrifuge, for concentration, or washing, or both ofthe cells/spores of th

46、e test organism(s).6.29 Markers, permanent labware marking pens.6.30 Sterile Polypropylene Centrifuge Tubes with Caps,50mL.6.31 Colony Counter, for example, Quebec Colony Counter.6.32 Sterile Disposable Gloves, for handling the carriers.6.33 Hemocytometer, for counting fungal conidia.6.34 Spectropho

47、tometer, for measuring turbidity of micro-bial suspensions.6.35 Bunsen Burner, for aseptic technique7. General Solutions and Reagents7.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of t

48、he Committee onAnalytical Reagents of the American Chemical Society (4).Other grades may be used (5), provided it is first ascertainedthat the reagent is of sufficiently high purity to permit its usewithout lessening the accuracy of the determination.7.2 Absolute AlcoholIn a 100-mL plastic or glass

49、beakerfor flame-sterilization of metallic forceps used to handlemembrane filters.7.3 Normal Saline (0.85 % NaCl; pH 7.2)To be used asan eluent and control fluid.7.4 Test MicrobicidePrepared at its use-dilution andbrought to the test temperature.7.5 Growth, Recovery Media and Media SupplementsTherequired types of materials (see below) can be purchased froma variety of sources specializing in laboratory supplies.7.6 MnSO4H2O, added to Columbia broth to promote the B.subtilis sporulation.7.7 Test Product Diluent, water with a standard hardness