ASTM E2197-2002 Standard Quantitative Disk Carrier Test Method for Determining the Bactericidal Virucidal Fungicidal Mycobactericidal and Sporicidal Activities of Liquid Chemical G.pdf

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1、Designation: E 2197 02Standard Quantitative Disk Carrier Test Method forDetermining the Bactericidal, Virucidal, Fungicidal,Mycobactericidal and Sporicidal Activities of LiquidChemical Germicides1This standard is issued under the fixed designation E 2197; the number immediately following the designa

2、tion indicates the 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 quantitative test method des

3、cribed here uses disks of stainless steel (1 cm in diameter) ascarriers. Because it employs the same basic set of materials and procedures to assess the ability ofliquid chemical germicides to inactivate vegetative bacteria, viruses, fungi, mycobacteria and bacterialspores (1,2)2it unifies the test

4、methodology against a wide array of microorganisms. Performancestandards for the categories of products to be tested, and the specific types of organism(s) to be usedmay vary depending on the regulatory agency. This basic test can also be adapted for use with othercarrier materials of similar dimens

5、ions.The development of this method was made possible with financial support from the AntimicrobialsDivision of the U.S. Environmental Protection Agency.1. Scope1.1 The method is designed to evaluate the ability of liquidchemical germicides to inactivate vegetative bacteria, viruses,fungi, mycobacte

6、ria and bacterial spores in the presence of asoil load (1,2) on disk carriers that represent environmentalsurfaces and medical devices. It is also designed to havesurvivors that can be compared to mean of no less than threecontrol carriers to determine if the performance standard hasbeen met. For pr

7、oper statistical evaluation of the results, thesize of the test inoculum should be sufficiently large to takeinto account both the performance standard and the experimen-tal variation in the results.1.2 The test protocol does not include any wiping or rubbingaction. It is, therefore, not designed fo

8、r testing germicide-soaked wipes.1.3 This test method should be performed by persons withtraining in microbiology in facilities designed and equipped forwork with infectious agents at the appropriate biosafety level(3).1.4 In this test method, metric units are used for allapplications, except for di

9、stance in which case inches are usedand metric units follow.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

10、.6 This standard does not purport to address all of 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 Do

11、cuments2.1 ASTM Standards:3D 1129 Terminology Relating to WaterD 1193 Specification for Reagent Grade WaterE 1054 Practices for Evaluating Inactivators of Antimicro-bial Agents Used in Disinfectant, Sanitizer, Antiseptic, orPreserved ProductsE 2111 Standard Quantitative Carrier Test Method to Evalu-

12、ate the Bactericidal, Fungicidal, Mycobactericidal andSporicidal Potencies of Liquid Chemical Germicides2.2 CFR Standard:1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and is the direct responsibility of Subcommittee E35.15 on AntimicrobialAgents.Current edition appro

13、ved April 10, 2002. Published June 2002.2The boldface numbers in parenthesis refer to the list of references at the 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 in

14、formation, refer 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.40 CFR, Part 160; 21 CFR, Part 5842.3 Other Documents:Disinfectants (Chapter 6), Official Methods of Analyses(

15、1998)5CAN/CGSB-2.161-97, Assessment of Efficacy of Antimi-crobial Agents for Use on Environmental Surfaces andMedical Devices63. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 carrier, nan inanimate surface or object inoculatedwith the test organism.3.1.2 eluate, nan eluent, whi

16、ch contains the recoveredorganism(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, na process to quench the antimicro-bial activity of a test formulation. This process may beachieved by d

17、ilution of the organism/test formulation mixtureand/or by adding to it one or more chemical neutralizers.3.1.5 soil load, na solution 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

18、extraneous substances.3.1.6 test formulation, na formulation that incorporatesantimicrobial ingredients.3.1.7 test organism, nan organism that has characteristicsthat allows it to be readily identified. It also may be referred toas a surrogate or a marker organism.4. Summary of Test Method4.1 Each d

19、isk (1 cm in diameter) receives 10 L of the testorganism with a soil load, dried, and is then placed on theinside bottom surface of a sterile, 15 to 20-mL-capacity vialprior to contact with 50 L of the use-dilution of test substance(germicide). The contact time and temperature may vary asrequired. C

20、ontrol carriers receive 50 L of a fluid harmless tothe test organism(s).4.2 For tests against vegetative bacteria, fungi, mycobacte-ria and bacteria spores, the test substance is then diluted/neutralized, and the inoculum eluted. The eluate and subse-quent rinses are membrane filtered. Culture plate

21、s with thefilters are incubated, colonies counted and log10reductions arecalculated.4.3 For tests with viruses, appropriate dilutions of the eluateare inoculated into suitable cell cultures, the cultures examinedfor cytopathology/infectious foci and log10calculated.5. Significance and Use5.1 The des

22、ign of this test minimizes any loss of viableorganisms through wash off, thus making it possible to producestatistically valid data using many fewer test carriers thanneeded for methods based on simple most probable number(MPN) estimates.5.2 The stringency in the test is provided by the use of a soi

23、lload, the microtopography of the carrier surface and the smallratio of disinfectant to surface area typical for many disinfec-tant applications. Thus the formulation under test is presentedwith a reasonable challenge while allowing for efficient recov-ery of the test organisms from the inoculated c

24、arriers with orwithout their exposure to the test formulation. The metal disksused in the basic test are also compatible with a wide variety ofactives.5.3 The design of the carriers makes it possible to place ontoeach precisely measured volume of the test organism (10 L)as well as the test formulati

25、on (50 L).5.4 The inoculum is placed at the centrer of each diskwhereas the volume of the test formulation covers nearly theentire disk surface thus eliminating the risk of any organismsremaining unexposed to the test formulation.5.5 The relatively small ratio of 1:5 between the volume ofthe inoculu

26、m and that of the test formulation closely reflectsmany field applications of liquid chemical germicides.5.6 In all tests other than those against viruses, the additionof 9.95 mL of an eluent/diluent gives a 1:200 dilution of thetest formulation immediately at the end of the contact time.While this

27、step in itself may be sufficient to arrest thegermicidal activity of most formulations, the test protocolpermits the addition of a specific neutralizer to the eluent/diluent, if required; the membrane filtration step also allowsprocessing of the entire eluate from the test carriers andtherefore the

28、capture and subsequent detection of even lownumbers of viable organisms that may be present. Subsequentrinsing of the membrane filters with normal saline also reducesthe risk of carrying any inhibitory residues over to the recoverymedium. Confirmation of neutralization of the test formulationis requ

29、ired by challenge with low numbers of the test organism.5.7 In tests against viruses, addition of 950 L of Earlesbalanced salt solution (EBSS) at the end of the contact timeachieves a 1:20 dilution of the test formulation while keepingthe volume of the eluate reasonably small to allow for thetitrati

30、on of most or all of the eluate in cell cultures. Confirma-tion of neutralization of the test formulation is required bychallenge of a residual disinfection load with low numbers ofinfective units of the test virus. Since the virus assay system isindirect, an additional step is required to demonstra

31、te that priorexposure of the appropriate cell line to any residual disinfectantor disinfectant/neutralizer mixture does not interfere with thedetection of a low level of virus challenge.5.8 The soil load for use in this test is a mixture of threetypes of proteins (high molecular weight proteins, low

32、 molecu-lar weight peptides and mucous material) designed to representthe body secretions, excretions or other extraneous substancesthat chemical germicides may encounter under field conditions.It is suitable for working with all types of test organismsincluded here. The components of the soil load

33、are readilyavailable and subject to much less variability than animal sera.5.9 If distilled water or other diluent is not to be specified onthe product label, the diluent is assumed to be tap water. Since4Available from Superintendent of Documents, U.S. Government PrintingOffice, Washington D.C. 204

34、02.5Available from AOAC International, Washington, DC.6Available from the Canadian General Standards Board, Ottawa, Ontario,Canada.E2197022the quality of tap water varies considerably both geographi-cally and temporally, this test method incorporates the use ofwater with a specified and documented l

35、evel of hardness toprepare use-dilutions of test products that require dilution inwater before use. The U.S. Environmental ProtectionAgencysScientific Advisory Panel (SAP) on Germicide Test Methodol-ogy has recommended the use of water with a standardhardness of 400 ppm as CaCO3.5.10 Depending on th

36、e label claim desired and the require-ments of the target regulatory agency, additional test organismsmay be used. In such cases, the details of the culture media andconditions must be validated and clearly specified in testreports.6. General Equipment and Labware6.1 Air Displacement Pipettes, Eppen

37、dorf or equivalent,100 to 1000 L with disposable tips.6.2 Analytical Balance, to weigh chemicals and to standard-ize inoculum delivery volumes by pipettes.6.3 Cell Culture Flasks and other plastic-ware for Viruses7,plastic cell culture flasks of 25 and 75-cm2capacity forculturing cells and for prepa

38、ring virus pools; 12-well or96-well plastic plates for titrating virus infectivity.6.4 Centrifuge, to allow for the sedimentation of the cells/spores of the test organism(s) for concentration, or washing, orboth.6.5 Colony Counter, for example, Quebec Colony Counter.6.6 Desiccator, recommended size

39、is 25 cm wide by 20 cmdeep, with an active desiccant for drying the inocula on thecarriers.6.7 Dissecting Microscope, for the screening of the metaldisks for damage to surface topography.6.8 Environmental Chamber or Incubator, to hold the car-riers at the desired test temperature.6.9 Filter Steriliz

40、ation System for Media and Reagents,amembrane or cartridge filtration system (0.22 m pore diam-eter) is required for sterilizing heat-sensitive solutions.6.10 Forceps, straight or curved, (1) with smooth tips tohandle membrane filters, and (2) to pick up the metal diskcarriers for placement in plast

41、ic vials.6.11 Freezers, a freezer at -20 6 2C is required for thestorage of media and additives. A second freezer at -70C orlower is required to store the stocks of test organisms.6.12 Glassware, 1-L flasks with a side-arm and appropriatetubing to capture the filtrates from 47-mm diameter membranefi

42、lters; 250-mL Erlenmeyer flasks for culture media.6.13 Hemocytometer, for counting fungal conidia, and or inthe preparation of suitable cell numbers for seeding cellmonolayers.6.14 Hot Air Oven, an oven at 60C to dry clean and sterileglassware.6.15 Incubators, an ordinary incubator and an anaerobici

43、ncubator. If only one ordinary incubator is available, itstemperature will require adjustment depending on the type oforganism under test; a CO2incubator to incubate cell culture ina5%CO2atmosphere.6.16 Inverted Microscope, an inverted microscope with103 eyepiece and 53,103, and 403 objectives to ex

44、aminecell cultures.6.17 Laminar Flow Cabinet, a Class II (Type A) biologicalsafety cabinet for this work. The procedures for the propermaintenance and use of such cabinets are given in Ref (3).6.18 Liquid Nitrogen Storage for Cells, a proper liquidnitrogen container and liquid nitrogen supply for cr

45、yopreser-vation of the stocks of cell lines.6.19 Magnet, strong enough to hold the disk carrier in placein the glass vial while the liquid is being poured out of it formembrane filtration.6.20 Magnetic Stir Plate and Stir Bars, large enough for a5-L beaker or Erlenmeyer flask for preparing culture m

46、edia orother solutions.6.21 Markers, permanent labware marking pens.6.22 Membrane Filtration System for Capture of the TestOrganisms other than Viruses, sterile 47-mm diameter mem-brane filters (0.22 or 0.45 m pore diameter) and glass, plasticor metal holders for such filters are required.6.23 pH Me

47、ter, to measure pH of buffers, eluents and testformulations.NOTE 1The method described here uses conventional membranefilters. The system with hydrophobic grid membrane filters (HGMF) mayalso be used for this purpose (4).6.24 Miscellaneous Laboratory Ware, pipette tips, plasticvials for storing cell

48、 and virus stocks, dilution tubes.6.25 Orbital Shaker, for shaking the broth cultures of B.subtilis during their incubation.6.26 Petri Plates (Pyrex glass) 150 mm in Diameter, forholding and autoclave sterilization of metal disks.6.27 Positive Displacement Pipette, a pipette and pipettetips fitted w

49、ith “plungers” that can dispense accurately 10-Lvolumes for inoculation of carriers without the aerosol genera-tion that occurs when air displacement pipettes are used.6.28 Refrigerator, a refrigerator at 4 6 2C for storage ofmedia, culture plates and reagents.6.29 Serological Pipettes, sterile reusable or single-usepipettes of 10.0, 5.0, and 1.0 mL capacity.6.30 Spectrophotometer, for measuring turbidity of micro-bial suspensions.6.31 Sterile Dispenser, 10 mL, for dispensing diluent/eluent.6.32 Sterile Disposable Gloves, for handling the carriers.6.33 Sterile Di