ASTM E2197-2011 Standard Quantitative Disk Carrier Test Method for Determining the Bactericidal Virucidal Fungicidal Mycobactericidal and Sporicidal Activities of Liquid Chemical G.pdf

1、Designation: E2197 11Standard Quantitative Disk Carrier Test Method forDetermining Bactericidal, Virucidal, Fungicidal,Mycobactericidal, and Sporicidal Activities of Chemicals1This standard is issued under the fixed designation E2197; the number immediately following the designation indicates the ye

2、ar 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 () indicates an editorial change since the last revision or reapproval.INTRODUCTIONThe quantitative test method described here uses disks

3、 of stainless steel (1 cm in diameter) ascarriers. It employs the same basic set of materials and procedures to assess the ability of liquidchemicals to inactivate vegetative bacteria, viruses, fungi, mycobacteria, and bacterial spores (1-7).2Performance standards for test substances, the level of w

4、ater hardness, the type and level of a soil load,the test organism(s), and other test conditions may vary depending on the target regulatory agency.This basic test can also be adapted for use with other carrier materials of similar dimensions.The development of this test method was made possible wit

5、h financial support from theAntimicrobials Division of the U.S. Environmental Protection Agency.1. Scope1.1 This test method is designed to evaluate the ability oftest substances to inactivate vegetative bacteria, viruses, fungi,mycobacteria, and bacterial spores (1-7) on disk carriers ofbrushed sta

6、inless steel that represent hard, nonporous environ-mental surfaces and medical devices. It is also designed to havesurvivors that can be compared to the mean of no less thanthree control carriers to determine if the performance standardhas been met. For proper statistical evaluation of the results,

7、the number of viable organisms in the test inoculum should besufficiently high to take into account both the performancestandard and the experimental variations in the results.1.2 The test protocol does not include any wiping or rubbingaction. It is, therefore, not designed for testing wipes.1.3 Thi

8、s test method should be performed by persons withtraining in microbiology in facilities designed and equipped forwork with infectious agents at the appropriate biosafety level(8).1.4 It is the responsibility of the investigator to determinewhether Good Laboratory Practice Regulations (GLPs) arerequi

9、red and to follow them where appropriate (40 CFR, Part160 for EPA submissions and 21 CFR, Part 58 for FDAsubmissions).1.5 In this test method, SI units are used for all applications,except for distance in which case inches are used and metricunits follow.1.6 This standard does not purport to address

10、 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 Documents2.1 ASTM Standards:3D1129 Terminology

11、 Relating to WaterD1193 Specification for Reagent WaterE2756 Terminology Relating toAntimicrobial andAntiviralAgents2.2 CFR Standard:421 CFR, Part 58 Laboratory Practice for Nonclinical Labo-ratory Studies40 CFR, Part 160 Good Laboratory Practice Standards3. Terminology3.1 DefinitionsFor definitions

12、 of general terms used inthis test method, refer to Terminology E2756.3.2 Definitions of Terms Specific to This Standard:1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Methods and is the directresponsibility of Subcommittee E35

13、.15 on Antimicrobial Agents.Current edition approved Jan. 1, 2011. Published March 2011. Originallyapproved in 2002. Last previous edition approved in 2002 as E2197 02. DOI:10.1520/E2197-11.2The boldface numbers in parenthesis refer to the list of references at the end ofthis standard.3For reference

14、d 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.4Available from U.S. Government Printing Office Superintendent of Documents

15、,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.2.1 carrier, nan inanimate surface or object inoculatedwith the test organism.3.2.2 eluate, nan

16、 eluent, which contains the recoveredorganism(s).3.2.3 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.3.2.4 neutralization, na process to quench the antimicro-bial activity of a test substance. This process may be ac

17、hievedby dilution of the organism/test substance mixture and/or byadding to it one or more chemical neutralizers.3.2.5 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, o

18、r other extraneous substances.3.2.6 test organism, nan organism that has characteristicsthat allow it to be readily identified. It also may be referred toas a surrogate, a simulant, or a marker organism.3.2.7 test substance, na formulation that incorporatesantimicrobial ingredients.4. Summary of Tes

19、t Method4.1 Each disk (1 cm in diameter) receives 10 L of the testorganism with a soil load. The inoculum is dried, and then thedisk is placed on the inside bottom surface of a sterile plasticvial prior to contact with 50 L of the use-dilution of testsubstance. The contact time and temperature may v

20、ary asrequired. Control carriers receive 50 L of a fluid harmless tothe test organism(s) and its host cells, if any, but are otherwisetreated in the same way as test carriers.4.2 For tests against vegetative bacteria, fungi, mycobacte-ria, and bacterial spores, the test substance is then neutralized

21、and the inoculum eluted. The eluate and subsequent rinses ofthe carrier and its vial are membrane filtered. Culture plateswith the filters are incubated, colonies counted, and log10reductions calculated.4.3 For tests with viruses, appropriate dilutions of the eluateare inoculated into suitable cell

22、cultures, the cultures areexamined for cytopathology/infectious foci, which are esti-mated as the most probable number (MPN) or counted as focior plaques, and log10are calculated.5. Significance and Use5.1 The design of this test eliminates any loss of viableorganisms through wash off, thus making i

23、t possible to producestatistically valid data using many fewer test carriers thanneeded for methods based on simple MPN estimates.5.2 The stringency in the test is provided by the use of a soilload, the microtopography of the brushed stainless steel carriersurface, and the smaller ratio of test subs

24、tance to surface areatypical for many disinfectant applications. Thus, the testsubstance being assessed is presented with a reasonable chal-lenge while allowing for efficient recovery of the test organ-isms from the inoculated carriers. The metal disks in the basictest are also compatible with a wid

25、e variety of actives.5.3 The design of the carriers makes it possible to place ontoeach a precisely measured volume of the test organism (10 L)as well as the control fluid or test substance (50 L).5.4 The inoculum is placed at the center of each diskwhereas the volumes of the test substance covers n

26、early theentire disk surface, thus virtually eliminating the risk of anyorganisms remaining unexposed.5.5 In all tests, other than those against viruses, the additionof 10 mL of an eluent/diluent gives a 1:200 dilution of the testsubstance immediately at the end of the contact time. Whilethis step i

27、n itself may be sufficient to arrest the microbicidalactivity of most actives, the test protocol permits the additionof a specific neutralizer to the eluent/diluent, if required.Except for viruses, the membrane filtration step also allowsprocessing of the entire eluate from the test carriers and,the

28、refore, the capture and subsequent detection of even lownumbers of viable organisms that may be present. Subsequentrinsing of the membrane filters with saline also reduces the riskof carrying any inhibitory residues over to the recoverymedium. Validation of the process of neutralization of the tests

29、ubstance is required by challenge with low numbers of the testorganism.5.6 In tests against viruses, addition of 1 mL of buffer at theend of the contact time achieves a 1:20 dilution of the testsubstance while keeping the volume of the eluate reasonablysmall to allow for the titration of most or all

30、 of the eluate in cellcultures. Confirmation of neutralization of the test substance isrequired by challenge of a residual disinfection load with lownumbers of infective units of the test virus. Since the virusassay system is indirect, an additional step is required todemonstrate that prior exposure

31、 of the appropriate cell line toany residual disinfectant or disinfectant/neutralizer mixturedoes not interfere with the detection of a low level of viruschallenge (See Appendix).NOTE 1In 5.5 and 5.6, volumes of 10 mL and 1 mL are recommendedinstead of 9.95 mL and 950 L, respectively, for ease of di

32、spensing theeluent.5.7 The soil load in this test is a mixture of three types ofproteins (high molecular weight proteins, low molecularweight peptides, and mucous material) designed to representbody secretions, excretions, or other extraneous substances thatmicrobicidal chemicals may encounter under

33、 field conditions.It is suitable for working with all types of test organismsincluded here. The components of the soil load are readilyavailable and subject to much less variability than animal sera.5.8 If distilled water or other diluent is not to be specified onthe product label, the diluent for t

34、he test substance is assumedto be tap water. Since the quality of tap water varies consid-erably both geographically and temporally, this test methodincorporates the use of water with a specified and documentedlevel of hardness to prepare use-dilutions of test substance thatrequire dilution in water

35、 before use. While water with ahardness of at least 300 ppm as CaCO3is recommendedconsult local regulations regarding use of hard water prior totesting.5.9 The Annex contains a list of those organisms that areoften used in assessing the microbicidal activities of disinfec-tants for use on environmen

36、tal surfaces or medical devices.Culture conditions for each organism are also included in theAnnex. Depending on the label claim(s) desired and therequirements of the target regulatory agency, one or more ofE2197 112the organisms listed may be selected for the testing. Iforganisms other than those l

37、isted are to be used (for example,in the dairy or brewing industries), a clear justification must beprovided and details of the culture media and growth condi-tions must be validated and clearly specified in test reports.6. General Equipment and Labware6.1 Air Displacement Pipettes, Eppendorf or equ

38、ivalent,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 Viruses,(see Note 2) plastic cell culture flasks of 25- and 75-cm2capacity for culturing cells and for pr

39、eparing virus pools;12-well or 96-well plastic plates for titrating virus infectivity.NOTE 2Plastic culture ware may be purchased from most laboratorysupply houses.6.4 Centrifuge, to allow for the sedimentation of the cells/spores of the test organism(s) for concentration, or washing, orboth.6.5 Col

40、ony Counter, for example, Quebec Colony Counter.6.6 Desiccator, recommended size 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 Inc

41、ubator, to hold the car-riers at the desired test temperature.6.9 Filter Sterilization 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 m

42、embrane filters, and (2) to pick up the metal diskcarriers for placement in plastic 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-ar

43、m and appropriatetubing to capture the filtrates from 47-mm diameter membranefilters; 250-mL Erlenmeyer flasks for culture media.6.13 Hemocytometer, for counting fungal conidia, and/orfor use in the preparation of suitable cell numbers for seedingmonolayers.6.14 Hot Air Oven, an oven at 60C to dry c

44、lean and sterileglassware.6.15 Incubators, an ordinary incubator, an anaerobic incu-bator, and a CO2incubator to incubate cell cultures in a 5 %CO2atmosphere. If only one ordinary incubator is available, itstemperature will require adjustment depending on the type oforganism under test.6.16 Inverted

45、 Microscope, an inverted microscope with103 eyepiece and 53,103, and 403 objectives to examinecell cultures.6.17 Laminar Flow Cabinet, a Class II (Type A) biologicalsafety cabinet. The procedures for the proper maintenance anduse of such cabinets are given in Ref (8).6.18 Liquid Nitrogen Storage for

46、 Cells, a proper liquidnitrogen container and liquid nitrogen supply for cryopreser-vation of the stocks of cell lines.6.19 Magnet, strong enough to hold the disk carrier in placein the plastic vial while the liquid is being poured out of it formembrane filtration.6.20 Magnetic Stir Plate and Stir B

47、ars, large enough for a5-L beaker or Erlenmeyer flask for preparing culture media orother solutions.6.21 Markers, for permanent marking of labware.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 diame

48、ter) and glass,plastic, or metal holders for such filters are required.6.23 pH Meter, to measure pH of buffers, eluents, and testformulations.6.24 Microwave Oven, to melt agar overlays.6.25 Miscellaneous Laboratory Ware, pipette tips, plasticvials for storing cell and viral stocks, dilution tubes.6.

49、26 Orbital Shaker, for shaking the broth cultures ofBacillus subtilis during their incubation.6.27 Petri Plates (Pyrex glass) 150 mm in diameter, forholding and autoclave sterilization of metal disks.6.28 Positive Displacement Pipette, a pipette and pipettetips fitted with “plungers” that can accurately dispense 10-Lvolumes for inoculation of carriers without the aerosol genera-tion that occurs when air displacement pipettes are used.6.29 Refrigerator, a refrigerator at 4 6 2C for storage ofmedia, culture plates and reagents.6.30 Serological Pipettes, ster