ASTM E2197-2017 Standard Quantitative Disk Carrier Test Method for Determining Bactericidal Virucidal Fungicidal Mycobactericidal and Sporicidal Activities of Chemicals《测定液体化学杀菌剂的杀.pdf

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1、Designation: E2197 11E2197 17Standard 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 indicate

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

3、es disks 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 le

4、vel of water 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 poss

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

6、rushed stainless steel that represent hard, nonporous environmentalsurfaces and medical devices. It is also designed to have survivors that can be compared to the mean of no less than three controlcarriers to determine if the performance standard has been met. For proper statistical evaluation of th

7、e results, the number of viableorganisms in the test inoculum should be sufficiently high to take into account both the performance standard and the experimentalvariations in the results.1.2 The test protocol does not include any wiping or rubbing action. It is, therefore, not designed for testing w

8、ipes.1.3 This test method should be performed by persons with training in microbiology in facilities designed and equipped for workwith infectious agents at the appropriate biosafety level (8).1.4 It is the responsibility of the investigator to determine whether Good Laboratory Practice Regulations

9、(GLPs) are requiredand to follow them where appropriate (40 CFR, Part 160 for EPA submissions and 21 CFR, Part 58 for FDA submissions).1.5 In this test method, SI units are used for all applications, except for distance in which case inches are used and metric unitsfollow.1.6 This standard does not

10、purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine theapplicability of regulatory limitations prior to use.1.7 This intern

11、ational standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Commi

12、ttee.2. Referenced Documents2.1 ASTM Standards:3A967/A967M Specification for Chemical Passivation Treatments for Stainless Steel Parts1 This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSu

13、bcommittee E35.15 on Antimicrobial Agents.Current edition approved Jan. 1, 2011Dec. 1, 2017. Published March 2011December 2017. Originally approved in 2002. Last previous edition approved in 20022011 asE2197 02.E2197 11. DOI: 10.1520/E2197-11.10.1520/E2197-17.2 The boldface numbers in parenthesis re

14、fer to the list of references at the end of this standard.3 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.Thi

15、s document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editio

16、ns as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1D1129 Terminology Relating to WaterD1193 Specificat

17、ion for Reagent WaterE2756 Terminology Relating to Antimicrobial and Antiviral Agents2.2 CFR Standard:421 CFR, Part 58 Laboratory Practice for Nonclinical Laboratory Studies40 CFR, Part 160 Good Laboratory Practice Standards2.3 CEN Standard:5EN 10088-2 1J/2J Stainless steels - Part 2: Technical deli

18、very conditions for sheet/plate and strip of corrosion resisting steelsfor general purposes3. Terminology3.1 DefinitionsFor definitions of general terms used in this test method, refer to Terminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 carrier, nan inanimate surface or obj

19、ect inoculated with the test organism.3.2.2 eluate, nan eluent, which contains the recovered organism(s).3.2.3 eluent, nany solution that is harmless to the test organism(s) and that is added to a carrier to recover the organism(s)in or on it.3.2.4 neutralization, na process to quench the antimicrob

20、ial activity of a test substance. This process may be achieved bydilution of the organism/test substance mixture and/or by adding to it one or more chemical neutralizers.3.2.5 soil load, na solution of one or more organic, or inorganic substances, or both, added to the suspension of the testorganism

21、 to simulate the presence of body secretions, excretions, or other extraneous substances.3.2.6 test organism, nan organism that has characteristics that allow it to be readily identified. It also may be referred to asa surrogate, a simulant, or a marker organism.3.2.7 test substance, na formulation

22、that incorporates antimicrobial ingredients.4. Summary of Test Method4.1 Each disk (1 cm in diameter) receives 10 L of the test organism with a soil load. The inoculum is dried, and then the diskis placed on the inside bottom surface of a sterile plastic vial prior to contact with 50 L of the use-di

23、lution of test substance. Thecontact time and temperature may vary as required. Control carriers receive 50 L of a fluid harmless to the test organism(s) andits host cells, if any, but are otherwise treated in the same way as test carriers.4.2 For tests against vegetative bacteria, fungi, mycobacter

24、ia, and bacterial spores, the test substance is then neutralized and theinoculum eluted. The eluate and subsequent rinses of the carrier and its vial are membrane filtered. Culture plates with the filtersare incubated, colonies counted, and log10 reductions calculated.4.3 For tests with viruses, app

25、ropriate dilutions of the eluate are inoculated into suitable cell cultures, the cultures are examinedfor cytopathology/infectious foci, which are estimated as the most probable number (MPN) or counted as foci or plaques, and log10are calculated.5. Significance and Use5.1 The design of this test eli

26、minates any loss of viable organisms through wash off, thus making it possible to producestatistically valid data using many fewer test carriers than needed for methods based on simple MPN estimates.5.2 The stringency in the test is provided by the use of a soil load, the microtopography of the brus

27、hed stainless steel carriersurface, and the smaller ratio of test substance to surface area typical for many disinfectant applications. Thus, the test substancebeing assessed is presented with a reasonable challenge while allowing for efficient recovery of the test organisms from theinoculated carri

28、ers. The metal disks in the basic test are also compatible with a wide variety of actives.5.3 The design of the carriers makes it possible to place onto each 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

29、 center of each disk whereas the volumes of the test substance covers nearly the entire disksurface, thus virtually eliminating the risk of any organisms remaining unexposed.5.5 In all tests, other than those against viruses, the addition of 10 mL of an eluent/diluent gives a 1:200 dilution of the t

30、estsubstance immediately at the end of the contact time. While this step in itself may be sufficient to arrest the microbicidal activity4 Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov

31、.5 Available from European Committee for Standardization (CEN), Avenue Marnix 17, B-1000, Brussels, Belgium, http:/www.cen.eu.E2197 172of most actives, the test protocol permits the addition of a specific neutralizer to the eluent/diluent, if required. Except for viruses,the membrane filtration step

32、 also allows processing of the entire eluate from the test carriers and, therefore, the capture andsubsequent detection of even low numbers of viable organisms that may be present. Subsequent rinsing of the membrane filterswith saline also reduces the risk of carrying any inhibitory residues over to

33、 the recovery medium. Validation of the process ofneutralization of the test substance is required by challenge with low numbers of the test organism.5.6 In tests against viruses, addition of 1 mLof buffer at the end of the contact time achieves a 1:20 dilution of the test substancewhile keeping the

34、 volume of the eluate reasonably small to allow for the titration of most or all of the eluate in cell cultures.Confirmation of neutralization of the test substance is required by challenge of a residual disinfection load with low numbers ofinfective units of the test virus. Since the virus assay sy

35、stem is indirect, an additional step is required to demonstrate that priorexposure of the appropriate cell line to any residual disinfectant or disinfectant/neutralizer mixture does not interfere with thedetection of a low level of virus challenge (See Appendix).NOTE 1In 5.5 and 5.6, volumes of 10 m

36、L and 1 mL are recommended instead of 9.95 mL and 950 L, respectively, for ease of dispensing the eluent.5.7 The soil load in this test is a mixture of three types of proteins (high molecular weight proteins, low molecular weightpeptides, and mucous material) designed to represent body secretions, e

37、xcretions, or other extraneous substances that microbicidalchemicals may encounter under field conditions. It is suitable for working with all types of test organisms included here. Thecomponents of the soil load are readily available and subject to much less variability than animal sera.5.8 If dist

38、illed water or other diluent is not to be specified on the product label, the diluent for the test substance is assumedto be tap water. Since the quality of tap water varies considerably both geographically and temporally, this test method incorporatesthe use of water with a specified and documented

39、 level of hardness to prepare use-dilutions of test substance that require dilutionin water before use. While water with a hardness of at least 300 ppm as CaCO3 is recommended consult local regulations regardinguse of hard water prior to testing.5.9 The Annex contains a list of those organisms that

40、are often used in assessing the microbicidal activities of disinfectants foruse on environmental surfaces or medical devices. Culture conditions for each organism are also included in theAnnex. Dependingon the label claim(s) desired and the requirements of the target regulatory agency, one or more o

41、f the organisms listed may beselected for the testing. If organisms other than those listed are to be used (for example, in the dairy or brewing industries), a clearjustification must be provided and details of the culture media and growth conditions must be validated and clearly specified intest re

42、ports.6. General Equipment and Labware6.1 Air Displacement Pipettes, Eppendorf or equivalent, 100 to 1000 L with disposable tips.6.2 Analytical Balance, to weigh chemicals and to standardize inoculum delivery volumes by pipettes.6.3 Cell Culture Flasks and Other Plastic-ware for Viruses, (see Note 2

43、) plastic cell culture flasks of 25- and 75-cm2 capacityfor culturing cells and for preparing virus pools; 12-well or 96-well plastic plates for titrating virus infectivity.NOTE 2Plastic culture ware may be purchased from most laboratory supply houses.6.4 Centrifuge, to allow for the sedimentation o

44、f the cells/spores of the test organism(s) for concentration, or washing, or both.6.5 Colony Counter, for example, Quebec Colony Counter.6.6 Desiccator, recommended size is 25 cm wide by 20 cm deep, with an active desiccant for drying the inocula on the carriers.6.7 Dissecting Microscope, for the sc

45、reening of the metal disks for damage to surface topography.6.8 Environmental Chamber or Incubator, to hold the carriers at the desired test temperature.6.9 Filter Sterilization System for Media and Reagents, a membrane or cartridge filtration system (0.22-m pore diameter) isrequired for sterilizing

46、 heat-sensitive solutions.6.10 Forceps, straight or curved, (1) with smooth tips to handle membrane filters, and (2) to pick up the metal disk carriers forplacement in plastic vials.6.11 Freezers, a freezer at 20 6 2C is required for the storage of media and additives. A second freezer at 70C or low

47、eris required to store the stocks of test organisms.6.12 Glassware, 1-L flasks with a side-arm and appropriate tubing to capture the filtrates from 47-mm diameter membranefilters; 250-mL Erlenmeyer flasks for culture media.6.13 Hemocytometer, for counting fungal conidia, and/or for use in the prepar

48、ation of suitable cell numbers for seedingmonolayers.6.14 Hot Air Oven, an oven at 60C to dry clean and sterile glassware.6.15 Incubators, an ordinary incubator, an anaerobic incubator, and a CO2 incubator to incubate cell cultures in a 5 % CO2atmosphere. If only one ordinary incubator is available,

49、 its temperature will require adjustment depending on the type of organismunder test.E2197 1736.16 Inverted Microscope, an inverted microscope with 10 eyepiece and 5, 10, and 40 objectives to examine cell cultures.6.17 Laminar Flow Cabinet, a Class II (Type A) biological safety cabinet. The procedures for the proper maintenance and useof such cabinets are given in Ref (8).6.18 Liquid Nitrogen Storage for Cells, a proper liquid nitrogen container and liquid nitrogen supply for cryopreservation of thestocks of cell lines.6.19 Magnet, strong enough to