1、Designation: E2111 12Standard Quantitative Carrier Test Method toEvaluate the Bactericidal, Fungicidal, Mycobactericidal, andSporicidal Potencies of Liquid Chemicals1This standard is issued under the fixed designation E2111; the number immediately following the designation indicates the year oforigi
2、nal 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 need for better tests to assess the microbicidal activity of
3、 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 potency of liqui
4、d 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 under more stri
5、ngent 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 devel-opment an
6、d 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 CFU after themand
7、atory 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 been met. To a
8、llow 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 titer of the t
9、est 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 method should b
10、e 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 unitsfollow.1.5 I
11、t 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 of thesafety conce
12、rns, 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 Relating to WaterD1193
13、 Specification for Reagent WaterE1054 Test Methods for Evaluation of Inactivators of An-timicrobial AgentsE2197 Quantitative Disk Carrier Test Method for Determin-ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,1This test method is under the jurisdiction of ASTM Committee E35 onPesticides,
14、 Antimicrobials, and Alternative Control Agents and is the directresponsibility of SubcommitteeE35.15 on Antimicrobial Agents.Current edition approved May 15, 2012. Published June 2012. Originallyapproved in 2000. Last previous edition approved in 2005 as E2111 05. DOI:10.1520/E211112.2The boldface
15、numbers in parentheses 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 information, refer to the standards Document Summary pag
16、e onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.and Sporicidal Activities of ChemicalsE2756 Terminology Relating toAntimicrobial andAntiviralAgents2.2 CFR Standards:440 CFR Part 16021 CFR Part 583. Terminology3.1
17、 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 contains 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
18、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 of the organism/test formulation mixture and/or byadding to it one or more chemical neutralizers. (Refer to TestMethods E1054 for further detai
19、ls3.1.4.1 DiscussionThis process may be achieved by dilu-tion of the organism/test formulation mixture or by adding to itone or more chemical neutralizers, 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 simul
20、ate the presence of body secretions,excretions, or other extraneous substances.3.1.6 test formulation, nformulation that incorporates an-timicrobial ingredients.3.1.7 test organism, napplied inoculum of an organismthat has characteristics that allows it to be readily identified. Italso may be referr
21、ed 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 potency of chemicals against vegetativebacteria, fungi, mycobacteria, as well as bacterial spores. It isdesigned primarily for testing formulations to be
22、used on hardenvironmental surfaces and medical devices. This test methoduses the flat inside bottom surface of glass vials as the carrier.Each vial receives 10 L of the test organism with or withouta soil load. The contamination of the inside surface of thecarrier with microaerosols is avoided by th
23、e use of glassinserts. The inoculum is dried and exposed to 1 mL of the testmicrobicide for the desired contact time at the recommendedtemperature; control 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 v
24、ial to dilute/neutralize the microbicide and any inoculum adhering to thecarrier surface is recovered using a magnetic stir bar with athreaded surface. The eluate is passed through a membranefilter, the carrier vial is then rinsed several times with eluent/diluent and the rinses are also passed thro
25、ugh the same filter.The total rinse volume is no less than 100 mL. Control and testeluates requiring dilution to get countable colonies are firstsubjected to a series of tenfold dilutions and the material fromsuitable dilutions is passed separately through membranefilters. Each filter is placed on t
26、he 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 incubationtemperature, colonies counted, and log10reductions in theviability titer of the test organism calculated.NOTE 1Do not soak the magnetic stir bars i
27、n ethanol or other solventsfor decontamination as this may damage the sealant on them.5. Significance and Use5.1 This test method is fully quantitative and it also avoidsany loss of viable organisms through wash off, making itpossible to produce statistically valid data using many fewertest and cont
28、rol carriers than other quantitative methods basedon most probable numbers (MPN).5.2 The design of the carriers makes it possible to place intoeach a precisely measured volume of the test suspension. Theuse of the threaded stir bars allows for efficient recovery of theinoculum even after its exposur
29、e for several hours to strongfixatives such as glutaraldehyde.5.3 The membrane filtration step allows processing of theentire eluate from the test carriers and therefore the capture andsubsequent detection of even low numbers of viable organismsthat may be present.5.4 This test can be performed with
30、 or without a soil load todetermine the effect of such loading on microbicide perfor-mance. Consult the target regulatory agency on the need,type(s), and acceptable level(s) of soil load prior to testing. Onetype of soil load (Quantitative Disk Carrier Test MethodE2197) to consider for this test is
31、a mixture of three types ofproteins (high molecular weight proteins, low molecularweight peptides, and mucous material) to represent the bodysecretions, excretions, or other extraneous substances thatchemical microbicides may encounter under field conditions. Itis suitable for working with the vario
32、us test organisms includedhere. The components of the soil load are readily available andsubject to much less variability than animal sera.5.5 If distilled water or other diluent is not to be specified onthe product label, the diluent for the test substance is assumedto be tap water. Since the quali
33、ty 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 before use. Consult the target regula-tory agency regarding
34、 the use and level of water hardness priorto testing.6. General Equipment and Labware6.1 Laminar Flow CabinetA Class II (Type A) biologicalsafety cabinet for this work. The procedures for the propermaintenance and use of such cabinets are given in Ref 3.6.2 IncubatorAn ordinary incubator and an anae
35、robicincubator. If only one ordinary incubator is available, itstemperature will require adjustment depending on the type oforganism under test.4Available from U.S. Government Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.E21111226.3 Sterili
36、zerAny 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 filtration system (0.22-m pore dia
37、m-eter) is required for sterilizing heat-sensitive solutions.6.5 Membrane Filtration System for Capture of the TestOrganismsSterile 47-mm diameter sterilizing membranefilters and glass, metal, or plastic holders for such filters arerequired. Membranes made from polyethersulfone (PES) arerecommended.
38、 Filter membranes with a pore diameter of 0.22m must be used when working with bacterial spores.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 and additives. A second freezer at 70C orlower
39、 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 glassware.6.11 Magnetic Stir Plate and Stir Bars
40、Large 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.FIG. 1 Components of a Carrier for the Quantitative Carrier TestE21111236.13
41、 Air Displacement PipettesEppendorf 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
42、to capture the filtrates from 47-mm diametermembrane filters; 250-mL Erlenmeyer flasks for culture media;100 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 SourceA vacuum pump, access to an in-house va
43、cuum 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-Bottomed Glass Vials, 20 mL, with regular andseptate
44、 caps (Fig. 1A). Flat-bottomed glass vials may bemanufactured such that the bottom of the vials is completelyflat with no ridges.56.21 Vials, wide-mouth, glass, 25 mL, for use as dilutionvials.6.22 Desiccator, recommended size is 25 cm wide by 20 cmdeep, with an active desiccant for drying the inocu
45、la 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.66.24 Magnet, strong enough to hold the threaded stir bar inplace in the glass carrier while the liquid is being poured out
46、 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 glass carriersduring inoculation with the test org
47、anism. Such inserts havebeen found to eliminate the deposition of microaerosols on theinside walls of the carriers. Glass inserts may be manufacturedaccording to Fig. 1C.76.28 Centrifuge, for concentration, or washing, or both ofthe cells/spores of the test organism(s).6.29 Markers, permanent labwar
48、e 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 Spectrophotometer, for measuring turbidity of micro-bial su
49、spensions.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 the 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 glas
