ASTM E2197-02
(Test Method)Standard Quantitative Disk Carrier Test Method for Determining the Bactericidal, Virucidal, Fungicidal, Mycobactericidal and Sporicidal Activities of Liquid Chemical Germicides
Standard Quantitative Disk Carrier Test Method for Determining the Bactericidal, Virucidal, Fungicidal, Mycobactericidal and Sporicidal Activities of Liquid Chemical Germicides
SCOPE
1.1 The method is designed to evaluate the ability of liquid chemical germicides to inactivate vegetative bacteria, viruses, fungi, mycobacteria and bacterial spores in the presence of a soil load (1,2) on disk carriers that represent environmental surfaces and medical devices. It is also designed to have survivors that can be compared to mean of no less than three control carriers to determine if the performance standard has been met. For proper statistical evaluation of the results, the size of the test inoculum should be sufficiently large to take into account both the performance standard and the experimental variation in the results.
1.2 The test protocol does not include any wiping or rubbing action. It is, therefore, not designed for testing germicide-soaked wipes.
1.3 This test method should be performed by persons with training in microbiology in facilities designed and equipped for work with infectious agents at the appropriate biosafety level ().
1.4 In this test method, metric units are used for all applications, except for distance in which case inches are used and metric units follow.
1.5 It is the responsibility of the investigator to determine whether Good Laboratory Practice Regulations (GLPs) are required and to follow them where appropriate (40 CFR, Part 160 for EPA submissions and 21 CFR, Part 58 for FDA submissions).
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation:E2197–02
Standard Quantitative Disk Carrier Test Method for
Determining the Bactericidal, Virucidal, Fungicidal,
Mycobactericidal and Sporicidal Activities of Liquid
Chemical Germicides
This standard is issued under the fixed designation E2197; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
The quantitative test method described here uses disks of stainless steel (1 cm in diameter) as
carriers. Because it employs the same basic set of materials and procedures to assess the ability of
liquidchemicalgermicidestoinactivatevegetativebacteria,viruses,fungi,mycobacteriaandbacterial
spores (1,2) it unifies the test methodology against a wide array of microorganisms. Performance
standards for the categories of products to be tested, and the specific types of organism(s) to be used
may vary depending on the regulatory agency. This basic test can also be adapted for use with other
carrier materials of similar dimensions.
The development of this method was made possible with financial support from theAntimicrobials
Division of the U.S. Environmental Protection Agency.
1. Scope 1.5 It is the responsibility of the investigator to determine
whether Good Laboratory Practice Regulations (GLPs) are
1.1 The method is designed to evaluate the ability of liquid
required and to follow them where appropriate (40 CFR, Part
chemical germicides to inactivate vegetative bacteria, viruses,
160 for EPA submissions and 21 CFR, Part 58 for FDA
fungi, mycobacteria and bacterial spores in the presence of a
submissions).
soil load (1,2) on disk carriers that represent environmental
1.6 This standard does not purport to address all of the
surfaces and medical devices. It is also designed to have
safety concerns, if any, associated with its use. It is the
survivors that can be compared to mean of no less than three
responsibility of the user of this standard to establish appro-
control carriers to determine if the performance standard has
priate safety and health practices and determine the applica-
been met. For proper statistical evaluation of the results, the
bility of regulatory limitations prior to use.
size of the test inoculum should be sufficiently large to take
intoaccountboththeperformancestandardandtheexperimen-
2. Referenced Documents
tal variation in the results.
2.1 ASTM Standards:
1.2 Thetestprotocoldoesnotincludeanywipingorrubbing
D1129 Terminology Relating to Water
action. It is, therefore, not designed for testing germicide-
D1193 Specification for Reagent Water
soaked wipes.
E1054 Test Methods for Evaluation of Inactivators of An-
1.3 This test method should be performed by persons with
timicrobial Agents
traininginmicrobiologyinfacilitiesdesignedandequippedfor
E2111 Quantitative Carrier Test Method to Evaluate the
work with infectious agents at the appropriate biosafety level
Bactericidal, Fungicidal, Mycobactericidal, and Sporicidal
(3).
Potencies of Liquid Chemical Microbicides
1.4 In this test method, metric units are used for all
2.2 CFR Standard:
applications, except for distance in which case inches are used
40 CFR, Part 160; 21 CFR, Part 58
and metric units follow.
2.3 Other Documents:
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides and is the direct responsibility of Subcommittee E35.15 onAntimicrobial For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Agents. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Current edition approved April 10, 2002. Published June 2002. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
E2197-02. the ASTM website.
2 4
The boldface numbers in parenthesis refer to the list of references at the end of Available from Superintendent of Documents, U.S. Government Printing
this standard. Office, Washington D.C. 20402.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2197–02
Disinfectants (Chapter 6), Official Methods of Analyses 5.2 Thestringencyinthetestisprovidedbytheuseofasoil
(1998) load, the microtopography of the carrier surface and the small
CAN/CGSB-2.161-97, Assessment of Efficacy of Antimi- ratio of disinfectant to surface area typical for many disinfec-
crobial Agents for Use on Environmental Surfaces and tant applications. Thus the formulation under test is presented
Medical Devices with a reasonable challenge while allowing for efficient recov-
ery of the test organisms from the inoculated carriers with or
3. Terminology without their exposure to the test formulation. The metal disks
usedinthebasictestarealsocompatiblewithawidevarietyof
3.1 Definitions of Terms Specific to This Standard:
actives.
3.1.1 carrier, n—an inanimate surface or object inoculated
5.3 Thedesignofthecarriersmakesitpossibletoplaceonto
with the test organism.
each precisely measured volume of the test organism (10 µL)
3.1.2 eluate, n—an eluent, which contains the recovered
as well as the test formulation (50 µL).
organism(s).
3.1.3 eluent, n—any solution that is harmless to the test 5.4 The inoculum is placed at the centrer of each disk
whereas the volume of the test formulation covers nearly the
organism(s) and that is added to a carrier to recover the
organism(s) in or on it. entire disk surface thus eliminating the risk of any organisms
remaining unexposed to the test formulation.
3.1.4 neutralization, n—a process to quench the antimicro-
bial activity of a test formulation. This process may be
5.5 The relatively small ratio of 1:5 between the volume of
achieved by dilution of the organism/test formulation mixture
the inoculum and that of the test formulation closely reflects
and/or by adding to it one or more chemical neutralizers.
many field applications of liquid chemical germicides.
3.1.5 soil load, n—a solution of one or more organic, or
5.6 In all tests other than those against viruses, the addition
inorganic substances, or both, added to the suspension of the
of 9.95 mL of an eluent/diluent gives a 1:200 dilution of the
test organism to simulate the presence of body secretions,
test formulation immediately at the end of the contact time.
excretions, or other extraneous substances.
While this step in itself may be sufficient to arrest the
3.1.6 test formulation, n—a formulation that incorporates
germicidal activity of most formulations, the test protocol
antimicrobial ingredients.
permits the addition of a specific neutralizer to the eluent/
3.1.7 test organism, n—an organism that has characteristics
diluent, if required; the membrane filtration step also allows
that allows it to be readily identified. It also may be referred to
processing of the entire eluate from the test carriers and
as a surrogate or a marker organism.
therefore the capture and subsequent detection of even low
numbers of viable organisms that may be present. Subsequent
4. Summary of Test Method
rinsing of the membrane filters with normal saline also reduces
theriskofcarryinganyinhibitoryresiduesovertotherecovery
4.1 Each disk (1 cm in diameter) receives 10 µL of the test
organism with a soil load, dried, and is then placed on the medium. Confirmation of neutralization of the test formulation
isrequiredbychallengewithlownumbersofthetestorganism.
inside bottom surface of a sterile, 15 to 20-mL-capacity vial
prior to contact with 50 µLof the use-dilution of test substance
5.7 In tests against viruses, addition of 950 µL of Earle’s
(germicide). The contact time and temperature may vary as balanced salt solution (EBSS) at the end of the contact time
required. Control carriers receive 50 µL of a fluid harmless to
achieves a 1:20 dilution of the test formulation while keeping
the test organism(s). the volume of the eluate reasonably small to allow for the
4.2 For tests against vegetative bacteria, fungi, mycobacte-
titration of most or all of the eluate in cell cultures. Confirma-
ria and bacteria spores, the test substance is then diluted/ tion of neutralization of the test formulation is required by
neutralized, and the inoculum eluted. The eluate and subse-
challenge of a residual disinfection load with low numbers of
quent rinses are membrane filtered. Culture plates with the
infective units of the test virus. Since the virus assay system is
filters are incubated, colonies counted and log reductions are
indirect, an additional step is required to demonstrate that prior
calculated.
exposureoftheappropriatecelllinetoanyresidualdisinfectant
4.3 For tests with viruses, appropriate dilutions of the eluate
or disinfectant/neutralizer mixture does not interfere with the
are inoculated into suitable cell cultures, the cultures examined
detection of a low level of virus challenge.
for cytopathology/infectious foci and log calculated.
10 5.8 The soil load for use in this test is a mixture of three
types of proteins (high molecular weight proteins, low molecu-
5. Significance and Use
larweightpeptidesandmucousmaterial)designedtorepresent
the body secretions, excretions or other extraneous substances
5.1 The design of this test minimizes any loss of viable
thatchemicalgermicidesmayencounterunderfieldconditions.
organismsthroughwashoff,thusmakingitpossibletoproduce
It is suitable for working with all types of test organisms
statistically valid data using many fewer test carriers than
included here. The components of the soil load are readily
needed for methods based on simple most probable number
available and subject to much less variability than animal sera.
(MPN) estimates.
5.9 Ifdistilledwaterorotherdiluentisnottobespecifiedon
the product label, the diluent is assumed to be tap water. Since
the quality of tap water varies considerably both geographi-
Available from AOAC International, Washington, DC.
cally and temporally, this test method incorporates the use of
Available from the Canadian General Standards Board, Ottawa, Ontario,
Canada. water with a specified and documented level of hardness to
E2197–02
prepare use-dilutions of test products that require dilution in 6.17 Laminar Flow Cabinet, a Class II (Type A) biological
water before use.The U.S. Environmental ProtectionAgency’s safety cabinet for this work. The procedures for the proper
ScientificAdvisory Panel (SAP) on Germicide Test Methodol- maintenance and use of such cabinets are given in Ref (3).
ogy has recommended the use of water with a standard
6.18 Liquid Nitrogen Storage for Cells, a proper liquid
hardness of 400 ppm as CaCO .
3 nitrogen container and liquid nitrogen supply for cryopreser-
5.10 Depending on the label claim desired and the require-
vation of the stocks of cell lines.
mentsofthetargetregulatoryagency,additionaltestorganisms
6.19 Magnet, strong enough to hold the disk carrier in place
maybeused.Insuchcases,thedetailsoftheculturemediaand
in the glass vial while the liquid is being poured out of it for
conditions must be validated and clearly specified in test
membrane filtration.
reports.
6.20 Magnetic Stir Plate and Stir Bars, large enough for a
5-L beaker or Erlenmeyer flask for preparing culture media or
6. General Equipment and Labware
other solutions.
6.1 Air Displacement Pipettes, Eppendorf or equivalent,
6.21 Markers, permanent labware marking pens.
100 to 1000 µL with disposable tips.
6.22 Membrane Filtration System for Capture of the Test
6.2 Analytical Balance, to weigh chemicals and to standard-
Organisms other than Viruses, sterile 47-mm diameter mem-
ize inoculum delivery volumes by pipettes.
7 brane filters (0.22 or 0.45 µm pore diameter) and glass, plastic
6.3 Cell Culture Flasks and other plastic-ware for Viruses ,
2 or metal holders for such filters are required.
plastic cell culture flasks of 25 and 75-cm capacity for
6.23 pH Meter, to measure pH of buffers, eluents and test
culturing cells and for preparing virus pools; 12-well or
formulations.
96-well plastic plates for titrating virus infectivity.
6.4 Centrifuge, to allow for the sedimentation of the cells/
NOTE 1—The method described here uses conventional membrane
spores of the test organism(s) for concentration, or washing, or
filters. The system with hydrophobic grid membrane filters (HGMF) may
both.
also be used for this purpose (4).
6.5 Colony Counter, for example, Quebec Colony Counter.
6.24 Miscellaneous Laboratory Ware, pipette tips, plastic
6.6 Desiccator, recommended size is 25 cm wide by 20 cm
vials for storing cell and virus stocks, dilution tubes.
deep, with an active desiccant for drying the inocula on the
6.25 Orbital Shaker, for shaking the broth cultures of B.
carriers.
subtilis during their incubation.
6.7 Dissecting Microscope, for the screening of the metal
6.26 Petri Plates (Pyrex glass) 150 mm in Diameter, for
disks for damage to surface topography.
holding and autoclave sterilization of metal disks.
6.8 Environmental Chamber or Incubator, to hold the car-
riers at the desired test temperature.
6.27 Positive Displacement Pipette, a pipette and pipette
6.9 Filter Sterilization System for Media and Reagents,a
tips fitted with “plungers” that can dispense accurately 10-µL
membrane or cartridge filtration system (0.22 µm pore diam-
volumes for inoculation of carriers without the aerosol genera-
eter) is required for sterilizing heat-sensitive solutions.
tion that occurs when air displacement pipettes are used.
6.10 Forceps, straight or curved, (1) with smooth tips to
6.28 Refrigerator, a refrigerator at 4 6 2°C for storage of
handle membrane filters, and (2) to pick up the metal disk
media, culture plates and reagents.
carriers for placement in plastic vials.
6.29 Serological Pipettes, sterile reusable or single-use
6.11 Freezers, a freezer at -20 6 2°C is required for the
pipettes of 10.0, 5.0, and 1.0 mL capacity.
storage of media and additives. A second freezer at -70°C or
6.30 Spectrophotometer, for measuring turbidity of micro-
lower is required to store the stocks of test organisms.
bial suspensions.
6.12 Glassware, 1-L flasks with a side-arm and appropriate
6.31 Sterile Dispenser, 10 mL, for dispensing diluent/
tubing to capture the filtrates from 47-mm diameter membrane
eluent.
filters; 250-mL Erlenmeyer flasks for culture media.
6.32 Sterile Disposable Gloves, for handling the carriers.
6.13 Hemocytometer, for counting fungal conidia, and or in
the preparation of suitable cell numbers for seeding cell 6.33 Sterile Disposable Plastic Petri Dishes,100by15mm.
monolayers.
6.34 Sterile Polypropylene Centrifuge Tubes with Caps,50
6.14 Hot Air Ove
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