ASTM E2197-17e1

Standard Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals

SIGNIFICANCE AND USE
5.1 The design of this test eliminates any loss of viable organisms through wash off, thus making it possible to produce statistically 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 brushed stainless steel carrier surface, and the smaller ratio of test substance to surface area typical for many disinfectant applications. Thus, the test substance being assessed is presented with a reasonable challenge while allowing for efficient recovery of the test organisms from the inoculated carriers. 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 μL) as well as the control fluid or test substance (50 μL).  
5.4 The inoculum is placed at the center of each disk whereas the volumes of the test substance covers nearly the entire disk surface, 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 test substance immediately at the end of the contact time. While this step in itself may be sufficient to arrest the microbicidal activity of 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 also allows processing of the entire eluate from the test carriers and, therefore, the capture and subsequent detection of even low numbers of viable organisms that may be present. Subsequent rinsing of the membrane filters with saline also reduces the risk of carrying any inhibitory residues over to the recovery medium. Validation of the process of neutralization of the test substance is required by challenge with low numbers of the te...
SCOPE
1.1 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 brushed stainless steel that represent hard, nonporous environmental surfaces and medical devices. It is also designed to have survivors that can be compared to the 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 number of viable organisms in the test inoculum should be sufficiently high to take into account both the performance standard and the experimental variations in the results.  
1.2 The test protocol does not include any wiping or rubbing action. It is, therefore, not designed for testing 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 (8).  
1.4 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).  
1.5 In this test method, SI units are used for all applications, except for distance in which case inches are used and metric units follow.  
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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World T...

General Information

• Status: Published
• Publication Date: 30-Nov-2017
• ICS: 07.100.01 - Microbiology in general
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Replaces: ASTM E2197-17 - Standard Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals
• Effective Date: 01-Dec-2017
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Nov-2019
• Refers: ASTM E2756-18 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Apr-2018
• Refers: ASTM A967/A967M-17 - Standard Specification for Chemical Passivation Treatments for Stainless Steel Parts
• Effective Date: 01-Jul-2017
• Refers: ASTM A967/A967M-13 - Standard Specification for Chemical Passivation Treatments for Stainless Steel Parts
• Effective Date: 15-Feb-2013
• Refers: ASTM E2756-10 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-May-2010
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• Refers: ASTM D1129-06ae1 - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1129-06a - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM D1129-06 - Standard Terminology Relating to Water
• Effective Date: 15-Feb-2006
• Refers: ASTM D1129-04 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2004
• Refers: ASTM D1129-04e1 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2004
• Refers: ASTM D1129-03a - Standard Terminology Relating to Water
• Effective Date: 10-Aug-2003

Overview

ASTM E2197-17e1 is the Standard Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals. Developed by ASTM International, this widely recognized standard provides scientifically sound methods to evaluate the microbicidal efficacy of chemical disinfectants on hard, nonporous surfaces typically found in clinical, laboratory, and public settings.

The method utilizes stainless steel disk carriers, inoculated with specific amounts of test organisms, to realistically simulate environmental surfaces or medical devices. This approach generates statistically valid data with fewer test carriers and is compatible with a wide variety of antimicrobial formulations.

Key Topics

• Broad Spectrum Efficacy: The standard covers evaluation of disinfectants against vegetative bacteria, viruses, fungi, mycobacteria, and bacterial spores, addressing a wide range of pathogens of public health concern.
• Stainless Steel Disk Carriers: Brushed stainless steel disks reflect common nonporous surfaces, providing realistic and reproducible test conditions.
• Quantitative Data: The method enables accurate measurement of the reductions in viable organisms, yielding statistically robust results for assessing chemical efficacy.
• Soil Load Challenge: Incorporates a soil load simulating organic contamination (e.g., proteins and mucin), increasing test rigor and ensuring realistic evaluation.
• Precise Applications: Suitable for testing microbicidal properties of liquids, but not intended for evaluating wipes or disinfectant towelettes.
• Neutralization and Recovery: The protocol includes steps for neutralizing microbicidal activity and efficiently recovering survivors, enhancing accuracy and reducing false negatives.
• Regulatory Compliance: Can be adapted to support regulatory submissions, with recommendations to follow GLP (Good Laboratory Practices) when needed.

Applications

ASTM E2197-17e1 is applicable in settings where objective, reliable evidence of disinfectant effectiveness is required. Principal uses include:

• Healthcare Facilities: Validating surface disinfectants for hospitals, surgical suites, and clinics to prevent healthcare-associated infections.
• Medical Device Industry: Testing sterilants and disinfectants intended for nonporous medical equipment.
• Public Health Laboratories: Assessing disinfectants used in laboratories, including those with biosafety requirements.
• Industrial & Institutional Hygiene: Supporting manufacturers and users in selecting products for use in food processing, pharmaceutical manufacturing, schools, and public spaces.
• Product Development & Regulatory Approval: Providing data necessary for EPA or FDA submissions for antimicrobial product registration or claim substantiation.

Related Standards

Several related standards and references support or complement ASTM E2197-17e1, including:

• ASTM A967/A967M - Specification for Chemical Passivation Treatments for Stainless Steel Parts, relevant for preparing stainless disk carriers.
• ASTM D1129 & ASTM D1193 - Related to reagent water and water terminology, ensuring consistency in sample preparation.
• ASTM E2756 - Terminology specific to antimicrobial and antiviral agents, for correct usage in reporting.
• 21 CFR, Part 58 - Good Laboratory Practice for Nonclinical Laboratory Studies (FDA).
• 40 CFR, Part 160 - Good Laboratory Practice Standards (EPA).
• EN 10088-2 - Standards for stainless steel materials used for carrier disks.

Practical Value

Adopting ASTM E2197-17e1 enhances confidence in disinfectant performance claims by providing a reliable, rigorous framework for quantitative testing. It supports infection prevention efforts and regulatory compliance, contributing directly to the safety of healthcare, laboratory, and public environments.

Keywords: ASTM E2197-17e1, quantitative disk carrier test, disinfectant efficacy, microbicidal activity, bactericidal, virucidal, fungicidal, mycobactericidal, sporicidal, surface disinfection, stainless steel disk, environmental surfaces, regulatory compliance, ASTM standard, medical device cleaning, laboratory biosafety, public health disinfection.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
´1
Designation: E2197 − 17
Standard Quantitative Disk Carrier Test Method for
Determining Bactericidal, Virucidal, Fungicidal,
Mycobactericidal, and Sporicidal Activities of Chemicals
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.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
ε NOTE—Sections 8.1 and 11.8 were editorially corrected in January 2018.
INTRODUCTION
The quantitative test method described here uses disks of stainless steel (1 cm in diameter) as
carriers. It employs the same basic set of materials and procedures to assess the ability of liquid
chemicals to inactivate vegetative bacteria, viruses, fungi, mycobacteria, and bacterial spores (1-7).
Performancestandardsfortestsubstances,thelevelofwaterhardness,thetypeandlevelofasoilload,
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 with financial support from the
Antimicrobials Division of the U.S. Environmental Protection Agency.
1. Scope required and to follow them where appropriate (40 CFR, Part
160 for EPA submissions and 21 CFR, Part 58 for FDA
1.1 This test method is designed to evaluate the ability of
submissions).
test substances to inactivate vegetative bacteria, viruses, fungi,
1.5 Inthistestmethod,SIunitsareusedforallapplications,
mycobacteria, and bacterial spores (1-7) on disk carriers of
except for distance in which case inches are used and metric
brushed stainless steel that represent hard, nonporous environ-
units follow.
mentalsurfacesandmedicaldevices.Itisalsodesignedtohave
survivors that can be compared to the mean of no less than
1.6 This standard does not purport to address all of the
three control carriers to determine if the performance standard
safety concerns, if any, associated with its use. It is the
has been met. For proper statistical evaluation of the results,
responsibility of the user of this standard to establish appro-
the number of viable organisms in the test inoculum should be
priate safety, health, and environmental practices and deter-
sufficiently high to take into account both the performance
mine the applicability of regulatory limitations prior to use.
standard and the experimental variations in the results.
1.7 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.2 Thetestprotocoldoesnotincludeanywipingorrubbing
ization established in the Decision on Principles for the
action. It is, therefore, not designed for testing wipes.
Development of International Standards, Guides and Recom-
1.3 This test method should be performed by persons with
mendations issued by the World Trade Organization Technical
traininginmicrobiologyinfacilitiesdesignedandequippedfor
Barriers to Trade (TBT) Committee.
work with infectious agents at the appropriate biosafety level
(8).
2. Referenced Documents
1.4 It is the responsibility of the investigator to determine
2.1 ASTM Standards:
whether Good Laboratory Practice Regulations (GLPs) are
A967/A967MSpecification for Chemical Passivation Treat-
ments for Stainless Steel Parts
D1129Terminology Relating to Water
D1193Specification for Reagent Water
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Dec. 1, 2017. Published December 2017. Originally
approved in 2002. Last previous edition approved in 2011 as E2197–11. DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/E2197-17E01. contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
The boldface numbers in parenthesis refer to the list of references at the end of Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
´1
E2197 − 17
E2756Terminology Relating toAntimicrobial andAntiviral 4.3 Fortestswithviruses,appropriatedilutionsoftheeluate
Agents are inoculated into suitable cell cultures, the cultures are
2.2 CFR Standard: examined for cytopathology/infectious foci, which are esti-
21 CFR, Part 58Laboratory Practice for Nonclinical Labo- mated as the most probable number (MPN) or counted as foci
ratory Studies or plaques, and log are calculated.
40 CFR, Part 160Good Laboratory Practice Standards
2.3 CEN Standard:
5. Significance and Use
EN 10088-2 1J/2JStainless steels - Part 2: Technical deliv-
5.1 The design of this test eliminates any loss of viable
ery conditions for sheet/plate and strip of corrosion
organismsthroughwashoff,thusmakingitpossibletoproduce
resisting steels for general purposes
statistically valid data using many fewer test carriers than
needed for methods based on simple MPN estimates.
3. Terminology
3.1 Definitions—Fordefinitionsofgeneraltermsusedinthis 5.2 Thestringencyinthetestisprovidedbytheuseofasoil
test method, refer to Terminology E2756. load,themicrotopographyofthebrushedstainlesssteelcarrier
surface, and the smaller ratio of test substance to surface area
3.2 Definitions of Terms Specific to This Standard:
typical for many disinfectant applications. Thus, the test
3.2.1 carrier, n—an inanimate surface or object inoculated
substance being assessed is presented with a reasonable chal-
with the test organism.
lenge while allowing for efficient recovery of the test organ-
3.2.2 eluate, n—an eluent, which contains the recovered
isms from the inoculated carriers. The metal disks in the basic
organism(s).
test are also compatible with a wide variety of actives.
3.2.3 eluent, n—any solution that is harmless to the test
5.3 Thedesignofthecarriersmakesitpossibletoplaceonto
organism(s) and that is added to a carrier to recover the
eachapreciselymeasuredvolumeofthetestorganism(10µL)
organism(s) in or on it.
as well as the control fluid or test substance (50 µL).
3.2.4 neutralization, n—a process to quench the antimicro-
5.4 The inoculum is placed at the center of each disk
bial activity of a test substance. This process may be achieved
whereas the volumes of the test substance covers nearly the
by dilution of the organism/test substance mixture and/or by
entire disk surface, thus virtually eliminating the risk of any
adding to it one or more chemical neutralizers.
organisms remaining unexposed.
3.2.5 soil load, n—a solution of one or more organic, or
inorganic substances, or both, added to the suspension of the
5.5 Inalltests,otherthanthoseagainstviruses,theaddition
test organism to simulate the presence of body secretions,
of 10 mLof an eluent/diluent gives a 1:200 dilution of the test
excretions, or other extraneous substances.
substance immediately at the end of the contact time. While
this step in itself may be sufficient to arrest the microbicidal
3.2.6 test organism, n—an organism that has characteristics
activity of most actives, the test protocol permits the addition
that allow it to be readily identified. It also may be referred to
of a specific neutralizer to the eluent/diluent, if required.
as a surrogate, a simulant, or a marker organism.
Except for viruses, the membrane filtration step also allows
3.2.7 test substance, n—a formulation that incorporates
processing of the entire eluate from the test carriers and,
antimicrobial ingredients.
therefore, the capture and subsequent detection of even low
4. Summary of Test Method numbers of viable organisms that may be present. Subsequent
rinsingofthemembranefilterswithsalinealsoreducestherisk
4.1 Each disk (1 cm in diameter) receives 10 µL of the test
of carrying any inhibitory residues over to the recovery
organism with a soil load. The inoculum is dried, and then the
medium. Validation of the process of neutralization of the test
disk is placed on the inside bottom surface of a sterile plastic
substanceisrequiredbychallengewithlownumbersofthetest
vial prior to contact with 50 µL of the use-dilution of test
organism.
substance. The contact time and temperature may vary as
required. Control carriers receive 50 µL of a fluid harmless to
5.6 Intestsagainstviruses,additionof1mLofbufferatthe
the test organism(s) and its host cells, if any, but are otherwise
end of the contact time achieves a 1:20 dilution of the test
treated in the same way as test carriers.
substance while keeping the volume of the eluate reasonably
smalltoallowforthetitrationofmostoralloftheeluateincell
4.2 For tests against vegetative bacteria, fungi,
cultures. Confirmation of neutralization of the test substance is
mycobacteria, and bacterial spores, the test substance is then
required by challenge of a residual disinfection load with low
neutralizedandtheinoculumeluted.Theeluateandsubsequent
numbers of infective units of the test virus. Since the virus
rinses of the carrier and its vial are membrane filtered. Culture
assay system is indirect, an additional step is required to
plateswiththefiltersareincubated,coloniescounted,andlog
demonstrate that prior exposure of the appropriate cell line to
reductions calculated.
any residual disinfectant or disinfectant/neutralizer mixture
does not interfere with the detection of a low level of virus
AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
challenge (See Appendix).
732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
NOTE1—In5.5and5.6,volumesof10mLand1mLarerecommended
www.access.gpo.gov.
Available from European Committee for Standardization (CEN), Avenue instead of 9.95 mL and 950 µL, respectively, for ease of dispensing the
Marnix 17, B-1000, Brussels, Belgium, http://www.cen.eu. eluent.
´1
E2197 − 17
5.7 The soil load in this test is a mixture of three types of 6.10 Forceps, straight or curved, (1) with smooth tips to
proteins (high molecular weight proteins, low molecular handle membrane filters, and (2) to pick up the metal disk
weight peptides, and mucous material) designed to represent carriers for placement in plastic vials.
bodysecretions,excretions,orotherextraneoussubstancesthat
6.11 Freezers, a freezer at –20 6 2°C is required for the
microbicidal chemicals may encounter under field conditions.
storage of media and additives. A second freezer at –70°C or
It is suitable for working with all types of test organisms
lower is required to store the stocks of test organisms.
included here. The components of the soil load are readily
6.12 Glassware, 1-L flasks with a side-arm and appropriate
available and subject to much less variability than animal sera.
tubing to capture the filtrates from 47-mm diameter membrane
5.8 Ifdistilledwaterorotherdiluentisnottobespecifiedon
filters; 250-mL Erlenmeyer flasks for culture media.
the product label, the diluent for the test substance is assumed
to be tap water. Since the quality of tap water varies consid- 6.13 Hemocytometer,forcountingfungalconidia,and/orfor
use in the preparation of suitable cell numbers for seeding
erably both geographically and temporally, this test method
incorporates the use of water with a specified and documented monolayers.
level of hardness to prepare use-dilutions of test substance that
6.14 Hot Air Oven, an oven at 60°C to dry clean and sterile
require dilution in water before use. While water with a
glassware.
hardness of at least 300 ppm as CaCO is recommended
6.15 Incubators, an ordinary incubator, an anaerobic
consult local regulations regarding use of hard water prior to
incubator, and a CO incubator to incubate cell cultures in a
testing. 2
5% CO atmosphere. If only one ordinary incubator is
5.9 The Annex contains a list of those organisms that are
available,itstemperaturewillrequireadjustmentdependingon
often used in assessing the microbicidal activities of disinfec-
the type of organism under test.
tants for use on environmental surfaces or medical devices.
6.16 Inverted Microscope,aninvertedmicroscopewith10×
Culture conditions for each organism are also included in the
eyepiece and 5×, 10×, and 40× objectives to examine cell
Annex. Depending on the label claim(s) desired and the
cultures.
requirements of the target regulatory agency, one or more of
the organisms listed may be selected for the testing. If
6.17 Laminar Flow Cabinet, a Class II (TypeA) biological
organisms other than those listed are to be used (for example,
safety cabinet. The procedures for the proper maintenance and
inthedairyorbrewingindustries),aclearjustificationmustbe
use of such cabinets are given in Ref (8).
provided and details of the culture media and growth condi-
6.18 Liquid Nitrogen Storage for Cells, a proper liquid
tions must be validated and clearly specified in test reports.
nitrogen container and liquid nitrogen supply for cryopreser-
vation of the stocks of cell lines.
6. General Equipment and Labware
6.19 Magnetic Stir Plate and Stir Bars, large enough for a
6.1 Air Displacement Pipettes, Eppendorf or equivalent,
5-L beaker or Erlenmeyer flask for preparing culture media or
100 to 1000 µL with disposable tips.
other solutions.
6.2 Analytical Balance,toweighchemicalsandtostandard-
6.20 Markers, for permanent marking of labware.
ize inoculum delivery volumes by pipettes.
6.21 Membrane Filtration System for Capture of the Test
6.3 Cell Culture Flasks and Other Plastic-ware for Viruses,
Organisms other than Viruses, sterile 47-mm diameter mem-
(see Note 2) plastic cell culture flasks of 25- and 75-cm
brane filters (0.22- or 0.45-µm pore diameter) and glass,
capacity for culturing cells and for preparing virus pools;
plastic, or metal holders for such filters are required.
12-well or 96-well plastic plates for titrating virus infectivity.
NOTE 2—Plastic culture ware may be purchased from most laboratory
6.22 pH Meter, to measure pH of buffers, eluents, and test
supply houses.
formulations.
6.4 Centrifuge, to allow for the sedimentation of the cells/
6.23 Microwave Oven, to melt agar overlays.
sporesofthetestorganism(s)forconcentration,orwashing,or
both.
6.24 Miscellaneous Laboratory Ware, pipette tips, plastic
vials for storing cell and viral stocks, dilution tubes.
6.5 Colony Counter, for example, Quebec Colony Counter.
6.25 Orbital Shaker, for shaking the broth cultures of
6.6 Desiccator, recommended size is 25 cm wide by 20 cm
Bacillus subtilis during their incubation.
deep, with an active desiccant for drying the inocula on the
carriers.
6.26 Petri Plates (Pyrex glass) 150 mm in diameter, for
holding and autoclave sterilization of metal disks.
6.7 Dissecting Microscope, for the screening of the metal
disks for damage to surface topography.
6.27 Positive Displacement Pipette, a pipette and pipette
tips fitted with “plungers” that can accurately dispense 10-µL
6.8 Environmental Chamber or Incubator, to hold the car-
riers at the desired test temperature. volumes for inoculation of carriers without the aerosol genera-
tion that occurs when air displacement pipettes are used.
6.9 Filter Sterilization System for Media and Reagents, a
membrane or cartridge filtration system (0.22-µm pore diam- 6.28 Refrigerator, a refrigerator at 4 6 2°C for storage of
eter) is required for sterilizing heat-sensitive solutions. media, culture plates and reagents.
´1
E2197 − 17
6.29 Serological Pipettes, sterile reusable or single-use supplements will vary depending on the cell line used. Eagle’s
pipettes of 10.0, 5.0, and 1.0 mL capacity. minimal essential medium (EMEM) with 5 to 10% fetal
bovine serum is used for growing a wide variety of cells.
6.30 Spectrophotometer, for measuring turbidity of micro-
Pleaserefertoothersourcesforfurtherdetailsonworkingwith
bial suspensions.
cellcultures (13)andviruses (14)andforpreparingviruspools
6.31 Sterile Dispenser,10mL,fordispensingdiluent/eluent.
to be used in virucidal tests (15).
6.32 Sterile Disposable Gloves, for handling the carriers.
NOTE 5—Material and reagents for cell culture and virology may be
purchased from biological supply houses.
6.33 Sterile Disposable Plastic Petri Dishes,100by15mm.
7.5 Phosphate Buffered Stock Solution—To prepare a stock
6.34 Sterile Polypropylene Centrifuge Tubes with Caps,
solution of phosphate buffer, dissolve 34.0 g of potassium
50-mL.
dihydrogen phosphate (KH PO ) in 500 mL of water. Adjust
2 4
6.35 Sterilizer, any steam sterilizer suitable for processing
pH to 7.2 6 0.2 with 0.1 N NaOH or 0.1 N HCl and bring to
culture media, reagents, and labware is acceptable. The steam
1000 mL with deionized water.
supplied to the sterilizer must be free from additives toxic to
7.6 Phosphate Buffered Saline (PBS),tobeusedasadiluent
the test organisms or cell cultures.
andwashforallorganismsexceptviruses;topreparePBS,add
6.36 Timer, any stopwatch that can be read in minutes and
1.25 mL of the stock solution and 8.75 g of NaCl to a
seconds.
volumetric flask, fill with deionized water to the 1000 mL
6.37 Vacuum Source,avacuumpump,accesstoanin-house
mark, and mix; adjust pH to 7.2 6 0.2, if necessary. Sterilize
vacuum line or a water faucet vacuum apparatus required to
by filtration or autoclaving.
pull the samples through the membrane filters.
7.7 Trypsin (1:250) for Work with Rotaviruses, to be added
6.38 Vials (Glass), wide-mouth, 20-mL, for use as dilution
atafinalconcentrationof5µg/mLtomaintenancemediawhen
vials.
making rotavirus pools or assaying for their infectivity.
NOTE 6—Trypsin preparations can vary in strength depending on the
6.39 Vials (Nalgene), wide-mouth, 30-mL, for holding the
6 supplier and the degree of purity, and the concentration specified here is
inoculated carriers to be exposed to the test formulation.
onlyaguide.Preliminarytestingmayberequiredtodeterminetheoptimal
concentration for the specific type of product being used.
6.40 Vortex Mixer, to vortex the eluate and rinsing fluid in
the carrier vials to ensure efficient recovery of the test
7.8 Test Substance, prepared at its use-dilution and brought
organism(s).
tothetesttemperature.Thenumberoflotsofthetestsubstance
NOTE 3—The method described here uses conventional membrane
tobeevaluated,andwhetheroneormoreofthemisagedornot
filters. The system with hydrophobic grid membrane filters (HGMF) may
to simulate the shelf-life to be claimed, will depend on the
also be used for this purpose (9).
target regulatory agency.
NOTE4—Itisimportanttoanalyzethewholesampletodetectandcount
anysurvivorsandensureconfidenceinthedatagenerated.Forthisreason,
7.9 Growth, Recovery Media, and Media Supplements, the
membrane filtration is usually superior to pour-plating or spread-plating,
required types of materials (see below) can be purchased from
whichnormallycanprocessonlyasmallfractionofthevolumesofeluates
in this method. a variety of sources specializing in laboratory supplies.
7.10 MnSO ·H O, added to diluted Columbia broth to
4 2
7. General Solutions and Reagents
promote B. subtilis sporulation.
7.1 Purity of Reagents, Reagent grade chemicals shall be
7.11 Test Substance Diluent, for test substances requiring
used in all tests. Unless otherwise indicated, it is intended that
dilution to obtain a use-dilution, water with a standardized and
all reagents conform to the specifications of the Committee on
specified level of hardness, as CaCO , shall be used as the
Analytical Reagents of the American Chemical Society (10). 3
diluent.
7.2 Other chemical grades may be used, provided it is first
7.12 Deionized Distilled Water (DDW), or equivalent high-
ascertained that the reagent is of sufficiently high purity to
quality water, for making reagent solutions and media. (See
permit its use without lessening the accuracy of the determi-
Terminology D1129 and Specification D1193.)
nation. For information on the testing of reagents not listed by
the American Chemical Society, see (11) and (12).
7.13 Plates of Recovery Media for Bacteria and Fungi,
media must be prepared and sterilized according to manufac-
7.3 AbsoluteAlcohol,ina100-mLplasticorglassbeakerfor
turer’s instructions and then aseptically dispensed into culture
flame-sterilization of metallic forceps used to handle mem-
plates.Sterilityandgrowthpromotionchecksofmediabatches
brane filters.
should always be performed as the included negative and
7.4 Cell Culture Media and Supplements for Working with
positive controls.
Viruses—(seeNote5)Culturemediaandthetypesandratiosof
7.14 Earle’s Balanced Salt Solution (EBSS), pH of 7.2 to
7.4. To be used as diluent and wash for virus titration.
Thesolesourceofsupplyoftheapparatus(Nalgenevials,Catalog#2118-0001)
known to the committee at this time is Nalge Nunc International, 75 Panorama
7.15 Tryptone, Bovine Serum Albumin (BSA), and Bovine
Creek Dr., Rochester, N.Y. 14625-2385. If you are aware of alternative suppliers,
Mucin,thethreeingredientsforthesoilload(Section9)canbe
please provide this information to ASTM International Headquarters. Your com-
purchasedfromavarietyofchemicalsuppliers.Thesamelevel
ments will receive careful consideration at a meeting of the responsible technical
committee, which you may attend. of yeast extract may be used in place of Tryptone.
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7.16 Eluent, PBS with 0.1% (w/v) Tween-80. The eluent screening under a dissecting microscope at a magnification of
maycontainadditionalingredientstoneutralizetheactive(s)in at least 20×. Discard those with visible damage to surface
the test substance. topography.
8.2 Preparation of the Carriers—Place a sheet of filter
8. Carriers
paper on the inside bottom surface of a glass petri dish (150
8.1 Stainless Steel Disks (1 cm in diameter and approxi-
mm in diameter) and lay out up to 20 clean disks on it.
mately 0.7 mm thick)—The disks are prepared from sheets of
Autoclave (45 min at 121°C) to sterilize the disks.
brushed stainless steel (AISI type 304). See Note 7.Aground
unidirectional finish obtained with 150 grit abrasive (AISI)
9. Soil Load
meeting the specifications of a No. 4 Finish (EN 10088-2
9.1 Thesoilloadtobeincorporatedinthesuspensionofthe
1J/2J)ispresentonbothsidesofthecarrierwithroundededges
test organism will consist of a mixture of the following stock
on the top side. The carriers should be passivated according to
solutions in PBS (pH 7.2):
Specification A967/A967M which requires soaking in a mild
acid bath (7% citric acid) for at least 20 min to remove any
9.2 Add0.5gofTryptoneoryeastextractto10mLofPBS.
impurities and accumulated debris from the disk surface. To
9.3 Add 0.5 g of BSA to 10 mL of PBS.
remove punching burrs from the edges of the discs, tumble in
a barrel together with ceramic chips and a cleanser. 9.4 Add 0.04 g of bovine mucin to 10 mL of PBS.
9.5 Prepare the solutions separately and sterilize by passage
NOTE 7—Initially 430 stainless steel was recommended and used in the
developmentofthemethod.While430steelmaystillbeusefulwithmany
through a 0.22 µm pore diameter membrane filter, aliquot, and
chemistries,recentdatahasshownapotentialincompatibilitywithcertain
store at either 4 6 2°C or –20 6 2°C.
formulations and diluents leading to corrosion of the 430 steel and
interferencewithefficacy.Useof304steelwillavoidthiscorrosioneffect.
9.6 To obtain 500 µL of the inoculum, add 25 µL of BSA,
100 µLof mucin, and 35 µLof Tryptone or yeast extract stock
8.1.1 Newdisksshouldbesoakedinadetergentsolutionfor
to 340 µL of the microbial suspension.
atleastonehourtodegreasethemandtheycanthenbewashed
NOTE 8—Animal sera, often used as a soil load, vary widely in their
andsterilizedbyautoclaving.Theycaneitherbeusedonceand
composition and may also contain microbial inhibitors. The soil load
discarded or used repeatedly with proper cleaning and steril-
mixture given above contains a level of protein roughly equal to that in
ization in between. Avoid extended soaking of the disks in
5% serum. Preliminary screening of albumin and mucin is recommended
water or aggressive chemicals to reduce risk of corrosion or
to ensure compatibility with test organism(s).
rusting.
8.1.2 If disks are to be reused, check each disk for pitting,
10. Preparation of Inocula
rust, other damage or accumulated debris before use by
10.1 This test method can be used with most species of
vegetative and spore-forming bacteria, viruses, fungi, and
mycobacteria. The Appendix lists the organisms most often
7 used. The number of CFU/mL of each freshly prepared and
The sole commercial source of supply of the stainless steel disks known to the
committee at this time is Catalog P/N 304-107 from Pegen Industries, Inc. (#2-200 homogenizedmicrobialtestsuspension,exceptviruses,maybe
Iber Road, Ottawa, ON Canada K2SOL5; www.pegenindustries.com), but most
estimated spectrophotometrically, based on a standard curve at
competent machine shops could prepare such discs from the specifications. If you
a specific wavelength, but should be confirmed by titration
are aware of alternative suppliers, please provide this information to ASTM
using membrane filtration or alternative methods.
Headquarters.Your comments will receive careful consideration at a meeting of the
responsible technical committee, which you may attend.
NOTE 1—(A) Stainless disk inoculated with 10 µL of the test suspension, (B) The disk with the dried inoculum placed at the bottom of the vial, and
(C) The disk with 50 µL of the test formulation over the dried inoculum.
FIG. 1 Inoculation and Handling of Stainless Steel Disks for Quantitative Carrier Test
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10.2 The concentration of the viable test organisms in the throughout the inoculation of a batch of carriers). Cover the
driedinoculumshouldbehighenoughtomeettherequiredtest petri plate with its lid.
substance performance criterion. In general, this number
11.2 Drying of the Inoculated Carriers—Place the petri
shouldnotbemorethan10×thedefinedperformancestandard.
plate inside a desiccator and remove the lid of the petri plate.
This should be confirmed in each test by determining the
Cover the desiccator and make sure that it is properly sealed.
numbers of viable organisms on the control carriers.
Attachtheoutletofthedesiccatortoavacuumsourceandstart
11. Carrier Test (see Fig. 2)
evacuationoftheairtoachieveavacuumof20-25in.mercury
(508-635 torr; 677-847 mbar; 68000-85000 Pascal). Leave the
11.1 Vortex the test suspension to evenly distribute cells,
disks in the evacuated desiccator at room temperature for two
spores, or virus particles. Withdraw 10 µL of the suspension
hours to dry.
with a positive displacement pipette and place it at the center
of each sterilized disk carrier without spreading the inoculum NOTE 9—To avoid contamination of carriers with other organisms, it is
recommended to dedicate a desiccator for virus work only.
(Fig. 1A). (For consistency, the same pipette tip can be used
NOTE 1—*For spread-plating, the eluate is subjected to 10-fold dilutions as required and 0.1 mL of appropriate dilutions is separately spread on the
surface of a suitable recovery medium, and incubated.
FIG. 2 Flow Chart Summary of Main Steps in Disk Carrier Test
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the challenge organism(s) under the specific test conditions. To obtain a
11.3 At the end of the drying period, remove the petri plate
more precise indication of log reduction in viability by the test
from the desiccator, cover the plate and observe the dried
substance, assay of additional ten-fold dilutions of the eluates may be
inoculum on each carrier. Discard any carrier in which the
necessary (see Fig. 2).
inoculum has run off the surface.
NOTE 12—Separate filters, but the same filtration unit, can be used for
agivencarrierprovidingthedilutionsarefilteredinorderstartingwiththe
11.4 Carefully pick up each disk and place it, with the
most dilute.
inoculated side up, on the inside bottom surface of a Nalgene
vial (Fig. 1B). These carriers are now ready for the test 12. Additional Controls in Virucidal Tests
procedure.
12.1 The need for cell cultures when working with viruses
NOTE10—Useatleastthreecarriersascontrolsineachtestandatleast
requires the incorporation of additional controls because either
five test carriers for each lot of the test substance.
the test substance or the neutralizer or a combination of both
11.5 Exposure of the Organism(s) to the Test Substance—
could alter the susceptibility of host cells to the virus under
Cover the dried inoculum on each disk carrier with 50 µL of
test.These controls must be run initially at least once and may
test substance (Fig. 1C) and hold the carriers at the desired
not be included in subsequent tests as long as the same cell
temperature for the recommended contact period. Immediately
line, virus, test substance and neutralizer are being used for
attheendofthecontacttime,add10mLoftheeluentaloneor
testing. The Appendix provides the details on these controls.
with a suitable neutralizer for all organisms except for viruses.
13. Calculating Log Reductions
When testing for virucidal activity, add 1.0 mL of the eluent
13.1 A method for determining log reductions in the
alone or with a suitable neutralizer to each vial containing the
viability titer of th
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