ASTM E2471-23

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Designation: E2471 − 05 (Reapproved 2016) E2471 − 23
Standard Test Method for
Using Seeded-Agar for the Screening Assessment of
Antimicrobial Activity In Carpets
This standard is issued under the fixed designation E2471; 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
Today’s modern commercial carpets (especially modular carpet tile) often incorporate antimicrobial
agents either in or on the face fibers or incorporated into the primary backing (attachment point of
carpet fiber to the backing structure). The American Association of Textile Chemists and Colorists
(AATCC) Method 174 permits both qualitative and quantitative antibacterial assessment and
antifungal assessment (qualitative only) of antimicrobial treatments in or on carpet. However, the
method is not suited for rapid screening of antimicrobials low in water solubility or that have slow
diffusion rates when incorporated into the carpet’s primary backing layer. The test method described
here provides a rapid screen of antimicrobial activity in or on carpets and allows for the simultaneous
assessment of multiple components of the carpet (not just the fibers).
1. Scope
1.1 This test method is designed to evaluate (qualitatively) the presence of antimicrobial activity in or on carpets. Use this test
method to qualitatively evaluate both antibacterial and antifungal activity.
1.2 Use half strength (nutrient and agar) tryptic soy agar as the inoculum vehicle for bacteria and half strength potato dextrose
agar as the inoculum vehicle for mold conidia. Use of half strength agars may reduce undue neutralization of an antimicrobial due
to excessive organic load.
1.3 This test method simultaneously evaluates (both visual and stereo-microscopic) antimicrobial activity both at the fiber layer
and at the primary backing layer of carpet.
1.4 Use this test method to assess the durability of the antimicrobial treatments on new carpets, and on those repeatedly
shampooed or exposed to in-use conditions.
1.5 Knowledge of microbiological techniques is required for the practice of this test method.
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 healthsafety, health, and environmental practices and determine
the applicability of regulatory limitations prior to use.
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 Nov. 1, 2016June 1, 2023. Published December 2016June 2023. Originally approved in 2005 as 2005. E2471 – 05.Last previous edition published
ɛ1
in 20112016 as E2471–05(2011)E2471 – 05 (2016). . DOI: 10.1520/E2471-05R16. 10.1520/E2471-23.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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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 Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 American Association of Textile Chemists and Colorists (AATCC) Standard:
Method 174-2007, Antimicrobial Activity Assessment of Carpets
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this test method, refer to Terminology E2756.
3.1.2 face fiber, n—the wear layer of the carpet; can be composed of nylon, polypropylene, wool, or other natural or synthetic
polymers. Typically, face fiber is tufted into a woven or non-woven scrim and then coated with latex to bond the face fiber securely
to the backing; this latex coated scrim forms the primary backing.
3.1.3 inoculum vehicle, n—carrier solution used to transport bacterial cells or mold conidia to the test substrate.
3.1.4 primary backing, n—the uppermost layer of carpet backing where carpet fiber bundles are physically attached at the base
to the backing structure. This layer is typically constructed of synthetic latex (ethylene vinyl acetate, styrene butadiene, or a
thermo-polymer; that is, ethylene vinyl acetate hot-melt adhesive).
3.1.5 seeded agar, n—a thin layer of molten (liquid) microbiological agar containing either bacterial cells or mold conidia (spores)
used to challenge a test substrate.
4. Summary of Test Method
4.1 Cut carpet samples into small rectangular pieces either via a carpet knife or mechanical die and press. Shave half of the face
fiber on each sample using electric hair clippers and arrange in sterile Petri dishes (typically with the shaven half of the sample
facing the center of the dish. Cool molten agars (full or partial complement) to 45 6 2°C45 °C 6 2 °C and inoculate with the
challenge bacteria or mold conidia. Following wrist action mixing, immerse samples into the seeded-molten agar, place into a Petri
dish and pour additional seeded agar into the dish to surround but not cover the test sample. Incubate the Petri dish for 24 to 72
h at 30 6 2°C.30 °C 6 2 °C. Visually and microscopically examine both at the face fiber and shaven (primary backing) layer for
inhibition of the challenge microorganisms. Report the presence of carpet surface inhibition (for low water soluble or slow
migrating antimicrobials) or zone of inhibition for water soluble antimicrobials.
5. Significance and Use
5.1 This test method provides for rapid screening of antimicrobial treatments located in or on the carpet face fiber or incorporated
into the backing structure of the carpet (or both).
5.2 This test method simulates actual use conditions that may occur on carpets (for example, food and beverage spills, soiling from
foot traffic, prolonged moisture exposure).
5.3 This test method provides a means to screen for activity and durability of an antimicrobial treatment under conditions of
organic loading.
5.4 This test method provides for the simultaneous assessment of multiple carpet components for antimicrobial activity.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Available from American Association of Textile Chemists and Colorists (AATCC), P.O. Box 12215, Research Triangle Park, NC 27709-2215, http://www.aatcc.org.
Technical Manual of the American Association of Textile Chemists and Colorists, 2000, Volume 75, American Association of Textile Chemists and Colorists.
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5.5 Carpets may be cleaned prior to testing with this test method in order to assess the durability of the antimicrobial effect.
6. Apparatus
6.1 Stereomicroscope, 10 to 70× objectives.
6.2 Erlenmeyer Flasks, 250 mL.
6.3 Sterile Petri Dishes, 150 mm.
6.4 Incubators, set at required temperatures (30 6 2°C and 37 6 2°C).(30 °C 6 2 °C and 37 °C 6 2 °C).
6.5 Autoclave.
6.6 Water Bath, capable of maintaining water at 45 6 2°C.45 °C 6 2 °C.
6.7 Test Tubes, 16 by 100 mm.16 mm by 100 mm.
6.8 Hot Plate with Stirrer.
6.9 Spectrophotometer.
6.10 Sterile Cuvettes.
6.11 Test Carpet.
6.12 Electric Hair Clippers (Oster Golden A5 or equivalent #30 Blade).
6.13 Canned Air (compressed air for surface dusting).
6.14 Sterile Petri Dishes, 100 mm.100 mm.
6.15 Carpet Knife (razor knife).
6.16 Mechanical Die (Optional), 2.5 by 3.8 cm.2.5 cm by 3.8 cm.
6.17 Hydraulic Press (Optional).
6.18 Sterile Funnel, with a glass wool plug.
6.19 Counting Chamber (hemocytometer).
6.20 Light Microscope, 10 and 40× objectives.
6.21 Disposable Examination Gloves.
7. Reagents
7.1 Media:
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7.1.1 Tryptic Soy Broth.
7.1.2 Tryptic Soy Agar.
7.1.3 Potato Dextrose Agar.
7.1.4 Sterile 0.85 % Saline, with 0.1 % polysorbate 80.
7.2 Test Organisms—Specific organisms are recommended; however, other microorganisms may be used to mimic those found in
a specific environment or those expected contaminants which may be present where the carpet is expected to perform.
7.2.1 Gram-positive bacteria Staphylococcus aureus ATCC 6538.
7.2.2 Gram-negative bacteria Serratia marcescens ATCC 14756.
7.2.3 Fungus Aspergillus niger ATCC 9642.
NOTE 1—Originally deposited as Aspergillus niger, the current ATCC designation is Aspergillus brasiliensis.
8. Procedure
8.1 Grow 18 hour tryptic soy broth cultures of Staphylococcus aureus at 37 6 2°C37 °C 6 2 °C and Serratia marcescens at 30
6 2°C.30 °C 6 2 °C. These cultures should originate from 18 to 24 h growth stock culture plates or agar slants. Origination from
glycerol stocks with two transfers is also permissible.
8.2 Prepare a suspension of fungal conidia by harvesting mature conidia from a 1 week old stock culture plate or slant grown at
30 6 2°C.30 °C 6 2 °C. Pour sterile 0.85 % saline with 0.1 % polysorbate 80 over the growth, agitate the liquid with a sterile glass
rod, and filter the hyphal fragments by pouring the suspension through a sterile funnel plugged with glass wool.
1 1
8.3 Prepare 200-mL200 mL lots of ⁄2 s
...