Designation: E2471 − 05 (Reapproved 2011)´1

Standard Test Method for

Using Seeded-Agar for the Screening Assessment of
Antimicrobial Activity In Carpets1
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.

´1 NOTE—Editorial changes were made throughout in April 2011.

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 priate safety and health practices and determine the applica-
1.1 This test method is designed to evaluate (qualitatively) bility of regulatory limitations prior to use.
the presence of antimicrobial activity in or on carpets. Use this 2. Referenced Documents
test method to qualitatively evaluate both antibacterial and
antifungal activity. 2.1 American Association of Textile Chemists and Colorists
(AATCC) Standard:
1.2 Use half strength (nutrient and agar) tryptic soy agar as Method 174-2007, Antimicrobial Activity Assessment of
the inoculum vehicle for bacteria and half strength potato Carpets2
dextrose agar as the inoculum vehicle for mold conidia. Use of
half strength agars may reduce undue neutralization of an 3. Terminology
antimicrobial due to excessive organic load. 3.1 Definitions:
1.3 This test method simultaneously evaluates (both visual 3.1.1 face fiber, n—the wear layer of the carpet; can be
and stereo-microscopic) antimicrobial activity both at the fiber composed of nylon, polypropylene, wool, or other natural or
layer and at the primary backing layer of carpet. synthetic polymers. Typically, face fiber is tufted into a woven
1.4 Use this test method to assess the durability of the or non-woven scrim and then coated with latex to bond the face
antimicrobial treatments on new carpets, and on those repeat- fiber securely to the backing; this latex coated scrim forms the
edly shampooed or exposed to in-use conditions. primary backing.
1.5 Knowledge of microbiological techniques is required 3.1.2 inoculum vehicle, n—carrier solution used to transport
for the practice of this test method. bacterial cells or mold conidia to the test substrate.
1.6 This standard does not purport to address all of the 3.1.3 primary backing, n—the uppermost layer of carpet
safety concerns, if any, associated with its use. It is the backing where carpet fiber bundles are physically attached at
responsibility of the user of this standard to establish appro- 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).
1
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides, Antimicrobials, and Alternative Control Methods and is the direct
2
responsibility of Subcommittee E35.15 on Antimicrobial Agents. Available from American Association of Textile Chemists and Colorists
Current edition approved March 1, 2011. Published April 2011. Originally (AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http://
approved in 2005 as E2471 – 05. DOI: 10.1520/E2471-05R11E01. www.aatcc.org.

Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States

1
 E2471 − 05 (2011)´1
3.1.4 seeded agar, n—a thin layer of molten (liquid) micro- 6.11 Test Carpet.
biological agar containing either bacterial cells or mold conidia 6.12 Electric Hair Clippers (Oster Golden A5 or equivalent
(spores) used to challenge a test substrate. #30 Blade).
4. Summary of Test Method 6.13 Canned Air (compressed air for surface dusting).
4.1 Cut carpet samples into small rectangular pieces either 6.14 Sterile Petri Dishes, 100 mm.
via a carpet knife or mechanical die and press. Shave half of 6.15 Carpet Knife (razor knife).
the face fiber on each sample using electric hair clippers and
arrange in sterile Petri dishes (typically with the shaven half of 6.16 Mechanical Die (Optional), 2.5 by 3.8 cm.
the sample facing the center of the dish. Cool molten agars (full 6.17 Hydraulic Press (Optional).
or partial complement) to 45 6 2°C and inoculate with the 6.18 Sterile Funnel, with a glass wool plug.
challenge bacteria or mold conidia. Following wrist action
mixing, immerse samples into the seeded-molten agar, place 6.19 Counting Chamber (hemocytometer).
into a Petri dish and pour additional seeded agar into the dish 6.20 Light Microscope, 10 and 40× objectives.
to surround but not cover the test sample. Incubate the Petri 6.21 Disposable Examination Gloves.
dish for 24 to 72 h at 30 6 2°C. Visually and microscopically
examine both at the face fiber and shaven (primary backing) 7. Reagents
layer for inhibition of the challenge microorganisms. Report
the presence of carpet surface inhibition (for low water soluble 7.1 Media:
or slow migrating antimicrobials) or zone of inhibition for 7.1.1 Tryptic Soy Broth.
water soluble antimicrobials.3 7.1.2 Tryptic Soy Agar.
7.1.3 Potato Dextrose Agar.
5. Significance and Use 7.1.4 Sterile 0.85 % Saline, with 0.1 % polysorbate 80.
5.1 This test method provides for rapid screening of anti- 7.2 Test Organisms—Specific organisms are recommended;
microbial treatments located in or on the carpet face fiber or however, other microorganisms may be used to mimic those
incorporated into the backing structure of the carpet (or both). found in a specific environment or those expected contami-
nants which may be present where the carpet is expected to
5.2 This test method simulates actual use conditions that
perform.
may occur on carpets (for example, food and beverage spills,
7.2.1 Gram-positive bacteria Staphylococcus aureus ATCC
soiling from foot traffic, prolonged moisture exposure).
6538.
5.3 This test method provides a means to screen for activity 7.2.2 Gram-negative bacteria Serratia marcescens ATCC
and durability of an antimicrobial treatment under conditions 14756.
of organic loading. 7.2.3 Fungus Aspergillus niger ATCC 9642.
5.4 This test method provides for the simultaneous assess- NOTE 1—Originally deposited as Aspergillus niger, the current ATCC
ment of multiple carpet components for antimicrobial activity. designation is Aspergillus brasiliensis.
5.5 Carpets may be cleaned prior to testing with this test
8. Procedure
method in order to assess the durability of the antimicrobial
effect. 8.1 Grow 18 hour tryptic soy broth cultures of Staphylococ-
cus aureus at 37 6 2°C and Serratia marcescens at 30 6 2°C.
6. Apparatus These cultures should originate from 18 to 24 h growth stock
6.1 Stereomicroscope, 10 to 70× objectives. culture plates or agar slants. Origination from glycerol stocks
with two transfers is also permissible.
6.2 Erlenmeyer Flasks, 250 mL.
8.2 Prepare a suspension of fungal conidia by harvesting
6.3 Sterile Petri Dishes, 150 mm.
mature conidia from a 1 week old stock culture plate or slant
6.4 Incubators, set at required temperatures (30 6 2°C and grown at 30 6 2°C. Pour sterile 0.85 % saline with 0.1 %
37 6 2°C). polysorbate 80 over the growth, agitate the liquid with a sterile
6.5 Autoclave. glass rod, and filter the hyphal fragments by pouring the
suspension through a sterile funnel plugged with glass wool.
6.6 Water Bath, capable of maintaining water at 45 6 2°C.
8.3 Prepare 200-mL lots of 1⁄2 strength tryptic soy and 1⁄2
6.7 Test Tubes, 16 by 100 mm. strength potato dextrose agars in 250-mL Erlenmeyer flasks
6.8 Hot Plate with Stirrer. and autoclave. Cool the molten agars to 45 6 2°C in a water
6.9 Spectrophotometer. bath.
6.10 Sterile Cuvettes. 8.4 Cut carpet samples (minimum of duplicate carpet
samples for each challenge organism) 2.5 by 3.8 cm.
3
8.5 With gloved hands, aseptically shear half of the fiber
Technical Manual of the American Association of Textile Chemists and
Colorists, 2000, Volume 75, American Association of Textile Chemists and from the 3.8-cm length of carpet using electric clippers. The
Colorists. sheared half of the carpet sample should have fibers no longer

2
 E2471 − 05 (2011)´1
than 2 6 1 mm. Use canned air to remove the loose fibers from layer and the shaven primary backing layer of the carpet
each carpet sample after shaving. samples for evidence of bacterial or fungal inhibition. Compare
8.6 Place carpet samples into 150-mm Petri dishes with the the observations to a non-treated control carpet or growth in
shaven half of each sample facing the center of the dish. distal areas of the Petri dish away from the carpet samples (that
Physically separate samples in the dish so they do not touch is, the center of dish).
one another. Each dish should contain replicates of the same 9.1.3 Key for reporting degree of microbial inhibition on the
sample versus a control (if available). carpet samples is as follows:
9.1.3.1 NI = bacterial or fungal growth on the sample; no
8.7 Standardize the bacterial inoculum to 1-2×107 CFU/mL. inhibition when compared to controls.
8.8 Standardize the suspension of fungal conidia to 1-2×106 9.1.3.2 CI = no bacterial or fungal growth directly on the
CFU/mL. surface on the sample; complete inhibition of the challenge
8.9 Inoculate 200-mL lots of cooled (45 6 2°C) tryptic soy microorganism when compared to controls.
agar with 2.0 mL of standardized bacterial inoculum (final cell 9.1.3.3 PI = partial inhibition of the bacterial or fungal
density 1-2×105 CFU/mL). Wrist-action mix the agar for 30 growth directly on the sample. Partial inhibition at 72 hours is
seconds. rated qualitatively as:
Low >50 but less than 100 % coverage of the sample
8.10 Inoculate 200-mL lots of cooled (45 6 2°C) potato Medium 10 to 50 % coverage of the sample
dextrose agar with 2.0 mL of fungal conidia suspension (final High <10 % coverage of the sample
conidial density 1-2×104 CFU/mL). Wrist-action mix the agar 9.1.3.4 CIZ = complete inhibition of the challenge micro-
for 30 seconds. organism with the presence of a zone of inhibition (average
8.11 Immerse each carpet sample into the seeded agar using size reported in mm).
flame sterilized forceps. Then place each sample into the Petri 9.1.4 Morphological confirmation of challenge mold via
dish as described in 8.6. Pour a sufficient amount of the seeded tape mount and examination with light microscopy at 400×
agar into the Petri dish to cover the bottom of the dish and to magnification is useful in the case of non-sterile samples.
surround but not cover the sample (20- to 25-mL molten agar
volume is typical). 10. Precision and Bias
8.12 Allow the seeded agar to gel around the carpet samples 10.1 Carpet with plush fibers (>10-mm pile height) or those
(10 min). constructed of wool and hemp may absorb an excess volume of
the seeded agar making them less likely to demonstrate
8.13 Incubate all samples at 30 6 2°C for 24 to 72 hours. meaningful fiber layer inhibition.
9. Report 10.2 Natural fiber carpets may have inherent bioburdens,
9.1 The report shall contain the following elements: which may influence results obtained for the recommended
9.1.1 Report gross examination of the fiber layer and shaven challenge microorganisms. In these cases autoclaving or irra-
primary backing layer for direct surface inhibition or a zone of diation of the carpet should reduce or eliminate contaminants.
inhibition surrounding the carpet sample at 24, 48, and 72
hours, or both. 11. Keywords
9.1.2 Report the results of a stereo-microscopic (10 to 30× 11.1 antimicrobial; bacteria; carpet; fungi; low solubility
magnification) inspection. Examine both the unshaven fiber preservative; mold

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