1、Designation: E2471 05 (Reapproved 2016)Standard Test Method forUsing Seeded-Agar for the Screening Assessment ofAntimicrobial Activity In Carpets1This standard is issued under the fixed designation E2471; the number immediately following the designation indicates the year oforiginal adoption or, in
2、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.INTRODUCTIONTodays modern commercial carpets (especially modular carpet tile) often incorporate
3、antimicrobialagents either in or on the face fibers or incorporated into the primary backing (attachment point ofcarpet fiber to the backing structure). The American Association of Textile Chemists and Colorists(AATCC) Method 174 permits both qualitative and quantitative antibacterial assessment and
4、antifungal assessment (qualitative only) of antimicrobial treatments in or on carpet. However, themethod is not suited for rapid screening of antimicrobials low in water solubility or that have slowdiffusion rates when incorporated into the carpets primary backing layer. The test method describedher
5、e provides a rapid screen of antimicrobial activity in or on carpets and allows for the simultaneousassessment of multiple components of the carpet (not just the fibers).1. Scope1.1 This test method is designed to evaluate (qualitatively)the presence of antimicrobial activity in or on carpets. Use t
6、histest method to qualitatively evaluate both antibacterial andantifungal activity.1.2 Use half strength (nutrient and agar) tryptic soy agar asthe inoculum vehicle for bacteria and half strength potatodextrose agar as the inoculum vehicle for mold conidia. Use ofhalf strength agars may reduce undue
7、 neutralization of anantimicrobial due to excessive organic load.1.3 This test method simultaneously evaluates (both visualand stereo-microscopic) antimicrobial activity both at the fiberlayer and at the primary backing layer of carpet.1.4 Use this test method to assess the durability of theantimicr
8、obial treatments on new carpets, and on those repeat-edly shampooed or exposed to in-use conditions.1.5 Knowledge of microbiological techniques is requiredfor the practice of this test method.1.6 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It
9、 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 American Association of Textile Chemists and Colorists(AATCC) Standard:Method 174-2007, Antimicr
10、obial Activity Assessment ofCarpets23. Terminology3.1 Definitions:3.1.1 face fiber, nthe wear layer of the carpet; can becomposed of nylon, polypropylene, wool, or other natural orsynthetic polymers. Typically, face fiber is tufted into a wovenor non-woven scrim and then coated with latex to bond th
11、e facefiber securely to the backing; this latex coated scrim forms theprimary backing.3.1.2 inoculum vehicle, ncarrier solution used to transportbacterial cells or mold conidia to the test substrate.3.1.3 primary backing, nthe uppermost layer of carpetbacking where carpet fiber bundles are physicall
12、y attached atthe base to the backing structure. This layer is typicallyconstructed of synthetic latex (ethylene vinyl acetate, styrenebutadiene, or a thermo-polymer; that is, ethylene vinyl acetatehot-melt adhesive).3.1.4 seeded agar, na thin layer of molten (liquid) micro-biological agar containing
13、 either bacterial cells or mold conidia(spores) used to challenge a test substrate.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edi
14、tion approved Nov. 1, 2016. Published December 2016. Originallyapproved in 2005 as E2471 05. Last previous edition published in 2011 asE247105(2011)1. DOI: 10.1520/E2471-05R16.2Available from American Association of Textile Chemists and Colorists(AATCC), P.O. Box 12215, Research Triangle Park, NC 27
15、709-2215, http:/www.aatcc.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States14. Summary of Test Method4.1 Cut carpet samples into small rectangular pieces eithervia a carpet knife or mechanical die and press. Shave half ofthe face fi
16、ber on each sample using electric hair clippers andarrange in sterile Petri dishes (typically with the shaven half ofthe sample facing the center of the dish. Cool molten agars (fullor partial complement) to 45 6 2C and inoculate with thechallenge bacteria or mold conidia. Following wrist actionmixi
17、ng, immerse samples into the seeded-molten agar, placeinto a Petri dish and pour additional seeded agar into the dishto surround but not cover the test sample. Incubate the Petridish for 24 to 72 h at 30 6 2C. Visually and microscopicallyexamine both at the face fiber and shaven (primary backing)lay
18、er for inhibition of the challenge microorganisms. Reportthe presence of carpet surface inhibition (for low water solubleor slow migrating antimicrobials) or zone of inhibition forwater soluble antimicrobials.35. Significance and Use5.1 This test method provides for rapid screening of anti-microbial
19、 treatments located in or on the carpet face fiber orincorporated into the backing structure of the carpet (or both).5.2 This test method simulates actual use conditions thatmay occur on carpets (for example, food and beverage spills,soiling from foot traffic, prolonged moisture exposure).5.3 This t
20、est method provides a means to screen for activityand durability of an antimicrobial treatment under conditionsof organic loading.5.4 This test method provides for the simultaneous assess-ment of multiple carpet components for antimicrobial activity.5.5 Carpets may be cleaned prior to testing with t
21、his testmethod in order to assess the durability of the antimicrobialeffect.6. Apparatus6.1 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 2C and37 6 2C).6.5 Autoclave.6.6 Water Bath, capable of
22、maintaining water at 45 6 2C.6.7 Test Tubes, 16 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 m
23、m.6.15 Carpet Knife (razor knife).6.16 Mechanical Die (Optional), 2.5 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. Reagents7.1 Media:7.1.1
24、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 OrganismsSpecific organisms are recommended;however, other microorganisms may be used to mimic thosefound in a specific environment or those expected contami-nants whic
25、h may be present where the carpet is expected toperform.7.2.1 Gram-positive bacteria Staphylococcus aureus ATCC6538.7.2.2 Gram-negative bacteria Serratia marcescens ATCC14756.7.2.3 Fungus Aspergillus niger ATCC 9642.NOTE 1Originally deposited as Aspergillus niger, the current ATCCdesignation is Aspe
26、rgillus brasiliensis.8. Procedure8.1 Grow 18 hour tryptic soy broth cultures of Staphylococ-cus aureus at 37 6 2C and Serratia marcescens at 30 6 2C.These cultures should originate from 18 to 24 h growth stockculture plates or agar slants. Origination from glycerol stockswith two transfers is also p
27、ermissible.8.2 Prepare a suspension of fungal conidia by harvestingmature conidia from a 1 week old stock culture plate or slantgrown at 30 6 2C. Pour sterile 0.85 % saline with 0.1 %polysorbate 80 over the growth, agitate the liquid with a sterileglass rod, and filter the hyphal fragments by pourin
28、g thesuspension through a sterile funnel plugged with glass wool.8.3 Prepare 200-mL lots of12 strength tryptic soy and12strength potato dextrose agars in 250-mL Erlenmeyer flasksand autoclave. Cool the molten agars to 45 6 2C in a waterbath.8.4 Cut carpet samples (minimum of duplicate carpetsamples
29、for each challenge organism) 2.5 by 3.8 cm.8.5 With gloved hands, aseptically shear half of the fiberfrom the 3.8-cm length of carpet using electric clippers. Thesheared half of the carpet sample should have fibers no longerthan 2 6 1 mm. Use canned air to remove the loose fibers fromeach carpet sam
30、ple after shaving.8.6 Place carpet samples into 150-mm Petri dishes with theshaven half of each sample facing the center of the dish.3Technical Manual of the American Association of Textile Chemists andColorists, 2000, Volume 75, American Association of Textile Chemists andColorists.E2471 05 (2016)2
31、Physically separate samples in the dish so they do not touchone another. Each dish should contain replicates of the samesample versus a control (if available).8.7 Standardize the bacterial inoculum to 1-2107CFU/mL.8.8 Standardize the suspension of fungal conidia to 1-2106CFU/mL.8.9 Inoculate 200-mL
32、lots of cooled (45 6 2C) tryptic soyagar with 2.0 mL of standardized bacterial inoculum (final celldensity 1-2105CFU/mL). Wrist-action mix the agar for 30seconds.8.10 Inoculate 200-mL lots of cooled (45 6 2C) potatodextrose agar with 2.0 mL of fungal conidia suspension (finalconidial density 1-2104C
33、FU/mL). Wrist-action mix the agarfor 30 seconds.8.11 Immerse each carpet sample into the seeded agar usingflame sterilized forceps. Then place each sample into the Petridish as described in 8.6. Pour a sufficient amount of the seededagar into the Petri dish to cover the bottom of the dish and tosurr
34、ound but not cover the sample (20- to 25-mL molten agarvolume is typical).8.12 Allow the seeded agar to gel around the carpet samples(10 min).8.13 Incubate all samples at 30 6 2C for 24 to 72 hours.9. Report9.1 The report shall contain the following elements:9.1.1 Report gross examination of the fib
35、er layer and shavenprimary backing layer for direct surface inhibition or a zone ofinhibition surrounding the carpet sample at 24, 48, and 72hours, or both.9.1.2 Report the results of a stereo-microscopic (10 to 30magnification) inspection. Examine both the unshaven fiberlayer and the shaven primary
36、 backing layer of the carpetsamples for evidence of bacterial or fungal inhibition. Comparethe observations to a non-treated control carpet or growth indistal areas of the Petri dish away from the carpet samples (thatis, the center of dish).9.1.3 Key for reporting degree of microbial inhibition on t
37、hecarpet samples is as follows:9.1.3.1 NI = bacterial or fungal growth on the sample; noinhibition when compared to controls.9.1.3.2 CI = no bacterial or fungal growth directly on thesurface on the sample; complete inhibition of the challengemicroorganism when compared to controls.9.1.3.3 PI = parti
38、al inhibition of the bacterial or fungalgrowth directly on the sample. Partial inhibition at 72 hours israted qualitatively as:Low 50 but less than 100 % coverage of the sampleMedium 10 to 50 % coverage of the sampleHigh 10-mm pile height) or thoseconstructed of wool and hemp may absorb an excess vo
39、lume ofthe seeded agar making them less likely to demonstratemeaningful fiber layer inhibition.10.2 Natural fiber carpets may have inherent bioburdens,which may influence results obtained for the recommendedchallenge microorganisms. In these cases autoclaving or irra-diation of the carpet should red
40、uce or eliminate contaminants.11. Keywords11.1 antimicrobial; bacteria; carpet; fungi; low solubilitypreservative; moldASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressl
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42、ither reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may atte
43、nd. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. I
44、ndividual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the Copyright Clearance Center, 222Rosewood Drive, Danvers, MA 01923, Tel: (978) 646-2600; http:/ 05 (2016)3
