1、Designation: E2471 05 (Reapproved 2011)1Standard 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.1NOTEEditorial changes were made throughout in April 2011.INTRODUCTIONTodays modern commercial
3、carpets (especially modular carpet tile) often incorporate 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 bot
4、h qualitative and quantitative antibacterial assessment andantifungal 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
5、carpets primary backing layer. The test method describedhere 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
6、 presence of antimicrobial activity in or on carpets. Use thistest 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 f
7、or mold conidia. Use ofhalf strength agars may reduce undue 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 U
8、se this test method to assess the durability of theantimicrobial 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
9、 of thesafety concerns, if any, associated with its use. It 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 Chemi
10、sts and Colorists(AATCC) Standard:Method 174-2007, Antimicrobial 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 wo
11、venor non-woven scrim and then coated with latex to bond the 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 laye
12、r of carpetbacking where carpet fiber bundles are physically 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 thi
13、n layer of molten (liquid) micro-biological agar containing 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 Methods and is the directresponsibilit
14、y of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved March 1, 2011. Published April 2011. Originallyapproved in 2005 as E2471 05. DOI: 10.1520/E2471-05R11E01.2Available from American Association of Textile Chemists and Colorists(AATCC), P.O. Box 12215, Research Triangle Park, NC
15、 27709, http:/www.aatcc.org.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.4. 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 fib
16、er 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 actionmixin
17、g, 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)laye
18、r 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 te
20、st 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 th
21、is testmethod in order to assess the durability of the antimicrobialeffect.6. Apparatus6.1 Stereomicroscope,10to703 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 mai
22、ntaining 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 mm.6
23、.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 403 objectives.6.21 Disposable Examination Gloves.7. Reagents7.1 Media:7.1.1 Tr
24、yptic 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 which
25、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 Asperg
26、illus 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 per
27、missible.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 pouring
28、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 fo
29、r 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 sampl
30、e after shaving.8.6 Place carpet samples into 150-mm Petri dishes with theshaven half of each sample facing the center of the dish.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 Standa
31、rdize the bacterial inoculum to 1-23107CFU/mL.8.8 Standardize the suspension of fungal conidia to1-23106CFU/mL.3Technical Manual of the American Association of Textile Chemists andColorists, 2000, Volume 75, American Association of Textile Chemists andColorists.E2471 05 (2011)128.9 Inoculate 200-mL
32、lots of cooled (45 6 2C) tryptic soyagar with 2.0 mL of standardized bacterial inoculum (final celldensity 1-23105CFU/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 1-2310
33、4CFU/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 tosu
34、rround 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 f
35、iber 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 303magnification) inspection. Examine both the unshaven fiberlayer and the shaven prim
36、ary 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 o
37、n thecarpet 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 = pa
38、rtial 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
39、 volume 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
40、reduce 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 expre
41、ssly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised
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