1、Designation: E 2471 05Standard Test Method forUsing Seeded-Agar for the Screening Assessment ofAntimicrobial Activity In Carpets1This standard is issued under the fixed designation E 2471; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revi
2、sion, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.INTRODUCTIONTodays modern commercial carpets (especially modular carpet tile) often incorporate antimicrobialag
3、ents 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 Colorists and Chemists(AATCC) Method 174 permits both qualitative and quantitative antibacterial assessment andantifungal asse
4、ssment (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 describedhere provides a ra
5、pid 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 thistest method
6、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 neutralization
7、 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 theantimicrobial treatment
8、s 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 is theresponsi
9、bility 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 Colorists and Chemists(AATCC) Standard:Method 174-1998, Antimicrobial Activity
10、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 the facefiber sec
11、urely 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 physically attached atth
12、e base to the backing structure. This layer is typically1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and is the direct responsibility of Subcommittee E35.15 on AntimicrobialAgents.Current edition approved Nov. 1, 2005. Published November 2005.2Available from America
13、n Association of Textile Chemists and Colorists(AATCC), One Davis Dr., P.O. Box 12215, Research Triangle Park, NC 27709-2215.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.constructed of synthetic latex (ethylene vinyl acetate, styr
14、enebutadiene, 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 either bacterial cells or mold conidia(spores) used to challenge a test substrate.4. Summary of Test Method4.1 Cut carpet samples
15、into small rectangular pieces eithervia a carpet knife or mechanical die and press. Shave half ofthe face fiber 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
16、 complement) to 45 6 2C and inoculate with thechallenge bacteria or mold conidia. Following wrist actionmixing, 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
17、 72 h at 30 6 2C. Visually and microscopicallyexamine both at the face fiber and shaven (primary backing)layer for inhibition of the challenge microorganisms. Reportthe presence of carpet surface inhibition (for low water solubleor slow migrating antimicrobials) or zone of inhibition forwater solubl
18、e antimicrobials.35. Significance and Use5.1 This test method provides for rapid screening of anti-microbial 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 carpe
19、ts (for example, food and beverage spills,soiling from foot traffic, prolonged moisture exposure).5.3 This test 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
20、 of multiple carpet components for antimicrobial activity.5.5 Carpets may be cleaned prior to testing with this 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
21、.4 Incubators, set at required temperatures (30 6 2C and37 6 2C).6.5 Autoclave.6.6 Water Bath, capable of 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
22、equivalent#30 Blade).6.13 Canned Air (compressed air for surface dusting).6.14 Sterile Petri Dishes, 100 mm.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).
23、6.20 Light Microscope, 10 and 403 objectives6.21 Disposable Latex Examination Gloves.7. Reagents7.1 Media: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 % Tween 80.7.2 Test OrganismsSpecific organisms are recommendedhowever other micro
24、organisms may be used to mimic thosefound in a specific environment or those expected contami-nants which 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 Aspergil
25、lus niger ATCC 9642.8. Procedure8.1 Grow 18 hour tryptic soy broth cultures of Staphylococ-cus aureus at 37 6 2C and Serratia marcescens in tryptic soybroth at 30 6 2C. These cultures should originate from 18 to24 h growth stock culture plates or on agar slants. Originationfrom glycerol stocks with
26、two transfers is also permissible.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 %Tween 80 over the growth, agitate the liquid with a sterile glassrod, and filter the hyphal fr
27、agments by pouring the suspensionthrough 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 duplica
28、te carpetsamples 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 fro
29、meach carpet sample after shaving.8.6 Place carpet samples into 150mm 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 avai
30、lable).8.7 Standardize the bacterial inoculum to 1-23107CFU/mL.3Technical Manual of the American Association of Textile Chemists andColorists, 2000, Volume 75, American Association of Textile Chemists andColorists.E24710528.8 Standardize the suspension of fungal conidia to1-23106CFU/mL.8.9 Inoculate
31、 200 mL 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 densit
32、y 1-23104CFU/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
33、and tosurround 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 o
34、f the fiber 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 shav
35、en primary 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 inhib
36、ition on 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
37、PI = partial 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
38、 excess volume ofthe seeded agar making them less likely to demonstratemeaningful fiber layer inhibition.10.2 Natural fiber carpets may have inherent bioburdens andmay influence results obtained for the recommended challengemicroorganisms. In these cases autoclaving or irradiation of thecarpet shoul
39、d 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 exp
40、ressly 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 revis
41、ed, either 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
42、 attend. 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. Individual 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).E2471053
