1、Designation: E2722 09E2722 14Standard Test Method forUsing Seeded-Agar for the Screening Assessment ofAntimicrobial Activity in Fabric and Air Filter Media1This standard is issued under the fixed designation E2722; the number immediately following the designation indicates the year oforiginal adopti
2、on or, in 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.INTRODUCTIONFabrics (woven, non-woven) and filters often incorporate a topical or poly
3、mer incorporatedantimicrobial agent to protect them from mold and bacteria. The American Association of TextileColorists and Chemists (AATCC) Method AATCC 1472004 and AATCC 1002004 permitqualitative and quantitative (respectively) assessment of fabric for antibacterial activity. AATCC302004 is an es
4、tablished antifungal method, qualitative only, of antimicrobial treatments in or onfabric. However, these methods are not well suited for rapid screening of antimicrobials low in watersolubility or that have slow diffusion rates when incorporated into a fabric back-coating layer. Thestandard method
5、described here provides a rapid screen of antimicrobial activity in or on fabric andfilter media and does not depend on a zone of inhibition to demonstrate a surface protective effect.1. Scope1.1 This test method is designed to evaluate qualitatively the presence of antibacterial and antifungal acti
6、vity in or on fabricsor air filter media.1.2 Use half-strength (nutrient and agar) tryptic soy agar as the inoculum vehicle for bacteria and half-strength potato dextroseagar as the inoculum vehicle for mold conidia. Use of half-strength agars may reduce undue neutralization of an antimicrobial duet
7、o excessive organic load.1.3 This test method permits evaluation, both visually and stereomicroscopically, of the antimicrobial activity of fabric or filtermedia.1.4 Use this test method to assess the durability of the antimicrobial treatments on new fabric or filter media, and on thoserepeatedly la
8、undered or exposed to in-use conditions.1.5 This test method may not be suited for covalently bonded (non-soluble or non-leaching) antimicrobials such as (that is,silane-modified quaternary ammonium pounds) or actives with limited migration or solubility.1.6 Knowledge of microbiological techniques i
9、s required for the practice of this test method.1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsib
10、ilityof the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1054 Test Methods for Evaluation of Inactivators of Antimicrobial AgentsE2756 Terminology Relating
11、to Antimicrobial and Antiviral Agents1 This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Nov. 15, 2009Aug. 1, 2014. Pub
12、lished December 2009September 2014. Originally approved in 2009. last previous edition approved in 2009 asE272209. DOI: 10.1520/E272209.10.1520/E272214.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Sta
13、ndardsvolume information, refer to the standards Document Summary page on the ASTM website.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possibl
14、e to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West
15、Conshohocken, PA 19428-2959. United States12.2 AATCC Documents:3AATCC 302004 Antifungal Activity, Assessment on Textile Materials: Mildew and Rot Resistance of Textile MaterialsAATCC 1002004 Antibacterial Finishes on Textile Materials: Assessment ofAATCC 1472004 Antibacterial Activity Assessment of
16、Textile Materials: Parallel Streak Method3. Terminology3.1 For definitions of terms used in this test method refer to Terminology E2756.3.2 Definitions:Definitions of Terms Specific to This Standard:3.2.1 back-coating, na film (typically synthetic latex) applied to the back side of certain textiles
17、to provide dimensionalstability.3.2.2 inoculum vehicle, ncarrier solution used to transport bacterial cells or mold conidia to the test substrate.3.2.3 seeded agar, nmolten (liquid) microbiological agar containing either bacterial cells or mold conidia (spores) used tochallenge a test substrate.3.2.
18、4 swatch, na small sample of fabric of a defined size.4. Summary of Test Method4.1 Using flame-sterilized scissors, cut fabric or media samples into 9 cm2 swatches.Arrange the swatches in sterile petri dishes.Cool molten agars to 45 6 2C and inoculate with the challenge bacteria or mold conidia. Fol
19、lowing wrist-action mixing, immerseswatches into the seeded-molten agar, allow excess agar to drain from the sample, and then place the swatch into a petri dish.Pipette 2 mL of seeded agar at the perimeter of the petri dish. This will be used as a viability control. Incubate the petri dish for48 to
20、72 h at 30 6 2C. Visually and microscopically examine at the surface of the fabric swatch for inhibition of the challengemicroorganisms. Report the presence and degree of fabric surface inhibition.5. Significance and Use5.1 This test method provides for rapid screening of antimicrobial treatments lo
21、cated in or on fabrics and air filter media.5.2 This test method simulates actual use conditions that may occur on fabrics, for example, food and beverage spills; soilingfrom body contact, that is, body oils, skin cells; prolonged moisture exposure.5.3 This test method provides a means to screen for
22、 activity and durability of an antimicrobial treatment under conditions oforganic loading.5.4 This test method provides for the simultaneous assessment of multiple fabric components, for example, fabric, componentfibers with polymer incorporated treatments, and back coating if present, for antimicro
23、bial activity.5.5 Fabrics or filter media may be cleaned prior to testing with this method 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 Petri dishes, 150 mm, sterile.6.4 Incubators, set at requi
24、red temperatures (30 6 2C and 36 6 1C)6.5 Autoclave.6.6 Water bath, capable of maintaining water at 45 6 2C6.7 Test tubes, 16 by 100 mm.6.8 Hot Plate, with stirrer.6.9 Spectrophotometer.6.10 Sterile cuvettes.6.11 Test fabric.6.12 Flame-sterilized scissors.6.13 Petri dishes, 100 mm, sterile.6.14 Ster
25、ile funnel, with a glass wool plug.3 Available from American Association of Textile Chemists and Colorists (AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http:/www.aatcc.org.E2722 1426.15 Counting chamber (hemocytometer).6.16 Light microscope, (10 and 40 objectives).6.17 Disposable latex
26、 examination gloves.6.18 Flame-sterilized forceps or hemostats.7. Reagents and Materials7.1 Media:7.1.1 Tryptic soy broth or nutrient broth.7.1.2 Tryptic soy agar or nutrient agar.7.1.3 Potato dextrose agar.7.1.4 Sterile 0.85 % saline with 0.1 % Tween 80.7.2 Test OrganismsSpecific species are recomm
27、ended, however, other microorganisms may be used to mimic those found ina specific environment, or those expected contaminants which may be present where the fabric is expected to perform.7.2.1 Gram-positive species Staphylococcus aureus ATCC 6538.7.2.2 Gram-negative species Serratia marcescens ATCC
28、 14756.7.2.3 Fungus: Aspergillus nigerbrasiliensis ATCC 9642.9642 (deposited as Aspergillus niger).8. Procedure8.1 Grow 18 h tryptic soy broth cultures of Staphylococcus aureus at 36 61C and Serratia marcescens at 30 6 2C. Thesecultures should originate from 18 to 24 h growth stock culture plates or
29、 agar slants.8.2 Prepare a suspension of fungal conidia by harvesting conidia from a 2-week-old stock culture plate or slant incubated at 306 2C. Pour sterile 0.85 % saline with 0.1 % Tween 80 (see Note 1) over the fungal mat, agitate the liquid with a sterile glass rod,and filter out hyphal fragmen
30、ts by pouring the suspension through a sterile funnel plugged with glass wool.NOTE 1Other surfactant agents may be chosen provided that they are non-damaging to the fungal conidia, and that they do not chemically neutralizethe antimicrobial of interest. Test Methods E1054 may be used to assess for n
31、eutralization potential.8.3 Prepare 200 mL lots of half-strength tryptic soy (20 g/L), or nutrient, and half-strength potato dextrose agars (19.5 g/L) in250 mL erlenmeyer flasks and autoclave. Cool the molten agars to 45 6 2C in a water bath. To minimize cross-contaminationand antimicrobial treatmen
32、t leachate, separate agar lots should be prepared for each fabric type or fabric treatment tested.8.4 Cut fabric or filter media samples to form 3.0 by 3.0 cm swatches. A minimum of triplicate swatches should be used foreach challenge organism.8.5 Place samples into 150 mm petri dishes such that the
33、y do not touch one another. Each dish should contain replicates of thesame sample and a control (if used).8.6 Standardize the bacterial inoculum to 1-2107 CFU/mL.8.7 Standardize the suspension of fungal conidia to 1-2106 CFU/mL.8.8 Inoculate 200 mL lots of cooled, half-strength (45 6 2C) tryptic soy
34、 agar, or nutrient agar, with 2.0 mL of standardizedbacterial inoculum (final cell density 1-2105 CFU/mL). Wrist-action mix (manually) the agar for 30 s.8.9 Inoculate 200 mL lots of cooled, half-strength (45 6 2C) potato dextrose agar with 2.0 mL of fungal conidia suspension(final conidial density 1
35、-2104 CFU/mL). Wrist-action mix the agar for 30 s.8.10 Immerse each sample into the half-strength seeded agar using flame sterilized forceps.Allow excess agar to drain from theswatch or wring out the swatch on the inner neck of the flask. Then place each sample into the petri dish as described in 8.
36、5. Fabricsamples with varying concentrations of the same antimicrobial treatment should be immersed from low to high concentration tominimize carry over and buildup in the agar.8.11 Drop 2 mL of seeded agar at the perimeter of the petri dish using a sterile pipette and pipette tip. This is to be use
37、d as theviability control.8.12 Allow the seeded agar to gel at the perimeter of the petri dishes (10 min).8.13 Incubate all samples at 30 6 2C for 48 to 72 h. An open 35 mm diameter tissue culture dish containing sterile deionizedwater may be placed beside the test specimens (inside the 150 mm dish)
38、 in order to maintain relative humidity during the test.9. Report9.1 The report shall contain the following elements:9.1.1 Report gross examination of the fabric for direct surface inhibition at 48 and 72 h.9.1.2 Report the results of a stereo-microscopic (10 to 30 magnification) inspection. Examine
39、 the surface of the fabric.Compare the observations to a non-treated control fabric or the viability control area located at the perimeter of the petri dish.E2722 1439.1.3 Key for reporting the presence and degree of bacteria or mold inhibition by the treated fabric samples is as follows:9.1.3.1 NI
40、= bacterial or fungal growth on the sample; no inhibition when compared to controls.9.1.3.2 CI = no bacterial or fungal growth directly on the surface on the sample; complete inhibition of the challengemicroorganism when compared to controls.9.1.3.3 PI = partial inhibition of the bacterial or fungal
41、 growth directly on the sample. Partial inhibition at 72 h is ratedqualitatively as:Low 50 but less than 100 % coverage of the sampleMedium 10 to 50 % coverage of the sampleHigh 10 % coverage of the sample9.1.4 Morphological confirmation of challenge mold via tape mount and examination with light mi
42、croscopy at 400magnification is useful in the case of non-sterile samples.10. Precision and Bias10.1 Highly lofted fabrics or filter media or those constructed of wool, cotton, and hemp may absorb an excess volume of theseeded agar making them less likely to demonstrate meaningful surface layer inhi
43、bition.10.2 Natural fiber fabrics may have inherent bioburdens and may influence results obtained for the recommended challengemicroorganisms. In these cases, autoclaving or irradiation of the fabric should reduce or eliminate contaminants.11. Keywords11.1 antimicrobial; bacteria; fabric; filter med
44、ia; fungi; low solubility preservative; mold; polymer incorporated preservative;textileASTM 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 expressly advised that determination of
45、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, either reapproved or withdrawn.Yo
46、ur 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 attend. If you feel that your comment
47、s 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 mul
48、tiple 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:/ 144
