1、Designation: E2722 09Standard 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 adoption or, i
2、n 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 polymer inco
3、rporatedantimicrobial agent to protect them from mold and bacteria. The American Association of TextileColorists and Chemists (AATCC) Method AATCC 1472004 and AATCC 1002004 permit qualita-tive and quantitative (respectively) assessment of fabric for antibacterial activity. AATCC 302004 isan establis
4、hed antifungal method, qualitative only, of antimicrobial treatments in or on fabric.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 descri
5、bed 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 thepresence of antibacterial and antifungal activity in
6、 or on fabricsor air filter media.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 of anantimicrobial due to excessi
7、ve organic load.1.3 This test method permits evaluation, both visually andstereomicroscopically, of the antimicrobial activity of fabric orfilter media.1.4 Use this test method to assess the durability of theantimicrobial treatments on new fabric or filter media, and onthose repeatedly laundered or
8、exposed to in-use conditions.1.5 This test method may not be suited for covalentlybonded (non-soluble or non-leaching) antimicrobials such assilane-modified quaternary ammonium compounds.1.6 Knowledge of microbiological techniques is requiredfor the practice of this test method.1.7 The values stated
9、 in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.8 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and h
10、ealth practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1054 Test Methods for Evaluation of Inactivators of An-timicrobial Agents2.2 AATCC Documents:3AATCC 302004 Antifungal Activity, Assessment on Tex-tile Materials: Mildew
11、 and Rot Resistance of TextileMaterialsAATCC 1002004 Antibacterial Finishes on Textile Mate-rials: Assessment ofAATCC 1472004 Antibacterial Activity Assessment ofTextile Materials: Parallel Streak Method3. Terminology3.1 Definitions:3.1.1 back-coating, na film (typically synthetic latex)applied to t
12、he back side of certain textiles to provide dimen-sional stability.3.1.2 inoculum vehicle, ncarrier solution used to transportbacterial cells or mold conidia to the test substrate.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the
13、 direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Nov. 15, 2009. Published December 2009. DOI:10.1520/E272209.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMS
14、tandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from American Association of Textile Chemists and Colorists(AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http:/www.aatcc.org.1Copyright ASTM International, 100 Barr Harbor Drive, PO B
15、ox C700, West Conshohocken, PA 19428-2959, United States.3.1.3 seeded agar, nmolten (liquid) microbiological agarcontaining either bacterial cells or mold conidia (spores) usedto challenge a test substrate.3.1.4 swatch, na small sample of fabric of a defined size.4. Summary of Test Method4.1 Using f
16、lame-sterilized scissors, cut fabric or mediasamples into 9 cm2swatches. Arrange the swatches in sterilepetri dishes. Cool molten agars to 45 6 2C and inoculate withthe challenge bacteria or mold conidia. Following wrist-actionmixing, immerse swatches into the seeded-molten agar, allowexcess agar to
17、 drain from the sample, and then place the swatchinto a petri dish. Pipette 2 mL of seeded agar at the perimeterof the petri dish. This will be used as a viability control.Incubate the petri dish for 48 to 72 h at 30 6 2C. Visually andmicroscopically examine at the surface of the fabric swatch forin
18、hibition of the challenge microorganisms. Report the pres-ence and degree of fabric surface inhibition.5. Significance and Use5.1 This test method provides for rapid screening of anti-microbial treatments located in or on fabrics and air filtermedia.5.2 This test method simulates actual use conditio
19、ns thatmay occur on fabrics, for example, food and beverage spills;soiling from body contact, that is, body oils, skin cells;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 Thi
20、s test method provides for the simultaneous assess-ment of multiple fabric components, for example, fabric,component fibers with polymer incorporated treatments, andback coating if present, for antimicrobial activity.5.5 Fabrics or filter media may be cleaned prior to testingwith this method in orde
21、r to assess the durability of theantimicrobial effect.6. Apparatus6.1 Stereomicroscope, (103 to 703 objectives).6.2 Erlenmeyer flasks, 250 mL.6.3 Petri dishes, 150 mm, sterile.6.4 Incubators, set at required temperatures (30 6 2C and36 6 1C)6.5 Autoclave.6.6 Water bath, capable of maintaining water
22、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 Sterile funnel, with a glass wool plug.6.15 Counting chamber (hemocytometer).6.16 Light microscope, (
23、103 and 403 objectives).6.17 Disposable latex 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.
24、7.2 Test OrganismsSpecific species are recommended,however, other microorganisms may be used to mimic thosefound in a specific environment, or those expected contami-nants which may be present where the fabric is expected toperform.7.2.1 Gram-positive species Staphylococcus aureus ATCC6538.7.2.2 Gra
25、m-negative species Serratia marcescens ATCC14756.7.2.3 Fungus: Aspergillus niger ATCC 9642.8. Procedure8.1 Grow 18 h tryptic soy broth cultures of Staphylococcusaureus at 36 61C and Serratia marcescens at 30 6 2C.These cultures should originate from 18 to 24 h growth stockculture plates or agar slan
26、ts.8.2 Prepare a suspension of fungal conidia by harvestingconidia from a 2-week-old stock culture plate or slant incu-bated at 30 6 2C. Pour sterile 0.85 % saline with 0.1 % Tween80 (see Note 1) over the fungal mat, agitate the liquid with asterile glass rod, and filter out hyphal fragments by pour
27、ing thesuspension through a sterile funnel plugged with glass wool.NOTE 1Other surfactant agents may be chosen provided that they arenon-damaging to the fungal conidia, and that they do not chemicallyneutralize the antimicrobial of interest. Test Methods E1054 may be usedto assess for neutralization
28、 potential.8.3 Prepare 200 mL lots of half-strength tryptic soy (20g/L), or nutrient, and half-strength potato dextrose agars (19.5g/L) in 250 mL erlenmeyer flasks and autoclave. Cool themolten agars to 45 6 2C in a water bath. To minimizecross-contamination and antimicrobial treatment leachate,sepa
29、rate agar lots should be prepared for each fabric type orfabric treatment tested.8.4 Cut fabric or filter media samples to form 3.0 by 3.0 cmswatches. A minimum of triplicate swatches should be used foreach challenge organism.8.5 Place samples into 150 mm petri dishes such that theydo not touch one
30、another. Each dish should contain replicatesof the same sample and a control (if used).8.6 Standardize the bacterial inoculum to 1-23107CFU/mL.8.7 Standardize the suspension of fungal conidia to1-23106CFU/mL.8.8 Inoculate 200 mL lots of cooled, half-strength (45 62C) tryptic soy agar, or nutrient ag
31、ar, with 2.0 mL ofstandardized bacterial inoculum (final cell density 1-23105CFU/mL). Wrist-action mix (manually) the agar for 30 s.8.9 Inoculate 200 mL lots of cooled, half-strength (45 62C) potato dextrose agar with 2.0 mL of fungal conidiasuspension (final conidial density 1-23104CFU/mL). Wrist-a
32、ction mix the agar for 30 s.E2722 0928.10 Immerse each sample into the half-strength seededagar using flame sterilized forceps. Allow excess agar to drainfrom the swatch or wring out the swatch on the inner neck ofthe flask. Then place each sample into the petri dish asdescribed in 8.5. Fabric sampl
33、es with varying concentrations ofthe same antimicrobial treatment should be immersed from lowto high concentration to minimize carry over and buildup in theagar.8.11 Drop 2 mL of seeded agar at the perimeter of the petridish using a sterile pipette and pipette tip. This is to be used asthe viability
34、 control.8.12 Allow the seeded agar to gel at the perimeter of thepetri dishes (10 min).8.13 Incubate all samples at 30 6 2C for 48 to 72 h. Anopen 35 mm diameter tissue culture dish containing steriledeionized water may be placed beside the test specimens(inside the 150 mm dish) in order to maintai
35、n relative humidityduring the test.9. Report9.1 The report shall contain the following elements:9.1.1 Report gross examination of the fabric for directsurface inhibition at 48 and 72 h.9.1.2 Report the results of a stereo-microscopic (10 to 303magnification) inspection. Examine the surface of the fa
36、bric.Compare the observations to a non-treated control fabric or theviability control area located at the perimeter of the petri dish.9.1.3 Key for reporting the presence and degree of bacteriaor mold inhibition by the treated fabric samples is as follows:9.1.3.1 NI = bacterial or fungal growth on t
37、he 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 = partial inhibition of the bacterial or fungalgrowth directly on the sample. Part
38、ial 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 viatape mount and examination with light microscopy at 4003magnification is usef
39、ul in the case of non-sterile samples.10. Precision and Bias10.1 Highly lofted fabrics or filter media or those con-structed of wool, cotton, and hemp may absorb an excessvolume of the seeded agar making them less likely to demon-strate meaningful surface layer inhibition.10.2 Natural fiber fabrics
40、may have inherent bioburdens andmay influence results obtained for the recommended challengemicroorganisms. In these cases, autoclaving or irradiation ofthe fabric should reduce or eliminate contaminants.11. Keywords11.1 antimicrobial; bacteria; fabric; filter media; fungi; lowsolubility preservativ
41、e; mold; polymer incorporated preserva-tive; 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 the validity of any such patent rig
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