142 TM 100-2004 AATCC Technical Manual/2010

Developed in 1961 by AATCC Commit-tee RA31; revised 1965, 1981, 1988(with title change), 1993, 1999; editori-ally revised 1969, 1971, 1974, 1985,2009; reaffirmed 1977, 1981, 1989,1998, 2008; editorially revised and re-affirmed 1986, 2004.

1. Purpose and Scope

1.1 This test method provides a quanti-tative procedure for the evaluation of thedegree of antibacterial activity. Assess-ment of antibacterial finishes on textilematerials is determined by the degree ofantibacterial activity intended in the useof such materials. If only bacteriostaticactivity (inhibition of multiplication) isintended, a qualitative procedure whichclearly demonstrates antibacterial activityas contrasted with lack of such activity byan untreated specimen may be accept-able. However, if bactericidal activity isintended or implied, quantitative evalua-tion is necessary. Quantitative evaluationalso provides a clearer picture for possi-ble uses of such treated textile materials.

2. Principle

2.1 Swatches of test and control textilematerials are tested qualitatively for anti-bacterial activity by AATCC Method 147,Antibacterial Activity Assessment ofTextile Materials: Parallel Streak Method.Those showing activity are evaluatedquantitatively. Test and control swatchesare inoculated with the test organisms.After incubation, the bacteria are elutedfrom the swatches by shaking in knownamounts of neutralizing solution. Thenumber of bacteria present in this liquid isdetermined, and the percentage reductionby the treated specimen is calculated.

3. Terminology

3.1 activity, n.—of an antibacterialagent, a measure of effectiveness of theagent.

3.2 antibacterial agent, n.—in tex-tiles, any chemical which kills bacteria(bactericide) or interferes with the multi-plication, growth or activity of bacteria(bacteriostat).

4. Safety Precautions

NOTE: These safety precautions arefor information purposes only. The pre-cautions are ancillary to the testing proce-dures and are not intended to be all inclu-sive. It is the user’s responsibility to usesafe and proper techniques in handling

materials in this test method. Manufac-turers MUST be consulted for specificdetails such as material safety data sheetsand other manufacturer’s recommenda-tions. All OSHA standards and rulesmust also be consulted and followed.

4.1 Both the qualitative and quantita-tive tests should be carried out by personswith training and experience in the use ofbacteriological techniques. The U.S. De-partment of Health and Human Servicespublication, Biosafety in Microbiologicaland Biomedical Laboratories, should beconsulted (see 13.1).

4.2 CAUTION: Some of the bacteriaused in this test are capable of infectinghumans and producing disease. There-fore, every necessary and reasonable pre-caution must be taken to eliminate thisrisk to the laboratory personnel and topersonnel in the associated environment.Wear protective clothing and respiratoryprotection that prevents penetration bythe bacteria.

4.3 Good laboratory practices shouldbe followed. Wear safety glasses in alllaboratory areas.

4.4 All chemicals should be handledwith care.

4.5 An eyewash/safety shower shouldbe located nearby for emergency use.

4.6 Sterilize all contaminated samplesand test materials prior to disposal.

4.7 Exposure to chemicals used in thisprocedure must be controlled at or belowlevels set by government authorities (e.g.,Occupational Safety and Health Adminis-tration’s [OSHA] permissible exposurelimits [PEL] as found in 29 CFR1910.1000 of January 1, 1989). In addition,the American Conference of Governmen-tal Industrial Hygienists (ACGIH) Thresh-old Limit Values (TLVs) comprised of timeweighted averages (TLV-TWA), short termexposure limits (TLV-STEL) and ceilinglimits (TLV-C) are recommended as a gen-eral guide for air contaminant exposurewhich should be met (see 13.2).

5. Limitations

5.1 For a qualitative, relatively quickand easily executed method to determineresidual antibacterial activity of textilematerials, refer to AATCC Method 147.

6. Test Organisms

6.1 Test bacteria.6.1.1 Staphylococcus aureus, American

Type Culture Collection No. 6538. Grampositive organism (see13.3).

6.1.2 Klebsiella pneumoniae, Ameri-

can Type Culture Collection No. 4352.Gram negative organism (see 13.3).

6.1.3 Other suitable species can also beused.

7. Culture Medium

7.1 Suitable broth/agar media are Nu-trient, Trypticase Soy and Brain-HeartInfusion.

Nutrient Broth:Peptone (Bacto-peptone)

(see 13.4) 5 gBeef extract (see 13.5) 3 gDistilled water to 1000 mL7.2 Heat to a boil to disperse ingre-

dients. Adjust to pH 6.8 ± 0.1 with 1Nsodium hydroxide (NaOH) solution. (Thisis not necessary if prepared, dehydratedmedium is used.)

7.3 Dispense in 10 mL amounts in con-ventional bacteriological culture tubes(i.e., 125 × 17 mm). Plug and sterilize at103 kPa (15 psi) for 15 min.

7.4 Nutrient agar. Add 1.5% bacterio-logical agar to nutrient (or appropriate)broth (see 7.1). Heat to boiling. CheckpH and adjust to 7.1 ± 0.1 using NaOHsolution if necessary. Dispense in 15 ± 1mL amounts in conventional bacteriolog-ical culture tubes. Plug and sterilize at103 kPa (15 psi) for 15 min. (May besterilized in 1000 mL borosilicate glassflasks and petri dishes poured from this.)

7.5 Slurry Inoculum Carrier (for hy-drophobic fabrics) (see 7.2 and 7.3):

Sodium Chloride 8.5 gAgar 3.0 gDistilled Water 1000 mL

8. Maintenance of Culture of TestOrganisms

8.1 Using a 4 mm inoculating loop,transfer the culture daily in nutrient (orappropriate medium) broth for not morethan two weeks. At the conclusion of twoweeks, make a fresh transplant from stockculture. Incubate cultures at 37 ± 2°C (99± 3°F) or other optimal temperature.

8.2 Maintain stock cultures on nutrientor appropriate agar slants. Store at 5 ±1°C (41 ± 2°F) and transfer once a monthto fresh agar (see 13.6).

9. Qualitative Test (Screening orPresumptive Test)

9.1 For detection of bacteriostatic ac-tivity use AATCC Method 147 on a testspecimen and control specimen using theorganisms referred to above. For demon-stration of bactericidal activity, proceedto the quantitative test described below.

AATCC Test Method 100-2004

Antibacterial Finishes on Textile Materials: Assessment of

Copyright © 2009 American Association of Textile Chemists and Colorists

AATCC Technical Manual/2010 TM 100-2004 143

10. Quantitative Test (Reference orConfirmatory Test)

10.1 Preparation. The following de-scription will be in terms of fabricswatches. Textile materials not in fabricform can likewise be tested with the ap-propriate modification.

10.1.1 Size and shape of treatedswatches: Cut circular swatches 4.8 ±0.1 cm (1.9 ± 0.03 in.) in diameter, fromthe test fabric (preferably with a steeldie). Stack the swatches in a 250 mLwide-mouth glass jar with screw cap.The number of swatches to be used isdependent on the fiber type and fabricconstruction. Use that amount of fabricwhich will absorb the 1.0 ± 0.1 mL ofinoculum, and leave no free liquid in thejar. For example, 4 swatches of cottonprint cloth will absorb 1 mL. The num-ber of swatches used per jar should bereported.

10.1.2 Controls. Swatches of the samefiber type and fabric construction as testsample but containing no antibacterialfinish (negative control).

10.1.3 Sterilization of samples. This isoptional. The method to be used dependson the type of fiber and finish. Cotton, ac-etate and many manmade fibers can besterilized in the autoclave. Wool can besterilized by ethylene oxide or by inter-mittent (fractional) sterilization in flow-ing steam. The latter is also least damag-ing to certain finishes. Report method ofsterilization, if used.

10.1.4 Size of inoculum per sample.Apply 1.0 ± 0.1 mL of an appropriate di-lution of a 24 h broth culture of the testorganism so that recovery from (1) un-treated control fabric swatches or (2)treated test fabric swatches at “0” contacttime (plated as soon as possible after in-oculation) will show counts of 1-2 × 105

organisms. The dilution of the test organ-ism should be made in nutrient (or appro-priate) broth (see 7.1, 7.5 and 13.7).

10.2 Procedure.10.2.1 Inoculation of fabrics. When

using Staphylococcus aureus, shake a 24h culture and let stand for 15-20 min be-fore preparing the inoculum.* Place theswatches separately in sterile petri dishesand use a microliter pipette to inoculatethem making sure that there is even dis-tribution of the inoculum (see 13.8).Transfer these swatches aseptically to thejar. Screw the jar tops on tightly to pre-vent evaporation.

10.2.2 As soon as possible after inocu-lation (“0” contact time), add 100 ± 1 mL

of neutralizing solution to each of the jarscontaining the inoculated untreated con-trol swatches, the inoculated treated testswatches and the uninoculated treatedtest swatches.

10.2.3 The neutralizing solution shouldinclude ingredients to neutralize the spe-cific antibacterial fabric treatment and totake care of any pH requirements of thefabrics (from finishes, antibacterialagents, etc.). The neutralizing solutionemployed should be reported (see 13.9).

10.2.4 Shake the jars vigorously forone minute. Make serial dilutions withwater and plate (in duplicate) on nutrient(or appropriate) agar. Dilutions of 100,101, 102 are usually suitable.

10.2.5 Incubation over contact periods.Incubate additional jars containing inocu-lated untreated control swatches andjars containing inoculated treated testswatches at 37 ± 2°C (99 ± 3°F) for 18-24 h. Similar jars may be incubated overother periods (e.g., 1 or 6 h) to provideinformation about the bactericidal activ-ity of the treatment over such periods.

10.2.6 Sampling of inoculated andincubated swatches. After incubation,add 100 ± 1 mL of neutralizing solutionto jars containing untreated controlswatches and to jars containing treatedtest swatches. Shake the jars vigorouslyfor one minute. Make serial dilutions andplate (in duplicate) on nutrient (or appro-priate) agar. Dilutions of 100, 101, 102 areusually suitable for treated test fabrics.Several different dilutions may be re-quired for untreated control fabrics de-pending on the incubation period.

10.2.7 Incubate all plates for 48 hat 37 ± 2°C (99 ± 3°F) or other optimaltemperature.

11. Evaluation

11.1 Report bacterial counts as thenumber of bacteria per sample (swatchesin jar) not as the number of bacteria permL of neutralizing solution. Report “0”counts at 100 dilution as “less than 100.”

11.2 Calculate percent reduction ofbacteria by the specimen treatments byone of the following formulas:

1) 100 (B – A)/B = R

where:R = % reductionA = the number of bacteria recovered

from the inoculated treated testspecimen swatches in the jar in-cubated over the desired contactperiod

B = the number of bacteria recoveredfrom the inoculated treated testspecimen swatches in the jar im-mediately after inoculation (at“0” contact time)

2) 100 (C – A)/C = R

where:C = the number of bacteria recovered

from the inoculated untreatedcontrol specimen swatches in thejar immediately after inoculation(at “0” contact time)

If “B and C” are not similar, the largernumber should be used. If “B” and “C”are not significantly different, (B + C)/2should be used as follows:

3) 100(D – A)/D = R

where:D = (B + C)/2

11.3 If an untreated control is not avail-able, use the following calculation whichallows for any background organismsthat might interfere with the test.

Bg = 100 [(B – E) – (A – F)/B – E]

where:A, B = (see 11.2)E = the number of bacteria initially

recovered from the uninocu-lated treated test sample (exist-ing background organisms)

F = The number of bacteria recov-ered from the uninoculated,pre-wet treated test sample afterincubation in the jar over thedesired contact period (existingbackground organisms aftercontact period)

Bg = background organisms

11.4 For a valid test there should be:(1) “0” colonies of test organism recov-ered from the uninoculated treated testspecimen swatches and (2) a significantincrease in the numbers of bacteria recov-ered from the inoculated untreated con-trol specimen swatches incubated for thespecified contact time over the numbersof bacteria recovered from the inoculateduntreated specimen swatches at “0” con-tact time (immediately after inoculation).This applies only if dilution was made inbroth (see 10.1.4 and 13.7).

11.5 Report percent reduction of bac-teria by the specimen treatment againsteach test organism.

11.6 The criterion for passing the testmust be determined by the interestedparties.

11.7 Report the dilution medium used.

12. Precision and Bias

12.1 Studies (see 13.10) indicate thefollowing within-laboratory precision ofthe Standard Plate Count (SPC) Test: (a)among-analyst variation of 18% and (b)within-analyst variation of 8%.

*Using a 1 mL pipette, pad the inoculum carefully ontothe fabric. If a strain of Pseudomonas that forms a pelli-cle is used, avoid including fragments of the pellicle inthe inoculum.

Copyright © 2009 American Association of Textile Chemists and Colorists

144 TM 100-2004 AATCC Technical Manual/2010

13. Notes and References

13.1 Publication available from U.S. De-partment of Health and Human Services CDC/NIH-HHS Publication No. (CDC) 84-8395;web site: www.hhs.gov.

13.2 Booklet available from PublicationsOffice, ACGIH, Kemper Woods Center, 1330Kemper Meadow Dr., Cincinnati OH 45240;tel: 513/742-2020; web site: www.acgih.org.

13.3 Available from American Type Cul-ture Collection (ATCC), P.O. Box 1549,Manassas VA 20108; tel: 703/365-2700; fax:703/365-2701; web site: www.atcc.org.

13.4 Bacto-Peptone may be obtained fromDifco Laboratories, 920 Henry St., Detroit MI48201.

13.5 Beef extract may be obtained fromBaltimore Biological Laboratories, 250 Schill-ing Cir., Cockeysville MD 21030; Difco Labo-ratories (address above); or Oxoid (USA) Ltd.,9017 Red Branch Rd., Columbia MD 21045.

13.6 Consistent and accurate testing re-quires maintenance of a pure, uncontaminated,nonmutant test culture. Avoid contaminationby use of good sterile technique in plating andtransferring. Avoid mutation by strict adher-ence to monthly stock transfers. Check culturepurity by making streak plates periodicallyand observing for single species-characteristictype of colonies.

13.7 The dilution of the test organism maybe prepared in sterile 0.85% saline solution orsuitable buffer if a steady-state culture is de-

sired during the contact period with a fabric orin the slurry inoculum carrier when hydropho-bic fabrics are being tested.

13.8 A surfactant may be added to the dilu-tion medium to enhance wetting of hydropho-bic fabrics. The surfactant must be shown not tocause a reduction in bacterial numbers, by priortesting at the intended use concentration. Reportthe use and concentration of surfactant used.

13.9 If sterile distilled water is used in theplace of a neutralizing solution, there will al-ways be the possibility that some of the bio-cide will be carried over.

13.10 Peeler, J. T.; Leslie, J. W.; Messer,J. W. Replicate counting errors by analystsand bacterial colony counters. J. Food Protec-tion, Vol. 45, 1982, pp 238-240.

Copyright © 2009 American Association of Textile Chemists and Colorists