AATCC TM100-2019
Test Method for Antibacterial Finishes on Textile Materials: Assessment of
1. Purpose and Scope
1.1 This test method provides a quanti-
tative procedure for the evaluation of the
degree of antibacterial activity. Assess-
ment of antibacterial finishes on textile
materials is determined by the degree of
antibacterial activity intended in the use
of such materials. If only bacteriostatic
activity (inhibition of multiplication) is
intended, a qualitative procedure which
clearly demonstrates antibacterial activity
as contrasted with lack of such activity by
an untreated specimen may be accept-
able. However, if bactericidal activity is
intended or implied, quantitative evalua-
tion is necessary. Quantitative evaluation
also provides a clearer picture for possi-
ble uses of such treated textile materials.
2. Principle
2.1 This test method provides a quanti-
tative procedure for the comparison and
evaluation of the degree of antibacterial
activity after a 24 h exposure to the test
bacteria on the test fabric compared di-
rectly against an untreated control. After
incubation, the bacterial challenge is
eluted from the swatches and enumerated
and a percent reduction by the fabric
specimen is calculated.
3. Terminology
3.1 activity, n.—of an antibacterial
agent, a measure of effectiveness of the
agent.
3.2 antibacterial agent, n.—any
chemical which kills bacteria (bacteri-
cide) or interferes with the multiplication,
growth or activity of bacteria (bacteri-
ostat).
4. Safety Precautions
4.1 The safety precautions specified in
the method are ancillary to the testing
procedures and are not intended to be all
inclusive.
4.2 It is the user’s responsibility to ref-
erence applicable safety data sheets, use
safe and proper techniques, and wear ap-
propriate personal protective equipment
in handling materials in this standard.
4.3 Users MUST be consult manufac-
turers for specific details such as safety
data sheets, equipment operating instruc-
tions, and other recommendations. Con-
sult and follow all applicable health and
safety regulations (e.g., OSHA standards
and rules).
4.4 This test should be performed only
174 AATCC TM100-2019
Copyright © 2020 American Association of Textile Chemists and Colorists
by appropriately trained personnel. The
U.S. Department of Health and Human
Services publication, Biosafety in Micro-
biological and Biomedical Laboratories,
should be consulted (see 13.1).
4.5 CAUTION: Some of the microor-
ganisms used in these tests are allergenic
and pathogenic; i.e., capable of infecting
humans and producing disease. There-
fore, every necessary and reasonable pre-
caution must be taken to eliminate this
risk to the laboratory personnel and to
personnel in the associated environment.
Wear protective clothing, respiratory pro-
tection, and impervious gloves when
working with the organisms.
4.6 Sterilize all contaminated samples
and test materials prior to disposal.
5. Test Organisms
5.1 Test bacteria:
5.1.1 Staphylococcus aureus, ATCC
No. 6538, Gram positive organism, CIP
4.83, DSM 799, NBRC 13276, NCIMB
9518 or equivalent strain (see 13.2 and
13.3).
5.1.2 Klebsiella pneumoniae, ATCC
No. 4352, Gram negative organism, CIP
104216, DSM 789, NBRC 13277,
NCIMB 10341 or equivalent strain (see
13.2 and 13.3).
5.1.3 Other suitable species can also be
used depending on the intended end-use
of the test sample.
6. Materials, Media and Reagents
6.1 Media and reagents. Suitable broth/
agar media are:
6.1.1 Nutrient broth/agar.
6.1.2 Trypticase Soy broth/agar.
6.1.3 Sterile distilled or deionized
water.
6.1.4 Triton X-100.
6.1.5 Neutralizing broth: Letheen
broth, Dey Engley broth or other appro-
priate for the antimicrobial compound be-
ing tested.
6.1.6 Suspension Medium, 1:20 (5%)
Nutrient broth or Trypticase Soy broth +
0.05% Triton X-100. Dilute the nutrient
broth (TSB) 1:20 with distilled or deion-
ized water containing 0.05% Triton X-
100. Sterilize by autoclaving at 121 ±
2°C. If not used immediately after prepa-
ration, store at 5-10°C.
6.2 Materials.
6.2.1 Incubator maintained at 37 ± 2°C
(99 ± 4°F).
6.2.2 Inoculating loop.
6.2.3 Bunsen Burner or equivalent.
6.2.4 Water bath maintained at 45-
AATCC Manual of International Test Methods and Procedures/2021
50°C (113-122°F).
6.2.5 Pipettes and tips: 10-1000 μL pi-
pettes and appropriate tips.
6.2.6 Culture tubes with non-screw
caps; minimum 10 mL capacity.
6.2.7 Petri dishes, 100 mm diam. × 15
mm deep, sterile.
6.2.8 Forceps, sterile.
6.2.9 Stereomicroscope, minimum 40 ×
magnification.
6.2.10 Ruler.
6.2.11 Analytical balance, capable of
measuring 10.0 ± 0.1 g.
6.2.12 Sterile specimen container with
screw cap, 150-mL capacity or similar.
6.2.13 Exam gloves, nitrile or other.
6.2.14 Vortex mixer.
6.2.15 Steam sterilizer or autoclave.
6.2.16 Viability control fabric (see 13.4).
7. Preparation of Bacterial Inocula
7.1 Grow a fresh 18-h shake culture of
each test specimen in sterile Tryptic Soy
(or Nutrient) broth at 37 ± 2°C and 150-
250 rpm prior to performing the test.
These cultures should originate from a
single colony selected from stock culture
plates or growth on agar slants. Fresh
stock plates should be generated weekly.
7.1.1 Each 18-h shake culture is then
diluted with suspension medium (6.1.6),
as appropriate for the estimated bacterial
concentration, to obtain a bacterial con-
centration that is between 1.0 × 105 and
3.0 × 105 CFU/mL. These suspensions
are used as the test inoculum. Prepared
test inoculum is used within 2 h of prepa-
ration and bacterial concentration should
be verified by appropriate enumeration
method.
8. Test Specimens
8.1 Preparation. The following descrip-
tion will be in terms of fabric swatches.
Textile materials not in fabric form can
likewise be tested with the appropriate
modification.
8.1.1 Size and shape of test swatches:
Cut circular swatches 4.8 ± 0.1 cm (1.9 ±
0.03 in.) in diameter or square swatches
3.8 × 3.8 ± 0.1 cm, from the test fabric.
Use the number of swatches needed to
equal 1.0 ± 0.1g. Stack the swatches in a
sterile specimen container with screw cap
or other appropriate closed container. The
number of swatches to be used is depen-
dent on the fiber type and fabric construc-
tion. The number of swatches used per jar
should be reported.
8.1.2 Untreated Controls. Swatches of
the same fiber type and fabric construc-
tion as test sample but containing no anti-
bacterial finish will be required if calcu-
lating percent reduction using formula
outlined in 10.2.
8.1.3 Viability Controls. Viability con-
trol fabric is required and should be
known to demonstrate > 1 log bacterial
growth as defined in 10.4.
8.1.4 Test specimens should not be
sterilized prior to testing. If sterilization
is performed, method and reason for ster-
ilization must be noted on the test report.
9. Procedure
9.1 Size of inoculum per sample. Ap-
ply 1.0 ± 0.1 mL of test inoculum (7.1.1)
so that recovery from (1) viability control
fabric swatches or (2) test fabric swatches
at “0” contact time (plated as soon as pos-
sible after inoculation) will show counts
of 1-3 × 105 organisms.
9.1.1 As soon as possible after inocula-
tion (“0” contact time), add 100 ± 1 mL
of neutralizing solution to each of the jars
containing the inoculated untreated con-
trol swatches, the inoculated test
swatches and the viability control fabric
swatches.
9.1.2 The neutralizing solution should
include ingredients to neutralize the spe-
cific antibacterial fabric treatment and to
take care of any pH requirements of the
fabrics (from finishes, antibacterial
agents, etc.). The neutralizing solution
employed should be reported.
9.1.3 Shake the jars vigorously for one
minute. Make serial dilutions with sterile
distilled water and plate (in duplicate) on
nutrient agar. Dilutions of 100, 101, 102
are usually suitable.
9.1.4 Incubation over contact periods.
Incubate additional jars containing inocu-
lated viability control swatches and jars
containing inoculated test swatches at 37
± 2°C (99 ± 3°F) for 24 h. Similar jars
may be incubated over other periods
(e.g., 1 or 6 h) to provide information
about the bactericidal activity of the treat-
ment over such periods.
9.1.5 Sampling of viability control and
test swatches. After incubation, add 100
± 1 mL of neutralizing solution to jars
containing viability control swatches and
to jars containing test swatches. Shake
the jars vigorously for 1 minute. Make
serial dilutions and plate (in duplicate) on
nutrient agar. Dilutions of 100, 101, 102
are usually suitable for treated test fab-
rics. Dilutions of 103 or 104 may be re-
quired for viability control and untreated
control swatches depending on the incu-
bation period.
9.1.6 Incubate all plates for 24-48 h at
37 ± 2°C (99 ± 3°F).
AATCC Manual of International Test Methods and Procedures/2021
Copyright © 2020 American Association of Textile Chemists and Colorists
10. Calculation
10.1 Report bacterial counts as the
number of bacteria per sample (swatches
in jar) not as the number of bacteria per
mL of neutralizing solution. Report “0”
counts at 100 dilution as “less than 100.”
10.2 Calculate percent reduction of
bacteria by the specimen treatments using
Eq. 1.
100(B – A)/B = R (Eq. 1)
where:
R = % reduction
A = the number of bacteria recovered
from the inoculated treated test
specimen swatches in the jar in-
cubated over the 24-h contact pe-
riod
B = the number of bacteria recovered
from the inoculated untreated test
specimen swatches in the jar in-
cubated over the 24-h contact pe-
riod.
10.3 If an untreated control is not avail-
able, use Eq. 2.
100(C – A)/C = R (Eq. 2)
where:
C = the number of bacteria recovered
from the inoculated, test speci-
men swatches in the jar immedi-
ately after inoculation (at “0”
contact time)
10.4 For a valid test there should be:
(1) “0” colonies of test organism recov-
ered from the uninoculated test specimen
swatches and (2) a significant (≥ 1 log)
increase in the numbers of bacteria recov-
ered from the inoculated viability control
specimen swatches incubated for the
specified contact time over the numbers
of bacteria recovered from the inoculated
viability specimen swatches at “0” con-
tact time (immediately after inoculation).
11. Report
11.1 Report percent reduction of bacte-
ria by the specimen treatment against
each test organism. Report should include
the calculation method used.
11.2 The criterion for passing the test
must be determined by the interested par-
ties.
11.3 All variables and materials used in
the test including sample size, steriliza-
tion and method, media used, neutralizer
used, and the dilution medium used.
12. Precision and Bias
12.1 Precision for this test method has
not been established. Until a precision
statement is generated for this test
method, caution should be used when
testing materials with this method. In
most cases the use of standard statistical
techniques in making any comparisons of
test results for either within-laboratory or
between-laboratory averages have been
found to be generally accepted.
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 ATCC is the American Type Culture
Collection (USA), P.O. Box 1549, Manassas
VA 20108; tel: +1.703.365.2700; fax: +1.703.
365.2701; web site: www.atcc.org. CIP is the
Pasteur Institute Collection (France), DSM is
the German Collection of Microorganisms
and Cell Cultures (Germany), NBRC is the
NITE Biological Resource Center (Japan),
NRRL is the Northern Regional Research Lab
(USA), NCIMB is the National Collection of
Industrial Bacteria (UK), and CUG is the Cul-
ture Collection University of Göteborg (Swe-
den). Equivalent bacteria strains obtained
from agencies of the World Federation of Cul-
ture Collection (WFCC) may be used by
agreement between the interested parties. The
strains used in the test shall be documented
with their supply source.
13.3 Consistent and accurate testing re-
quires maintenance of a pure, uncontaminated,
nonmutant test culture. Avoid contamination
by use of good sterile technique in plating and
transferring. Avoid mutation by strict adher-
ence to monthly stock transfers. Check culture
purity by making streak plates periodically
and observing for single species-characteristic
type of colonies.
13.4 A suitable viability control fabric
known to demonstrate > 1 log growth under
the standard conditions in this method has
been identified and can be purchased through
the International Antimicrobial Council
(www.amcouncil.org).
14. History
14.1 Last revised 2019 to remove several
issues that led to confusion among users. Am-
biguity was reduced by defining the parame-
ters for use of untreated viability controls,
concentration of nutrient in inoculum me-
dium, inoculum preparation, fabric swatch
preparation, sample sterilization, and final test
report contents.
14.2 Revised 2012. Editorially revised
2010, 2009. Reaffirmed 2008. Editorially re-
vised and reaffirmed 2004. Revised 1999. Re-
affirmed 1998. Revised 1993. Reaffirmed
1989. Revised (with title change) 1988. Edito-
rially revised and reaffirmed 1986. Editorially
revised 1985. Revised 1981. Reaffirmed 1977.
Editorially revised 1974, 1971, 1969. Revised
1965.
14.3 Developed in 1961 by AATCC Com-
mittee RA31.
AATCC TM100-2019 175