This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles

for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

Designation: E2149 − 20

Standard Test Method for

Determining the Antimicrobial Activity of Antimicrobial
Agents Under Dynamic Contact Conditions1
This standard is issued under the fixed designation E2149; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.

1. Scope 1.8 This standard may involve hazardous materials, opera-

1.1 This test method is designed to evaluate the antimicro- tions and equipment. This standard does not purport to address
bial activity of antimicrobial-treated specimens under dynamic all of the safety concerns, if any, associated with its use. It is
contact conditions. This dynamic shake flask test was devel- the responsibility of the user of this standard to establish
oped for routine quality control and screening tests in order to appropriate safety, health, and environmental practices and
overcome difficulties in using classical antimicrobial test meth- determine the applicability of regulatory limitations prior to
ods to evaluate substrate-bound antimicrobials. These difficul- use.
ties include ensuring contact of inoculum to treated surface (as 1.9 This international standard was developed in accor-
in AATCC TM100), flexibility of retrieval at different contact dance with internationally recognized principles on standard-
times, use of inappropriately applied static conditions (as in ization established in the Decision on Principles for the
AATCC TM147), sensitivity, and reproducibility. Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
1.2 This test method allows for the ability to evaluate many Barriers to Trade (TBT) Committee.
different types of treated substrates and a wide range of
microorganisms. Treated substrates used in this test method 2. Referenced Documents
can be subjected to a wide variety of physical/chemical stresses 2.1 ASTM Standards:2
or manipulations and allows for the versatility of testing the E1054 Test Methods for Evaluation of Inactivators of Anti-
effect of contamination due to such things as hard water, microbial Agents
proteins, blood, serum, various chemicals, and other contami- E2756 Terminology Relating to Antimicrobial and Antiviral
nants. Agents
1.3 Surface antimicrobial activity is determined by compar- E2922 Guide for The Use of Standard Test Methods and
ing results from the test sample to controls run simultaneously. Practices for Evaluating Antibacterial Activity on Textiles
2.2 AATCC Documents:3
1.4 This test method may not be appropriate for all types of
AATCC TM147 Antibacterial Activity Assessment of Tex-
antimicrobial-treated articles or antimicrobial agents. The
tile Materials: Parallel Streak Method
proper test methodology should be determined based on
AATCC TM100 Antibacterial Finishes on Textile Materials:
antimicrobial mode of action and end-use expectations (Guide
Assessment of
E2922)
1.5 Proper neutralization of all antimicrobials must be 3. Terminology
confirmed using Test Methods E1054. 3.1 For definitions of terms used in this test method, refer to
1.6 This test method should be performed only by those E2756 Standard Terminology Relating to Antimicrobial and
trained in microbiological techniques. Antiviral Agents.
1.7 The values stated in SI units are to be regarded as 4. Summary of Test Method
standard. No other units of measurement are included in this 4.1 The antimicrobial activity of some substrate-bound,
standard. antimicrobial agents is dependent upon direct contact of

2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
1
This test method is under the jurisdiction of ASTM Committee E35 on contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct Standards volume information, refer to the standard’s Document Summary page on
responsibility of Subcommittee E35.15 on Antimicrobial Agents. the ASTM website.
3
Current edition approved Sept. 15, 2020. Published October 2020. Originally Available from American Association of Textile Chemists and Colorists
approved in 2001. Last previous edition approved in 2013 as E2149 – 13a. DOI: (AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http://
10.1520/E2149-20. www.aatcc.org.

Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
Copyright by ASTM Int'l (all rights reserved); Wed Oct 20 20:20:55 EDT 2021
1
Downloaded/printed by
() pursuant to License Agreement. No further reproductions authorized.
 E2149 − 20
microbes with the surface of the textile containing the active 6.6 Spectrophotometer, capable of measuring an absorbance
chemical agent. This is particularly true for quaternary-silane of 475 nm.
type agents. This test determines the antimicrobial activity of a 6.7 Sterile serological pipettes, capable of 50 and 10 mL
treated specimen by shaking samples of surface-bound mate- capacity.
rials in a concentrated bacterial suspension for a one hour
contact time. The suspension is serially diluted both before and 6.8 Sterilizer, any suitable steam sterilizer producing the
after contact and cultured. The number of viable organisms conditions of sterility.
from the suspension is determined and the percent reduction 6.9 Vortex mixer, to vortex dilution tubes during serial
(or log10 reduction) is calculated by comparing retrievals from dilutions.
appropriate controls. 6.10 Water bath, for short term storage of liquefied agar
media, capable of maintaining 45 to 50 °C.
5. Significance and Use
5.1 Substrate–bonded, antimicrobial agents are not typically 7. Reagents
free to diffuse into their environment under normal conditions 7.1 Buffer Solution—The following solution is prepared
of use. This test method ensures good contact between the from reagent-grade chemicals. For buffer stock solution
bacteria and the treated fiber, fabric, or other substrate, by (0.25M KH2PO4): Prepare a fresh stock solution at least once
constant agitation of the test specimen in a challenge suspen- every 6 months as follows: Weigh 34 6 0.1 g of potassium
sion during the test period. dihydrogen phosphate into a 1000 mL beaker. Add 500 mL of
5.2 The metabolic state of the challenge species can directly distilled water. Adjust pH to 7.2 6 0.1 with a dilute solution of
affect measurements of the effectiveness of particular antimi- NaOH. Dilute to 1000 mL; transfer to a flask and store at 4 °C.
crobial agents or concentrations of agents. The susceptibility of For working buffer solution (0.3mM KH2PO4): Prepare a fresh
the species to particular biocides could be altered depending on solution at least once every 2 months as follows: Transfer 1 6
its life stage (cycle). One-hour contact time in a buffer solution 0.01 mL of stock buffer solution with a sterile pipette to flask
allows for metabolic stasis in the population. This test method containing 800 mL of distilled water. Cap, sterilize and store at
standardizes both the growth conditions of the challenge room temperature.
species and substrate contact times to reduce the variability 7.2 Media:
associated with growth phase of the microorganism. 7.2.1 Tryptic Soy Broth, prepared according to manufactur-
5.3 Leaching of an antimicrobial is dependent upon the test er’s directions.
conditions being utilized and the ultimate end use of the 7.2.2 Plate Count Agar, prepared according to manufactur-
product. Additional testing may be required to determine if a er’s directions.
compound is substrate-bound in all conditions or during the 7.3 Wetting Agent Surfactant—Agents must be shown by
end use of the product. prior testing at the intended use concentration not to cause a
5.4 This test method cannot determine if a compound is reduction or increase in bacterial numbers. DC Q2-52114 at
leaching into solution or is immobilized on the substrate. This 0.01 % final dilution of working buffer solution has been
test method is only intended to determine efficacy as described shown to be effective.
in subsequent portions of the method.
8. Test Organism
5.5 The test is suitable for evaluating stressed or modified
specimens, when accompanied by adequate controls. 8.1 Escherichia coli, American Type Culture Collection No.
NOTE 1—Stresses may include laundry, wear and abrasion, radiation 25922.
and steam sterilization, UV exposure, solvent manipulation, temperature 8.1.1 Cultures of the test organism should be maintained
susceptibility, or similar physical or chemical manipulation. according to good microbiological practice and checked for
purity on a routine basis. Consistent and accurate testing
6. Apparatus requires maintenance of a pure, uncontaminated test culture.
6.1 Air displacement pipettes, Eppendorf or equivalent, 100 Avoid contamination by use of good sterile technique in plating
to 1000 µL with disposable tips. and transferring. Avoid mutation or reversion by strict adher-
ence to monthly stock transfers. Check culture purity by
6.2 Analytical balance, to weigh chemicals and substrates
making streak plates periodically, observing for colonies char-
and to standardize inoculum delivery volumes by pipettes.
acteristic of Escherichia coli, and Gram-staining.
6.3 Glassware:
NOTE 2—This test method was developed and validated using ATCC
6.3.1 Contact Flask, 250 mL Erlenmeyer flask, capped, No. 25922 as the test organism. If an alternative culture is used, the results
autoclavable. must be reported as having been obtained using a modified test method. If
6.3.2 Test tubes, 18 × 150 mm rimless bacteriological test the test method is modified in any way, the report must also include a
tubes used for growing test organisms and for serial dilution.
6.4 Incubator, capable of maintaining a temperature of 35 6 4
The sole supplier of DC Q2-5211 known to the committee at this time is Dow
2 °C. Corning, Midland, MI. If you are aware of alternative suppliers, please provide this
information to ASTM International Headquarters. Your comments will receive
6.5 Shaker, wrist action, capable of aggressive agitation of careful consideration at a meeting of the responsible technical committee,1 which
bacteria and substrate solutions. you may attend.

Copyright by ASTM Int'l (all rights reserved); Wed Oct 20 20:20:55 EDT 2021
2
Downloaded/printed by
() pursuant to License Agreement. No further reproductions authorized.
 E2149 − 20
detailed description of all modifications made, including, but not limited only” sample for the series being run. Add 50 6 0.5 mL of
to: test organisms, media, buffer, contact time, bacterial concentration, etc. working dilution of bacterial inoculum prepared in 10.2 to each
9. Parameters flask.
9.1 Surface preparation or conditioning must be specified. 12.3 Determine bacterial concentration of solution at the
Prior manipulation of the specimen may be required in order to “0” time by performing serial dilutions and standard plate
demonstrate maximum activity in a desired time frame and count techniques from the “inoculum only” sample flask
must be reported and compared to identically handled controls. 12.4 Place the test and control specimen in their individual
9.2 The weight, size, and material of construction of speci- flasks. No series of test flasks should be large enough to require
men must be specified. more than 5 min, post-contact, between the first and last serial
dilution.
9.3 Specimens should be prepared such that they can
maximize agitation and are reflective of a recordable ratio of 12.5 Place the series of flasks on the wrist-action shaker.
surface area to test titer. Shake at maximum stroke for 1 h 6 5 min. Immediately serial
dilute and plate each sample out in triplicate, as was done for
10. Preparation of Bacterial Inoculum the “0” contact time subgroup (12.3).
10.1 Grow a fresh 18 h shake culture of Escherichia coli in NOTE 4—Residual bacterial retention in/on specimen could be tested
sterile Tryptic Soy Broth at 35 6 2°C prior to performing the using appropriate retrieval techniques such as agar imprint tests or buffer
test. extraction and plate count.
NOTE 5—Filter solutions in which samples have degraded during
10.2 Dilute the culture with the sterile buffer solution until shaking. Whatman filter paper Type 1 has been found to be appropriate for
the solution has an absorbance of 0.28 6 0.02 at 475 nm, as this step. Contents of the “inoculum only” flask must be treated in the
measured spectrophotometrically. This has a concentration of same manner.
1.5-3.0 × 108 CFU/mL. Dilute appropriately into sterile buffer NOTE 6—This test method was developed and validated using a 1-hour
solution to obtain a final concentration of 1.5-3.0 × 105 contact time. Alternate contact times have been used depending on the end
use of the product and specific antimicrobial mode of action. If alternative
CFU/mL. This solution will be the working bacterial dilution. contact times are used, the results must be reported as having been
obtained using a modified test method.
11. Test Specimen
12.6 Allow all the Petri dishes from both subsets to incubate
11.1 Preparation of Test Specimen:
for at 35 6 2 °C for 24 h.
11.1.1 Fabric and Paper—Samples are selected on weight
basis and weighed to 1.0 6 0.1 g. 12.7 Count the colonies in Petri dish. Record the values,
11.1.2 Powder and Granular Material—Weigh to 1.0 6 average the triplicate Petri dish numbers and convert the
0.1 g. The material must settle after shaking so that no average to colony-forming units per millilitre (CFU/mL).
specimen interferes with the retrieval and counting techniques. NOTE 7—The presence of the original test organism may be confirmed
11.1.3 Other Solids (Surface Treatment)—Reduce the solid by Gram stains and colony morphology.
in size to fit into the flask or use a sterile wide-mouth bottle. NOTE 8—When the number of colony-forming units per mL is less than
Use a specimen that gives 4 in.2 (25.8 cm2) of treated surface 30 on the lowest dilution plate, report the recovered CFU/mL as “<30”,
area. Specimen may also be selected on weight basis, 60.1 g, determine the percent reduction and report the reduction as “greater than”
the percent found.
at the discretion of the investigator. Care must be exercised
during shaking not to break the flask or bottle. The untreated 12.8 Calculate percent or log bacterial reduction.
specimen of the solid must not absorb the solution. If appro- 12.8.1 If the average CFU/mL values for the untreated
priate to the nature of the test specimen, it can be mounted as control and the “inoculum only” flask agree within 15 % after
a seal for the test container so that only the treated surface is in specified contact time, or if an untreated control is not
direct contact with the inoculum. available, calculate percent reduction of the organisms result-
ing from treated sample (A) directly compared to “inoculum
NOTE 3—Solids anticipated in this part of the method are plastics, glass
beads or chips, ceramics, metal chips, or similar hard surfaces. Sample only” flask after specified contact time (B) using the following
mass can vary from 0.5g-2.0g depending on sample composition. formula. Results can be presented in either percent reduction
However, the precision and bias statement has been developed using a one when measuring CFU/mL or as Log10 bacterial reduction when
gram sample of a textile material only. There are no data to support a calculating mean log10 density of bacteria.
precision and bias statement for other sample masses or sample types at
this point. Include in the report the use of alternate sample mass or type, B2A
in addition to any other modifications of temperature, method of dynamic Reduction, % ~ CFU/mL! 5 3 100
B
contact, etc. Log10 bacteria reduction 5 Log10 ~ B ! 2 Log10 ~ A !
12. Procedure for Determining Antimicrobial Activity where:
12.1 Prepare the specimen to be tested as described in A = CFU per millilitre for the flask containing the treated
Section 11. One treated piece of each specimen is required. substrate after the specified contact time, and
One untreated piece of each specimen of identical composition B = CFU per millilitre for the “inoculum only” flask after the
is highly recommended for each series of specimen tested. specified contact time.
12.2 Prepare one sterile 250 mL screw-cap Erlenmeyer flask 12.8.2 If the untreated control (if present) and the “inoculum
for each treated and untreated specimen, and one “inoculum only” flask do not agree within 15 %, calculate the percent

Copyright by ASTM Int'l (all rights reserved); Wed Oct 20 20:20:55 EDT 2021
3
Downloaded/printed by
() pursuant to License Agreement. No further reproductions authorized.
 E2149 − 20
reduction of organisms from treated sample (A) directly com- philic material). An ANOVA model was fit with random effects
pared to the untreated control (C). to determine the repeatability and reproducibility of the log
C2A density of organisms in the inocula, the repeatability and
Reduction, % ~ CFU/mL! 5 3 100 reproducibility of the organisms associated with the untreated
C
Log10 bacteria reduction 5 Log10 ~ C ! 2 Log10 ~ A ! controls, and the repeatability and reproducibility of the log
reductions of organisms associated with the treated sample
where: results.
A = CFU per millilitre for the flask containing the treated 13.1.2 For the inoculum for this protocol, the repeatability
substrate after the specified contact time, and standard deviation was 0.28; and the reproducibility standard
C = CFU per millilitre for the flask containing the untreated deviation was also 0.28, with 0.00 % of the variability due to
substrate after the specified contact time. among lab sources.
12.9 Record and report the value to the nearest one- 13.1.3 For the untreated control material for this protocol,
hundredth percent or log10 reduction of bacteria. Whether the repeatability standard deviation was 0.21; and the repro-
reduction calculations are based on values from an untreated ducibility standard deviation was 0.29, with 50 % of the
control or inoculum control shall be indicated on report. variability due to among lab sources.
NOTE 9—Utilizting a one hour time point, if no untreated control 13.1.4 The repeatability and reproducibility of the log
substrate is available, and the counts for the flask containing the reductions (calculated with respect to the inoculum) for each
“inoculum only” control after specified contact time (C) are not within 15 sample type are summarized in the Table 1.
% of original count, repeat the test. If longer time frames are utilized, such
13.1.5 The repeatability and reproducibility of the log
as 24h, it is possible to have growth within the “inoculum only” flask that
would exceed the allowable 15%, in this case the test would not be reductions (calculated with respect to the untreated controls)
repeated. for each sample type are summarized in Table 2.
13.1.6 For the high efficacy hydrophobic and hydrophilic
13. Precision and Bias5 samples considered, the method was statistically significantly
13.1 Precision: responsive to the increase in efficacy compared to the low
13.1.1 An interlaboratory study (ASTM ILS #715) of this efficacy sample.
test method was conducted at ten laboratories testing four
13.2 Bias—Since an accepted reference value is not
sample types (an untreated control, a low efficacy material, a
available, randomization is used whenever possible to reduce
high efficacy hydrophobic material, and a high efficacy hydro-
the potential for systematic bias.
5
Supporting data have been filed at ASTM International Headquarters and may 14. Keywords
be obtained by requesting Research Report RR:E35-1007. Contact ASTM Customer
Service at service@astm.org. 14.1 antimicrobial; antibacterial; shake flask test; textile

TABLE 1 Repeatability and Reproducibility Standard Deviations as a Function of the LR with Respect to the Inocula at the T1 Time
Point for the Three Sample Types Tested
Sample Description Mean LR Repeatability Among lab Reproducibility
SD % SD
2 low efficacy 1.70 0.3581 13 % 0.8775
high efficacy,
3 5.40 0.7042 63 % 1.15
hydrophobic
high efficacy,
4 5.98 0.3186 24 % 0.3643
hydrophilic

Copyright by ASTM Int'l (all rights reserved); Wed Oct 20 20:20:55 EDT 2021
4
Downloaded/printed by
() pursuant to License Agreement. No further reproductions authorized.
 E2149 − 20
TABLE 2 Repeatability and Reproducibility Standard Deviations as a Function of the LR with Respect to the Untreated Controls for the
Three Sample Types Tested.
Sample Description Mean LR Repeatability SD Among lab % Reproducibility SD
2 low efficacy 1.40 0.2111 93% 0.8255
high efficacy,
3 5.10 0.6940 62% 1.13
hydrophobic
4 high efficacy, hydrophilic 5.68 0.2369 57% 0.3596

ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentioned
in this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the risk
of 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 and
if not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standards
and should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the
responsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you should
make 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 above
address or at 610-832-9585 (phone), 610-832-9555 (fax), or service@astm.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, 222
Rosewood Drive, Danvers, MA 01923, Tel: (978) 646-2600; http://www.copyright.com/

Copyright by ASTM Int'l (all rights reserved); Wed Oct 20 20:20:55 EDT 2021
5
Downloaded/printed by
() pursuant to License Agreement. No further reproductions authorized.