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: E2180 − 18

Standard Test Method for

Determining the Activity of Incorporated Antimicrobial
Agent(s) In Polymeric or Hydrophobic Materials1
This standard is issued under the fixed designation E2180; 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.

INTRODUCTION

Polymeric materials such as vinyl pool liners, shower curtains, and various medical devices are
treated frequently with incorporated or bound antimicrobial agents. Practices G21 is used to determine
the ability of polymer materials to resist microbial attack or staining (see also Practice E1428);
however, none of the methods permit quantitative evaluations of incorporated antimicrobial activity.2
These antimicrobials typically require contact with the microbial cell for maximal activity. When
aqueous based bacterial inoculum suspensions are applied onto a preservative-treated plastic or other
hydrophobic material, the surface tension of the polymer often causes the inocula suspension to dome.
Bacteria within the drops of inoculum may not contact the treated surface if the challenged surface
does not dry, or upon drying, cells may become layered. This test standard involves an agar slurry
inoculum vehicle that provides a relatively uniform contact of the inocula with antimicrobial-treated
hydrophobic surfaces.

1. Scope mination of quantitative differences in antimicrobial activity

1.1 This test method is designed to evaluate (quantitatively) between untreated plastics or polymers and those with bound
the antimicrobial effectiveness of agents incorporated or bound or incorporated low water-soluble antimicrobial agents. Com-
into or onto mainly flat (two dimensional) hydrophobic or parisons between the numbers of survivors on preservative-
polymeric surfaces. The method focuses primarily on assessing treated and control hydrophobic surfaces may also be made.
antibacterial activity; however, other microorganisms such as 1.4 The procedure also permits determination of “shelf-life”
yeast and fungal conidia may be tested using this method. or long term durability of an antimicrobial treatment which
1.2 The vehicle for the inoculum is an agar slurry which may be achieved through testing both non-washed and washed
reduces the surface tension of the saline inoculum carrier and samples over a time span.
allows formation of a “pseudo-biofilm,” providing more even 1.5 Knowledge of microbiological techniques is required
contact of the inoculum with the test surface. for these procedures.
NOTE 1—This test method facilitates the testing of hydrophobic 1.6 The values stated in SI units are to be regarded as
surfaces by utilizing cells held in an agar slurry matrix. This test method,
as written, is inappropriate to determine efficacy against biofilm cells, standard. No other units of measurement are included in this
which are different both genetically and metabolically than planktonic standard.
cells used in this test.
1.7 This standard does not purport to address all of the
1.3 This method can confirm the presence of antimicrobial safety concerns, if any, associated with its use. It is the
activity in plastics or hydrophobic surfaces and allows deter- responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter-
1
This test method is under the jurisdiction of ASTM Committee E35 on mine the applicability of regulatory limitations prior to use.
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
responsibility of Subcommittee E35.15 on Antimicrobial Agents. 1.8 This international standard was developed in accor-
Current edition approved May 1, 2018. Published May 2018. Originally dance with internationally recognized principles on standard-
approved in 2001. Last previous edition approved in 2018 as E2180 – 07(2017). ization established in the Decision on Principles for the
DOI: 10.1520/E2180-18.
2
Development of International Standards, Guides and Recom-
Price, D. L., Sawant, A. D., and Ahearn, D. G., “Assessment of the antimicro-
bial activity of an insoluble quaternary amine complex in plastics,” J. Industr. mendations issued by the World Trade Organization Technical
Microbiol, Vol 8, No. 2, 1991, pp. 83–89. Barriers to Trade (TBT) Committee.

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); Fri Jun 26 08:24:41 EDT 2020
1
Downloaded/printed by
(UFRJ) Universidade Federal do Rio de Janeiro ((UFRJ) Universidade Federal do Rio de Janeiro) pursuant to License Agreement. No further reproductions authorized.
 E2180 − 18
2. Referenced Documents 5. Significance and Use
3
2.1 ASTM Standards: 5.1 This method can be used to evaluate effectiveness of
E1054 Test Methods for Evaluation of Inactivators of Anti- incorporated/bound antimicrobials in hydrophobic materials
microbial Agents such as plastics, epoxy resins, as well as other hard surfaces.
E1428 Test Method for Evaluating the Performance of 5.2 The aqueous based bacterial inoculum remains in close,
Antimicrobials in or on Polymeric Solids Against Staining uniform contact in a “pseudo-biofilm” state with the treated
by Streptomyce species (A Pink Stain Organism) material. The percent reduction in the surviving populations of
G21 Practice for Determining Resistance of Synthetic Poly- challenge bacterial cells at 24 h versus those recovered from a
meric Materials to Fungi non-treated control is determined.
3. Terminology 5.3 The hydrophobic substrate may be repeatedly tested
over time for assessment of persistent antimicrobial activity.
3.1 Definitions:
3.1.1 agar slurry, n—a semi-gelatinous liquid formed when 6. Apparatus
3 g/L agar-agar is added to a 0.85 % saline solution. 6.1 Erlenmeyer Flask, 250 mL.
3.1.2 inoculum, n—in microbiology, a specimen comprised 6.2 Petri Dishes, (15 × 100 mm), sterile.
of living spores, bacteria, single celled organisms, or other live
materials, yeast or the multicellular filamentous fungi, or 6.3 Colony Counter.
combination of two or more types of microorganisms, that are 6.4 Specimen Cups, (120 mL), sterile or equivalent sterile
introduced into a test medium or onto a specimen to be tested equipment for extraction.
in order to investigate the lability of the medium or specimen 6.5 Pipetters, (1000 µL) positive displacement.
to support microbial growth or to investigate its antimicrobial
properties. 6.6 Pipette Tips, sterile.
3.1.3 inoculum vehicle, n—the carrier solution used to 6.7 Test Tubes, 16 × 100 mm.
transport the carrier solution used to transport the inoculum to 6.8 Incubator, set at required temperature (25-35 6 2 °C).
a given sample or object.
6.9 Autoclave.
3.1.4 neutralizing recovery broth, n—liquid growth media
6.10 Water Bath, capable of maintaining water at 45 6 2 °C.
used to inactivate the effects of the test antimicrobial agent.
6.11 Sterile Cotton Swabs.
4. Summary of Test Method 6.12 Sonic Bath, 47 Khz, cleaning non-cavitating.
4.1 This method involves inoculation of a molten (45 °C) 6.13 Vortex Mixer.
agar slurry with a standardized culture of bacterial cells.
6.14 pH Meter.
4.2 A thin layer of the inoculated agar slurry (0.5-1.0 mL) is
6.15 Hot Plate, with stirrer.
pipetted onto the test and untreated control material (triplicate
samples minimum). 6.16 Spectrophotometer, set at 600 nm.
4.3 After the specified contact time (24 h commonly used), 6.17 Sterile Cuvettes.
surviving microorganisms are recovered via elution of the agar 6.18 Test Materials, sterile if specified by interested parties.
slurry inoculum from the test substrate into neutralizing broth
6.19 Cell Counting Chamber.
and extracted via methods that provide complete removal of
the inoculum from the test article (examples include 7. Reagents
sonication, vortexing, and/or manual extraction, that is, stom-
7.1 Media:
acher).
7.1.1 Tryptic Soy Broth, or appropriate broth.
4.4 Serial dilutions are made, then pour or spread plates are 7.1.2 Tryptic Soy Agar, or appropriate agar.
made of each dilution. Agar plates and dilution broths are 7.1.3 Neutralizing Broth, appropriate for the antimicrobial
incubated for 48 62 h at a specified temperature dependent compound tested.(See Practice E1054.)
upon the optimal temperature for test organism. 7.1.4 Agar-agar.
4.5 Bacterial colonies from each dilution series are counted 7.1.5 NaCl.
and recorded. 7.1.6 Sterile Deionized Water.
7.1.7 Sterile 0.85 % Saline Dilution Blanks, 9.0 mL in 16 ×
4.6 Calculation of percent reduction of bacteria from treated 100 mm test tubes or appropriate dilution buffer (such as
versus untreated samples is made. phosphate buffer or Butterfield’s buffer).
7.2 Test Organisms—Specific organisms are recommended
3
but choice of organism should be relevant to the environment
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
in which the product will perform.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on 7.2.1 Gram-positive bacteria Staphylococcus aureus ATCC
the ASTM website. 6538.

Copyright by ASTM Int'l (all rights reserved); Fri Jun 26 08:24:41 EDT 2020
2
Downloaded/printed by
(UFRJ) Universidade Federal do Rio de Janeiro ((UFRJ) Universidade Federal do Rio de Janeiro) pursuant to License Agreement. No further reproductions authorized.
 E2180 − 18
7.2.2 Gram-negative bacteria Pseudomonas aeruginosa within the incubator should be at or above 75 %. This can be
ATCC 15442 or Klebsiella pneumoniae ATCC 4352. accomplished with open reservoirs of certain saturated salt in
7.2.3 Other microorganisms such as yeast or fungal conidia water solutions.4
may also be tested using this procedure. Exposure periods may 8.11 Make serial dilutions (as described in 8.12 – 8.16) of
be modified (up to 96 h) to address more resistant microorgan- the agar slurry recovered immediately from “0” h control
isms. samples and spread or pour plate each dilution to determine
cfu/mL recoverable at time “0 h.”
8. Procedure
8.12 Following the specified contact time, aseptically re-
8.1 Grow 18 h bacterial cultures (three transfers) at a move the incubation period control samples and incubation
specified temperature dependent upon the optimal temperature period treated samples from the petri dishes to 120 mL
for the test organism in tryptic soy or appropriate broth. These specimen cups or other suitable container containing a suffi-
cultures should originate from 18-24 h growth coming from cient volume of neutralizing broth to form an initial 1:10 or
stock culture plates or growth on agar slants. 1:100 dilution of the original inoculum.
8.2 Prepare the agar slurry by dissolving 0.85 g NaCl and 8.13 Place the specimen cups containing the recovered test
0.3 g agar-agar in 100 mL of deionized water. Heat with samples into a non-cavitating sonic bath and sonicate for 1
stirring on a hot plate until the agar dissolves. One agar slurry min.
should be prepared for each organism tested.
8.14 Sonication should be followed by 1 min of vigorous
8.3 Sterilize the agar slurry by autoclaving for 15 min at 121 mechanical vortexing. This should facilitate the complete
6 2 °C, 15 psi., then equilibrate at 45 6 2 °C. release of the agar slurry from the sample. Note that the test
8.4 Prepare 3.0 × 3.0 cm square samples of the treated and surface may be imprint cultured onto tryptic soy agar following
control test materials (sample minimum of triplicates for “0” h sonication and vortexing in order to determine release effi-
controls, incubation period treated samples and incubation ciency of the inoculum from the treated surface.
period control samples for each test organism). 8.15 Perform serial dilutions of the initial neutralizing broth
sufficient to include one dilution beyond the original inoculum
8.5 Place each sample into a sterile 15 × 100 mm petri dish.
(as determined in 8.11).
8.6 Adjust bacterial broth cultures to 1-5×108 cells/mL with
8.16 Spread or pour plate each dilution into tryptic soy agar
a spectrophotometer or cell counting chamber.
or other appropriate agar (for example, Sabourauds agar for
8.7 Dip a cotton swab into sterile 0.85 % saline (with or fungi) and incubate plates at an optimal temperature for the test
without a non-inhibitory levels of a surfactant) and pre-wet the organism for 48 6 2 h.
test sample. This will aid in dispersing the agar slurry evenly 8.17 Count and record colony numbers for each dilution
on the sample. plate.
8.8 Place 1.0 mL of standardized culture (1-5×108 cells/mL)
into the 100 mL agar slurry equilibrated at 45 6 2 °C. The final 9. Calculation
concentration should be 1-5×106 cells/mL in the molten agar 9.1 Determine the geometric mean of the number of organ-
slurry. isms recovered from the triplicate incubation period control
8.9 Pipet 0.5-1.0 mL of inoculated agar slurry onto the test and incubation period treated samples by the following equa-
and control samples. Slow, gentle application at a low angle of tion:
incidence relative to the sample will aid in formation of a film ~ Log10X 1 1Log10X 2 1Log10X 3 !
no more than 1 mm in depth. If the inoculum volume and/or geometric mean 5 (1)
3
surface area of the sample are modified, the inoculum volume
where:
should be adjusted to provide an agar slurry 1 mm in depth
over the entire sample surface. The inoculum volume and X = number of organisms recovered from the incubation
surface area tested should be reported. period control or incubation period treated samples.

NOTE 2—For substrates found to be extremely hydrophobic, addition of

9.2 Percent Reduction—Use the following equation to cal-
non-inhibitory levels of a surfactant to the agar slurry may also aid in culate the percent reduction:
surface wetting properties. Alternatively, the slurry may be cooled to 25 6 ~ a 2 b ! 3 100
2 °C then applied to the polymer surface. Application of pre-cooled agar % reduction 5 (2)
a
slurry has also been shown to aid in the dispersion of the inoculum over
an extremely hydrophobic test surface. where:
8.10 Allow the agar slurry inoculum to gel and then place a = the antilog of the geometric mean of organisms recov-
the samples in an incubator at a specified temperature depen- ered from the incubation period control samples (as
dent upon the optimal temperature for the test organism or one determined in 9.1), and
which mimics the temperature in which the test substrate will
be utilized for the specified exposure period (usually 24 6 2 h).
Low humidity in the incubator can cause drying of the agar 4
Corey, J. In Beuchat, L.R. ed., Food and Beverage Mycology, AVI Publishing
slurry inoculum on the samples, therefore relative humidity Co., Inc., Westport, CT, p. 52.

Copyright by ASTM Int'l (all rights reserved); Fri Jun 26 08:24:41 EDT 2020
3
Downloaded/printed by
(UFRJ) Universidade Federal do Rio de Janeiro ((UFRJ) Universidade Federal do Rio de Janeiro) pursuant to License Agreement. No further reproductions authorized.
 E2180 − 18

b = the antilog of the geometric mean of the number of 11. Keywords

organisms recovered from the incubation period treated 11.1 agar slurry; hydrophobic surface; percent bacterial
samples (as determined in 9.1). reduction; plastics; polymers; quantitative antibacterial assay
10. Precision and Bias
10.1 A precision and bias statement has not been developed
for this method at this time.

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); Fri Jun 26 08:24:41 EDT 2020
4
Downloaded/printed by
(UFRJ) Universidade Federal do Rio de Janeiro ((UFRJ) Universidade Federal do Rio de Janeiro) pursuant to License Agreement. No further reproductions authorized.