ASTM E2180-24

Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) In Polymeric or Hydrophobic Materials

SIGNIFICANCE AND USE
5.1 This method can be used to evaluate effectiveness of incorporated/bound antimicrobials in hydrophobic materials such as plastics, epoxy resins, as well as other hard surfaces.  
5.2 The aqueous based bacterial inoculum remains in close, uniform contact in a “pseudo-biofilm” state with the treated material. The percent reduction in the surviving populations of challenge bacterial cells at 24 h versus those recovered from a non-treated control is determined.  
5.3 The hydrophobic substrate may be repeatedly tested over time for assessment of persistent antimicrobial activity.
SCOPE
1.1 This test method is designed to evaluate (quantitatively) the antimicrobial effectiveness of agents incorporated or bound into or onto mainly flat (two dimensional) hydrophobic or polymeric surfaces. The method focuses primarily on assessing antibacterial activity; however, other microorganisms such as yeast and fungal conidia may be tested using this method.  
1.2 The vehicle for the inoculum is an agar slurry which reduces the surface tension of the saline inoculum carrier and allows formation of a “pseudo-biofilm,” providing more even contact of the inoculum with the test surface.
Note 1: This test method facilitates the testing of hydrophobic surfaces by utilizing cells held in an agar slurry matrix. This test method, as written, is inappropriate to determine efficacy against biofilm cells, which are different both genetically and metabolically than planktonic cells used in this test.  
1.3 This method can confirm the presence of antimicrobial activity in plastics or hydrophobic surfaces and allows determination of quantitative differences in antimicrobial activity between untreated plastics or polymers and those with bound or incorporated low water-soluble antimicrobial agents. Comparisons between the numbers of survivors on preservative-treated and control hydrophobic surfaces may also be made.  
1.4 The procedure also permits determination of “shelf-life” or long term durability of an antimicrobial treatment which may be achieved through testing both non-washed and washed samples over a time span.  
1.5 Knowledge of microbiological techniques is required for these procedures.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 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.

General Information

• Status: Published
• Publication Date: 31-Mar-2024
• ICS: 07.100.01 - Microbiology in general
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Replaces: ASTM E2180-18 - Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) In Polymeric or Hydrophobic Materials
• Effective Date: 01-Apr-2024
• Referred By: ASTM E3031-20 - Standard Practice for Determination of Antibacterial Activity on Ceramic Surfaces
• Effective Date: 01-Apr-2024
• Referred By: ASTM E3160-18 - Standard Test Method for Quantitative Evaluation of the Antibacterial Properties of Porous Antibacterial Treated Articles
• Effective Date: 01-Apr-2024
• Referred By: ASTM E2922-23 - Standard Guide for Use of Standard Test Methods and Practices for Evaluating Antibacterial Activity on Textiles
• Effective Date: 01-Apr-2024
• Referred By: ASTM E1115-11(2017) - Standard Test Method for Evaluation of Surgical Hand Scrub Formulations
• Effective Date: 01-Apr-2024
• Referred By: ASTM E3371-22 - Standard Test Method for Measuring the Ability of a Synthetic Polymeric Material to Resist Bacterial Adherence
• Effective Date: 01-Apr-2024

Overview

ASTM E2180-24 is a standardized test method developed by ASTM International for quantitatively assessing the activity of incorporated antimicrobial agents in polymeric or hydrophobic materials, including plastics and epoxy resins. This method is primarily designed to evaluate antibacterial effectiveness but can also be applied to other microorganisms such as yeast and fungi. By utilizing an agar slurry-based inoculum, this standard ensures uniform “pseudo-biofilm” contact between the test microorganism and the treated surface, enabling reliable measurement of antimicrobial activity over specified exposure periods.

Key Topics

• Purpose: Quantitative evaluation of antimicrobial agents that are incorporated into or bound onto flat, hydrophobic, or polymeric surfaces.
• Scope: Applicable to materials such as plastics, resins, and other hard hydrophobic surfaces. The test typically measures antibacterial activity but can be adapted for yeast and fungal conidia.
• Test Method:
  • Utilizes an agar slurry as an inoculum vehicle, reducing surface tension to provide even contact between microorganisms and test surfaces.
  • After a specified incubation period (most commonly 24 hours), the number of surviving microbial cells is determined and compared between treated and untreated samples.
  • The percent reduction in surviving populations quantifies antimicrobial efficacy.
  • Allows for repeated testing to assess long-term or “shelf-life” durability of antimicrobial treatments.
• Microbial Testing: Standard bacterial strains recommended include Staphylococcus aureus and Pseudomonas aeruginosa, with flexibility to include relevant fungi or yeast.
• Requirements: Users of this standard should have foundational microbiological techniques and follow appropriate safety and environmental protocols.

Applications

ASTM E2180-24 is widely utilized in various industries for product development, quality assurance, and regulatory compliance, particularly where antimicrobial performance is critical. Typical applications include:

• Consumer Products: Validation of antimicrobial polymers in items such as shower curtains, vinyl pool liners, and kitchenware.
• Medical Devices: Evaluation of antimicrobial properties in hard surfaces or coatings of devices that are susceptible to microbial contamination.
• Building Materials: Assessment of treated surfaces in tiles, countertops, or wall panels designed to resist microbial growth.
• Industrial and Food Processing Equipment: Testing of antimicrobial-treated plastics and resins used in settings where hygienic surfaces are essential.
• Durability Assessment: Determining the long-term effectiveness (“shelf-life”) of antimicrobial treatments after repeated washings or exposure to environmental conditions.
• Comparative Analysis: Benchmarks new or improved antimicrobial materials against untreated controls to demonstrate enhanced performance.

By following ASTM E2180-24, manufacturers, researchers, and regulatory bodies achieve consistent, reproducible, and quantitative data on the antibacterial or antifungal activity of hydrophobic materials.

Related Standards

• ASTM E1054: Practices for Evaluation of Inactivators of Antimicrobial Agents
• ASTM E1428: Test Method for Evaluating Performance of Antimicrobials in or on Polymeric Solids (now withdrawn)
• ASTM E2756: Terminology Relating to Antimicrobial and Antiviral Agents
• ASTM G21: Practice for Determining Resistance of Synthetic Polymeric Materials to Fungi

These related standards provide additional guidance on terminology, test organisms, materials, and alternative methods for evaluating antimicrobial performance in polymeric products.

Keywords: ASTM E2180-24, antimicrobial activity, polymeric materials, hydrophobic surfaces, plastics, antibacterial testing, quantitative assay, agar slurry, biofilm, durability testing, shelf-life, standard test method.

Standards Content (Sample)

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 − 24
Standard Test Method for
Determining the Activity of Incorporated Antimicrobial
Agent(s) In Polymeric or Hydrophobic Materials
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.
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
between untreated plastics or polymers and those with bound
1.1 This test method is designed to evaluate (quantitatively)
or incorporated low water-soluble antimicrobial agents. Com-
the antimicrobial effectiveness of agents incorporated or bound
parisons between the numbers of survivors on preservative-
into or onto mainly flat (two dimensional) hydrophobic or
treated and control hydrophobic surfaces may also be made.
polymeric surfaces. The method focuses primarily on assessing
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
may be achieved through testing both non-washed and washed
1.2 The vehicle for the inoculum is an agar slurry which
samples over a time span.
reduces the surface tension of the saline inoculum carrier and
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,
standard. No other units of measurement are included in this
as written, is inappropriate to determine efficacy against biofilm cells,
standard.
which are different both genetically and metabolically than planktonic
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-
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
1.8 This international standard was developed in accor-
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
dance with internationally recognized principles on standard-
Current edition approved April 1, 2024. Published April 2024. Originally
approved in 2001. Last previous edition approved in 2018 as E2180 – 18. DOI:
ization established in the Decision on Principles for the
10.1520/E2180-24.
Development of International Standards, Guides and Recom-
Price, D. L., Sawant, A. D., and Ahearn, D. G., “Assessment of the antimicro-
mendations issued by the World Trade Organization Technical
bial activity of an insoluble quaternary amine complex in plastics,” J. Industr.
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
E2180 − 24
2. Referenced Documents 4.5 Bacterial colonies from each dilution series are counted
3 and recorded.
2.1 ASTM Standards:
E1054 Practices for Evaluation of Inactivators of Antimicro- 4.6 Calculation of percent reduction of bacteria from treated
bial Agents versus untreated samples is made.
E1428 Test Method for Evaluating the Performance of
Antimicrobials in or on Polymeric Solids Against Staining 5. Significance and Use
by Streptomyce species (A Pink Stain Organism) (With-
5.1 This method can be used to evaluate effectiveness of
drawn 2024)
incorporated/bound antimicrobials in hydrophobic materials
E2756 Terminology Relating to Antimicrobial and Antiviral
such as plastics, epoxy resins, as well as other hard surfaces.
Agents
5.2 The aqueous based bacterial inoculum remains in close,
G21 Practice for Determining Resistance of Synthetic Poly-
uniform contact in a “pseudo-biofilm” state with the treated
meric Materials to Fungi
material. The percent reduction in the surviving populations of
challenge bacterial cells at 24 h versus those recovered from a
3. Terminology
non-treated control is determined.
3.1 Definitions:
5.3 The hydrophobic substrate may be repeatedly tested
3.1.1 For definitions of terms used in this standard refer to
over time for assessment of persistent antimicrobial activity.
Terminology E2756.
3.1.2 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.
3.1.3 inoculum, n—in microbiology, a specimen comprised
of living spores, bacteria, single celled organisms, or other live
6.2 Petri Dishes, (15 mm by 100 mm), sterile.
materials, yeast or the multicellular filamentous fungi, or
6.3 Colony Counter.
combination of two or more types of microorganisms, that are
introduced into a test medium or onto a specimen to be tested 6.4 Specimen Cups, (120 mL), sterile or equivalent sterile
in order to investigate the lability of the medium or specimen equipment for extraction.
to support microbial growth or to investigate its antimicrobial
6.5 Pipetters, (1000 μL) positive displacement.
properties.
6.6 Pipette Tips, sterile.
3.1.4 inoculum vehicle, n—the carrier solution used to
6.7 Test Tubes, 16 mm by 100 mm.
transport the inoculum to a given sample or object.
6.8 Incubator, set at required temperature (25 °C to 35 °C 6
3.1.5 neutralizing recovery broth, n—liquid growth media
2 °C).
used to inactivate the effects of the test antimicrobial agent.
6.9 Autoclave.
4. Summary of Test Method
6.10 Water Bath, capable of maintaining water at 45 °C 6
4.1 This method involves inoculation of a molten (45 °C)
2 °C.
agar slurry with a standardized culture of bacterial cells.
6.11 Sterile Cotton Swabs.
4.2 A thin layer of the inoculated agar slurry (0.5 mL to
6.12 Sonic Bath, 47 Khz, cleaning non-cavitating.
1.0 mL) is pipetted onto the test and untreated control material
(triplicate samples minimum). 6.13 Vortex Mixer.
4.3 After the specified contact time (24 h commonly used),
6.14 pH Meter.
surviving microorganisms are recovered via elution of the agar
6.15 Hot Plate, with stirrer.
slurry inoculum from the test substrate into neutralizing broth
6.16 Spectrophotometer, set at 600 nm.
and extracted via methods that provide complete removal of
the inoculum from the test article (examples include
6.17 Sterile Cuvettes.
sonication, vortexing, and/or manual extraction, that is, stom-
6.18 Test Materials, sterile if specified by interested parties.
acher).
6.19 Cell Counting Chamber.
4.4 Serial dilutions are made, then pour or spread plates are
made of each dilution. Agar
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E2180 − 18 E2180 − 24
Standard Test Method for
Determining the Activity of Incorporated Antimicrobial
Agent(s) In Polymeric or Hydrophobic Materials
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.
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
1.1 This test method is designed to evaluate (quantitatively) the antimicrobial effectiveness of agents incorporated or bound into
or onto mainly flat (two dimensional) hydrophobic or polymeric surfaces. The method focuses primarily on assessing antibacterial
activity; however, other microorganisms such as yeast and fungal conidia may be tested using this method.
1.2 The vehicle for the inoculum is an agar slurry which reduces the surface tension of the saline inoculum carrier and allows
formation of a “pseudo-biofilm,” providing more even contact of the inoculum with the test surface.
NOTE 1—This test method facilitates the testing of hydrophobic surfaces by utilizing cells held in an agar slurry matrix. This test method, as written, is
inappropriate to determine efficacy against biofilm cells, which are different both genetically and metabolically than planktonic cells used in this test.
1.3 This method can confirm the presence of antimicrobial activity in plastics or hydrophobic surfaces and allows determination
of quantitative differences in antimicrobial activity between untreated plastics or polymers and those with bound or incorporated
low water-soluble antimicrobial agents. Comparisons between the numbers of survivors on preservative-treated and control
hydrophobic surfaces may also be made.
This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved May 1, 2018April 1, 2024. Published May 2018April 2024. Originally approved in 2001. Last previous edition approved in 2018 as
E2180 – 07E2180 – 18.(2017). DOI: 10.1520/E2180-18.10.1520/E2180-24.
Price, D. L., Sawant, A. D., and Ahearn, D. G., “Assessment of the antimicrobial activity of an insoluble quaternary amine complex in plastics,” J. Industr. Microbiol,
Vol 8, No. 2, 1991, pp. 83–89.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2180 − 24
1.4 The procedure also permits determination of “shelf-life” or long term durability of an antimicrobial treatment which may be
achieved through testing both non-washed and washed samples over a time span.
1.5 Knowledge of microbiological techniques is required for these procedures.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
1.8 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.
2. Referenced Documents
2.1 ASTM Standards:
E1054 Practices for Evaluation of Inactivators of Antimicrobial Agents
E1428 Test Method for Evaluating the Performance of Antimicrobials in or on Polymeric Solids Against Staining by
Streptomyce species (A Pink Stain Organism) (Withdrawn 2024)
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
G21 Practice for Determining Resistance of Synthetic Polymeric Materials to Fungi
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this standard refer to Terminology E2756.
3.1.2 agar slurry, n—a semi-gelatinous liquid formed when 3 g/L agar-agar is added to a 0.85 % saline solution.
3.1.3 inoculum, n—in microbiology, a specimen comprised of living spores, bacteria, single celled organisms, or other live
materials, yeast or the multicellular filamentous fungi, or combination of two or more types of microorganisms, that are introduced
into a test medium or onto a specimen to be tested in order to investigate the lability of the medium or specimen to support
microbial growth or to investigate its antimicrobial properties.
3.1.4 inoculum vehicle, n—the carrier solution used to transport the carrier solution used to transport the inoculum to a given
sample or object.
3.1.5 neutralizing recovery broth, n—liquid growth media used to inactivate the effects of the test antimicrobial agent.
4. Summary of Test Method
4.1 This method involves inoculation of a molten (45 °C) agar slurry with a standardized culture of bacterial cells.
4.2 A thin layer of the inoculated agar slurry (0.5-1.0 mL) (0.5 mL to 1.0 mL) is pipetted onto the test and untreated control
material (triplicate samples minimum).
4.3 After the specified contact time (24 h commonly used), surviving microorganisms are recovered via elution of the agar slurry
inoculum from the test substrate into neutralizing broth and extracted via methods that provide complete removal of the inoculum
from the test article (examples include sonication, vortexing, and/or manual extraction, that is, stomacher).
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
The last approved version of this historical standard is referenced on www.astm.org.
E2180 − 24
4.4 Serial dilutions are made, then pour or spread plates are made of each dilution. Agar plates and dilution broths are incubated
for 48 62 48 h 6 2 h at a specified temperature dependent upon the optimal temperature for test organism.
4.5 Bacterial colonies from each dilution series are counted and recorded.
4.6 Calculation of percent reduction of bacteria from treated versus untreated samples is made.
5. Significance and Use
5.1 This method can be used to evaluate effectiveness of incorporated/bound antimicrobials in hydrophobic materials such as
plastics, epoxy resins, as well as other hard surfaces.
5.2 The aqueous based bacterial inoculum remains in close, uniform contact in a “pseudo-biofilm” state with the treated material.
The percent reduction in the surviving populations of challenge bacterial cells at 24 h versus those recovered from a non-treated
control is determined.
5.3 The hydrophobic substrate may be repeatedly tested over time for assessment of persistent antimicrobial activity.
6. Apparatus
6.1 Erlenmeyer Flask, 250 mL.
6.2 Petri Dishes, (15 ×(15 mm by 100 mm), sterile.
6.3 Colony Counter.
6.4 Specimen Cups, (120 mL), sterile or equivalent sterile equipment for extraction.
6.5 Pipetters
...