ASTM E2180-01

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Designation:E2180–01
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 E 2180; 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 (e) 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. PracticesG21 andG22 are used
to determine the ability of polymer materials to resist microbial attack or staining (see also Practice
E 1428); 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 1.5 Knowledge of microbiological techniques is required
for these procedures.
1.1 This test method is designed to evaluate (quantitatively)
1.6 This standard does not purport to address all of the
the antimicrobial effectiveness of agents incorporated or bound
safety concerns, if any, associated with its use. It is the
into or onto mainly flat (two dimensional) hydrophobic or
responsibility of the user of this standard to establish appro-
polymericsurfaces.Themethodfocusesprimarilyonassessing
priate safety and health practices and determine the applica-
antibacterial activity; however, other microorganisms such as
bility of regulatory limitations prior to use.
yeast and fungal conidia may be tested using this method.
1.2 The vehicle for the inoculum is an agar slurry which
2. Referenced Documents
reduces the surface tension of the saline inoculum carrier and
2.1 ASTM Standards:
allows formation of a “pseudo-biofilm,” providing more even
E 1054 Practice for Evaluation of Inactivators of Antimi-
contact of the inoculum with the test surface.
crobial Agents used in Disinfectant, Sanitizer, Antiseptic,
1.3 This method can confirm the presence of antimicrobial
or Preserved Products
activity in plastics or hydrophobic surfaces and allows deter-
E 1428 Test Method for Evaluating the Performance of
mination of quantitative differences in antimicrobial activity
Antimicrobials in or on Polymeric SolidsAgainst Staining
between untreated plastics or polymers and those with bound
by Streptoverticillium reticulum (A Pink Stain Organism)
or incorporated low water-soluble antimicrobial agents. Com-
G21 Practice for Determining Resistance of Synthetic
parisons between the numbers of survivors on preservative-
Polymeric Materials to Fungi
treated and control hydrophobic surfaces may also be made.
G22 Practice for Determining Resistance of Synthetic
1.4 The procedure also permits determination of “shelf-life”
Polymeric Materials to Bacteria
or long term stability of an antimicrobial treatment which may
be achieved through testing both non-washed and washed
3. Terminology
samples over a time span.
3.1 Definitions:
3.1.1 agar slurry, n—a semi-gelatinous liquid formed when
3 g/L agar-agar is added to a 0.85 % saline solution.
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Dec. 10, 2001. Published February 2002. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Price, D.L., A.D. Sawant, and D.G. Ahearn. 1991. Assessment of the antimi- contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
crobial activity of an insoluble quaternary amine complex in plastics. J. Industr. Standards volume information, refer to the standard’s Document Summary page on
Microbiol. Vol 8 (No.2):83-89. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2180–01
3.1.2 inoculum vehicle, n—the carrier solution used to 6.17 Sterile Cuvettes.
transport bacterial cells to a test surface. Samples include 6.18 Test Materials, sterile if specified by interested parties.
saline, nutrient broth, tryptic soy broth, agar slurry, or other 6.19 Cell Counting Chamber.
buffers that maintain bacterial viability.
7. Reagents
3.1.3 neutralizing recovery broth, n—liquid growth media
7.1 Media:
used to inactivate the effects of the test antimicrobial agent.
7.1.1 Tryptic Soy Broth, or appropriate broth.
7.1.2 Tryptic Soy Agar, or appropriate agar.
4. Summary of Test Method
7.1.3 Neutralizing Broth, appropriate for the antimicrobial
4.1 This method involves inoculation of a molten (45°C)
compound tested.(See Practice E 1054.)
agar slurry with a standardized culture of bacterial cells.
7.1.4 Agar-agar.
4.2 Athin layer of the inoculated agar slurry (0.5-1.0 mL) is
7.1.5 NaCl.
pipetted onto the test and untreated control material (triplicate
7.1.6 Sterile Deionized Water.
samples minimum).
7.1.7 Sterile 0.85 % Saline Dilution Blanks, 9.0 mLin 16 3
4.3 After the specified contact time (24 h commonly used),
100 mm test tubes or appropriate dilution buffer (such as
surviving microorganisms are recovered via elution of the agar
phosphate buffer or Butterfield’s buffer).
slurry inoculum from the test substrate into neutralizing broth
7.2 Test Organisms—Specific organisms are recommended
and extracted via methods that provide complete removal of
but choice of organism should be relevant to the environment
the inoculum from the test article (examples include sonica-
in which the product will perform.
tion, vortexing, and/or manual extraction, that is, stomacher).
7.2.1 Gram-positive bacteria Staphylococcus aureus ATCC
4.4 Serial dilutions are made, then pour or spread plates are
6538.
made of each dilution. Agar plates and dilution broths are
7.2.2 Gram-negative bacteria Pseudomonas aeruginosa
incubated for 48 6 2 h at a specified temperature dependent
ATCC 15442 or Klebsiella pneumoniae ATCC 4352.
upon the optimal temperature for test organism.
7.2.3 Other microorganisms such as yeast or fungal conidia
4.5 Bacterial colonies from each dilution series are counted
may also be tested using this procedure. Exposure periods may
and recorded.
be modified (up to 96 h) to address more resistant microorgan-
4.6 Calculation of percent reduction of bacteria from treated
isms.
versus untreated samples is made.
8. Procedure
5. Significance and Use
8.1 Grow 18 h bacterial cultures (three transfers) at a
5.1 This method can be used to evaluate effectiveness of
specified temperature dependent upon the optimal temperature
incorporated/bound antimicrobials in hydrophobic materials
for the test organism in tryptic soy or appropriate broth. These
such as plastics
...