ASTM E2180-07(2012)
(Test Method)Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) In Polymeric or Hydrophobic Materials
Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) In Polymeric or Hydrophobic Materials
SIGNIFICANCE AND USE
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.
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.
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 and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: E2180 − 07 (Reapproved 2012)
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.Anumber 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
treatedfrequentlywithincorporatedorboundantimicrobialagents.PracticesG21isusedtodetermine
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
hydrophobicmaterial,thesurfacetensionofthepolymeroftencausestheinoculasuspensiontodome.
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 between untreated plastics or polymers and those with bound
or incorporated low water-soluble antimicrobial agents. Com-
1.1 This test method is designed to evaluate (quantitatively)
parisons between the numbers of survivors on preservative-
theantimicrobialeffectivenessofagentsincorporatedorbound
treated and control hydrophobic surfaces may also be made.
into or onto mainly flat (two dimensional) hydrophobic or
polymericsurfaces.Themethodfocusesprimarilyonassessing 1.4 Theprocedurealsopermitsdeterminationof“shelf-life”
antibacterial activity; however, other microorganisms such as or long term durability of an antimicrobial treatment which
yeast and fungal conidia may be tested using this method. may be achieved through testing both non-washed and washed
samples over a time span.
1.2 The vehicle for the inoculum is an agar slurry which
reduces the surface tension of the saline inoculum carrier and 1.5 Knowledge of microbiological techniques is required
allows formation of a “pseudo-biofilm,” providing more even for these procedures.
contact of the inoculum with the test surface.
1.6 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
NOTE 1—This test method facilitates the testing of hydrophobic
surfaces by utilizing cells held in an agar slurry matrix. This test method,
standard.
as written, is inappropriate to determine efficacy against biofilm cells,
1.7 This standard does not purport to address all of the
which are different both genetically and metabolically than planktonic
safety concerns, if any, associated with its use. It is the
cells used in this test.
responsibility of the user of this standard to establish appro-
1.3 This method can confirm the presence of antimicrobial
priate safety and health practices and determine the applica-
activity in plastics or hydrophobic surfaces and allows deter-
bility of regulatory limitations prior to use.
mination of quantitative differences in antimicrobial activity
2. Referenced Documents
2.1 ASTM Standards:
This test method is under the jurisdiction of ASTM Committee E35 on
E1054Test Methods for Evaluation of Inactivators of Anti-
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
microbial Agents
Current edition approved April 1, 2012. Published June 2012. Originally
approved in 2001. Last previous edition approved in 2007 as E2180–07. DOI:
10.1520/E2180-07R12. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Price, D. L., Sawant,A. D., andAhearn, D. G., “Assessment of the antimicro- contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
bial 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, 1991, pp. 83–89. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2180 − 07 (2012)
E1428Test Method for Evaluating the Performance of 6.4 Specimen Cups, (120 mL), sterile or equivalent sterile
AntimicrobialsinoronPolymericSolidsAgainstStaining equipment for extraction.
by Streptoverticillium reticulum (A Pink Stain Organism)
6.5 Pipetters, (1000 µL) positive displacement.
G21Practice for Determining Resistance of Synthetic Poly-
6.6 Pipette Tips, sterile.
meric Materials to Fungi
6.7 Test Tubes, 16 × 100 mm.
3. Terminology
6.8 Incubator, set at required temperature (25-35 6 2°C).
3.1 Definitions:
6.9 Autoclave.
3.1.1 agar slurry, n—a semi-gelatinous liquid formed when
6.10 Water Bath,capableofmaintainingwaterat45 62°C.
3 g/L agar-agar is added to a 0.85% saline solution.
6.11 Sterile Cotton Swabs.
3.1.2 inoculum vehicle, n—the carrier solution used to
transport bacterial cells to a test surface. Samples include
6.12 Sonic Bath, 47 Khz, cleaning non-cavitating.
saline, nutrient broth, tryptic soy broth, agar slurry, or other
6.13 Vortex Mixer.
buffers that maintain bacterial viability.
6.14 pH Meter.
3.1.3 neutralizing recovery broth, n—liquid growth media
used to inactivate the effects of the test antimicrobial agent. 6.15 Hot Plate, with stirrer.
6.16 Spectrophotometer, set at 600 nm.
4. Summary of Test Method
6.17 Sterile Cuvettes.
4.1 This method involves inoculation of a molten (45°C)
6.18 Test Materials, sterile if specified by interested parties.
agar slurry with a standardized culture of bacterial cells.
6.19 Cell Counting Chamber.
4.2 Athinlayeroftheinoculatedagarslurry(0.5-1.0mL)is
pipetted onto the test and untreated control material (triplicate
7. Reagents
samples minimum).
7.1 Media:
4.3 After the specified contact time (24 h commonly used),
7.1.1 Tryptic Soy Broth, or appropriate broth.
survivingmicroorganismsarerecoveredviaelutionoftheagar
7.1.2 Tryptic Soy Agar, or appropriate agar.
slurry inoculum from the test substrate into neutralizing broth
7.1.3 Neutralizing Broth, appropriate for the antimicrobial
and extracted via methods that provide complete removal of
compound tested.(See Practice E1054.)
the inoculum from the test article (examples include sonica-
7.1.4 Agar-agar.
tion, vortexing, and/or manual extraction, that is, stomacher).
7.1.5 NaCl.
4.4 Serial dilutions are made, then pour or spread plates are
7.1.6 Sterile Deionized Water.
made of each dilution. Agar plates and dilution broths are
7.1.7 Sterile 0.85 % Saline Dilution Blanks, 9.0 mLin 16 ×
incubated for 48 6 2 h at a specified temperature dependent
100 mm test tubes or appropriate dilution buffer (such as
upon the optimal temperature for test organism.
phosphate buffer or Butterfield’s buffer).
4.5 Bacterial colonies from each dilution series are counted
7.2 Test Organisms—Specific organisms are recommended
and recorded.
but choice of organism should be relevant to the environment
4.6 Calculationofpercentreductionofbacteriafromtreated
in which the product will perform.
versus untreated samples is made.
7.2.1 Gram-positive bacteria Staphylococcus aureus ATCC
6538.
5. Significance and Use
7.2.2 Gram-negative
...
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