ASTM E2180-07(2017)
(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
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 and health 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.
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Designation: E2180 − 07 (Reapproved 2017)
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 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-
theantimicrobialeffectivenessofagentsincorporatedorbound
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.
polymericsurfaces.Themethodfocusesprimarilyonassessing
antibacterial activity; however, other microorganisms such as
1.4 Theprocedurealsopermitsdeterminationof“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 and health practices and determine the applica-
This test method is under the jurisdiction of ASTM Committee E35 on
bility 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, 2017. Published April 2017. Originally
approved in 2001. Last previous edition approved in 2012 as E2180–07(2012).
ization established in the Decision on Principles for the
DOI: 10.1520/E2180-07R17.
Development of International Standards, Guides and Recom-
Price, D. L., Sawant,A. D., andAhearn, 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 − 07 (2017)
2. Referenced Documents challenge bacterial cells at 24 h versus those recovered from a
3 non-treated control is determined.
2.1 ASTM Standards:
E1054Test Methods for Evaluation of Inactivators of Anti-
5.3 The hydrophobic substrate may be repeatedly tested
microbial Agents over time for assessment of persistent antimicrobial activity.
E1428Test Method for Evaluating the Performance of
AntimicrobialsinoronPolymericSolidsAgainstStaining
6. Apparatus
by Streptomyce species (A Pink Stain Organism)
6.1 Erlenmeyer Flask, 250 mL.
G21Practice for Determining Resistance of Synthetic Poly-
6.2 Petri Dishes, (15 × 100 mm), sterile.
meric Materials to Fungi
6.3 Colony Counter.
3. Terminology
6.4 Specimen Cups, (120 mL), sterile or equivalent sterile
3.1 Definitions:
equipment for extraction.
3.1.1 agar slurry, n—a semi-gelatinous liquid formed when
6.5 Pipetters, (1000 µL) positive displacement.
3 g/L agar-agar is added to a 0.85% saline solution.
6.6 Pipette Tips, sterile.
3.1.2 inoculum vehicle, n—the carrier solution used to
transport bacterial cells to a test surface. Samples include
6.7 Test Tubes, 16 × 100 mm.
saline, nutrient broth, tryptic soy broth, agar slurry, or other
6.8 Incubator, set at required temperature (25-35 6 2°C).
buffers that maintain bacterial viability.
6.9 Autoclave.
3.1.3 neutralizing recovery broth, n—liquid growth media
used to inactivate the effects of the test antimicrobial agent.
6.10 Water Bath,capableofmaintainingwaterat45 62°C.
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)
agar slurry with a standardized culture of bacterial cells.
6.13 Vortex Mixer.
4.2 Athinlayeroftheinoculatedagarslurry(0.5-1.0mL)is
6.14 pH Meter.
pipetted onto the test and untreated control material (triplicate
6.15 Hot Plate, with stirrer.
samples minimum).
6.16 Spectrophotometer, set at 600 nm.
4.3 After the specified contact time (24 h commonly used),
6.17 Sterile Cuvettes.
survivingmicroorganismsarerecoveredviaelutionoftheagar
slurry inoculum from the test substrate into neutralizing broth
6.18 Test Materials, sterile if specified by interested parties.
and extracted via methods that provide complete removal of
6.19 Cell Counting Chamber.
the inoculum from the test article (examples include
sonication, vortexing, and/or manual extraction, that is, stom-
7. Reagents
acher).
7.1 Media:
4.4 Serial dilutions are made, then pour or spread plates are
7.1.1 Tryptic Soy Broth, or appropriate broth.
made of each dilution. Agar plates and dilution broths are
7.1.2 Tryptic Soy Agar, or appropriate agar.
incubated for 48 6 2 h at a specified temperature dependent
7.1.3 Neutralizing Broth, appropriate for the antimicrobial
upon the optimal temperature for test organism.
compound tested.(See Practice E1054.)
4.5 Bacterial colonies from each dilution series are counted
7.1.4 Agar-agar.
and recorded.
7.1.5 NaCl.
4.6 Calculationofpercentreductionofbacteriafromtreated
7.1.6 Sterile Deionized Water.
versus untreated samples is made.
7.1.7 Sterile 0.85 % Saline Dilution Blanks, 9.0 mLin 16 ×
100 mm test tubes or appropriate dilution buffer (such as
5. Significance and Use
phosphate buffer or Butterfield’s buffer).
5.1 This method can be used t
...
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 − 07 (Reapproved 2012) E2180 − 07 (Reapproved 2017)
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.
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.
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.
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 April 1, 2012April 1, 2017. Published June 2012April 2017. Originally approved in 2001. Last previous edition approved in 20072012 as
E2180 – 07.E2180 – 07(2012). DOI: 10.1520/E2180-07R12.10.1520/E2180-07R17.
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 − 07 (2017)
2. Referenced Documents
2.1 ASTM Standards:
E1054 Test Methods 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)
G21 Practice for Determining Resistance of Synthetic Polymeric Materials to Fungi
3. Terminology
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.
3.1.2 inoculum vehicle, n—the carrier solution used to transport bacterial cells to a test surface. Samples include saline, nutrient
broth, tryptic soy broth, agar slurry, or other buffers that maintain bacterial viability.
3.1.3 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) 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).
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 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 × 100 mm), sterile.
6.3 Colony Counter.
6.4 Specimen Cups, (120 mL), sterile or equivalent sterile equipment for extraction.
6.5 Pipetters, (1000 μL) positive displacement.
6.6 Pipette Tips, sterile.
6.7 Test Tubes, 16 × 100 mm.
6.8 Incubator, set at required temperature (25-35 6 2°C).
6.9 Autoclave.
6.10 Water Bath, capable of maintaining water at 45 6 2°C.
6.11 Sterile Cotton Swabs.
6.12 Sonic Bath, 47 Khz, cleaning non-cavitating.
6.13 Vortex Mixer.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book o
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