ASTM E2799-12
(Test Method)Standard Test Method for Testing Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm using the MBEC Assay
Standard Test Method for Testing Disinfectant Efficacy against <i>Pseudomonas aeruginosa</i> Biofilm using the MBEC Assay
SIGNIFICANCE AND USE
Vegetative biofilm bacteria are phenotypically different from suspended planktonic cells of the same genotype. Biofilm growth reactors are engineered to produce biofilms with specific characteristics. Altering either the engineered system or operating conditions will modify those characteristics. The goal in biofilm research and efficacy testing is to choose the growth reactor that generates the most relevant biofilm for the particular study.
The purpose of this test method is to direct a user in how to grow, treat, sample and analyze a Pseudomonas aeruginosa biofilm using the MBEC Assay. Microscopically, the biofilm is sheet-like with few architectural details as seen in Harrison et al (6). The MBEC Assay was originally designed as a rapid and reproducible assay for evaluating biofilm susceptibility to antibiotics (2). The engineering design allows for the simultaneous evaluation of multiple test conditions, making it an efficient method for screening multiple disinfectants or multiple concentrations of the same disinfectant. Additional efficiency is added by including the neutralizer controls within the assay device. The small well volume is advantageous for testing expensive disinfectants, or when only small volumes of the disinfectant are available.
SCOPE
1.1 This test method specifies the operational parameters required to grow and treat a Pseudomonas aeruginosa biofilm in a high throughput screening assay known as the MBEC (trademarked) (Minimum Biofilm Eradication Concentration) Physiology and Genetics Assay. The assay device consists of a plastic lid with ninety-six (96) pegs and a corresponding receiver plate with ninety-six (96) individual wells that have a maximum 200-μL working volume. Biofilm is established on the pegs under batch conditions (that is, no flow of nutrients into or out of an individual well) with gentle mixing. The established biofilm is transferred to a new receiver plate for disinfectant efficacy testing. The reactor design allows for the simultaneous testing of multiple disinfectants or one disinfectant with multiple concentrations, and replicate samples, making the assay an efficient screening tool.
1.2 This test method defines the specific operational parameters necessary for growing a Pseudomonas aeruginosa biofilm, although the device is versatile and has been used for growing, evaluating and/or studying biofilms of different species as seen in Refs (1-4).
1.3 Validation of disinfectant neutralization is included as part of the assay.
1.4 This test method describes how to sample the biofilm and quantify viable cells. Biofilm population density is recorded as log10 colony forming units per surface area. Efficacy is reported as the log10 reduction of viable cells.
1.5 Basic microbiology training is required to perform this assay.
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 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.
1.8 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:E2799 −12
Standard Test Method for
Testing Disinfectant Efficacy against Pseudomonas
1
aeruginosa Biofilm using the MBEC Assay
This standard is issued under the fixed designation E2799; 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.
1. Scope corded as log colony forming units per surface area. Efficacy
10
is reported as the log reduction of viable cells.
10
1.1 This test method specifies the operational parameters
required to grow and treat a Pseudomonas aeruginosa biofilm 1.5 Basic microbiology training is required to perform this
in a high throughput screening assay known as the MBEC assay.
2
(trademarked) (Minimum Biofilm Eradication Concentration)
1.6 The values stated in SI units are to be regarded as
Physiology and GeneticsAssay.The assay device consists of a
standard. No other units of measurement are included in this
plastic lid with ninety-six (96) pegs and a corresponding
standard.
receiver plate with ninety-six (96) individual wells that have a
1.7 ASTM International takes no position respecting the
maximum 200-µL working volume. Biofilm is established on
validity of any patent rights asserted in connection with any
the pegs under batch conditions (that is, no flow of nutrients
item mentioned in this standard. Users of this standard are
into or out of an individual well) with gentle mixing. The
expressly advised that determination of the validity of any such
established biofilm is transferred to a new receiver plate for
3,4
patent rights, and the risk of infringement of such rights, are
disinfectant efficacy testing. The reactor design allows for
entirely their own responsibility.
the simultaneous testing of multiple disinfectants or one
disinfectant with multiple concentrations, and replicate 1.8 This standard does not purport to address all of the
samples, making the assay an efficient screening tool.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
1.2 This test method defines the specific operational param-
priate safety and health practices and determine the applica-
eters necessary for growing a Pseudomonas aeruginosa
bility of regulatory limitations prior to use.
biofilm, although the device is versatile and has been used for
growing, evaluating and/or studying biofilms of different
2. Referenced Documents
5
species as seen in Refs (1-4).
6
2.1 ASTM Standards:
1.3 Validation of disinfectant neutralization is included as
E1054Test Methods for Evaluation of Inactivators of Anti-
part of the assay.
microbial Agents
1.4 This test method describes how to sample the biofilm
2.2 Other Standards:
and quantify viable cells. Biofilm population density is re-
Method 9050 C.1.a Buffered Dilution Water Preparation
according to Eaton et al (5)
1
3. Terminology
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
3.1 Definitions:
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
3.1.1 biofilm, n—microorganisms living in a self-organized
Current edition approved April 1, 2012. Published June 2012. Originally
approved in 2011. Last previous edition approved in 2011 as E2799–11. DOI:
community attached to surfaces, interfaces, or each other,
10.1520/E2799–12.
embedded in a matrix of extracellular polymeric substances of
2
TheMBECtrademarkisheldbyInnovotech,Inc.,Edmonton,Alberta,Canada.
microbial origin, while exhibiting altered phenotypes with
3
The sole source of supply of the apparatus known to the committee at this time
is Innovotech Inc., Edmonton, Alberta, Canada. If you are aware of alternative respect to growth rate and gene transcription.
suppliers, please provide this information to ASTM International Headquarters.
3.1.1.1 Discussion—Biofilmsmaybecomprisedofbacteria,
Your comments will receive careful consideration at a meeting of the responsible
fungi, algae, protozoa, viruses, or infinite combinations of
1
technical committee, which you may attend.
4
TheMBECAssayiscoveredbyapatent.Interestedpartiesareinvitedtosubmit
information regarding the identification of an alternative(s) to this patented item to
6
theASTM International Headquarters. Your comments will receive careful consid- For referenced ASTM standards, visit the ASTM website, www.astm.org, or
1
erationatameetingoftheresponsibletechnicalcommittee, whichyoumayattend. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
5
The boldface numbers in parentheses refer to a list of references at the end of Standards volume information, refer to th
...
This document is not anASTM standard and is intended only to provide the user of anASTM 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:E2799–11 Designation: E2799 – 12
Standard Test Method for
Testing Disinfectant Efficacy against Pseudomonas
1
aeruginosa Biofilm using the MBEC Assay
This standard is issued under the fixed designation E2799; 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.
1. Scope
1.1 ThistestmethodspecifiestheoperationalparametersrequiredtogrowandtreataPseudomonasaeruginosabiofilminahigh
2
throughput screening assay known as the MBEC (trademarked) (Minimum Biofilm Eradication Concentration) Physiology and
Genetics Assay. The assay device consists of a plastic lid with ninety-six (96) pegs and a corresponding receiver plate with
ninety-six (96) individual wells that have a maximum 200-µL working volume. Biofilm is established on the pegs under batch
conditions (that is, no flow of nutrients into or out of an individual well) with gentle mixing. The established biofilm is transferred
3, 4
to a new receiver plate for disinfectant efficacy testing. The reactor design allows for the simultaneous testing of multiple
disinfectants or one disinfectant with multiple concentrations, and replicate samples, making the assay an efficient screening tool.
1.2 This test method defines the specific operational parameters necessary for growing a Pseudomonas aeruginosa biofilm,
although the device is versatile and has been used for growing, evaluating and/or studying biofilms of different species as seen in
5
Refs (1-4).
1.3 Validation of disinfectant neutralization is included as part of the assay.
1.4 This test method describes how to sample the biofilm and quantify viable cells. Biofilm population density is recorded as
log colony forming units per surface area. Efficacy is reported as the log reduction of viable cells.
10 10
1.5 Basic microbiology training is required to perform this assay.
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 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.
1.8 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.
2. Referenced Documents
6
2.1 ASTM Standards:
E1054 Test Methods for Evaluation of Inactivators of Antimicrobial Agents
2.2 Other Standards:
Method 9050 C.1.a Buffered Dilution Water Preparation according to Eaton et al (5)
3. Terminology
3.1 Definitions:
3.1.1 biofilm, n—microorganisms living in a self-organized, cooperative self-organized community attached to surfaces,
interfaces, or each other, embedded in a matrix of extracellular polymeric substances of microbial origin, while exhibiting an
1
This test method is under the jurisdiction ofASTM Committee E35 on Pesticides,Antimicrobials, andAlternative Control MethodsAgents and is the direct responsibility
of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved April 1, 2011. Published April 2011. DOI: 10.1520/E2799–11.
CurrenteditionapprovedApril1,2012.PublishedJune2012.Originallyapprovedin2011.Lastpreviouseditionapprovedin2011asE2799 – 11.DOI:10.1520/E2799–12.
2
The MBEC trademark is held by Innovotech, Inc., Edmonton, Alberta, Canada.
3
The sole source of supply of the apparatus known to the committee at this time is Innovotech Inc., Edmonton,Alberta, Canada. If you are aware of alternative suppliers,
please provide this information toASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee,
which you may attend.
4
The MBECAssay is covered by a patent. Interested parties are invited to submit information regarding the identification of an alternative(s) to this patented item to the
ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee, which you may attend.
5
The boldface numbers in parentheses refer to a l
...
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