ASTM E2647-20

Standard Test Method for Quantification of <emph type="bdit">Pseudomonas aeruginosa</emph > Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow

SIGNIFICANCE AND USE
5.1 Vegetative biofilm bacteria are phenotypically different from suspended 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.  
5.2 The purpose of this test method is to direct a user in how to grow, sample, and analyze a P. aeruginosa biofilm under low fluid shear and close to the air/liquid interface using the DFR. The P. aeruginosa biofilm that grows has a smooth appearance that varies across the coupon surface and is loosely attached. Microscopically, the biofilm is sheet-like with few architectural details. This laboratory biofilm could represent those found on produce sprayers, on food processing conveyor belts, on catheters, in lungs with cystic fibrosis, and oral biofilms, for example. The biofilm generated in the DFR is also suitable for efficacy testing. After the 54 h growth phase is complete, the user may add the treatment in situ or harvest the coupons and treat them individually. Research has shown that P. aeruginosa biofilms grown in the DFR were less tolerant to disinfection than biofilms grown under high shear conditions.5
SCOPE
1.1 This test method specifies the operational parameters required to grow a repeatable2 Pseudomonas aeruginosa  biofilm close to the air/liquid interface in a reactor with a continuous flow of nutrients under low fluid shear conditions. The resulting biofilm is representative of generalized situations where biofilm exists at the air/liquid interface under low fluid shear rather than representative of one particular environment.  
1.2 This test method uses the drip flow biofilm reactor. The drip flow biofilm reactor (DFR) is a plug flow reactor with laminar flow resulting in low fluid shear. The reactor is versatile and may also be used for growing and/or characterizing biofilms of different species, although this will require changing the operational parameters to optimize the method based upon the growth requirements of the new organism.  
1.3 This test method describes how to sample and analyze biofilm for viable cells. Biofilm population density is recorded as log colony forming units per surface area.  
1.4 Basic microbiology training is required to perform this test method.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 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.  
1.7 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-2020
• ICS: 13.060.30 - Sewage water
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Refers: ASTM D5465-16(2020) - Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Jul-2020

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:E2647 −20
Standard Test Method for
Quantification of Pseudomonas aeruginosa Biofilm Grown
Using Drip Flow Biofilm Reactor with Low Shear and
1
Continuous Flow
This standard is issued under the fixed designation E2647; 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 Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
1.1 This test method specifies the operational parameters
2 Barriers to Trade (TBT) Committee.
required to grow a repeatable Pseudomonas aeruginosa bio-
film close to the air/liquid interface in a reactor with a
2. Referenced Documents
continuous flow of nutrients under low fluid shear conditions.
3
2.1 ASTM Standards:
The resulting biofilm is representative of generalized situations
D5465 Practices for Determining Microbial Colony Counts
where biofilm exists at the air/liquid interface under low fluid
from Waters Analyzed by Plating Methods
shear rather than representative of one particular environment.
2.2 Other Standard:
1.2 This test method uses the drip flow biofilm reactor. The
Method 9050 C.1.a Buffered Dilution Water Preparation,
drip flow biofilm reactor (DFR) is a plug flow reactor with
4
according to Rice et al
laminar flow resulting in low fluid shear. The reactor is
versatile and may also be used for growing and/or character-
3. Terminology
izing biofilms of different species, although this will require
3.1 Definitions:
changing the operational parameters to optimize the method
3.1.1 biofilm, n—microorganisms living in a self-organized,
based upon the growth requirements of the new organism.
cooperativecommunityattachedtosurfaces,interfaces,oreach
1.3 This test method describes how to sample and analyze
other, embedded in a matrix of extracellular polymeric sub-
biofilm for viable cells. Biofilm population density is recorded
stances of microbial origin, while exhibiting an altered pheno-
as log colony forming units per surface area.
type with respect to growth rate and gene transcription.
1.4 Basic microbiology training is required to perform this
3.1.1.1 Discussion—Biofilmsmaybecomprisedofbacteria,
test method.
fungi, algae, protozoa, viruses, or infinite combinations of
these microorganisms. The qualitative characteristics of a
1.5 The values stated in SI units are to be regarded as
biofilm (including, but not limited to, population density,
standard. No other units of measurement are included in this
taxonomic diversity, thickness, chemical gradients, chemical
standard.
composition,consistency,andothermaterialsinthematrixthat
1.6 This standard does not purport to address all of the
arenotproducedbythebiofilmmicroorganisms)arecontrolled
safety concerns, if any, associated with its use. It is the
by the physicochemical environment in which it exists.
responsibility of the user of this standard to establish appro-
3.1.2 coupon, n—biofilm sample surface.
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use. 3.1.3 chamber, n—reactor base containing four rectangular
1.7 This international standard was developed in accor- wells or channels.
dance with internationally recognized principles on standard-
3.1.4 channel, n—one of four rectangular wells in reactor
ization established in the Decision on Principles for the
chamber (base) where coupon is placed.
1 3
This test method is under the jurisdiction of ASTM Committee E35 on For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
responsibility of Subcommittee E35.15 on Antimicrobial Agents. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved April 1, 2020. Published May 2020. Originally the ASTM website.
4
approved in 2008. Last previous edition approved in 2013 as E2647 – 13. DOI: Rice,E.W.,Baird,R.B.andEaton,A.D.,A.E.,Eds.,StandardMethodsforthe
10.1520/E2647-20. Examination of Water and Waste Water, 19th Edition, American Public Health
2
Ellison, S. L. R., Rosslein, M., andWilliams,A., Eds., Quantifying Uncertainty Association, American Water Works Association, Water Environment Federation,
in Analytical Measurement, 2nd Edition, Eurachem, 2000. Washington, DC, 2017.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Bo
...

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: E2647 − 13 E2647 − 20
Standard Test Method for
Quantification of Pseudomonas aeruginosa Biofilm Grown
Using Drip Flow Biofilm Reactor with Low Shear and
1
Continuous Flow
This standard is issued under the fixed designation E2647; 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
2
1.1 This test method specifies the operational parameters required to grow a repeatable Pseudomonas aeruginosa biofilm close
to the air/liquid interface in a reactor with a continuous flow of nutrients under low fluid shear conditions. The resulting biofilm
is representative of generalized situations where biofilm exists at the air/liquid interface under low fluid shear rather than
representative of one particular environment.
1.2 This test method uses the drip flow biofilm reactor. The drip flow biofilm reactor (DFR) is a plug flow reactor with laminar
flow resulting in low fluid shear. The reactor is versatile and may also be used for growing and/or characterizing biofilms of
different species, although this will require changing the operational parameters to optimize the method based upon the growth
requirements of the new organism.
1.3 This test method describes how to sample and analyze biofilm for viable cells. Biofilm population density is recorded as
log colony forming units per surface area.
1.4 Basic microbiology training is required to perform this test method.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.7 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
3
2.1 ASTM Standards:
D5465 Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
2.2 Other Standard:
4
Method 9050 C.1.a Buffered Dilution Water Preparation, according to EatonRice et al
3. Terminology
3.1 Definitions:
3.1.1 biofilm, n—microorganisms living in a self-organized, cooperative community attached to surfaces, interfaces, or each
other, embedded in a matrix of extracellular polymeric substances of microbial origin, while exhibiting an altered phenotype with
respect to growth rate and gene transcription.
1
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, 2013April 1, 2020. Published May 2013May 2020. Originally approved in 2008. Last previous edition approved in 20082013 as
E2647 – 08.E2647 – 13. DOI: 10.1520/E2647-13.10.1520/E2647-20.
2
Ellison, S. L. R., Rosslein, M., and Williams, A., Eds., Quantifying Uncertainty in Analytical Measurement, 2nd Edition, Eurachem, 2000.
3
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.
4
Eaton Rice, A. D., Clesceri, L. S., and Greenberg,E.W., Baird, R.B. and Eaton, A. D., A. E., Eds., Standard Methods for the Examination of Water and Waste Water,
19th Edition, American Public Health Association, American Water Works Association, Water Environment Federation, Washington, DC, 19952017.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

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E2647 − 20
3.1.1.1 Discussion—
Biofilms may be comprised of bacteria, fungi, algae, protozoa, viruses, or infinite combinations of these microorganisms. The
qualitative characteristics of a biofilm (including, but not limited to, population density, taxonomic diversity, thickness, chemical
gradien
...