ASTM E2647-13
(Test Method)Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow
Standard Test Method for Quantification of <i>Pseudomonas aeruginosa</i> 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 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 reactor. The drip flow 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 and health practices and determine the applicability of regulatory limitations prior to use.
General Information
Relations
Buy Standard
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:E2647 −13
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. Referenced Documents
3
1.1 This test method specifies the operational parameters 2.1 ASTM Standards:
2
required to grow a repeatable Pseudomonas aeruginosa bio- D5465 Practice for Determining Microbial Colony Counts
film close to the air/liquid interface in a reactor with a from Waters Analyzed by Plating Methods
continuous flow of nutrients under low fluid shear conditions.
2.2 Other Standard:
The resulting biofilm is representative of generalized situations
Method 9050 C.1.a Buffered Dilution Water Preparation,
4
where biofilm exists at the air/liquid interface under low fluid
according to Eaton et al
shear rather than representative of one particular environment.
3. Terminology
1.2 This test method uses the drip flow reactor. The drip
flow reactor (DFR) is a plug flow reactor with laminar flow 3.1 Definitions:
resulting in low fluid shear. The reactor is versatile and may 3.1.1 biofilm, n—microorganisms living in a self-organized,
also be used for growing and/or characterizing biofilms of cooperativecommunityattachedtosurfaces,interfaces,oreach
different species, although this will require changing the other, embedded in a matrix of extracellular polymeric sub-
operational parameters to optimize the method based upon the stances of microbial origin, while exhibiting an altered pheno-
growth requirements of the new organism. type with respect to growth rate and gene transcription.
3.1.1.1 Discussion—Biofilms may be comprised of bacteria,
1.3 This test method describes how to sample and analyze
fungi, algae, protozoa, viruses, or infinite combinations of
biofilm for viable cells. Biofilm population density is recorded
these microorganisms. The qualitative characteristics of a
as log colony forming units per surface area.
biofilm (including, but not limited to, population density,
1.4 Basic microbiology training is required to perform this
taxonomic diversity, thickness, chemical gradients, chemical
test method.
composition,consistency,andothermaterialsinthematrixthat
1.5 The values stated in SI units are to be regarded as arenotproducedbythebiofilmmicroorganisms)arecontrolled
standard. No other units of measurement are included in this by the physicochemical environment in which it exists.
standard.
3.1.2 coupon, n—biofilm sample surface.
1.6 This standard does not purport to address all of the
3.1.3 chamber, n—reactor base containing four rectangular
safety concerns, if any, associated with its use. It is the
wells or channels.
responsibility of the user of this standard to establish appro-
3.1.4 channel, n—one of four rectangular wells in reactor
priate safety and health practices and determine the applica-
chamber (base) where coupon is placed.
bility of regulatory limitations prior to use.
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 service@astm.org. 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, 2013. Published May 2013. Originally the ASTM website.
4
approved in 2008. Last previous edition approved in 2008 as E2647 – 08. DOI: Eaton,A. D., Clesceri, L. S., and Greenberg,A. E., Eds., Standard Methods for
10.1520/E2647-13. the 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, 1995.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
E2647−13
4. Summary of Test Method 6.7 Vortex—Anyvortexthatwillensureproperagitationand
mixing of culture tubes.
4.1 This test method is used for growing a repeatable P.
aeruginosa biofilm in a drip flow reactor. The biofilm is 6.8 Homogenizer—Any capable of mixing at
established by operating the reactor in batch mode (no flow of 20 500 6 5000 r/min in a 50 mL volume.
nutrients) for
...
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 − 08 E2647 − 13
Standard Test Method for
Quantification of a Pseudomonas aeruginosa Biofilm Grown
Using a 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 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 different species of
biofilms.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 and health practices and determine the applicability of regulatory
limitations prior to use.
2. Referenced Documents
3
2.1 ASTM Standards:
D5465 Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
2.2 Other Standard:
4
Method 9050 C.1.a Buffered Dilution Water PreparationPreparation, according to Eaton 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 Oct. 1, 2008April 1, 2013. Published October 2008May 2013. Originally approved in 2008. Last previous edition approved in 2008 as
E2647 – 08. DOI: 10.1520/E2647-08.10.1520/E2647-13.
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 service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
4
Eaton, A. D., Clesceri, L. S., and Greenberg, 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, 1995.
3.1.1.1 Discussion—
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
E2647 − 13
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
gradients, chemical composition, consistency, and other materials in the matrix that are not produced by the biofilm
microorganisms) are controlled by the physicochemical environment in which it exists.
3.1.2 coupon, n—biofilm sample surface.
3.1.3 chamber, n—reactor base containing four rectangular wells or channels.
3.1.4 channel, n—one of four rectangular wells in reactor chamber
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.