ASTM E2196-12
(Test Method)Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with Medium Shear and Continuous Flow Using Rotating Disk Reactor
Standard Test Method for Quantification of <i>Pseudomonas aeruginosa</i> Biofilm Grown with Medium Shear and Continuous Flow Using Rotating Disk Reactor
SIGNIFICANCE AND USE
Bacteria that exist in a biofilm are phenotypically different from suspended cells of the same genotype. The study of biofilm in the laboratory requires protocols that account for this difference. Laboratory biofilms are engineered in growth reactors designed to produce a specific biofilm type. Altering system parameters will correspondingly result in a change in the biofilm. The purpose of this method is to direct a user in the laboratory study of biofilms by clearly defining each system parameter. This method will enable a person to grow, sample, and analyze a laboratory biofilm.
SCOPE
1.1 This test method is used for growing a reproducible (1) Pseudomonas aeruginosa biofilm in a continuously stirred tank reactor (CSTR) under medium shear conditions. In addition, the test method describes how to sample and analyze biofilm for viable cells.
1.2 Although this test method was created to mimic conditions within a toilet bowl, it can be adapted for the growth and characterization of varying species of biofilm (rotating disk reactorrepeatability and relevance (2)).
1.3 This test method describes how to sample and analyze biofilm for viable cells. Biofilm population density is recorded as log10 colony forming units per surface area (rotating disk reactorefficacy test method (3)).
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.
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Designation:E2196 −12
Standard Test Method for
Quantification of Pseudomonas aeruginosa Biofilm Grown
with Medium Shear and Continuous Flow Using Rotating
1
Disk Reactor
This standard is issued under the fixed designation E2196; 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 from Waters Analyzed by Plating Methods
2
2.2 Other Standards:
1.1 Thistestmethodisusedforgrowingareproducible (1)
Method 9050 C.1.aBuffered Dilution Water Preparation (4)
Pseudomonasaeruginosabiofilminacontinuouslystirredtank
reactor (CSTR) under medium shear conditions. In addition,
3. Terminology
the test method describes how to sample and analyze biofilm
3.1 biofilm, n—– microorganisms living in a self-organized
for viable cells.
community attached to surfaces, interfaces, or each other,
1.2 Although this test method was created to mimic condi-
embedded in a matrix of extracellular polymeric substances of
tions within a toilet bowl, it can be adapted for the growth and
microbial origin, while exhibiting altered phenotypes with
characterization of varying species of biofilm (rotating disk
respect to growth rate and gene transcription.
reactor—repeatability and relevance (2)).
3.1.1 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 (rotating disk biofilm, including, but not limited to, population density,
10
reactor—efficacy test method (3)). taxonomic diversity, thickness, chemical gradients, chemical
composition,consistency,andothermaterialsinthematrixthat
1.4 Basic microbiology training is required to perform this
arenotproducedbythebiofilmmicroorganisms,arecontrolled
test method.
by the physiochemical environment in which it exists.
1.5 The values stated in SI units are to be regarded as
3.2 coupon, n—biofilm sample surface.
standard. No other units of measurement are included in this
standard.
4. Summary of Test Method
1.6 This standard does not purport to address all of the
4.1 This test method is used for growing a reproducible
safety concerns, if any, associated with its use. It is the
Pseudomonas aeruginosa biofilm in a rotating disk reactor.
responsibility of the user of this standard to establish appro-
The biofilm is established by operating the reactor in batch
priate safety and health practices and determine the applica-
mode (no flow) for 24 h. Steady state growth (attachment is
bility of regulatory limitations prior to use.
equal to detachment) is reached while the reactor operates for
an additional 24 h with continuous flow of the nutrients. The
2. Referenced Documents
residence time of the nutrients in the reactor is set to select for
3
2.1 ASTM Standards:
biofilm growth, and is species and reactor parameter specific.
D5465Practice for Determining Microbial Colony Counts
During the entire 48 h, the biofilm is exposed to continuous
fluidshearfromtherotationofthedisk.Attheendofthe48h,
biofilm accumulation is quantified by removing coupons from
1
This test method is under the jurisdiction of ASTM Committee E35 on
the disk, harvesting the biofilm from the coupon surface,
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
responsibility of Subcommittee E35.15 on Antimicrobial Agents. disaggregating the clumps, then diluting and plating for viable
Current edition approved April 1, 2012. Published June 2012. Originally
cell enumeration.
approved in 2002. Last previous edition approved in 2007 as E2196–07. DOI:
10.1520/E2196-12.
5. Significance and Use
2
The boldface numbers in parentheses refer to a list of references at the end of
this standard.
5.1 Bacteria that exist in a biofilm are phenotypically
3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
differentfromsuspendedcellsofthesamegenotype.Thestudy
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
of biofilm in the laboratory requires protocols that account for
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. this difference. Laboratory biofilms are engineered in growth
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
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E2196−12
reactors designed to produce a specific biofilm type. Altering 6.19 Silicone Tubing, two sizes of tubing: one with inner
system par
...
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:E2196–07 Designation: E2196 – 12
Standard Test Method for
Quantification of a Quantification of Pseudomonas
aeruginosa Biofilm Grown with Medium Shear and
1
Continuous Flow Using a Rotating Disk Reactor
This standard is issued under the fixed designation E2196; 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
1.1This test method is used for growing a repeatable
2
1.1 This test method is used for growing a reproducible (1) Pseudomonas aeruginosa biofilm in a continuously stirred flow
reactor. In addition, the test method describes how to sample and analyze biofilm for viable cells.
1.2In this test method, biofilm population density is recorded as log colony forming units per surface area.
1.3Basic microbiology training is required to perform this test method. This standard does not claim to address all of the safety
problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety practices and
determine the applicability of regulatory limitations prior to use. biofilm in a continuously stirred tank reactor (CSTR) under
medium shear conditions. In addition, the test method describes how to sample and analyze biofilm for viable cells.
1.2 Although this test method was created to mimic conditions within a toilet bowl, it can be adapted for the growth and
characterization of varying species of biofilm (rotating disk reactor—repeatability and relevance (2)).
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 (rotating disk reactor—efficacy test method (3)).
10
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
2.1 Other Standards:
Buffered Dilution Water Preparation—Method 9050 C.1a
3
2.1 ASTM Standards:
Rotating Disk Reactor—Repeatability and Relevance
Rotating Disk Reactor—Efficacy Test Method
D5465 Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
2.2 Other Standards:
Method 9050 C.1.a Buffered Dilution Water Preparation (4)
3. Terminology
3.1 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
altered phenotypes with respect to growth rate and gene transcription.
3.1.1 Discussion—Biofilms may be comprised of bacteria, fungi, algae, protozoa, viruses, or infinite combinations of these
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 approvedApril 1, 2007.2012. Published May 2007.June 2012. Originally approved in 2002. Last previous edition approved in 20022007 as E2196 – 027.
DOI: 10.1520/E2196-07.10.1520/E2196-12.
2
Ellison, S.L.R., M. Rosslein, A. Williams. (Eds.) 2000. Quantifying Uncertainty in Anyalytical Measurement, 2nd Edition. Eurachem.
2
The boldface numbers in parentheses refer to a list of references at the end of this standard.
3
Eaton,A.D., L.S. Clesceri, Rice, E.W.,A.E. Greenberg. (Eds.) Standard Methods for the Examination of Water and Waste Water , 21st Edition.American Public Health
Association, American Water Works Association, Water Environment Federation. Washington D.C., 2005.
3
For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM 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.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
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