ASTM E2562-22

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

SIGNIFICANCE AND USE
5.1 Bacteria that exist in biofilms are phenotypically different from suspended cells of the same genotype. Research has shown that biofilm bacteria are more difficult to kill than suspended bacteria (5, 7). 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. For example, research has shown that biofilm grown under high shear is more difficult to kill than biofilm grown under low shear (5, 8). The purpose of this test method is to direct a user in the laboratory study of a Pseudomonas aeruginosa biofilm by clearly defining each system parameter. This test method will enable an investigator to grow, sample, and analyze a Pseudomonas aeruginosa biofilm grown under high shear. The biofilm generated in the CDC Biofilm Reactor is also suitable for efficacy testing. After the 48 h growth phase is complete, the user may add the treatment in situ or remove the coupons and treat them individually.
SCOPE
1.1 This test method specifies the operational parameters required to grow a reproducible (1)2 Pseudomonas aeruginosa ATCC 700888 biofilm under high shear. The resulting biofilm is representative of generalized situations where biofilm exists under high shear rather than being representative of one particular environment.  
1.2 This test method uses the Centers for Disease Control and Prevention (CDC) Biofilm Reactor. The CDC Biofilm Reactor is a continuously stirred tank reactor (CSTR) with high wall shear. Although it was originally designed to model a potable water system for the evaluation of Legionella pneumophila (2), the reactor is versatile and may also be used for growing and/or characterizing biofilm of varying species (3-5).  
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.  
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.  
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: 30-Apr-2022
• ICS: 07.100.01 - Microbiology in general
• 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: E2562 − 22
Standard Test Method for
Quantification of Pseudomonas aeruginosa Biofilm Grown
with High Shear and Continuous Flow using CDC Biofilm
1
Reactor
This standard is issued under the fixed designation E2562; 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* mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.1 This test method specifies the operational parameters
2
required to grow a reproducible (1) Pseudomonas aeruginosa
2. Referenced Documents
ATCC 700888 biofilm under high shear. The resulting biofilm
3
2.1 ASTM Standards:
is representative of generalized situations where biofilm exists
D5465 Practices for Determining Microbial Colony Counts
under high shear rather than being representative of one
from Waters Analyzed by Plating Methods
particular environment.
E2756 Terminology Relating to Antimicrobial and Antiviral
1.2 This test method uses the Centers for Disease Control
Agents
and Prevention (CDC) Biofilm Reactor. The CDC Biofilm
2.2 Other Standards:
Reactor is a continuously stirred tank reactor (CSTR) with high
Method 9050 C.1.a Buffered Dilution Water Preparation
wall shear. Although it was originally designed to model a
according to Baird et al (6)
potable water system for the evaluation of Legionella pneumo-
phila (2), the reactor is versatile and may also be used for
3. Terminology
growing and/or characterizing biofilm of varying species (3-5).
3.1 For definitions of terms used in this standard refer to
1.3 This test method describes how to sample and analyze Terminology E2756.
biofilm for viable cells. Biofilm population density is recorded
3.2 Definitions of Terms Specific to This Standard:
as log colony forming units per surface area.
10
3.2.1 biofilm, n—microorganisms living in a self-organized
1.4 Basic microbiology training is required to perform this
community attached to surfaces, interfaces, or each other,
test method.
embedded in a matrix of extracellular polymeric substances of
microbial origin, while exhibiting altered phenotypes with
1.5 The values stated in SI units are to be regarded as
respect to growth rate and gene transcription.
standard. No other units of measurement are included in this
3.2.1.1 Discussion—Biofilms may be comprised of bacteria,
standard.
fungi, algae, protozoa, viruses, or infinite combinations of
1.6 This standard does not purport to address all of the
these microorganisms. The qualitative characteristics of a
safety concerns, if any, associated with its use. It is the
biofilm, including, but not limited to, population density,
responsibility of the user of this standard to establish appro-
taxonomic diversity, thickness, chemical gradients, chemical
priate safety, health, and environmental practices and deter-
composition, consistency, and other materials in the matrix that
mine the applicability of regulatory limitations prior to use.
are not produced by the biofilm microorganisms, are controlled
1.7 This international standard was developed in accor-
by the physicochemical environment in which it exists.
dance with internationally recognized principles on standard-
3.2.2 coupon, n—biofilm sample surface.
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
4. Summary of Test Method
4.1 This test method is used for growing a reproducible
Pseudomonas aeruginosa ATCC 700888 biofilm in a CDC
1
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct Biofilm Reactor. The biofilm is established by operating the
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved May 1, 2022. Published May 2022. Originally
3
approved in 2007. Last previous edition approved in 2017 as E2562 – 17. DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/E2562-22. contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
2
The boldface numbers in parentheses refer to a list of references at the end of Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 -----------------
...

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: E2562 − 17 E2562 − 22
Standard Test Method for
Quantification of Pseudomonas aeruginosa Biofilm Grown
with High Shear and Continuous Flow using CDC Biofilm
1
Reactor
This standard is issued under the fixed designation E2562; 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 Scope*
2
1.1 This test method specifies the operational parameters required to grow a reproducible (1) Pseudomonas aeruginosa ATCC
700888 biofilm under high shear. The resulting biofilm is representative of generalized situations where biofilm exists under high
shear rather than being representative of one particular environment.
1.2 This test method uses the Centers for Disease Control and Prevention (CDC) Biofilm Reactor. The CDC Biofilm Reactor is
a continuously stirred tank reactor (CSTR) with high wall shear. Although it was originally designed to model a potable water
system for the evaluation of Legionella pneumophila(2), the reactor is versatile and may also be used for growing and/or
characterizing biofilm of varying species (3-5).
1.3 This test method describes how to sample and analyze biofilm for viable cells. Biofilm population density is recorded as log
10
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.
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:
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, 2017May 1, 2022. Published May 2017May 2022. Originally approved in 2007. Last previous edition approved in 20122017 as
E2562 – 12.E2562 – 17. DOI: 10.1520/E2562-17.10.1520/E2562-22.
2
The boldface numbers in parentheses refer to a list of references at the end of this standard.
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.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
E2562 − 22
D5465 Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 Other Standards:
Method 9050 C.1.a Buffered Dilution Water Preparation according to RiceBaird et al (6)
3. Terminology
3.1 For definitions of terms used in this standard refer to Terminology E2756.
3.2 Definitions:Definitions of Terms Specific to This Standard:
3.2.1 biofilm, n—microorganisms living in a self-organized community attached to surfaces, interfaces, or each other, embedded
in a matrix of extracellular polymeric substances of microbial origin, while exhibiting altered phenotypes with respect to growth
rate and gene transcription.
3.2.1.1 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
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.
3.2.2 coupon, n—biofilm sample surf
...