ASTM E3161-21

Standard Practice for Preparing a <emph type="bdit">Pseudomonas aeruginosa</emph > or <emph type="bdit">Staphylococcus aureus</emph> Biofilm using the 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 (4, 5). 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 practice is to direct a user in the growth of a P. aeruginosa or S. aureus biofilm by clearly defining the operational parameters to grow a biofilm that can be assessed for efficacy using the Standard Test Method for Evaluating Disinfectant Efficacy Against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method (E2871).  
5.2 Operating the CDC Biofilm Reactor at the conditions specified in this method generates biofilm at log densities (log10 CFU per coupon) ranging from 8.0 to 9.5 for P. aeruginosa and 7.5 to 9.0 for S. aureus. These levels of biofilm are anticipated on surfaces conducive to biofilm formation such as the conditions outlined in this method.  
5.2.1 To achieve an S. aureus biofilm with a population comparable to that for P. aeruginosa using the bacterial liquid growth medium conditions specified here, the S. aureus biofilm must be grown at 36 °C ±2 °C rather than at room temperature (21 °C ±2 °C).
SCOPE
1.1 This practice specifies the parameters for growing a Pseudomonas aeruginosa (ATCC 15442) or Staphylococcus aureus (ATCC 6538) biofilm that can be used for disinfectant efficacy testing using the Test Method for Evaluating Disinfectant Efficacy Against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method (E2871) or in an alternate method capable of accommodating the coupons used in the CDC Biofilm Reactor. The resulting biofilm is representative of generalized situations where biofilm exist on hard, non-porous surfaces under shear rather than being representative of one particular environment. Additional bacteria may be grown using the basic procedure outlined in this document, however, alternative preparation procedures for frozen stock cultures and biofilm generation (for example, medium concentrations, baffle speed, temperature, incubation times, coupon types, etc.) may be necessary.  
1.2 This practice uses the CDC Biofilm Reactor created by the Centers for Disease Control and Prevention (1).2 The CDC Biofilm Reactor is a continuously stirred tank reactor (CSTR) with high wall shear. The reactor is versatile and may also be used for growing or characterizing various species of biofilm, or both (2-4) provided appropriate adjustments are made to the growth media and operational parameters of the reactor.  
1.3 Basic microbiology training is required to perform this practice.  
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this practice.  
1.5 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.  
1.6 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-Oct-2021
• ICS: 07.100.01 - Microbiology in general
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

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:E3161 −21
Standard Practice for
Preparing a Pseudomonas aeruginosa or Staphylococcus
1
aureus Biofilm using the CDC Biofilm Reactor
This standard is issued under the fixed designation E3161; 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* priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
1.1 This practice specifies the parameters for growing a
1.6 This international standard was developed in accor-
Pseudomonas aeruginosa (ATCC 15442) or Staphylococcus
dance with internationally recognized principles on standard-
aureus (ATCC 6538) biofilm that can be used for disinfectant
ization established in the Decision on Principles for the
efficacy testing using theTest Method for Evaluating Disinfec-
Development of International Standards, Guides and Recom-
tantEfficacyAgainst Pseudomonas aeruginosaBiofilmGrown
mendations issued by the World Trade Organization Technical
inCDCBiofilmReactorUsingSingleTubeMethod(E2871)or
Barriers to Trade (TBT) Committee.
in an alternate method capable of accommodating the coupons
used in the CDC Biofilm Reactor. The resulting biofilm is
2. Referenced Documents
representative of generalized situations where biofilm exist on
3
2.1 ASTM Standards:
hard, non-porous surfaces under shear rather than being repre-
E2756Terminology Relating toAntimicrobial andAntiviral
sentative of one particular environment. Additional bacteria
Agents
may be grown using the basic procedure outlined in this
E2871Test Method for Determining Disinfectant Efficacy
document, however, alternative preparation procedures for
Against Biofilm Grown in the CDC Biofilm Reactor
frozen stock cultures and biofilm generation (for example,
Using the Single Tube Method
medium concentrations, baffle speed, temperature, incubation
times, coupon types, etc.) may be necessary.
3. Terminology
1.2 This practice uses the CDC Biofilm Reactor created by 3.1 Definitions:
2
the Centers for Disease Control and Prevention (1). The CDC 3.1.1 For definition of terms used in this method refer to
Biofilm Reactor is a continuously stirred tank reactor (CSTR) Terminology E2756.
with high wall shear. The reactor is versatile and may also be 3.1.2 batch phase, n—establishment of the biofilm by oper-
ating the reactor without the flow of nutrients (batch phase
used for growing or characterizing various species of biofilm,
orboth (2-4)providedappropriateadjustmentsaremadetothe growth medium), but with mixing.
growth media and operational parameters of the reactor.
3.1.3 biofilm, n—microorganisms living in a self-organized
community attached to surfaces, interfaces, or each other,
1.3 Basic microbiology training is required to perform this
embedded in a matrix of extracellular polymeric substances of
practice.
microbial origin, while exhibiting altered phenotypes with
1.4 Units—The values stated in SI units are to be regarded
respect to growth rate and gene transcription.
asstandard.Nootherunitsofmeasurementareincludedinthis
3.1.4 continuously stirred tank reactor (CSTR) phase,
practice.
n—establishment of a steady state biofilm population achieved
1.5 This standard does not purport to address all of the
with the continuous flow of nutrients (continuous flow growth
safety concerns, if any, associated with its use. It is the
medium) in a glass vessel.
responsibility of the user of this standard to establish appro-
3.1.5 coupon, n—biofilm sample surface.
4. Summary of Practice
1
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides,
4.1 This practice is used for growing a P. aeruginosa or S.
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
aureus biofilm in the CDC Biofilm Reactor. The biofilm is
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Nov. 1, 2021. Published January 2022. Originally
3
approved in 2018. Last previous edition approved in 2018 as E3161–18. DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/E3161–21. contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
2
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof 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 Cons
...

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: E3161 − 18 E3161 − 21
Standard Practice for
Preparing a Pseudomonas aeruginosa or Staphylococcus
1
aureus Biofilm using the CDC Biofilm Reactor
This standard is issued under the fixed designation E3161; 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*
1.1 This practice specifies the parameters for growing a Pseudomonas aeruginosa (ATCC 15442) or Staphylococcus aureus
(ATCC 6538) biofilm that can be used for disinfectant efficacy testing using the Test Method for Evaluating Disinfectant Efficacy
Against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method (E2871) or in an alternate
method capable of accommodating the coupons used in the CDC Biofilm Reactor. The resulting biofilm is representative of
generalized situations where biofilm exist on hard, non-porous surfaces under shear rather than being representative of one
particular environment. Additional bacteria may be grown using the basic procedure outlined in this document, however,
alternative preparation procedures for frozen stock cultures and biofilm generation (for example, medium concentrations, baffle
speed, temperature, incubation times, coupon types, etc.) may be necessary.
2
1.2 This practice uses the CDC Biofilm Reactor created by the Centers for Disease Control and Prevention (1). The CDC Biofilm
Reactor is a continuously stirred tank reactor (CSTR) with high wall shear. The reactor is versatile and may also be used for
growing or characterizing various species of biofilm, or both (2-4) provided appropriate adjustments are made to the growth media
and operational parameters of the reactor.
1.3 Basic microbiology training is required to perform this practice.
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this practice.
1.5 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.
1.6 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:
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
1
This practice 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, 2018Nov. 1, 2021. Published June 2018January 2022. Originally approved in 2018. Last previous edition approved in 2018 as E3161–18.
DOI: 10.1520/E3161–18.10.1520/E3161–21.
2
The boldface numbers in parentheses refer to the 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

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E3161 − 21
E2871 Test Method for Determining Disinfectant Efficacy Against Biofilm Grown in the CDC Biofilm Reactor Using the Single
Tube Method
3. Terminology
3.1 Definitions:
3.1.1 For definition of terms used in this method refer to Terminology E2756.
3.1.2 batch phase, n—establishment of the biofilm by operating the reactor without the flow of nutrients (batch phase growth
medium), but with mixing.
3.1.3 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.
3.1.4 continuously stirred tank reactor (CSTR) phas
...