Standard Test Method for Evaluating Disinfectant Efficacy Against <i>Pseudomonas aeruginosa</i> Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method

SIGNIFICANCE AND USE
5.1 Vegetative biofilm bacteria are phenotypically different from suspended planktonic cells of the same genotype. Biofilm growth reactors are engineered to produce biofilms with specific characteristics (2). Altering either the engineered system or operating conditions will modify those characteristics as well as the physicochemical environment. The goal in biofilm research and efficacy testing is to choose the growth reactor and operating conditions that generate the most relevant biofilm for the particular study.  
5.2 The test method was developed using Pseudomonas aeruginosa ATCC 15442 biofilm grown on borosilicate glass coupons in the CDC Biofilm Reactor and liquid disinfectants. Efficacy data developed using other bacteria, different shear, different coupons, or other standardized biofilm reactor systems, and/or other forms of disinfectants may result in different log10 reduction (LR) values and repeatability and reproducibility standard deviations.  
5.3 The efficacy test was designed to determine the log10 reduction in bacteria after exposure to a disinfectant in a closed system.  
5.4 The test method was developed using 50-mL conical tubes. The conical geometry allows for disinfectant exposure to biofilm on all surfaces of the coupon.  
5.5 Each efficacy test includes a single contact time and temperature for three untreated control coupons (exposed to buffered dilution water) and three treated coupons (per disinfectant/concentration combination).
SCOPE
1.1 This test method specifies the operational parameters required to perform a quantitative liquid disinfectant efficacy test against biofilm bacteria.  
1.2 The test method was developed using a Pseudomonas aeruginosa biofilm grown in the CDC Biofilm Reactor (Test Method E2562), modified to include borosilicate glass coupons as a hard nonporous surface and P. aeruginosa ATCC 15442.  
1.3 Disinfectant preparation and contact time are used in the assessment according to the manufacturer’s instructions for use.  
1.4 The test method uses a closed system to treat biofilm. A coupon is placed in a single tube for the treatment, neutralization, and sampling steps to prevent the loss of cells.  
1.5 Verification of disinfectant neutralization is determined prior to conducting the test method.  
1.6 This test method describes how to sample and analyze treated and untreated control biofilms for viable cells. Biofilm population density is recorded as log10 colony-forming units per coupon. Efficacy is reported as a log10 reduction of viable cells.  
1.7 Basic microbiology training is required to perform this assay.  
1.8 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.9 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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ASTM E2871-13 - Standard Test Method for Evaluating Disinfectant Efficacy Against <i>Pseudomonas aeruginosa</i> Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method
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REDLINE ASTM E2871-13 - Standard Test Method for Evaluating Disinfectant Efficacy Against <i>Pseudomonas aeruginosa</i> Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method
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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:E2871 −13
Standard Test Method for
Evaluating Disinfectant Efficacy Against Pseudomonas
aeruginosa Biofilm Grown in CDC Biofilm Reactor Using
1
Single Tube Method
This standard is issued under the fixed designation E2871; 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
2
2.1 ASTM Standards:
1.1 This test method specifies the operational parameters
required to perform a quantitative liquid disinfectant efficacy E1054 Test Methods for Evaluation of Inactivators of Anti-
test against biofilm bacteria. microbial Agents
E2562 Test Method for Quantification of Pseudomonas
1.2 The test method was developed using a Pseudomonas
aeruginosa Biofilm Grown with High Shear and Continu-
aeruginosa biofilm grown in the CDC Biofilm Reactor (Test
ous Flow using CDC Biofilm Reactor
MethodE2562),modifiedtoincludeborosilicateglasscoupons
2.2 Other Standards:
as a hard nonporous surface and P. aeruginosa ATCC 15442.
Method 9050 C.1.a Buffered Dilution Water Preparation
1.3 Disinfectantpreparationandcontacttimeareusedinthe
3
according to Eaton et al (1)
assessment according to the manufacturer’s instructions for
use.
3. Terminology
1.4 The test method uses a closed system to treat biofilm.A
3.1 Definitions:
coupon is placed in a single tube for the treatment,
3.1.1 biofilm, n—microorganisms living in a self-organized
neutralization, and sampling steps to prevent the loss of cells.
community attached to surfaces, interfaces, or each other,
embedded in a matrix of extracellular polymeric substances of
1.5 Verification of disinfectant neutralization is determined
microbial origin, while exhibiting altered phenotypes with
prior to conducting the test method.
respect to growth rate and gene transcription.
1.6 This test method describes how to sample and analyze
3.1.1.1 Discussion—Biofilm may be comprised of bacteria,
treated and untreated control biofilms for viable cells. Biofilm
fungi, algae, protozoa, viruses, or infinite combinations of
population density is recorded as log colony-forming units
10
these microorganisms. The qualitative characteristics of a
per coupon. Efficacy is reported as a log reduction of viable
10
biofilm including, but not limited to, population density,
cells.
taxonomic diversity, thickness, chemical gradients, chemical
1.7 Basic microbiology training is required to perform this
composition,consistency,andothermaterialsinthematrixthat
assay.
arenotproducedbythebiofilmmicroorganisms,arecontrolled
by the physicochemical environment in which it exists.
1.8 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
3.1.2 contact time, n—predeterminedtimethatthebiofilmis
standard.
exposed to the activity of a disinfectant.
3.1.3 coupon, n—biofilm growth surface.
1.9 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
3.1.4 disinfectant, n—a chemical that destroys vegetative
responsibility of the user of this standard to establish appro-
forms of microorganisms, but does not ordinarily kill bacterial
priate safety and health practices and determine the applica-
spores.
bility of regulatory limitations prior to use.
3.2 Acronyms:
1 2
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 Oct. 1, 2013. Published November 2013. Originally the ASTM website.
3
approved in 2012. Last previous edition approved in 2012 as E2871–12. DOI: The boldface numbers in parentheses refer to a list of references at the end of
10.1520/E2871–13. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
E2871−13
3.2.1 ATCC—American Type Culture Collection. 6.3 Test tube rack, any capable of holding 50-mL conical
centrifuge tubes.
3.2.2 CDC—Centers for Disease Control and Prevention.
6.4 Micropipettes, continuously adjustable pipettes with
3.2.3 CFU—colony-forming unit.
volume capacity of 100 µL and 1000 µL.
4. Summary of Test Method
6.5 Sterile pipette tips, 100-µL and 1000-µL volumes.
4.1 This test method describes the use of the single tube
6.6 Bunsen burner, us
...

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: E2871 − 12 E2871 − 13
Standard Test Method for
Evaluating Disinfectant Efficacy againstAgainst
Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm
1
Reactor usingUsing Single Tube Method
This standard is issued under the fixed designation E2871; 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
1.1 This test method specifies the operational parameters required to perform a quantitative liquid disinfectant efficacy test
against biofilm bacteria.
1.2 The test method was developed using a Pseudomonas aeruginosa biofilm grown in the CDC Biofilm Reactor (Test Method
E2562), modified to include borosilicate glass coupons as a hard nonporous surface and P. aeruginosa ATCC 15442.
1.3 Disinfectant preparation and contact time are used in the assessment according to the manufacturer’s instructions for use.
1.4 The test method uses a closed system to treat biofilm. A coupon is placed in a single tube for the treatment, neutralization,
and sampling steps to prevent the loss of cells.
1.5 Verification of disinfectant neutralization is determined prior to conducting the test method.
1.6 This test method describes how to sample and analyze treated and untreated control biofilms for viable cells. Biofilm
population density is recorded as log colony-forming units per coupon. Efficacy is reported as a log reduction of viable cells.
10 10
1.7 Basic microbiology training is required to perform this assay.
1.8 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.9 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
2.1 ASTM Standards:
E1054 Test Methods for Evaluation of Inactivators of Antimicrobial Agents
E2562 Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow using
CDC Biofilm Reactor
2.2 Other Standards:
3
Method 9050 C.1.a Buffered Dilution Water Preparation according to Eaton et al (1)
3. Terminology
3.1 Definitions:
3.1.1 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.
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, 2012Oct. 1, 2013. Published June 2012November 2013. Originally approved in 2012. Last previous edition approved in 2012 as
E2871–12. DOI: 10.1520/E2871–12.10.1520/E2871–13.
2
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.
3
The boldface numbers in parentheses refer to a list of references at the end of this standard.
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 ----------------------
E2871 − 13
Biofilm 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 contact time, n—predetermined time that the biofilm is exposed to the activity of a disinfectant.
3.1.3 coupon, n—biofilm growth surface.
3.1.4 disinfectant, n—a chemical that destroys vegetative forms of microorganisms, but does not ordinarily kill bacterial spores.
3.2 Acronyms:
3.2.1 ATCC—American Type Culture Collection.
3.2.2 CDC—Centers for Disease Control an
...

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