ASTM E2647-20

Standard Test Method for Quantification of <emph type="bdit">Pseudomonas aeruginosa</emph > 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 that varies across the coupon surface 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 biofilm reactor. The drip flow biofilm 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, health, and environmental 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: 31-Mar-2020
• ICS: 13.060.30 - Sewage water
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Replaces: ASTM E2647-13 - Standard Test Method for Quantification of <i>Pseudomonas aeruginosa</i> Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow
• Effective Date: 01-Apr-2020
• Refers: ASTM D5465-16(2020) - Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Jul-2020
• Refers: ASTM D5465-16 - Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Jun-2016
• Refers: ASTM D5465-93(2012) - Standard Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Jun-2012
• Refers: ASTM D5465-93(2004) - Standard Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Jun-2004
• Refers: ASTM D5465-93(1998) - Standard Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 10-Dec-1998

Overview

ASTM E2647-20: Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow provides a standardized laboratory procedure for growing, sampling, and analyzing Pseudomonas aeruginosa biofilms. This method utilizes a drip flow biofilm reactor (DFR) to simulate environments where biofilm forms at the air/liquid interface under low shear conditions, making the results applicable to a wide range of real-world scenarios.

This ASTM standard supports research and testing efforts by ensuring reproducible results when cultivating and quantifying biofilms of P. aeruginosa, a common and clinically significant bacterium. It enables the comparison of various antimicrobial treatments and supports regulatory and industrial applications where biofilm management is required.

Key Topics

• Biofilm Growth and Analysis: The standard details the operational parameters necessary to grow repeatable biofilms close to the air/liquid interface under continuous nutrient flow and low-fluid shear.
• Drip Flow Biofilm Reactor (DFR): Describes set-up, usage, and maintenance of the DFR, a plug flow reactor providing controlled low-shear conditions.
• Sampling and Quantification: Explains the procedures for sampling the biofilm from coupons, preparation, and quantification via colony forming unit (CFU) counts per surface area.
• Practical Microbiological Techniques: Emphasizes the use of sterile techniques, standard microbiological media, and the requirement for trained personnel.
• Reproducibility and Reliability: Outlines methodological features designed for repeatability and consistency across laboratories.
• Versatility: Notes adaptability of the DFR for other biofilm species, with suitable changes to operational parameters.

Applications

The ASTM E2647-20 standard test method is widely applicable in both industrial and clinical settings, as well as in academic research:

• Medical Devices: Useful for evaluating biofilm formation on surfaces such as catheters and implants, simulating conditions found in the human body, including the lungs or oral cavity.
• Food Processing: Supports assessment of biofilm on surfaces like produce sprayers and conveyor belts, contributing to hygiene and safety in the food industry.
• Antimicrobial Efficacy Testing: Enables comparison of the susceptibility of P. aeruginosa biofilms to disinfectants and cleaning protocols under defined laboratory conditions.
• Environmental Microbiology: Facilitates the study of biofilm growth in water systems and other settings where low-shear conditions prevail.
• Regulatory Compliance: Provides a standardized and internationally recognized method for biofilm quantification, supporting regulatory submissions and efficacy claims.

Related Standards

The following standards and methods are relevant for users of ASTM E2647-20:

• ASTM D5465: Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods.
• Standard Methods for the Examination of Water and Waste Water (APHA/AWWA/WEF): Commonly referenced for the preparation of buffered dilution water.
• Biofilm Quantification Protocols: Related methods for sampling and enumeration of microbial populations from surface-attached communities.

Practical Value

Implementing ASTM E2647-20 ensures the production of consistent, reproducible biofilm samples for the quantification of viable Pseudomonas aeruginosa cells, vital for:

• Evaluating disinfection protocols
• Supporting product development and validation
• Reducing variability in research findings
• Enhancing laboratory and industrial biofilm management

This standard supports the global harmonization of biofilm research and testing, promoting public health, product safety, and innovation in industries impacted by biofilm formation.

Keywords: ASTM E2647-20, Pseudomonas aeruginosa, biofilm quantification, drip flow biofilm reactor, low shear, continuous flow, antimicrobial efficacy, biofilm sampling, laboratory standard, CFU enumeration.

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:E2647 −20
Standard Test Method for
Quantification of Pseudomonas aeruginosa Biofilm Grown
Using Drip Flow Biofilm Reactor with Low Shear and
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 Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
1.1 This test method specifies the operational parameters
2 Barriers to Trade (TBT) Committee.
required to grow a repeatable Pseudomonas aeruginosa bio-
film close to the air/liquid interface in a reactor with a
2. Referenced Documents
continuous flow of nutrients under low fluid shear conditions.
2.1 ASTM Standards:
The resulting biofilm is representative of generalized situations
D5465 Practices for Determining Microbial Colony Counts
where biofilm exists at the air/liquid interface under low fluid
from Waters Analyzed by Plating Methods
shear rather than representative of one particular environment.
2.2 Other Standard:
1.2 This test method uses the drip flow biofilm reactor. The
Method 9050 C.1.a Buffered Dilution Water Preparation,
drip flow biofilm reactor (DFR) is a plug flow reactor with
according to Rice et al
laminar flow resulting in low fluid shear. The reactor is
versatile and may also be used for growing and/or character-
3. Terminology
izing biofilms of different species, although this will require
3.1 Definitions:
changing the operational parameters to optimize the method
3.1.1 biofilm, n—microorganisms living in a self-organized,
based upon the growth requirements of the new organism.
cooperativecommunityattachedtosurfaces,interfaces,oreach
1.3 This test method describes how to sample and analyze
other, embedded in a matrix of extracellular polymeric sub-
biofilm for viable cells. Biofilm population density is recorded
stances of microbial origin, while exhibiting an altered pheno-
as log colony forming units per surface area.
type with respect to growth rate and gene transcription.
1.4 Basic microbiology training is required to perform this
3.1.1.1 Discussion—Biofilmsmaybecomprisedofbacteria,
test method.
fungi, algae, protozoa, viruses, or infinite combinations of
these microorganisms. The qualitative characteristics of a
1.5 The values stated in SI units are to be regarded as
biofilm (including, but not limited to, population density,
standard. No other units of measurement are included in this
taxonomic diversity, thickness, chemical gradients, chemical
standard.
composition,consistency,andothermaterialsinthematrixthat
1.6 This standard does not purport to address all of the
arenotproducedbythebiofilmmicroorganisms)arecontrolled
safety concerns, if any, associated with its use. It is the
by the physicochemical environment in which it exists.
responsibility of the user of this standard to establish appro-
3.1.2 coupon, n—biofilm sample surface.
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use. 3.1.3 chamber, n—reactor base containing four rectangular
1.7 This international standard was developed in accor- wells or channels.
dance with internationally recognized principles on standard-
3.1.4 channel, n—one of four rectangular wells in reactor
ization established in the Decision on Principles for the
chamber (base) where coupon is placed.
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 [e-mail]. 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, 2020. Published May 2020. Originally the ASTM website.
approved in 2008. Last previous edition approved in 2013 as E2647 – 13. DOI: Rice,E.W.,Baird,R.B.andEaton,A.D.,A.E.,Eds.,StandardMethodsforthe
10.1520/E2647-20. Examination of Water and Waste Water, 19th Edition, American Public Health
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, 2017.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2647−20
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 biofilm reactor (DFR). The 6.8 Homogenizer—Any capable of mixing at
biofilm is established by operating the reactor in batch mode 20 500 6 5000 r/min in a 50 mL volume.
(no flow of nutrients) for 6 h.Amature biofilm forms while the
6.9 Homogenizer Probe—Any capable of mixing at
reactor operates for an additional 48 h with a continuous flow
20 500 6 5000 r/min in a 50 mL volume and with a gap
(CF) of nutrients. During continuous flow, the biofilm experi-
between the rotor and stator of 0.25 mm. Both disposable
ences very low shear caused by the gravity flow of media
probes and probes that can withstand autoclaving or other
dripping onto a surface set at a 10° angle.At the end of the 54
means of sterilization are acceptable.
h, biofilm accumulation is quantified by removing coupons
6.10 Bunsen Burner—Used to flame sterilize inoculating
from the reactor channels, rinsing the coupons to remove the
loop and other instruments.
planktonic cells, scraping the biofilm from the coupon surface,
disaggregating the clumps, then diluting and plating for viable
6.11 95 % Ethanol—Used to flame sterilize hemostats or
cell enumeration. forceps.
6.12 Stainless Steel Hemostat Clamp or Forceps—For asep-
5. Significance and Use
tic handling of coupons.
5.1 Vegetative biofilm bacteria are phenotypically different
6.13 Pipetter—Continuouslyadjustablepipettewithvolume
from suspended cells of the same genotype. Biofilm growth
capability of 1 mL.
reactors are engineered to produce biofilms with specific
6.14 Analytical Balance—Sensitive to 0.01 g.
characteristics. Altering either the engineered system or oper-
ating conditions will modify those characteristics.
6.15 Sterilizers—Any steam sterilizer capable of producing
the conditions of sterilization.
5.2 Thepurposeofthistestmethodistodirectauserinhow
togrow,sample,andanalyzeaP.aeruginosabiofilmunderlow
6.16 Colony Counter—Any one of several types may be
fluid shear and close to the air/liquid interface using the DFR.
used. A hand tally for the recording of the bacterial count is
The P. aeruginosa biofilm that grows has a smooth appearance
recommended if manual counting is done.
that varies across the coupon surface and is loosely attached.
6.17 Peristaltic Pump—Four pump heads capable of hold-
Microscopically,thebiofilmissheet-likewithfewarchitectural
ing tubing with inner diameter (ID) 3.1 mm and outer diameter
details. This laboratory biofilm could represent those found on
(OD) 3.2 mm and operating at a flow rate of 200 mL per hour.
produce sprayers, on food processing conveyor belts, on
catheters, in lungs with cystic fibrosis, and oral biofilms, for
6.18 Environmental Shaker—Capable of maintaining a tem-
example. The biofilm generated in the DFR is also suitable for perature of 35 6 2 °C.
efficacy testing. After the 54 h growth phase is complete, the
6.19 Tubing—Two sizes of silicone tubing: one with ID 3.1
user may add the treatment in situ or harvest the coupons and
mm and OD 3.2 mm and the other with ID 7.9 mm and OD 9.5
treat them individually. Research has shown that P. aeruginosa
mm. Both sizes must withstand sterilization.
biofilms grown in the DFR were less tolerant to disinfection
6.20 Glass Flow Break—Any that will connect with tubing
than biofilms grown under high shear conditions.
of ID 3.1 mm and withstands sterilization.
6. Apparatus 6.20.1 Clamp—Used to hold flow break, extension clamp
with 0.5 cm minimum grip size.
6.1 Polytetrafluoroethylene (PTFE), Metal, or Rubber
6.20.2 Clamp Stand—Height no less than 76.2 cm, used
Spatulas—Sterile, for scraping biofilm from coupon surface.
with clamp to suspend glass flow break vertically and stabilize
6.2 Inoculating Loop.
tubing.
6.3 Petri Dish—100 by 15 mm, plastic, sterile, and empty
6.21 Reactor Components —A schematic of the drip flow
for transporting coupons from reactor to work station.
reactor is shown in Fig. 1. Fig. 2 is a picture of the assembled
system.
6.4 Culture Tubes and Culture Tube Closures—Any with a
6.21.1 Chamber (Base)—15.24 by 15.88 cm anodized
volume capability of 10 mL and a minimum diameter of 16
aluminum, polysulfone or polyethylene terephthalate (PET)
mm. Recommended size is 16 by 125 mm borosilicate glass
chamber with four 3.05- by 10.16-cm channels and four
with threaded opening.
1.27-cm barbed effluent ports (one at the end of each channel).
6.5 Glass Beakers—Sterile, any with a volume capacity of
During CF, the reactor sits on a stand to provide a 10° angle.
100 mL containing 45 mL sterile buffered water.
Each channel contains two pegs to guide coupon placement.
6.6 Conical Centrifuge Tubes—Sterile, any with 50 mL
volume capacity containing 45 mL sterile buffered water.
The sole source of supply of the drip flow biofilm reactor apparatus known to
the committee at this time is BioSurface Technologies, Corp., Bozeman, MT,
www.biofilms.biz. If you are aware of alternative suppliers, please provide this
Buckingham-Meyer, K., Goeres, D. M., and Hamilton, M. A., “Comparative information to ASTM International Headquarters. Your comments will receive
Evaluation of Biofilm Disinfectant EfficacyTests,” J. Microbiological Methods,Vol careful consideration at a meeting of the responsible technical committee, which
70, 2007, pp. 236–244. you may attend. Alternatively, the user may build the DFR apparatus.
E2647−20
FIG. 1Expanded View of the Drip Flow Reactor
FIG. 2Drip Flow Reactor Laboratory Set-Up in Continuous Flow Operation
E2647−20
6.21.2 Top—FourO-ringfittedpolycarbonatetopseachwith 9. Preparation of Apparatus
two threaded holes for nylon screws to secure to reactor
9.1 Reactor Set-Up:
chamber (base). Two ports, one for Mininert™ valve and
9.1.1 Use new coupons (glass microscope slides) for every
another for bacterial air vent attachment.
experiment. Only handles slides with gloved hands.
6.21.3 Mininert™ Valves—Fitintoeachtopasinfluentports
9.1.2 Insert a coupon into each
...

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 − 13 E2647 − 20
Standard Test Method for
Quantification of Pseudomonas aeruginosa Biofilm Grown
Using Drip Flow Biofilm Reactor with Low Shear and
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
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 biofilm 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 safety, health, and healthenvironmental 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
2.1 ASTM Standards:
D5465 Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
2.2 Other Standard:
Method 9050 C.1.a Buffered Dilution Water Preparation, according to EatonRice 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.
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, 2013April 1, 2020. Published May 2013May 2020. Originally approved in 2008. Last previous edition approved in 20082013 as
E2647 – 08.E2647 – 13. DOI: 10.1520/E2647-13.10.1520/E2647-20.
Ellison, S. L. R., Rosslein, M., and Williams, A., Eds., Quantifying Uncertainty in Analytical Measurement, 2nd Edition, Eurachem, 2000.
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.
Eaton Rice, A. D., Clesceri, L. S., and Greenberg,E.W., Baird, R.B. and Eaton, A. D., 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, 19952017.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2647 − 20
3.1.1.1 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.
3.1.2 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 (base) where coupon is placed.
4. Summary of Test Method
4.1 This test method is used for growing a repeatable P. aeruginosa biofilm in a drip flow reactor. biofilm reactor (DFR). The
biofilm is established by operating the reactor in batch mode (no flow of nutrients) for 6 h. A mature biofilm forms while the reactor
operates for an additional 48 h with a continuous flow (CF) of nutrients. During continuous flow, the biofilm experiences very low
shear caused by the gravity flow of media dripping onto a surface set at a 10° angle. At the end of the 54 h, biofilm accumulation
is quantified by removing coupons from the reactor channels, rinsing the coupons to remove the planktonic cells, scraping the
biofilm from the coupon surface, disaggregating the clumps, then diluting and plating for viable cell enumeration.
5. 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 that
varies across the coupon surface 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.
6. Apparatus
6.1 Tetrafluoroethylene (TFE),Polytetrafluoroethylene (PTFE), Metal, or Rubber Spatulas—Sterile, for scraping biofilm from
coupon surface.
6.2 Inoculating Loop.
6.3 Petri Dish—100 by 15 mm, plastic, sterile, and empty for transporting coupons from reactor to work station.
6.4 Culture Tubes and Culture Tube Closures—Any with a volume capability of 10 mL and a minimum diameter of 16 mm.
Recommended size is 16 by 125 mm borosilicate glass with threaded opening.
6.5 Glass Beakers—Sterile, any with a volume capacity of 100 mL containing 45 mL sterile buffered water.
6.6 Conical Centrifuge Tubes—Sterile, any with 50-mL50 mL volume capacity containing 45 mL sterile buffered water.
6.7 Vortex—Any vortex that will ensure proper agitation and mixing of culture tubes.
6.8 Homogenizer—Any capable of mixing at 20 500 6 5000 r/min in a 50 mL volume.
6.9 Homogenizer Probe—Any capable of mixing at 20 500 6 5000 r/min in a 50 mL volume and with a gap between the rotor
and stator of 0.25 mm. Both disposable probes and probes that can withstand autoclaving or other means of sterilization are
acceptable.
6.10 Bunsen Burner—Used to flame sterilize inoculating loop and other instruments.
6.11 95 % Ethanol—Used to flame sterilize hemostats or forceps.
6.12 Stainless Steel Hemostat Clamp or Forceps—For aseptic handling of coupons.
6.13 Pipetter—Continuously adjustable pipette with volume capability of 1 mL.
Buckingham-Meyer, K., Goeres, D. M., and Hamilton, M. A., “Comparative Evaluation of Biofilm Disinfectant Efficacy Tests,” J. Microbiological Methods, Vol 70,
2007, pp. 236–244.
E2647 − 20
6.14 Analytical Balance—Sensitive to 0.01 g.
6.15 Sterilizers—Any steam sterilizer capable of producing the conditions of sterilization.
6.16 Colony Counter—Any one of several types may be used. A hand tally for the recording of the bacterial count is
recommended if manual counting is done.
6.17 Peristaltic Pump—Four pump heads capable of holding tubing with inner diameter (ID) 3.1 mm and outer diameter (OD)
3.2 mm and operating at a flow rate of 200 mL per hour.
6.18 Environmental Shaker—Capable of maintaining a temperature of 35 6 2°C.2 °C.
6.19 Tubing—Two sizes of silicone tubing: one with ID 3.1 mm and OD 3.2 mm and the other with ID 7.9 mm and OD 9.5
mm. Both sizes must withstand sterilization.
6.20 Glass Flow Break—Any that will connect with tubing of ID 3.1 mm and withstands sterilization.
6.20.1 Clamp—Used to hold flow break, extension clamp with 0.5 cm minimum grip size.
6.20.2 Clamp Stand—Height no less than 76.2 cm, used with clamp to suspend glass flow break vertically and stabilize tubing.
6.21 Reactor Components —A schematic of the drip flow reactor is shown in Fig. 1. Fig. 2 is a picture of the assembled system.
FIG. 1 Expanded View of the Drip Flow Reactor
The sole source of supply of the drip flow biofilm reactor apparatus known to the committee at this time is BioSurface Technologies, Corp., Bozeman, MT,
www.imt.net/~mitbst.www.biofilms.biz. If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will
receive careful consideration at a meeting of the responsible technical committee, which you may attend. Alternatively, the user may build the DFR apparatus.
E2647 − 20
FIG. 2 Drip Flow Reactor Laboratory Set-Up in Continuous Flow Operation
6.21.1 Chamber (Base)—15.24 by 15.88 cm polysulfone anodized aluminum, polysulfone or polyethylene terephthalate (PET)
chamber with four 3.05- by 10.16-cm channels and four 1.27-cm barbed effluent ports (one at the end of each channel). The
underside holds four adjustable inserts (legs) providing a 10° angle for continuous flow conditions.During CF, the reactor sits on
a stand to provide a 10° angle. Each channel contains two pegs to guide coupon placement.placement.
E2647 − 20
6.21.2 Top—Four O-ring fitted polycarbonate tops each with two threaded holes for nylon screws to secure to reactor chamber
(base). Two ports, one for MininertMininert™ valve and another for bacterial air vent attachment.
6.21.3 MininertMininert™ Valves—Fit into each top as influent ports to allow inoculation and media line attachment.
6.21.4 Luer Lock Connectors with 3.1 mm Hose Barb—Used to connect needles to the tubing.
6.21.5 Needle—1 in., 21 gauge, to fit into MininertMininert™ port.
6.21.6 Glass Coupons—Four new rectangular glass microscope slides (or other similar shaped material) with a top surface area
of 18.75 cm (25 by 75 by 1 mm).
6.21.7 TFE Thread Seal Tape—To prevent leakage from effluent port connector.
6.22 Carboys—Two 10 to 20 L autoclavable carboys for waste and nutrients.
6.22.1 Carboy Lids—(Note 1)—One carboy lid with at least two barbed fittings to accommodate tubing ID 3.1 mm (one for
nutrient line and one for bacterial air vent). One carboy lid with at least two 1-cm holes bored in the same fashion (one for effluent
waste and one for bacterial air vent).
NOTE 1—Carboy tops can be purchased with fittings.
6.23 Bacterial Air Vent (Filter)—Autoclavable 0.2 μm pore size, to be attached into tubing on waste and nutrie
...