ASTM E2562-22

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
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*
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
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
2.1 ASTM Standards:
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.
The boldface numbers in parentheses refer to a list of references at the end of this standard.
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.
*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
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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 surface.
4. Summary of Test Method
4.1 This test method is used for growing a reproducible Pseudomonas aeruginosa ATCC 700888 biofilm in a CDC Biofilm
Reactor. The biofilm is established by operating the reactor in batch mode (no flow of the nutrients) for 24 h. A steady state
population is reached while the reactor operates for an additional 24 h with a continuous flow of the nutrients. The residence time
of the nutrients in the reactor is set to select for biofilm growth, and is species and reactor parameter specific. During the entire
48 h, the biofilm is exposed to continuous fluid shear from the rotation of a baffled stir bar. Controlling the rate at which the baffle
turns determines the intensity of the shear stress to which the coupons are exposed. At the end of the 48 h, biofilm accumulation
is quantified by removing coupons from suspended rods, harvesting the biofilm from the coupon surface by scraping the biofilm
from the coupon, homogenizing the removed biofilm to disaggregate the clumps, and diluting and plating for viable cell
enumeration.
5. 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.
6. Apparatus
6.1 Wooden Applicator Sticks—sterile.
6.2 Inoculating Loop.
6.3 Petri Dish—100 mm by 15 mm, plastic, sterile, and empty to put beneath rod while sampling.
6.4 Culture Tubes and Culture Tube Closures—any with a volume capacity of 10 mL and a minimum diameter of 16 mm.
Recommended size is 16 mm by 125 mm borosilicate glass with threaded opening.
6.5 Pipette—continuously adjustable pipetter with volume capacity of 1 mL.
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6.6 Vortex—any vortex that will ensure proper agitation and mixing of culture tubes.
6.7 Homogenizer—any that can mix at 20 500 r/min 6 5000 r/min in a 5 mL to 10 mL volume.
6.8 Homogenizer Probe—any that can mix at 20 500 r/min 6 5000 r/min in a 5 to 10 mL volume and can withstand autoclaving
or other means of sterilization.
6.9 Sonicating Water Bath—any cavitating sonicating bath that operates at 45 kHz to 50 kHz for cleaning coupons.
6.10 Bunsen Burner—used to flame inoculating loop and other instruments.
6.11 Stainless Steel Hemostat Clamp—with curved tip.
NOTE 1—Alternatively, a coupon manipulating tool may be used.
6.12 Environmental Shaker—that can maintain a temperature of 3636 °C 6 2°C.2 °C.
6.13 Analytical Balance—sensitive to 0.01 g.
6.14 Sterilizer—any steam sterilizer that can produce the conditions of sterilization is acceptable.
6.15 Colony Counter—any one of several types may be used, such as the Quebec, Buck, and Wolfhuegel. A hand tally for the
recording of the bacterial count is recommended if manual counting is done.
6.16 Peristaltic Pump—pump head that can hold tubing with inner diameter 3.1 mm and outer diameter 3.2 mm.
6.17 Digital Magnetic Stir Plate—top plate 10.16 cm × 10.16 cm, that can rotate at 125 r/min 6 5 r/min.
6.18 Silicone Tubing—twothree sizes of tubing: one with inner diameter 3.1 mm and outer diameter 3.2 mm, and the other one
with inner diameter 7.9 mm and outer diameter 9.5 mm. Bothmm, and one with inner diameter 8 mm and outer diameter 11 mm.
All sizes must withstand sterilization.
6.19 Norprene Tubing—inner diameter 3.1 mm and outer diameter 3.2 mm.
6.20 Glass Flow Break—any that will connect with tubing of inner diameter 3.1 mm and withstand 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
above reactor.
6.20.3 Laboratory Screw Clamp—used to clamp effluent tubing during batch growth.
6.21 Reactor Components.
The sole source of supply of the apparatus (coupon manipulating tool) known to the committee at this time is Biosurface Technologies, Corp., 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. The user may also build the holder.
Trademarked by the Saint-Gobain Performance Plastics Corporation.
The sole source of supply of the apparatus (CDC Biofilm Reactor) known to the committee at this time is BioSurface Technologies, Corp. 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. The user may also build the reactor.
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FIG. 1 Expanded Schematic of Reactor Top
6.21.1 Berzelius Borosilicate Glass Tall Beaker—1000 mL without pour spout, 9.59.5 cm 6 0.5 cm diameter. Barbed outlet spout
added at 400400 mL 6 20 mL mark. Angle the spout 3030° to 45° to ensure drainage. Spout should accommodate flexible tubing
with an inner diameter of 8 to 11 mm.
NOTE 2—The rods (see 6.21.3) and baffle (see 6.21.6) will displace approximately 50 mL of liquid when system is completely assembled. Therefore, an
outlet spout at the 400 mL mark will result in approximately a 350 mL operating volume. The user should confirm the actual liquid volume in the reactor,
when the rods and baffle are in place and the stir plate is turned on, before use. The measured operating volume is used to calculate an exact pump flow
rate.
6.21.2 Reactor Top—Fig. 1. Ultra-high molecular weight (UHMW) polyethylene top (10.1 cm diameter tapering to 8.33 cm)
equipped with a minimum of three holes accommodating 10 cm pieces of stainless steel or other rigid autoclavable tubing with
outside diameter of 55 mm to 8 mm for media inlet, air exchange, and inoculation port. Center hole, 1.27 cm diameter, to
accommodate the glass rod used to support the baffle assembly. Eight rod holes, 1.905 cm diameter, notched to accommodate
stainless steel rod alignment pin (0.236 cm outside diameter).
6.21.3 Polypropylene Rods—Fig. 2. Eight polypropylene rods, 21.08 cm long, machined to hold three coupons (see 6.21.4) at the
immersed end. Three 316 stainless steel set screws imbedded in side to hold coupons in place. Rods fit into holes in reactor top
and lock into preformed notches with alignment pin.
6.21.4 Twenty-four Cylindrical Polycarbonate Coupons—with a diameter of 1.271.27 cm 6 0.013 cm, thickness of approximately
3.0 mm.
6.21.5 Small Allen Wrench—for loosening set screws.
6.21.6 Stir Blade Assembly (Baffled Stir Bar)—Fig. 3. PTFE blade (5.61 cm) fitted into cylindrical PTFE holder (8.13 cm) and held
in place with a magnetic stir bar (2.54 cm). PTFE holder fits onto a glass rod (15.8 cm), fitted into the reactor top. The glass rod
is held in place with a compression fitting and acts as a support for the moving blade assembly.
6.22 Carboys—two 20 L autoclavable carboys, to be used for waste and nutrients.
6.22.1 Two Carboy Lids—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).
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FIG. 2 Expanded Schematic of Rod and Coupons
FIG. 3 Expanded Schematic of Baffled Stir Bar
NOTE 3—Carboy tops can be purchased with fittings.
6.22.2 Bacterial Air Vent (Filter)—autoclavable, 0.2 μm pore size, to be spliced into tubing on waste carboy, nutrient carboy, and
reactor top; recommended diameter 37 mm.
6.23 Fig. 4 illustrates a schematic of the assembled system.
7. Reagents and Materials
7.1 Purity of Water—All reference to water as diluent or reagent shall mean distilled water or water of equal purity.
7.2 Culture Media:
7.2.1 Bacterial Liquid Growth Broth—Tryptic Soy Broth (TSB) is recommended.
NOTE 4—Two different TSB concentrations are used in the test method, 300 mg/L for the inoculum and batch reactor operation, and 100 mg/L for the
continuous flow reactor operation.
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FIG. 4 Schematic of Complete
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