ASTM E2647-08
(Test Method)Standard Test Method for Quantification of a Pseudomonas aeruginosa Biofilm Grown Using a Drip Flow Biofilm Reactor with Low Shear and Continuous Flow
Standard Test Method for Quantification of a <span class="italic">Pseudomonas aeruginosa</span> Biofilm Grown Using a Drip Flow Biofilm Reactor with Low Shear and Continuous Flow
SIGNIFICANCE AND USE
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.
The purpose of this test method is to direct a user in how to grow, sample and analyze a Pseudomonas aeruginosa biofilm under low fluid shear and close to the air/liquid interface using the drip flow reactor. The Pseudomonas aeruginosa biofilm that grows has a smooth appearance 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 drip flow reactor 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 drip flow reactor were less resistant to disinfection than biofilms grown under high shear conditions.
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 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 different species of biofilms.
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.
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Designation:E2647 −08
StandardTest Method for
Quantification of a Pseudomonas aeruginosa Biofilm Grown
Using a 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 D5465 Practice for Determining Microbial Colony Counts
from Waters Analyzed by Plating Methods
1.1 This test method specifies the operational parameters
2.2 Other Standard:
required to grow a repeatable Pseudomonas aeruginosa bio-
Method 9050 C.1.a Buffered Dilution Water Preparation
film close to the air/liquid interface in a reactor with a
continuous flow of nutrients under low fluid shear conditions.
3. Terminology
The resulting biofilm is representative of generalized situations
where biofilm exists at the air/liquid interface under low fluid 3.1 Definitions:
shear rather than representative of one particular environment. 3.1.1 biofilm, n—microorganisms living in a self-organized,
cooperativecommunityattachedtosurfaces,interfaces,oreach
1.2 This test method uses the drip flow biofilm reactor. The
other, embedded in a matrix of extracellular polymeric sub-
drip flow reactor (DFR) is a plug flow reactor with laminar
stances of microbial origin, while exhibiting an altered pheno-
flow resulting in low fluid shear. The reactor is versatile and
type with respect to growth rate and gene transcription.
may also be used for growing and/or characterizing different
3.1.1.1 Discussion—Biofilms may be comprised of bacteria,
species of biofilms.
fungi, algae, protozoa, viruses, or infinite combinations of
1.3 This test method describes how to sample and analyze
these microorganisms. The qualitative characteristics of a
biofilm for viable cells. Biofilm population density is recorded
biofilm (including, but not limited to, population density,
as log colony forming units per surface area.
taxonomic diversity, thickness, chemical gradients, chemical
composition,consistency,andothermaterialsinthematrixthat
1.4 Basic microbiology training is required to perform this
test method. arenotproducedbythebiofilmmicroorganisms)arecontrolled
by the physicochemical environment in which it exists.
1.5 The values stated in SI units are to be regarded as
3.1.2 coupon, n—biofilm sample surface.
standard. No other units of measurement are included in this
standard.
3.1.3 chamber, n—reactor base containing four rectangular
wells or channels.
1.6 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
3.1.4 channel, n—one of four rectangular wells in reactor
responsibility of the user of this standard to establish appro-
chamber (base) where coupon is placed.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. 4. Summary of Test Method
4.1 This test method is used for growing a repeatable
2. Referenced Documents
Pseudomonasaeruginosabiofilminadripflowbiofilmreactor.
2.1 ASTM Standards:
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
This test method is under the jurisdiction of ASTM Committee E35 on
continuous flow of nutrients. During continuous flow, the
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
biofilm experiences very low shear caused by the gravity flow
Current edition approved Oct. 1, 2008. Published October 2008. DOI: 10.1520/
of media dripping onto a surface set at a 10° angle.At the end
E2647-08.
Ellison, S. L. R., Rosslein, M., andWilliams,A., Eds., Quantifying Uncertainty
in Analytical Measurement, 2nd Edition, Eurachem, 2000.
3 4
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Eaton,A. D., Clesceri, L. S., and Greenberg,A. E., Eds., Standard Methods for
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM the Examination of Water and Waste Water, 19th Edition, American Public Health
Standards volume information, refer to the standard’s Document Summary page on Association, American Water Works Association, Water Environment Federation,
the ASTM website. Washington, DC, 1995.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2647−08
of the 54 h, biofilm accumulation is quantified by removing rotorandstatorof0.25mm.Bothdisposableprobesandprobes
coupons from the reactor channels, rinsing the coupons to that can withstand autoclaving or other means of sterilization
remove the planktonic cells, scraping the biofilm from the are acceptable.
coupon surface, disaggregating the clumps, then diluting and
6.10 Bunsen Burner—Used to flame sterilize inoculating
plating for viable cell enumeration.
loop and other instruments.
6.11 95 % Ethanol—Used to flame sterilize hemostats or
5. Significance and Use
forceps.
5.1 Vegetative biofilm bacteria are phenotypically different
6.12 Stainless Steel Hemostat Clamp or Forceps—For asep-
from suspended cells of the same genotype. Biofilm growth
tic handling of coupons.
reactors are engineered to produce biofilms with specific
characteristics. Altering either the engineered system or oper-
6.13 Pipetter—Continuouslyadjustablepipettewithvolume
ating conditions will modify those characteristics.
capability of 1 mL.
5.2 Thepurposeofthistestmethodistodirectauserinhow
6.14 Analytical Balance—Sensitive to 0.01 g.
to grow, sample and analyze a Pseudomonas aeruginosa
6.15 Sterilizers—Any steam sterilizer capable of producing
biofilm under low fluid shear and close to the air/liquid
the conditions of sterilization.
interface using the drip flow reactor.The Pseudomonas aerugi-
nosabiofilmthatgrowshasasmoothappearanceandisloosely 6.16 Colony Counter—Any one of several types may be
used. A hand tally for the recording of the bacterial count is
attached. Microscopically, the biofilm is sheet-like with few
architectural details. This laboratory biofilm could represent recommended if manual counting is done.
those found on produce sprayers, on food processing conveyor
6.17 Peristaltic Pump—Four pump heads capable of hold-
belts, on catheters, in lungs with cystic fibrosis and oral
ing tubing with inner diameter (ID) 3.1 mm and outer diameter
biofilms, for example. The biofilm generated in the drip flow
(OD) 3.2 mm and operating at a flow rate of 200 mL per hour.
reactor is also suitable for efficacy testing. After the 54 h
6.18 Environmental Shaker—Capable of maintaining a tem-
growth phase is complete, the user may add the treatment in
perature of 35 6 2°C.
situ or harvest the coupons and treat them individually.
Research has shown that P. aeruginosa biofilms grown in the
6.19 Tubing—Two sizes of silicone tubing: one with ID 3.1
drip flow reactor were less resistant to disinfection than
mm and OD 3.2 mm and the other with ID 7.9 mm and OD 9.5
biofilms grown under high shear conditions.
mm. One size of Norprene tubing with an ID of 1.6 mm. All
tubing must withstand sterilization.
6. Apparatus
6.20 Glass Flow Break—Any that will connect with tubing
6.1 TFE-fluorocarbon, Metal, or Rubber Spatulas—Sterile,
of ID 3.1 mm and withstands sterilization.
for scraping biofilm from coupon surface.
6.20.1 Clamp—Used to hold flow break, extension clamp
with 0.5-cm minimum grip size.
6.2 Inoculating Loop.
6.20.2 Clamp Stand—Height no less than 76.2 cm, used
6.3 PetriDish—100by15mm,plastic,sterileandemptyfor
with clamp to suspend glass flow break vertically and stabilize
transporting coupons from reactor to work station.
tubing.
6.4 Culture Tubes and Culture Tube Closures—Sterile, any
6.21 Reactor Components —A schematic of the drip flow
with a volume capacity of 10 mL and a minimum diameter of
reactor is shown in Fig. 1. Fig. 2 is a picture of the assembled
16 mm. Recommended size is 16 by 125 mm borosilicate glass
system.
with threaded opening.
6.21.1 Chamber (Base)—15.24 by 15.88 cm polysulfone
6.5 Glass Beakers—Sterile, any with a volume capacity of
chamber with four 3.05 by 10.16-cm channels and four
100 mL containing 45 mL sterile buffered water.
1.27-cm barbed effluent ports (one at the end of each channel).
The underside holds four adjustable inserts (legs) providing a
6.6 Conical-Bottom Sterile Disposable Plastic Centrifuge
10° angle for continuous flow conditions. Each channel con-
Tubes—Any with a volume capacity of 50 mL. Fill each with
tains two pegs to guide coupon placement.
45 mL sterile buffered water.
6.21.2 Top—FourO-ringfittedpolycarbonatetopseachwith
6.7 Vortex—Anyvortexthatwillensureproperagitationand
two threaded holes for nylon screws to secure to reactor
mixing of culture tubes.
chamber (base). Two ports, one for Mininert valve and another
6.8 Homogenizer—Any capable of mixing at 20 500 6
for bacterial air vent attachment.
5000 rpm in a 50 mL volume.
6.9 Homogenizer Probe—Any capable of mixing at 20 500
6 5000 rpm in a 50 mL volume and with a gap between the 6
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. If you are aware of alternative suppliers, please provide this
information to ASTM International Headquarters. Your comments will receive
Buckingham-Meyer, K., Goeres, D. M., and Hamilton, M. A., “Comparative careful consideration at a meeting of the responsible technical committee, which
Evaluation of Biofilm Disinfectant Efficacy Tests,” J. Microbiological Methods, 70, you may attend. Alternatively, the user may build the Drip Flow Biofilm Reactor
2007, pp. 236-244. apparatus.
E2647−08
FIG. 1Expanded View of the Drip Flow Reactor
FIG. 2Drip Flow Reactor Laboratory Set-Up in Continuous Flow Operation
6.21.3 Mininert Valves—Fitintoeachtopasinfluentportsto carboy (recommended diameter is 37 mm) and each reactor
allow inoculation and media line attachment. channel top with (recommended diameter is 15 mm).
6.21.4 Needles—1 in., 21 gauge, to fit into Mininert port.
7. Reagents and Materials
6.21.5 Glass Coupons—Four new rectangular glass micro-
scope slides (or other similar shaped material) with a top
7.1 Purity of Water—All reference to water as diluent or
surface area of 18.75 cm (25 by 75 by 1 mm).
reagent shall mean distilled water or water of equal purity.
6.21.6 TFE-fluorocarbon Thread Seal Tape—To prevent
7.2 Culture Media:
leakage from effluent port connector.
7.2.1 Bacterial Liquid Growth Broth—Tryptic Soy Broth
6.22 Carboys—Two 10-20 Lautoclavable carboys for waste
(TSB) is recommended.
and nutrients.
NOTE 2—Two differentTSB conc
...
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