ASTM E2196-02
(Test Method)Standard Test Method for Quantification of a Pseudomonas aeruginosa Biofilm Grown with Shear and Continuous Flow Using a Rotating Disk Reactor
Standard Test Method for Quantification of a <i>Pseudomonas aeruginosa</i> Biofilm Grown with Shear and Continuous Flow Using a Rotating Disk Reactor
SCOPE
1.1 This test method is used for growing a repeatable Pseudomonas aeruginosa biofilm in a continuously stirred flow reactor. In addition, the test method describes how to sample and analyze biofilm for viable cells.
1.2 In this test method, biofilm population density is recorded as log colony forming units per surface area.
1.3 Basic microbiology training is required to perform this test method. This standard does not claim to address all of the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety practices and determine the applicability of regulatory limitations prior to use.
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Designation: E 2196 – 02
Standard Test Method for
Quantification of a Pseudomonas aeruginosa Biofilm Grown
with Shear and Continuous Flow Using a Rotating Disk
Reactor
This standard is issued under the fixed designation E 2196; 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 (e) indicates an editorial change since the last revision or reapproval.
NOTE 1—Biofilm is a dynamic, self-organized accumulation of micro-
1. Scope
organisms and microbial and environmental by-products that is deter-
1.1 This test method is used for growing a repeatable
mined by the environment in which it lives. Definition 3.1 does not
Pseudomonasaeruginosabiofilminacontinuouslystirredflow
capture every biofilm type that is known to exist.
reactor. In addition, the test method describes how to sample
3.2 coupon—biofilm sample surface.
and analyze biofilm for viable cells.
1.2 In this test method, biofilm population density is re-
4. Summary of Test Method
corded as log colony forming units per surface area.
4.1 This test method is used for growing a repeatable
1.3 Basic microbiology training is required to perform this
Pseudomonas aeruginosa biofilm in a rotating disk reactor.
test method. This standard does not claim to address all of the
The biofilm is established by operating the reactor in batch
safety problems associated with its use. It is the responsibility
mode (no flow) for 24 h. A steady state growth (attachment is
of the user of this standard to establish appropriate safety
equal to detachment) is reached while the reactor operates for
practices and determine the applicability of regulatory limita-
an additional 24 h with continuous flow of the nutrients. The
tions prior to use.
residence time of the nutrients in the reactor is set to select for
biofilm growth, and is species and reactor parameter specific.
2. Referenced Documents
Duringtheentire48h,thebiofilmexperiencescontinuousfluid
2.1 Other Standards:
shear from the rotation of the disk. At the end of the 48 h,
Buffered Dilution Water Preparation—Method 9050 C.1a
biofilm accumulation is quantified by removing coupons from
Rotating Disk Reactor—Repeatability and Relevance
the disk, scraping the biofilm from the coupon surface,
Rotating Disk Reactor—Efficacy Test Method
disaggregating the clumps, then diluting and plating for viable
cell enumeration.
3. Terminology
3.1 biofilm—an accumulation of bacterial cells immobilized
5. Significance and Use
onasubstratumandembeddedinanorganicpolymermatrixof
5.1 Bacteria that exist in a biofilm are phenotypically
microbial origin.
different from suspended cells of the same genotype.The study
of biofilm in the laboratory requires protocols that account for
this difference. Laboratory biofilms are engineered in growth
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides and Alternative Control Agents and is the direct responsibility of
reactors designed to produce a specific biofilm type. Altering
Subcommittee E35.15 on Antimicrobial Agents.
system parameters will correspondingly result in a change in
Current edition approved April 10, 2002. Published August 2002.
thebiofilm.Thepurposeofthismethodistodirectauserinthe
Ellison, S.L.R., M. Rosslein, A. Williams. (Eds.) 2000. Quantifying Uncer-
tainty in Anyalytical Measurement, 2nd Edition. Eurachem. laboratory study of biofilms by clearly defining each system
Eaton,A.D., L.S. Clesceri,A.E. Greenberg. (Eds.) 1995. Standard Methods for
parameter. This method will enable a person to grow, sample
the Examination of Water and Waste Water, 19th Edition. American Public Health
and analyze a laboratory biofilm.
Association, American Water Works Association, Water Environment Federation.
Washington D.C.
6. Apparatus
Zelver, N., M. Hamilton, B. Pitts, D. Goeres, D. Walker, P. Sturman, J.
Heersink. 1999. Methods for measuring antimicrobial effects on biofilm bacteria:
6.1 Wooden Applicator Sticks, sterile.
from laboratory to field. In: Doyle, R.J. (Ed.), Methods in Enzymology-Biofilms Vol
6.2 Inoculating Loop.
310, Academic Press, San Diego, CA, pp. 608-628.
6.3 Petri Dish, 100 by 15 mm, plastic, sterile and empty to
Zelver, N., M. Hamilton, D. Goeres, J. Heersink. 2001. Development of a
Standardized Antibiofilm Test. In: Doyle, R.J. (Ed.), Methods in Enzymology-
hold rotor while sampling.
Biofilms Vol 337, Academic Press, San Diego, CA, pp. 363-376.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2196–02
confirm the actual liquid volume in the reactor, when the rotor is in place,
6.4 Culture Tubes and Culture Tube Closures, any with a
before use. The measured volume is used to calculate an exact pump flow
volume capability of 10 mL and diameter no less than 6 cm.
rate.
Recommended size is 16 by 125 mm borosilicate glass with
threaded opening.
6.22.2 Reactor Top,size15rubberormachinedstopper,3to
6.5 Pipetter, continuously adjustable pipetter with volume
4 holes bored through stopper to accommodate 6 cm pieces of
capability of 1 mL.
fire-polished glass tubing or other suitable rigid autoclavable
6.6 Vortex, any vortex that will ensure proper agitation and
tubing with OD 4 to 6 mm. Another hole can be added to the
mixing of culture tubes.
stopper to contain an inoculum port. The inoculum port
6.7 Homogenizer, any capable of mixing at 20 500 6 5000
consists ofa6cm piece of fire-polished glass tubing or other
rpmina5to10mL volume.
suitable rigid autoclavable tubing fitted with a septum, as
6.8 Homogenizer Probe, any capable of mixing at 20 500 6
shown in Fig. 1.
5000 rpm ina5to10mL volume and can withstand 8
6.22.3 Rotor or Disk, nominal 1.6 mm thick Teflon sheet
autoclaving or other means of sterilization.
cut into a disk with a diameter of 7.0 6 0.2 cm containing 6
6.9 Sonicator, any noncavitating sonicating bath that oper-
evenly spaced holes with a diameter of 1.27 6 0.1 cm. The
ates at 50 to 60 hertz.
center of each hole is located 2.55 6 0.03 cm from the center
6.10 Syringe, sterile, 1 mL syringe used during reactor
of the disk. 4.5 to 7.0 mm thick Viton sheet, or other suitable
inoculation.
autoclavable material, cut into a disk with a diameter of 7.0 6
6.10.1 Needle, sterile, 22 gauge needle used with syringe to
0.2 cm containing 6 evenly spaced holes with a diameter of
inoculate reactor.
1.27 6 0.15 cm (the holes in the Viton are aligned with the
6.11 Bunsen Burner, used to flame inoculating loop and
holes in the Teflon) and a small hole in the center to house the
other instruments.
disk retrieving port. Teflon washer with disk retrieving port.
6.12 Stainless Steel Dissecting Tools.
Four nylon screws. Teflon coated 4.0 by 1.4 cm starhead
6.13 Stainless Steel Hemostat Clamp with Curved Tip.
magnetic stir bar, set flush against Teflon disk and with holes
6.14 Environmental Shaker, capable of maintaining tem-
drilled for assembly with nylon screws. The Teflon ridges on
perature of 35 6 2°C.
one side of the magnet may be shaved to provide a flush
6.15 Analytical Balance, sensitive to 0.01 g.
mounting surface. Assemble the pieces conforming to the
6.16 Sterilizers, any steam sterilizer capable of producing
general details shown in Fig. 2.
the conditions of sterilization is acceptable.
6.22.4 Cylindrical Polycarbonate Coupons, with a diameter
6.17 Colony Counter, any one of several types may be used,
of 1.27 6 0.013 cm and a height of 1.5 to 4.0 mm.
suchastheQuebec,Buck,andWolfhuegel.Ahandtallyforthe
6.23 Carboys, two 15 to 20 L autoclavable carboys, to be
recording of the bacterial count is recommended if manual
used for waste and nutrients.
counting is done.
6.23.1 Carboy Lids, two carboy lids: one carboy lid with at
6.18 PeristalticPump,pumpheadcapableofholdingtubing
least 2 barbed fittings to accommodate tubing ID 3.1 mm (one
with ID 3.1 mm and OD 3.2 mm.
6.19 Magnetic Stir Plate, top plate 10.16 by 10.16 cm, for nutrient line and one for bacterial air vent). One carboy lid
with at least 2-1 cm holes bored in the same fashion (one for
capable of rotating at 200 6 100 rpm.
6.20 Silicon Tubing, two sizes of tubing: one with ID 3.1 effluent waste and one for bacterial air vent).
mm and OD 3.2 mm and the other with ID 7.9 mm and OD 9.5
6.23.2 Bacterial Air Vent, autoclavable 0.2 µm pore size, to
mm. Both sizes must withstand sterilization.
be spliced into tubing on waste carboy, nutrient carboy and
6.21 Glass Flow Break, any that will connect with tubing of
reactor top, recommended diameter 37 mm.
ID 3.1 mm and withstand sterilization.
6.21.1 Clamp, used to hold flow break, extension clamp
7. Reagents and Materials
with 0.5-cm minimum grip size.
7.1 Purity of Water—All reference to water as diluent or
6.21.2 Clamp Stand, height no less than 76.2 cm, used with
reagent shall mean distilled water or water of equal purity.
clamp to suspend glass flow break vertically and stabilize
7.2 Culture Media:
tubing above reactor.
7.2.1 Bacterial Liquid Growth Broth, soybean-casein digest
6.22 Reactor Components .
medium, or an equivalent general bacterial growth medium.
6.22.1 Berzelius Pyrex Beaker, 1000 mL without pour
Tryptic soy broth is recommended.
spout, 9.5 6 0.5 cm diameter. Pyrex barbed outlet spout added
at 250 mL 6 15 mL mark at 30 to 45° angle, spout should
7.2.2 Bacterial Plating Medium,R2Aagarisrecommended.
accommodate silicon tubing with an ID of 8 to 11 mm.
NOTE 3—Media concentration in this protocol differs from the manu-
NOTE 2—The rotor, described in 6.22.3, will displace approximately 50
facturer’s recommendation. Two different concentrations are used in the
mL of liquid. Therefore, an outlet spout at the 250 mL mark will result in
protocol, 300 mg/L for the inoculum and batch reactor operation and 30
approximately a 200 mL operating volume. The user is encouraged to
mg/L for the continuous flow reactor operation.
rpm may be measured using a strobe light.
7 8
Rotating disk reactor is available commercially from BioSurface Technologies, Nominal implies that the manufacturer’s tolerance is acceptable.
Corp. www.imt.net/~mitbst, or the user may build the reactor. Carboy tops can b
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