1、Designation: E 2647 08Standard Test Method forQuantification of a Pseudomonas aeruginosa Biofilm GrownUsing a Drip Flow Biofilm Reactor with Low Shear andContinuous Flow1This standard is issued under the fixed designation E 2647; the number immediately following the designation indicates the year of
2、original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method specifies the operational parametersrequir
3、ed to grow a repeatable2Pseudomonas aeruginosa bio-film close to the air/liquid interface in a reactor with acontinuous flow of nutrients under low fluid shear conditions.The resulting biofilm is representative of generalized situationswhere biofilm exists at the air/liquid interface under low fluid
4、shear rather than representative of one particular environment.1.2 This test method uses the drip flow biofilm reactor. Thedrip flow reactor (DFR) is a plug flow reactor with laminarflow resulting in low fluid shear. The reactor is versatile andmay also be used for growing and/or characterizing diff
5、erentspecies of biofilms.1.3 This test method describes how to sample and analyzebiofilm for viable cells. Biofilm population density is recordedas log colony forming units per surface area.1.4 Basic microbiology training is required to perform thistest method.1.5 The values stated in SI units are t
6、o be regarded asstandard. No other units of measurement are included in thisstandard.1.6 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices an
7、d determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D 5465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating Methods2.2 Other Standard:Method 9050 C.1.a Buffered Dilution Water Preparation43. Terminology3.1 De
8、finitions:3.1.1 biofilm, nmicroorganisms living in a self-organized,cooperative community attached to surfaces, interfaces, or eachother, embedded in a matrix of extracellular polymeric sub-stances of microbial origin, while exhibiting an altered pheno-type with respect to growth rate and gene trans
9、cription.3.1.1.1 DiscussionBiofilms may be comprised of bacte-ria, fungi, algae, protozoa, viruses, or infinite combinations ofthese microorganisms. The qualitative characteristics of abiofilm (including, but not limited to, population density,taxonomic diversity, thickness, chemical gradients, chem
10、icalcomposition, consistency, and other materials in the matrix thatare not produced by the biofilm microorganisms) are controlledby the physicochemical environment in which it exists.3.1.2 coupon, nbiofilm sample surface.3.1.3 chamber, nreactor base containing four rectangularwells or channels.3.1.
11、4 channel, none of four rectangular wells in reactorchamber (base) where coupon is placed.4. Summary of Test Method4.1 This test method is used for growing a repeatablePseudomonas aeruginosa biofilm in a drip flow biofilm reactor.The biofilm is established by operating the reactor in batchmode (no f
12、low of nutrients) for 6 h. A mature biofilm formswhile the reactor operates for an additional 48 h with acontinuous flow of nutrients. During continuous flow, thebiofilm experiences very low shear caused by the gravity flowof media dripping onto a surface set at a 10 angle. At the endof the 54 h, bi
13、ofilm accumulation is quantified by removingcoupons from the reactor channels, rinsing the coupons toremove the planktonic cells, scraping the biofilm from the1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibilit
14、y ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2008. Published October 2008.2Ellison, S. L. R., Rosslein, M., and Williams,A., Eds., Quantifying Uncertaintyin Analytical Measurement, 2nd Edition, Eurachem, 2000.3For referenced ASTM standards, visit the ASTM website,
15、 www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4Eaton, A. D., Clesceri, L. S., and Greenberg, A. E., Eds., Standard Methods forthe Examination of Water and Waste W
16、ater, 19th Edition, American Public HealthAssociation, American Water Works Association, Water Environment Federation,Washington, DC, 1995.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.coupon surface, disaggregating the clumps, the
17、n diluting andplating for viable cell enumeration.5. Significance and Use5.1 Vegetative biofilm bacteria are phenotypically differentfrom suspended cells of the same genotype. Biofilm growthreactors are engineered to produce biofilms with specificcharacteristics. Altering either the engineered syste
18、m or oper-ating conditions will modify those characteristics.5.2 The purpose of this test method is to direct a user in howto grow, sample and analyze a Pseudomonas aeruginosabiofilm under low fluid shear and close to the air/liquidinterface using the drip flow reactor. The Pseudomonas aerugi-nosa b
19、iofilm that grows has a smooth appearance and is looselyattached. Microscopically, the biofilm is sheet-like with fewarchitectural details. This laboratory biofilm could representthose found on produce sprayers, on food processing conveyorbelts, on catheters, in lungs with cystic fibrosis and oralbi
20、ofilms, for example. The biofilm generated in the drip flowreactor is also suitable for efficacy testing. After the 54 hgrowth phase is complete, the user may add the treatment insitu or harvest the coupons and treat them individually.Research has shown that P. aeruginosa biofilms grown in thedrip f
21、low reactor were less resistant to disinfection thanbiofilms grown under high shear conditions.56. Apparatus6.1 TFE-fluorocarbon, Metal, or Rubber SpatulasSterile,for scraping biofilm from coupon surface.6.2 Inoculating Loop.6.3 Petri Dish100 by 15 mm, plastic, sterile and emptyfor transporting coup
22、ons from reactor to work station.6.4 Culture Tubes and Culture Tube ClosuresSterile, anywith a volume capacity of 10 mL and a minimum diameter of16 mm. Recommended size is 16 by 125 mm borosilicate glasswith threaded opening.6.5 Glass BeakersSterile, any with a volume capacity of100 mL containing 45
23、 mL sterile buffered water.6.6 Conical-Bottom Sterile Disposable Plastic CentrifugeTubesAny with a volume capacity of 50 mL. Fill each with45 mL sterile buffered water.6.7 VortexAny vortex that will ensure proper agitationand mixing of culture tubes.6.8 HomogenizerAny capable of mixing at 20 500 650
24、00 rpm in a 50 mL volume.6.9 Homogenizer ProbeAny capable of mixing at 20 5006 5000 rpm in a 50 mL volume and with a gap between therotor and stator of 0.25 mm. Both disposable probes and probesthat can withstand autoclaving or other means of sterilizationare acceptable.6.10 Bunsen BurnerUsed to fla
25、me sterilize inoculatingloop and other instruments.6.11 95 % EthanolUsed to flame sterilize hemostats orforceps.6.12 Stainless Steel Hemostat Clamp or ForcepsFor asep-tic handling of coupons.6.13 PipetterContinuously adjustable pipette with vol-ume capability of 1 mL.6.14 Analytical BalanceSensitive
26、 to 0.01 g.6.15 SterilizersAny steam sterilizer capable of producingthe conditions of sterilization.6.16 Colony CounterAny one of several types may beused. A hand tally for the recording of the bacterial count isrecommended if manual counting is done.6.17 Peristaltic PumpFour pump heads capable of h
27、old-ing 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 ShakerCapable of maintaining a tem-perature of 35 6 2C.6.19 TubingTwo sizes of silicone tubing: one with ID 3.1mm and OD 3.2 mm and the other with ID 7.9 mm
28、 and OD 9.5mm. One size of Norprene tubing with an ID of 1.6 mm. Alltubing must withstand sterilization.6.20 Glass Flow BreakAny that will connect with tubingof ID 3.1 mm and withstands sterilization.6.20.1 ClampUsed to hold flow break, extension clampwith 0.5-cm minimum grip size.6.20.2 Clamp Stand
29、Height no less than 76.2 cm, usedwith clamp to suspend glass flow break vertically and stabilizetubing.6.21 Reactor Components6A schematic of the drip flowreactor is shown in Fig. 1. Fig. 2 is a picture of the assembledsystem.6.21.1 Chamber (Base)15.24 by 15.88 cm polysulfonechamber with four 3.05 b
30、y 10.16-cm channels and four1.27-cm barbed effluent ports (one at the end of each channel).The underside holds four adjustable inserts (legs) providing a10 angle for continuous flow conditions. Each channel con-tains two pegs to guide coupon placement.6.21.2 TopFour O-ring fitted polycarbonate tops
31、eachwith two threaded holes for nylon screws to secure to reactorchamber (base). Two ports, one for Mininert valve and anotherfor bacterial air vent attachment.6.21.3 Mininert ValvesFit into each top as influent portsto allow inoculation and media line attachment.6.21.4 Needles1 in., 21 gauge, to fi
32、t into Mininert port.6.21.5 Glass CouponsFour new rectangular glass micro-scope slides (or other similar shaped material) with a topsurface area of 18.75 cm2(25 by 75 by 1 mm).6.21.6 TFE-fluorocarbon Thread Seal TapeTo preventleakage from effluent port connector.6.22 CarboysTwo 10-20 Lautoclavable c
33、arboys for wasteand nutrients.6.22.1 Carboy Lids(Note 1)One carboy lid with at leasttwo barbed fittings to accommodate tubing ID 3.1 mm (one for5Buckingham-Meyer, K., Goeres, D. M., and Hamilton, M. A., “ComparativeEvaluation of Biofilm Disinfectant Efficacy Tests,” J. Microbiological Methods, 70,20
34、07, pp. 236-244.6The sole source of supply of the Drip Flow Biofilm Reactor apparatus knownto the committee at this time is BioSurface Technologies, Corp., Bozeman, MT, If you are aware of alternative suppliers, please provide thisinformation to ASTM International Headquarters. Your comments will re
35、ceivecareful consideration at a meeting of the responsible technical committee, whichyou may attend. Alternatively, the user may build the Drip Flow Biofilm Reactorapparatus.E2647082nutrient line and one for bacterial air vent). One carboy lid withat least two 1-cm holes bored in the same fashion (o
36、ne foreffluent waste and one for bacterial air vent).NOTE 1Carboy tops can be purchased with fittings.6.23 Bacterial Air Vent (Filter)Autoclavable 0.2 mi-crometer pore size, to be attached into tubing on waste andnutrient carboy (recommended diameter is 37 mm) and eachreactor channel top with (recom
37、mended diameter is 15 mm).7. Reagents and Materials7.1 Purity of WaterAll reference to water as diluent orreagent shall mean distilled water or water of equal purity.7.2 Culture Media:7.2.1 Bacterial Liquid Growth BrothTryptic Soy Broth(TSB)7is recommended.NOTE 2Two different TSB concentrations are
38、used in the test method,3000 mg/L for the inoculum and batch reactor operation and 270 mg/L forthe continuous flow reactor operation.7.2.2 Bacterial Plating MediumR2A agar7is recom-mended.7.3 Buffered Water0.0425 g/L KH2PO4distilled water,filter sterilized and 0.405 g/L MgCl6H2O distilled water, fil
39、tersterilized (prepared according to Method 9050 C.1a).8. Culture/Inoculum Preparation8.1 Pseudomonas aeruginosa (ATCC 700888) is the organ-ism used in this test. Aseptically remove an isolated colonyfrom an R2A plate and inoculate into 100 mL of sterilebacterial liquid growth broth (3000 mg TSB/L).
40、 Incubatebacterial suspension in an environmental shaker at 35 6 2Cfor 20 to 24 hours. Viable bacterial density should equal 108CFU/mL and may be checked by serial dilution and plating.9. Preparation of Apparatus9.1 Reactor Set-up:7Atlas, R. M., Parks, L. C., Eds., Handbook of Microbiological Media,
41、 2nd ed.,CRC Press, Boca Raton, FL, 1997.FIG. 1 Expanded View of the Drip Flow ReactorFIG. 2 Drip Flow Reactor Laboratory Set-Up in Continuous Flow OperationE26470839.1.1 Use new coupons (glass microscope slides) for everyexperiment.9.1.2 Insert a coupon into each reactor channel by position-ing the
42、 top of the slide directly under the influent media portand allowing the end of the slide to rest on the pegs at thebottom of the channel.9.1.3 Place channel lids onto base and loosely screw innylon screws.9.1.4 Attach a bacterial air vent (15 mm diameter) to eachchannel lid.9.1.5 Splice the glass f
43、low break into the media tubing linethat will be positioned near the carboy top.9.1.6 Configure the media tubing so that four individuallines result. Splice a 12 to 15 cm piece of Norprene tubing intoeach line of silicone tubing, as shown in Fig. 2. The Norprenetubing is fed through the pump heads d
44、uring continuous flow(10.4.1).9.1.7 Wrap each effluent barbed fitting with TFE-fluorocarbon tape and insert into end port. Tightly clamp eacheffluent line using metal screw clamps.9.2 Sterilizing the Reactor System:9.2.1 Remove the adjustable legs from the reactor chamber.Wrap all exposed tubing end
45、s and openings with aluminum foiland place assembled reactor into an autoclave tray. Coverentire tray with aluminum foil.9.2.2 Sterilize the reactor system for 20 min on liquid cycle.10. Procedure10.1 The Batch Phase:10.1.1 Place the cooled reactor in a level position on thebench top.10.1.2 Clamp ea
46、ch channels effluent tubing and asepticallyadd 15 mL of sterile 3000 mg TSB/L (see 7.2.1)and1mLofinoculum (see 8.1) to each channel. Tighten each channel lidsecurely with nylon screws.10.1.3 Incubate the reactor system in batch mode at roomtemperature (21 6 2C) for 6 h, in the level position.10.1.4
47、Remove foil from the effluent tubing and attach endinto a waste carboy. Do not unclamp until continuous flowphase.10.2 Preparation for Continuous Flow Phase:10.2.1 Prepare continuous flow nutrient broth by addingsterilized bacterial liquid growth medium to 20 L sterilereagent grade water so that fin
48、al concentration is equal to 270mg TSB/L (see 7.2.1). Dissolve and sterilize the broth in asmaller volume of water to prevent caramelization. Asepticallypour the concentrated medium into the carboy of sterile waterto make a total of 20 L.10.3 Adjust Reactor Angle (Fig. 3):10.3.1 Measure a, the lengt
49、h of the reactor base, in cm.10.3.2 Angle (x) is 10.10.3.3 Calculate y using the following equation:y 5 asinx!# (1)where:a = measured length of reactor base in cm,x = angle (10), andy = difference between length of b and length of c in cm.10.3.4 Decide upon the lengths of b and c to obtain therequired difference (y). Use the following equation:y 5 b c (2)where:b = measured length (cm) from bottom corner of influentend of reactor base to laboratory surface,c = measured length (cm) from bottom corner of effluent
