1、Designation: E2647 08E2647 13Standard 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 E2647; the number immediately following the designation indicates the y
2、ear oforiginal 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 parameters
3、 required to grow a repeatable2 Pseudomonas aeruginosa biofilm closeto the air/liquid interface in a reactor with a continuous flow of nutrients under low fluid shear conditions. The resulting biofilmis representative of generalized situations where biofilm exists at the air/liquid interface under l
4、ow fluid shear rather thanrepresentative 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 flowresulting in low fluid shear. The reactor is versatile and may also be used for growing and/or character
5、izing different species ofbiofilms.biofilms of different species, although this will require changing the operational parameters to optimize the method basedupon the growth requirements of the new organism.1.3 This test method describes how to sample and analyze biofilm for viable cells. Biofilm pop
6、ulation density is recorded aslog 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 pur
7、port to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D546
8、5 Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods2.2 Other Standard:Method 9050 C.1.a Buffered Dilution Water PreparationPreparation, according to Eaton et al43. Terminology3.1 Definitions:3.1.1 biofilm, nmicroorganisms living in a self-organized, cooperative
9、 community attached to surfaces, interfaces, or eachother, embedded in a matrix of extracellular polymeric substances of microbial origin, while exhibiting an altered phenotype withrespect to growth rate and gene transcription.3.1.1.1 Discussion1 This test method is under the jurisdiction of ASTM Co
10、mmittee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2008April 1, 2013. Published October 2008May 2013. Originally approved in 2008. Last previous edition approved in
11、2008 asE2647 08. DOI: 10.1520/E2647-08.10.1520/E2647-13.2 Ellison, S. L. R., Rosslein, M., and Williams, A., Eds., Quantifying Uncertainty in Analytical Measurement, 2nd Edition, Eurachem, 2000.3 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at se
12、rviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.4 Eaton, A. D., Clesceri, L. S., and Greenberg, A. E., Eds., Standard Methods for the Examination of Water and Waste Water, 19th Edition, American Public HealthAssocia
13、tion, American Water Works Association, Water Environment Federation, Washington, DC, 1995.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. Becauseit may not be technically possibl
14、e to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West
15、Conshohocken, PA 19428-2959. United States1Biofilms may be comprised of bacteria, fungi, algae, protozoa, viruses, or infinite combinations of these microorganisms. Thequalitative characteristics of a biofilm (including, but not limited to, population density, taxonomic diversity, thickness, chemica
16、lgradients, chemical composition, consistency, and other materials in the matrix that are not produced by the biofilmmicroorganisms) are controlled by the physicochemical environment in which it exists.3.1.2 coupon, nbiofilm sample surface.3.1.3 chamber, nreactor base containing four rectangular wel
17、ls or channels.3.1.4 channel, none of four rectangular wells in reactor chamber (base) where coupon is placed.4. Summary of Test Method4.1 This test method is used for growing a repeatable PseudomonasP. aeruginosa biofilm in a drip flow biofilm reactor. Thebiofilm is established by operating the rea
18、ctor in batch mode (no flow of nutrients) for 6 h.Amature biofilm forms while the reactoroperates for an additional 48 h with a continuous flow of nutrients. During continuous flow, the biofilm experiences very low shearcaused by the gravity flow of media dripping onto a surface set at a 10 angle. A
19、t the end of the 54 h, biofilm accumulation isquantified by removing coupons from the reactor channels, rinsing the coupons to remove the planktonic cells, scraping the biofilmfrom the coupon surface, disaggregating the clumps, then diluting and plating for viable cell enumeration.5. Significance an
20、d Use5.1 Vegetative biofilm bacteria are phenotypically different from suspended cells of the same genotype. Biofilm growth reactorsare engineered to produce biofilms with specific characteristics. Altering either the engineered system or operating conditions willmodify those characteristics.5.2 The
21、 purpose of this test method is to direct a user in how to grow, sample, and analyze a PseudomonasP. aeruginosa biofilmunder low fluid shear and close to the air/liquid interface using the drip flow reactor. DFR. The PseudomonasP. aeruginosa biofilmthat grows has a smooth appearance and is loosely a
22、ttached. 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 lungswith cystic fibrosis, and oral biofilms, for example. The biofilm generated in the drip
23、 flow reactor DFR is also suitable for efficacytesting.After the 54 h growth phase is complete, the user may add the treatment inin situ situ or harvest the coupons and treat themindividually. Research has shown that P. aeruginosa biofilms grown in the drip flow reactor DFR were less resistanttolera
24、nt todisinfection than biofilms grown under high shear conditions.56. Apparatus6.1 TFE-fluorocarbon, Tetrafluoroethylene (TFE), Metal, or Rubber SpatulasSterile, for scraping biofilm from couponsurface.6.2 Inoculating Loop.6.3 Petri Dish100 by 15 mm, plastic, sterile, and empty for transporting coup
25、ons from reactor to work station.6.4 Culture Tubes and Culture Tube ClosuresSterile, any Any with a volume capacitycapability of 10 mL and a minimumdiameter of 16 mm. Recommended size is 16 by 125 mm borosilicate glass with threaded opening.6.5 Glass BeakersSterile, any with a volume capacity of 100
26、 mL containing 45 mL sterile buffered water.6.6 Conical-Bottom Sterile Disposable Plastic Conical Centrifuge TubesAny Sterile, any with a50-mL volume capacity of 50mL. Fill each with containing 45 mL sterile buffered water.6.7 VortexAny vortex that will ensure proper agitation and mixing of culture
27、tubes.6.8 HomogenizerAny capable of mixing at 20 500 6 5000 rpm 20 500 6 5000 r/min in a 50 mL 50 mL volume.6.9 Homogenizer ProbeAny capable of mixing at 20 500 6 5000 rpm 20 500 6 5000 r/min in a 50 mL 50 mL volume andwith a gap between the rotor and stator of 0.25 mm. 0.25 mm. Both disposable prob
28、es and probes that can withstand autoclavingor other means of sterilization are acceptable.6.10 Bunsen BurnerUsed to flame sterilize inoculating loop and other instruments.6.11 95 % EthanolUsed to flame sterilize hemostats or forceps.6.12 Stainless Steel Hemostat Clamp or ForcepsFor aseptic handling
29、 of coupons.6.13 PipetterContinuously adjustable pipette with volume capability of 1 mL.6.14 Analytical BalanceSensitive to 0.01 g.5 Buckingham-Meyer, K., Goeres, D. M., and Hamilton, M. A., “Comparative Evaluation of Biofilm Disinfectant Efficacy Tests,” J. Microbiological Methods, 70, 2007,pp. 236
30、-244.Buckingham-Meyer, K., Goeres, D. M., and Hamilton, M.A., “Comparative Evaluation of Biofilm Disinfectant Efficacy Tests,” J. Microbiological Methods, Vol70, 2007, pp. 236244.E2647 1326.15 SterilizersAny steam sterilizer capable of producing the conditions of sterilization.6.16 Colony CounterAny
31、 one of several types may be used. A hand tally for the recording of the bacterial count isrecommended if manual counting is done.6.17 Peristaltic PumpFour 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 ho
32、ur.6.18 Environmental ShakerCapable of maintaining a temperature of 35 6 2C.6.19 TubingTwo 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 9.5mm. One size of Norprene tubing with an ID of 1.6 mm. All tubing Both sizes must withstand sterilization.6.20 G
33、lass Flow BreakAny that will connect with tubing of ID 3.1 mm and withstands sterilization.6.20.1 ClampUsed to hold flow break, extension clamp with 0.5-cm 0.5 cm minimum grip size.6.20.2 Clamp StandHeight no less than 76.2 cm, used with clamp to suspend glass flow break vertically and stabilize tub
34、ing.6.21 Reactor Components6Aschematic of the drip flow reactor is shown in Fig. 1. Fig. 2 is a picture of the assembled system.6.21.1 Chamber (Base)15.24 by 15.88 cm polysulfone chamber with four 3.053.05- by 10.16-cm channels and four 1.27-cmbarbed effluent ports (one at the end of each channel).
35、The underside holds four adjustable inserts (legs) providing a 10 angle forcontinuous flow conditions. Each channel contains two pegs to guide coupon placement.6.21.2 TopFour O-ring fitted polycarbonate tops each with two threaded holes for nylon screws to secure to reactor chamber(base). Two ports,
36、 one for Mininert valve and another for bacterial air vent attachment.6.21.3 Mininert ValvesFit into each top as influent ports to allow inoculation and media line attachment.6.21.4 Luer Lock Connectors with 3.1 mm Hose BarbUsed to connect needles to the tubing.6.21.5 NeedlesNeedle1 in., 21 gauge, t
37、o fit into Mininert port.6.21.6 Glass CouponsFour new rectangular glass microscope slides (or other similar shaped material) with a top surface areaof 18.75 cm2 (25 by 75 by 1 mm).6.21.7 TFE-fluorocarbonTFE Thread Seal TapeTo prevent leakage from effluent port connector.6.22 CarboysTwo 10-20 10 to 2
38、0 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 fornutrient line and one for bacterial air vent). One carboy lid with at least two 1-cm holes bored in the same fashion (one for effluentwa
39、ste and one for bacterial air vent).NOTE 1Carboy tops can be purchased with fittings.6.23 Bacterial Air Vent (Filter)Autoclavable 0.2 micrometerm pore size, to be attached into tubing on waste and nutrientcarboy (recommended diameter is 37 mm) and each reactor channel top with (recommended diameter
40、is 15 mm).6 The sole source of supply of the Drip Flow Biofilm Reactordrip flow biofilm reactor apparatus known to the committee at this time is BioSurface Technologies, Corp.,Bozeman, MT, If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters.
41、 Your comments willreceive careful consideration at a meeting of the responsible technical committee, which you may attend. Alternatively, the user may build the Drip Flow Biofilm ReactorDFR apparatus.FIG. 1 Expanded View of the Drip Flow ReactorE2647 1337. Reagents and Materials7.1 Purity of WaterA
42、ll 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 BrothTryptic Soy Broth (TSB)7 is recommended.NOTE 2Two different TSB concentrations are used in the test method, 3000 mg/L for the inoculum and batch reacto
43、r operation and 270 mg/L forthe continuous flow reactor operation.7.2.2 Bacterial Plating MediumR2A agar7 is recommended.7.3 Buffered Water0.0425 g/L KH2PO4 distilled water, filter sterilized and 0.405 g/L MgCl6H2O distilled water, filtersterilized (prepared according to Method 9050 C.1a).8. Culture
44、/Inoculum Preparation8.1 Pseudomonas aeruginosa (ATCC 700888) is the organism used in this test. Aseptically remove an isolated colony from anR2A plate and inoculate into 100 mL of sterile bacterial liquid growth broth (3000 mg TSB/L). Incubate bacterial suspension inan environmental shaker at 35 6
45、2C for 20 to 24 hours.h. Viable bacterial density should equal 108 CFU/mL and may be checkedby serial dilution and plating.9. Preparation of Apparatus9.1 Reactor Set-up:Set-Up:9.1.1 Use new coupons (glass microscope slides) for every experiment.9.1.2 Insert a coupon into each reactor channel by posi
46、tioning the top of the slide directly under the influent media port andallowing the end of the slide to rest on the pegs at the bottom of the channel.9.1.3 Place channel lids onto base and loosely screw in nylon screws.9.1.4 Attach a bacterial air vent (15 mm diameter) to each channel lid.lid near t
47、he effluent port.9.1.5 Splice the glass flow break into the media tubing line that will be positioned near the carboy top.9.1.6 Configure the media tubing so that four individual lines result. Splice a 12 to 15 cm piece of Norprene tubing into eachline of silicone tubing, Feed each of the four lines
48、 through a pump head and attach to each channel as shown in Fig. 2. TheNorprene tubing is fed through the pump heads during continuous flow (10.4.1).9.1.7 Wrap each effluent barbed fitting with TFE-fluorocarbonTFE tape and insert into end port. Tightly clamp each effluent lineusing metal screw clamp
49、s.9.2 Sterilizing the Reactor System:9.2.1 Remove the adjustable legs from the reactor chamber. Wrap all exposed tubing ends and openings with aluminum foil andplace assembled reactor into an autoclave tray. Cover entire tray with aluminum foil.9.2.2 Sterilize the reactor system for 20 min on liquid cycle.7 Atlas, R. M., Parks, L. C., Eds., Handbook of Microbiological Media, 2nd ed., CRC Press, Boca Raton, FL, 1997.FIG. 2 Drip Flow Reactor Laboratory Set-Up in Continuous Flow OperationE2647 134NOTE 3Tight
