1、Designation:E256207 Designation: E2562 12Standard Test Method forQuantification of Pseudomonas aeruginosa Biofilm Grownwith High Shear and Continuous Flow using CDC BiofilmReactor1This standard is issued under the fixed designation E2562; the number immediately following the designation indicates th
2、e year 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. Scope1.1This test method specifies the operational paramete
3、rs required to grow a repeatable Pseudomonas aeruginosa biofilm underhigh shear1.1 This test method specifies the operational parameters required to grow a reproducible (1).2Pseudomonas aeruginosa biofilmunder high shear. The resulting biofilm is representative of generalized situations where biofil
4、m exists under high shear rather thanbeing representative of one particular environment.1.2 This test method uses the Centers for Disease Control and Prevention (CDC) biofilm reactor.Biofilm Reactor. The CDCbiofilm reactorBiofilm Reactor is a continuously stirred flowtank reactor (CSTR) with high wa
5、ll shear. Although it was originallydesigned to model a potable water system for the evaluation of Legionella pneumophila (2), the reactor is versatile and may alsobe used for growing and/or characterizing biofilm of varying species (3 and 43-5).1.3 This test method describes how to sample and analy
6、ze biofilm for viable cells. Biofilm population density is recorded aslog10colony forming units per surface area.1.4 Basic microbiology training is required to perform this test method.1.5The values stated in SI units are to be regarded as the standard. The values given in parentheses are for inform
7、ation only.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 responsibilityof the user of this standard to
8、establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D5465 Practice for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods2.2 Other Standards:Method 9050 C.1aBuffere
9、d Dilution Water Preparation Method 9050 C.1.a Buffered Dilution Water Preparation according toEaton et al (6)3. Terminology3.1 Definitions:3.1.1 biofilm, nmicroorganisms living in a self-organized, cooperative self-organized community attached to surfaces,interfaces, or each other, embedded in a ma
10、trix of extracellular polymeric substances of microbial origin, while exhibiting analtered phenotypes with respect to growth rate and gene transcription.3.1.1.1 DiscussionBiofilms may be comprised of bacteria, fungi, algae, protozoa, viruses, or infinite combinations of thesemicroorganisms. The qual
11、itative 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 thebiofilm microorganisms), are controlled by the physicochemica
12、l environment in which it exists.3.1.2 coupon, nbiofilm sample surface.1This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approv
13、ed April 1, 2007. Published May 2007. DOI: 10.1520/E2562-07.Current edition approvedApril 1, 2012. Published June 2012. Originally approved in 2007. Last previous edition approved in 2007 as E2562 07. DOI: 10.1520/E2562-12.2The boldface numbers in parentheses refer to a list of references at the end
14、 of this standard.3For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.1This document is not an ASTM standard an
15、d 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 possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only
16、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 Conshohocken, PA 19428-2959, United States.4. Summary of Test Method4.1 This test method is used for growing a repeatablereproducib
17、le Pseudomonas aeruginosa biofilm in a CDC biofilmreactor.Biofilm Reactor. The biofilm is established by operating the reactor in batch mode (no flow of the nutrients) for 24 h. Asteady state population is reached while the reactor operates for an additional 24 h with continuous flow of the nutrient
18、s. Theresidence time of the nutrients in the reactor is set to select for biofilm growth, and is species and reactor parameter specific. Duringthe entire 48 h, the biofilm is exposed to continuous fluid shear from the rotation of a baffled stir bar. Controlling the rate at whichthe baffle turns dete
19、rmines the intensity of the shear stress to which the coupons are exposed. At the end of the 48 h, biofilmaccumulation is quantified by removing coupons from suspended rods, scrapingharvesting the biofilm from the coupon surface,disaggregating the clumps, and diluting and plating for viable cell enu
20、meration.5. Significance and Use5.1 Bacteria that exist in biofilms are phenotypically different from suspended cells of the same genotype. Research has shownthat biofilm bacteria are more difficult to kill than suspended bacteria (5, 7). Laboratory biofilms are engineered in growth reactorsdesigned
21、 to produce a specific biofilm type. Altering system parameters will correspondingly result in a change in the biofilm. Forexample, research has shown that biofilm grown under high shear is more difficult to kill than biofilm grown under low shear (6(5,8). The purpose of this test method is to direc
22、t a user in the laboratory study of a Pseudomonas aeruginosa biofilm by clearlydefining each system parameter. This test method will enable an investigator to grow, sample, and analyze a Pseudomonasaeruginosa biofilm grown under high shear. The biofilm generated in the CDC biofilm reactorBiofilm Rea
23、ctor is also suitable forefficacy testing. After the 48 h growth phase is complete, the user may add the treatment in situ or harvest the coupons and treatthem individually.6. Apparatus6.1 Wooden Applicator Sticks, sterile. sterile.6.2 Inoculating Loop. Inoculating Loop.6.3 Petri Dish, 100 by 15 mm,
24、 plastic, sterile and empty to put beneath rod while sampling. 100- by 15-mm, plastic, sterile,and empty to put beneath rod while sampling.6.4 Culture Tubes and Culture Tube Closures, any any with a volume capacity of 10 mL and a minimum diameter of 16 mm.Recommended size is 16- by 125-mm borosilica
25、te glass with threaded opening.6.5 PipetterCcontinuously adjustable pipetter with volume capabilitycapacity of 1 mL.6.6 VortexAnyany vortex that will ensure proper agitation and mixing of culture tubes.6.7 HomogenizerAnyany that can mix at 20 500 6 5000 r/min ina5to10mLvolume.6.8 Homogenizer ProbeAn
26、yany that can mix at 20 500 6 5000 r/min in a 5- to 10-mL volume and can withstandautoclaving or other means of sterilization.6.9 SonicatorAny noncavitating sonicating bath that operates at 50 to 60 Hz. Sonicating Water Bathany cavitatingsonicating bath that operates at 50 to 60 Hz.6.10 Bunsen Burne
27、r, usedused to flame inoculating loop and other instruments.6.11 Stainless Steel Hemostat Clamp, with curved tip. with curved tip.NOTE 1Alternatively, a coupon holder4may be used.6.12 Environmental Shaker, that that can maintain a temperature of 3536 6 2C.6.13 Analytical Balance, sensitive to 0.01 g
28、. sensitive to 0.01 g.6.14 SterilizersAnyany steam sterilizer that can produce the conditions of sterilization is acceptable.6.15 Colony CounterAnyany one of several types may be used, such as the Quebec, Buck, and Wolfhuegel. A hand tallyfor the recording of the bacterial count is recommended if ma
29、nual counting is done.6.16 Peristaltic PumpPump head that can hold tubing with ID 3.1 mm and OD 3.2 mm. pump head that can hold tubingwith inner diameter 3.1 mm and outer diameter 3.2 mm.6.17 Magnetic Stir PlateTop plate 10.16 3 10.16 cm, that can rotate at 125 6 60 r/min.NOTE1A digital stir plate i
30、s recommended. Digital Magnetic Stir Platetop plate 10.16 3 10.16 cm, that can rotate at 125 6 5 r/min.6.18 Silicone TubingTwotwo sizes of tubing: one with ID inner diameter 3.1 mm and OD outer diameter 3.2 mm, and theother with ID inner diameter 7.9 mm and ODouter diameter 9.5 mm. Both sizes must w
31、ithstand sterilization.6.19 Norprene Tubinginner diameter 3.1 mm and outer diameter 3.2 mm.6.20 Glass Flow BreakAny that will connect with tubing of ID 3.1 mm and withstands sterilization.4Eaton, A.D., Clesceri, L.S., Rice, E.W., Greenberg, A.E., (Eds.) Standard Methods for the Examination of Water
32、and Waste Water , 21st Edition, American Public HealthAssociation, American Water Works Association, Water Environment Federation, Washington D.C., 2005.4The sole source of supply of the apparatus (coupon holder) known to the committee at this time is Biosurface Technologies, Corp., www.biofilms.biz
33、. If you are awareof alternative suppliers, please provide this information toASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsibletechnical committee, which you may attend. The user may also build the holder.E2562 1226.19.1any that will co
34、nnect with tubing of inner diameter 3.1 mm and withstand sterilization.6.20.1 ClampUsed to hold flow break, extension clamp with 0.5-cm minimum grip size.6.19.26.20.2 Clamp StandHeightheight no less than 76.2 cm, used with clamp to suspend glass flow break vertically andstabilize tubing above reacto
35、r.6.206.21 Reactor Components.56.20.16.21.1 Berzelius Pyrex or Kimax Borosilicate Glass Tall Beaker, 1000 1000 mL without pour spout, 9.5- 6 0.5-cmdiameter. Pyrex/Kimax barbed Barbed outlet spout added at 400- 6 20-mL mark. Angle the spout 30 to 45 to ensure drainage.Spout should accommodate flexibl
36、e tubing with an ID inner diameter of 8 to 11 mm.NOTE 2The rods, described in rods (see 6.20.36.21.3) and baffle (see 6.20.56.21.6) will displace approximately 50 mL of liquid when system iscompletely assembled. Therefore, an outlet spout at the 400-mL mark will result in approximately a 350-mL oper
37、ating volume. The user is encouragedto 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 measuredvolume is used to calculate an exact pump flow rate.6.20.26.21.2 Reactor TopSee Fig. 1. Ultra-high molecular w
38、eight (UHMW) polyethylene top (10.1-cm diameter taperingto 8.33 cm) equipped with 3 a minimum of three holes accommodating 10-cm pieces of stainless steel or other rigid autoclavabletubing with OD outside diameter of 5 to 8 mm for media inlet, air exchange, and inoculation port. Center hole, 1.27-cm
39、 diameter,to accommodate the glass rod used to support the baffle assembly. Eight rod holes, 1.905-cm diameter, notched to accommodatestainless steel rod alignment pin (0.236 cm OD).(0.236-cm outside diameter).6.20.36.21.3 Polypropylene RodsSee Fig. 2. Eight polypropylene rods, 21.08-cm long, machin
40、ed to hold three coupons(see 6.20.46.21.4) at the immersed end. Three 316 stainless steel set screws imbedded in side to hold coupons in place. Rods fitinto holes in reactor top and lock into preformed notches with alignment pin.6.20.46.21.4 Twenty-four Cylindrical Polycarbonate Couponswith a diamet
41、er of 1.27 6 0.013 cm, thickness of approxi-mately 3.0 mm.6.20.56.21.5 Small Allen Wrench, for loosening set screws.6.20.6for loosening set screws.6.21.6 Stir Blade Assembly (Baffled Stir Bar)See Fig. 3. PTFE blade (5.61-cm) fitted into cylindrical PTFE holder(8.13-cm) and held in place with a magne
42、tic stir bar (2.54-cm). PTFE holder fits onto a glass rod (15.8-cm), fitted into the reactortop. The glass rod is held in place with a compression fitting and acts as a support for the moving blade assembly.6.216.22 CarboysTwotwo 20-L autoclavable carboys, to be used for waste and nutrients.6.21.16.
43、22.1 Two Carboy LidsOne 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 effluentwaste and one for bacterial air vent).5The sole source of
44、 supply of the apparatus (CDC Biofilm reactor)Reactor) known to the committee at this time is BioSurface Technologies, Corp. www.biofilms.biz. Ifyou are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideratio
45、n at a meetingof the responsible technical committee, which you may attend. The user may also build the reactor.FIG. 1 Expanded Schematic of Reactor TopE2562 123NOTE 3Carboy tops can be purchased with fittings.6.21.26.22.2 Bacterial Air Vent (Filter)Aautoclavable, 0.2-m pore size, to be spliced into
46、 tubing on waste carboy, nutrient carboy,and reactor top; recommended diameter 37 mm.6.226.23 Fig. 4 illustrates a schematic of the assembled system.7. Reagents and Materials7.1 Purity of WaterAll reference to water as diluent or reagent shall mean distilled water or water of equal purity.7.2 Cultur
47、e Media:7.2.1 Bacterial Liquid Growth BrothTryptic Soy Broth (TSB) is recommended.7.2.2Bacterial Plating MediumR2A Agar is recommended.NOTE 4Two different TSB concentrations are used in the test method, 300 mg/L for the inoculum and batch reactor operation, and 100 mg/L for thecontinuous flow reacto
48、r operation.7.2.2 Bacterial Plating MediumR2A Agar is recommended.7.3 Buffered Water0.0425 g/l KH0.0425 g/LKH2PO4distilled water, filter sterilized, and 0.405 g/lg/L MgCl6H2O distilledwater, filter sterilized (prepared according to Method 9050 C.1.a(6).8. Culture Preparation8.1 Pseudomonas aeruginos
49、aATCC 700888 is the organism used in this test.Aseptically remove 3 to 5 an isolated colonies withFIG. 2 Expanded Schematic of Rod and CouponsFIG. 3 Expanded Schematic of Baffled Stir BarE2562 124the same morphology colony from an R2A plate and place into 100 mL of sterile TSB (300 mg/L). Incubate bacterial suspensionin an environmental shaker at 3536 6 2C for 20 to 2422 6 2 h. Viable bacterial density should equal 108CFU/mL, and may bechecked by serial dilution and plating.9. Reactor Preparation9.1 Preparation o
