1、Designation: E 2562 07Standard Test Method forQuantification of Pseudomonas aeruginosa Biofilm Grownwith High Shear and Continuous Flow using CDC BiofilmReactor1This standard is issued under the fixed designation E 2562; the number immediately following the designation indicates the year oforiginal
2、adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method specifies the operational parametersrequired to gr
3、ow a repeatable Pseudomonas aeruginosa biofilmunder high shear (1).2The resulting biofilm is representative ofgeneralized situations where biofilm exists under high shearrather than representative of one particular environment.1.2 This test method uses the Centers for Disease Controland Prevention (
4、CDC) biofilm reactor. The CDC biofilmreactor is a continuously stirred flow reactor with high wallshear. Although it was originally designed to model a potablewater system for the evaluation of Legionella pneumophila (2),the reactor is versatile and may also be used for growing and/orcharacterizing
5、biofilm of varying species (3 and 4).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
6、 units are to be regarded as thestandard. The values given in parentheses are for informationonly.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
7、 practices and 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 Standards:Method 9050 C.1a Buffered Dilution Water Preparation43. Term
8、inology3.1 Definitions: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 a
9、nd gene transcription.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 gr
10、adients, chemicalcomposition, 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.4. Summary of Test Method4.1 This test method is used for growing
11、 a repeatablePseudomonas aeruginosa biofilm in a CDC biofilm reactor.The biofilm is established by operating the reactor in batchmode (no flow of the nutrients) for 24 h. A steady statepopulation is reached while the reactor operates for an addi-tional 24 h with continuous flow of the nutrients. The
12、 residencetime of the nutrients in the reactor is set to select for biofilmgrowth, and is species and reactor parameter specific. Duringthe entire 48 h, the biofilm is exposed to continuous fluid shearfrom the rotation of a baffled stir bar. Controlling the rate atwhich the baffle turns determines t
13、he intensity of the shearstress to which the coupons are exposed. At the end of the 48h, biofilm accumulation is quantified by removing couponsfrom suspended rods, scraping the biofilm from the coupon1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Contr
14、ol Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2007. Published May 2007.2The boldface numbers in parentheses refer to a list of references at the end ofthis standard.3For referenced ASTM standards, visit the ASTM website, ww
15、w.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., Rice, E.W., Greenberg, A.E., (Eds.) StandardMethods for the Examination of Water and Wast
16、e Water , 21st Edition, AmericanPublic Health Association, American Water Works Association, Water EnvironmentFederation, Washington D.C., 2005.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.surface, disaggregating the clumps, and d
17、iluting and plating forviable cell enumeration.5. Significance and Use5.1 Bacteria that exist in biofilm are phenotypically differentfrom suspended cells of the same genotype. Research hasshown that biofilm bacteria are more difficult to kill thansuspended bacteria (5). Laboratory biofilms are engin
18、eered ingrowth reactors designed to produce a specific biofilm type.Altering system parameters will correspondingly result in achange in the biofilm. For example, research has shown thatbiofilm grown under high shear is more difficult to kill thanbiofilm grown under low shear (6). The purpose of thi
19、s testmethod is to direct a user in the laboratory study of aPseudomonas aeruginosa biofilm by clearly defining eachsystem parameter. This test method will enable an investigatorto grow, sample, and analyze a Pseudomonas aeruginosabiofilm grown under high shear. The biofilm generated in theCDC biofi
20、lm reactor is also suitable for efficacy testing. Afterthe 48 h growth phase is complete, the user may add thetreatment in situ or harvest the coupons and treat themindividually.6. Apparatus6.1 Wooden Applicator Sticks, sterile.6.2 Inoculating Loop.6.3 Petri Dish, 100 by 15 mm, plastic, sterile and
21、empty toput beneath rod while sampling.6.4 Culture Tubes and Culture Tube Closures, any with avolume capacity of 10 mL and a minimum diameter of 16 mm.Recommended size is 16 by 125 mm borosilicate glass withthreaded opening.6.5 PipetterContinuously adjustable pipetter with volumecapability of 1 mL.6
22、.6 VortexAny vortex that will ensure proper agitationand mixing of culture tubes.6.7 HomogenizerAny that can mix at 20 500 6 5000r/min ina5to10mLvolume.6.8 Homogenizer ProbeAny that can mix at 20 500 65000 r/min ina5to10mLvolume and can withstandautoclaving or other means of sterilization.6.9 Sonica
23、torAny noncavitating sonicating bath that op-erates at 50 to 60 Hz.6.10 Bunsen Burner, used to flame inoculating loop andother instruments.6.11 Stainless Steel Hemostat Clamp, with curved tip.6.12 Environmental Shaker, that can maintain a temperatureof 35 6 2C.6.13 Analytical Balance, sensitive to 0
24、.01 g.6.14 SterilizersAny steam sterilizer that can produce theconditions of sterilization is acceptable.6.15 Colony CounterAny one of several types may beused, such as the Quebec, Buck, and Wolfhuegel. A hand tallyfor the recording of the bacterial count is recommended ifmanual counting is done.6.1
25、6 Peristaltic PumpPump head that can hold tubingwith ID 3.1 mm and OD 3.2 mm.6.17 Magnetic Stir PlateTop plate 10.16 3 10.16 cm, thatcan rotate at 125 6 60 r/min.NOTE 1A digital stir plate is recommended.6.18 Silicone TubingTwo sizes of tubing: one with ID 3.1mm and OD 3.2 mm and the other with ID 7
26、.9 mm and OD 9.5mm. Both sizes must withstand sterilization.6.19 Glass Flow BreakAny that will connect with tubingof ID 3.1 mm and withstands sterilization.6.19.1 ClampUsed to hold flow break, extension clampwith 0.5 cm minimum grip size.6.19.2 Clamp StandHeight no less than 76.2 cm, usedwith clamp
27、to suspend glass flow break vertically and stabilizetubing above reactor.6.20 Reactor Components.56.20.1 Berzelius Pyrex or Kimax Tall Beaker, 1000 mLwithout pour spout, 9.5 6 0.5 cm diameter. Pyrex/Kimaxbarbed outlet spout added at 400 6 20 mL mark. Angle thespout 30 to 45 to ensure drainage. Spout
28、 should accommodateflexible tubing with an ID of 8 to 11 mm.NOTE 2The rods, described in 6.20.3 and baffle (6.20.5) will displaceapproximately 50 mL of liquid when system is completely assembled.Therefore, an outlet spout at the 400 mL mark will result in approximatelya 350 mL operating volume. The
29、user is encouraged to confirm the actualliquid volume in the reactor, when the rods and baffle are in place, beforeuse. The measured volume is used to calculate an exact pump flow rate.6.20.2 Reactor TopSee Fig. 1. Ultra-high molecularweight (UHMW) polyethylene top (10.1 cm diameter taperingto 8.33
30、cm) equipped with 3 holes accommodating 10 cmpieces of stainless steel or other rigid autoclavable tubing withOD of 5 to 8 mm for media inlet, air exchange, and inoculationport. Center hole, 1.27 cm diameter, to accommodate the glass5The sole source of supply of the apparatus (CDC Biofilm reactor) k
31、nown to thecommittee at this time is BioSurface Technologies, Corp. www.biofilms.biz. If youare aware of alternative suppliers, please provide this information to ASTMInternational Headquarters. Your comments will receive careful consideration at ameeting of the responsible technical committee,1whic
32、h you may attend. The usermay also build the reactor.FIG. 1 Expanded Schematic of Reactor TopE2562072rod used to support the baffle assembly. Eight rod holes, 1.905cm diameter, notched to accommodate stainless steel rodalignment pin (0.236 cm OD).6.20.3 Polypropylene RodsSee Fig. 2. Eight polypropy-
33、lene rods, 21.08 cm long, machined to hold three coupons (see6.20.4) at the immersed end. 316 stainless steel set screwsimbedded in side to hold coupons in place. Rods fit into holesin reactor top and lock into preformed notches.6.20.4 Twenty-four Cylindrical Polycarbonate Couponswith a diameter of
34、1.27 6 0.013 cm, thickness of approxi-mately 3.0 mm.6.20.5 Small Allen Wrench, for loosening set screws.6.20.6 Stir Blade Assembly (Baffled Stir Bar)See Fig. 3.PTFE blade (5.61 cm) fitted into cylindrical PTFE holder (8.13cm) and held in place with a magnetic stir bar (2.54 cm). PTFEholder fits onto
35、 a glass rod (15.8 cm), fitted into the reactor top.The glass rod is held in place with a compression fitting andacts as a support for the moving blade assembly.6.21 CarboysTwo 20-L autoclavable carboys, to be usedfor waste and nutrients.6.21.1 Two Carboy LidsOne carboy lid with at least twobarbed f
36、ittings to accommodate tubing ID 3.1 mm (one fornutrient line and one for bacterial air vent). One carboy lid withat least two 1-cm holes bored in the same fashion (one foreffluent waste and one for bacterial air vent).NOTE 3Carboy tops can be purchased with fittings.6.21.2 Bacterial Air Vent (Filte
37、r)Autoclavable 0.2 mpore size, to be spliced into tubing on waste carboy, nutrientcarboy and reactor top; recommended diameter 37 mm.6.22 Fig. 4 illustrates a schematic of the assembled system.7. Reagents and Materials7.1 Purity of WaterAll reference to water as diluent orreagent shall mean distille
38、d water or water of equal purity.7.2 Culture Media:7.2.1 Bacterial Liquid Growth BrothTryptic Soy Broth(TSB) is recommended.7.2.2 Bacterial Plating MediumR2A Agar is recom-mended.NOTE 4Two different TSB concentrations are used in the test method,300 mg/L for the inoculum and batch reactor operation
39、and 100 mg/L forthe continuous flow reactor operation.7.3 Buffered Water0.0425 g/l KH2PO4distilled water,filter sterilized and 0.405 g/l MgCl6H2O distilled water, filtersterilized (prepared according to Method 9050 C.1a).8. Culture Preparation8.1 Pseudomonas aeruginosaATCC 700888 is the organismused
40、 in this test. Aseptically remove 3 to 5 isolated colonieswith the same morphology from an R2A plate and place into100 mL of sterile TSB (300 mg/L). Incubate bacterial suspen-sion in an environmental shaker at 35 6 2C for 20 to 24 h.Viable bacterial density should equal 108CFU/mL, and may bechecked
41、by serial dilution and plating.9. Reactor Preparation9.1 Preparation of Polycarbonate Coupons:NOTE 5Coupons can be used once and discarded or used repeatedlywith proper cleaning and sterilization between each use. Check eachcoupon for scratching, chipping, other damage or accumulated debrisbefore ea
42、ch use by screening under a dissecting microscope at amagnification of at least 203. Discard those with visible damage tosurface topography.9.1.1 Sonicate coupons for 30 s in a 1:100 dilution oflaboratory soap and tap water. The soapy water must com-pletely cover the coupons.9.1.2 Rinse coupons with
43、 reagent water and sonicate for 30s in reagent water.9.1.3 Repeat rinsing and sonication with reagent water untilno soap is left on the coupon. Once the coupons are clean, caremust be taken to prevent oils and other residue from contami-nating the surface.9.1.4 Place a coupon into each hole in the r
44、eactor rods,leaving the top of the coupon flush with the inside rod surface.Tighten set screw.FIG. 2 Expanded Schematic of Rod and Coupons FIG. 3 Expanded Schematic of Baffled Stir BarE25620739.1.5 Place rods into reactor top loosely (not yet fitted intonotches).9.2 Preparation of Reactor Top:9.2.1
45、Place baffle onto glass rod suspended from the reactortop.9.2.2 Place assembled top into the reactor beaker.9.2.3 Connect the bacterial air vent (filter) by fitting the ventto a small section of appropriately sized tubing, and attach toone of the rigid tubes on the reactor top.9.2.4 The glass flow b
46、reak is spliced into the nutrient tubingline near the reactor top.NOTE 6The other ports on the reactor top may be adapted foranaerobic use, dilution water, or treatment solutions as necessary.9.3 Sterilization of the Reactor System:9.3.1 With the overflow (waste) line clamped, add 500 mLnon-sterile
47、batch culture media (300 mg/L TSB) to the reactorand securely fasten the reactor top to the beaker beforesterilization. Be sure to allow for pressure escape.9.3.2 Cover the end of the nutrient tubing that connects tothe nutrient carboy and the end of the overflow (waste) tubingwith aluminum foil. Co
48、ver any extra openings on the reactortop with aluminum foil.9.3.3 Sterilize the reactor system for 20 min on the liquidcycle of a steam sterilizer.10. Procedure10.1 The Batch Phase:10.1.1 Place cooled reactor onto a stir plate.10.1.2 Clamp flow break in upright position; leave othertubing clamped an
49、d foiled.10.1.3 Secure the rods by setting the alignment pins into thenotches on the reactor top.10.1.4 Inoculate the reactor with 1 mL of bacteria from theculture prepared previously (see Section 8.1).Aseptically injectthe inoculum into the beaker through one of the availablereactor top rigid tubes using a pipette with a sterile tip.10.1.5 Turn on the magnetic stir plate. The rotation speedshould equal 125 revolutions per minute. The reactor systemincubates in batch mode at room temperature (21 6 2C) for 24h.NOTE 7The speed at which the baffled
