1、Designation: E2562 17Standard 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 the year oforiginal ad
2、option 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 parametersrequired to grow
3、a reproducible (1)2Pseudomonas aeruginosaATCC 700888 biofilm under high shear. The resulting biofilmis representative of generalized situations where biofilm existsunder high shear rather than being representative of oneparticular environment.1.2 This test method uses the Centers for Disease Control
4、and Prevention (CDC) Biofilm Reactor. The CDC BiofilmReactor is a continuously stirred tank reactor (CSTR) with highwall shear. Although it was originally designed to model apotable water system for the evaluation of Legionella pneumo-phila (2), the reactor is versatile and may also be used forgrowi
5、ng and/or characterizing biofilm of varying species (3-5).1.3 This test method describes how to sample and analyzebiofilm for viable cells. Biofilm population density is recordedas log10colony forming units per surface area.1.4 Basic microbiology training is required to perform thistest method.1.5 T
6、he values stated in SI units are to 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-pri
7、ate safety and health practices and determine the applica-bility of regulatory limitations prior to use.1.7 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of Internat
8、ional Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:3D5465 Practices for Determining Microbial Colony Countsfrom Waters Analyzed by Plating Methods2.2 Other Standards:Method 9050 C.1.
9、a Buffered Dilution Water Preparationaccording to Rice et al (6)3. Terminology3.1 Definitions:3.1.1 biofilm, nmicroorganisms living in a self-organizedcommunity attached to surfaces, interfaces, or each other,embedded in a matrix of extracellular polymeric substances ofmicrobial origin, while exhibi
10、ting altered phenotypes withrespect to growth rate and gene transcription.3.1.1.1 DiscussionBiofilms may be comprised of bacteria,fungi, algae, protozoa, viruses, or infinite combinations ofthese microorganisms. The qualitative characteristics of abiofilm, including, but not limited to, population d
11、ensity,taxonomic diversity, thickness, chemical gradients, 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 T
12、est Method4.1 This test method is used for growing a reproduciblePseudomonas aeruginosa ATCC 700888 biofilm in a CDCBiofilm Reactor. The biofilm is established by operating thereactor in batch mode (no flow of the nutrients) for 24 h. Asteady state population is reached while the reactor operates fo
13、ran additional 24 h with a continuous flow of the nutrients. Theresidence time of the nutrients in the reactor is set to select for1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcomm
14、ittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2017. Published May 2017. Originallyapproved in 2007. Last previous edition approved in 2012 as E2562 12. DOI:10.1520/E2562-17.2The boldface numbers in parentheses refer to a list of references at the end ofthis standard.3For ref
15、erenced ASTM standards, visit the ASTM website, 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.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, Wes
16、t Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Tr
17、ade Organization Technical Barriers to Trade (TBT) Committee.1biofilm growth, and is species and reactor parameter specific.During the entire 48 h, the biofilm is exposed to continuousfluid shear from the rotation of a baffled stir bar. Controllingthe rate at which the baffle turns determines the in
18、tensity of theshear stress to which the coupons are exposed.At the end of the48 h, biofilm accumulation is quantified by removing couponsfrom suspended rods, harvesting the biofilm from the couponsurface by scraping the biofilm from the coupon, homogeniz-ing the removed biofilm to disaggregate the c
19、lumps, anddiluting and plating for viable cell enumeration.5. Significance and Use5.1 Bacteria that exist in biofilms are phenotypically differ-ent from suspended cells of the same genotype. Research hasshown that biofilm bacteria are more difficult to kill thansuspended bacteria (5, 7). Laboratory
20、biofilms are engineeredin growth 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 (5, 8
21、). The purpose of this 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 gene
22、rated in theCDC Biofilm Reactor is also suitable for efficacy testing.Afterthe 48 h growth phase is complete, the user may add thetreatment in situ or remove the coupons and treat themindividually.6. Apparatus6.1 Wooden Applicator Stickssterile.6.2 Inoculating Loop.6.3 Petri Dish100 by 15 mm, plasti
23、c, sterile, and empty toput beneath rod while sampling.6.4 Culture Tubes and Culture Tube Closuresany 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 Pipettecontinuously adjustable pipetter with volumecapacit
24、y of 1 mL.6.6 Vortexany vortex that will ensure proper agitation andmixing of culture tubes.6.7 Homogenizerany that can mix at 20 500 6 5000 r/minina5to10mLvolume.6.8 Homogenizer Probeany that can mix at 20 500 65000 r/min ina5to10mLvolume and can withstandautoclaving or other means of sterilization
25、.6.9 Sonicating Water Bathany cavitating sonicating baththat operates at 45 to 50 kHz for cleaning coupons.6.10 Bunsen Burnerused to flame inoculating loop andother instruments.6.11 Stainless Steel Hemostat Clampwith curved tip.NOTE 1Alternatively, a coupon manipulating tool4may be used.6.12 Environ
26、mental Shakerthat can maintain a tempera-ture of 36 6 2C.6.13 Analytical Balancesensitive to 0.01 g.6.14 Sterilizerany 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 ha
27、nd tallyfor the recording of the bacterial count is recommended ifmanual counting is done.6.16 Peristaltic Pumppump head that can hold tubingwith inner diameter 3.1 mm and outer diameter 3.2 mm.6.17 Digital Magnetic Stir Platetop plate 10.16 10.16cm, that can rotate at 125 6 5 r/min.6.18 Silicone Tu
28、bingtwo sizes of tubing: one with innerdiameter 3.1 mm and outer diameter 3.2 mm, and the otherwith inner diameter 7.9 mm and outer diameter 9.5 mm. Bothsizes must withstand sterilization.6.19 Norprene5Tubinginner diameter 3.1 mm and outerdiameter 3.2 mm.6.20 Glass Flow Breakany that will connect wi
29、th tubingof inner diameter 3.1 mm and withstand sterilization.6.20.1 ClampUsed to hold flow break, extension clampwith 0.5 cm minimum grip size.6.20.2 Clamp Standheight no less than 76.2 cm, used withclamp to suspend glass flow break vertically and stabilizetubing above reactor.6.20.3 Laboratory Scr
30、ew Clampused to clamp effluenttubing during batch growth.6.21 Reactor Components.66.21.1 Berzelius Borosilicate Glass Tall Beaker1000 mLwithout pour spout, 9.5 6 0.5 cm diameter. Barbed outlet spoutadded at 400 6 20 mL mark. Angle the spout 30 to 45 toensure drainage. Spout should accommodate flexib
31、le tubingwith an inner diameter of 8 to 11 mm.NOTE 2The rods (see 6.21.3) and baffle (see 6.21.6) will displaceapproximately 50 mL of liquid when system is completely assembled.Therefore, an outlet spout at the 400 mLmark will result in approximatelya 350 mL operating volume. The user should confirm
32、 the actual liquidvolume in the reactor, when the rods and baffle are in place and the stirplate is turned on, before use. The measured operating volume is used tocalculate an exact pump flow rate.6.21.2 Reactor TopFig. 1. Ultra-high molecular weight(UHMW) polyethylene top (10.1 cm diameter tapering
33、 to 8.334The sole source of supply of the apparatus (coupon manipulating tool) knownto the committee at this time is Biosurface Technologies, Corp., www.biofilms.biz.If you are aware of alternative suppliers, please provide this information to ASTMInternational Headquarters. Your comments will recei
34、ve careful consideration at ameeting of the responsible technical committee,1which you may attend. The usermay also build the holder.5Trademarked by the Saint-Gobain Performance Plastics Corporation.6The sole source of supply of the apparatus (CDC Biofilm Reactor) known to thecommittee at this time
35、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,1which you may attend. The usermay also
36、 build the reactor.E2562 172cm) equipped with a minimum of three holes accommodating10 cm pieces of stainless steel or other rigid autoclavabletubing with outside diameter of 5 to 8 mm for media inlet, airexchange, and inoculation port. Center hole, 1.27 cm diameter,to accommodate the glass rod used
37、 to support the baffleassembly. Eight rod holes, 1.905 cm diameter, notched toaccommodate stainless steel rod alignment pin (0.236 cmoutside diameter).6.21.3 Polypropylene RodsFig. 2. Eight polypropylenerods, 21.08 cm long, machined to hold three coupons (see6.21.4) at the immersed end. Three 316 st
38、ainless steel setscrews imbedded in side to hold coupons in place. Rods fit intoholes in reactor top and lock into preformed notches withalignment pin.6.21.4 Twenty-four Cylindrical Polycarbonate Couponswith a diameter of 1.27 6 0.013 cm, thickness of approxi-mately 3.0 mm.6.21.5 Small Allen Wrenchf
39、or loosening set screws.6.21.6 Stir Blade Assembly (Baffled Stir Bar)Fig. 3. PTFEblade (5.61 cm) fitted into cylindrical PTFE holder (8.13 cm)and held in place with a magnetic stir bar (2.54 cm). PTFEholder fits onto a glass rod (15.8 cm), fitted into the reactor top.The glass rod is held in place w
40、ith a compression fitting andacts as a support for the moving blade assembly.6.22 Carboystwo 20 L autoclavable carboys, to be usedfor waste and nutrients.6.22.1 Two Carboy LidsOne carboy lid with at least twobarbed fittings to accommodate tubing ID 3.1 mm (one fornutrient line and one for bacterial
41、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.6.22.2 Bacterial Air Vent (Filter)autoclavable, 0.2 mpore size, to be spliced into tubing on waste carboy, nutrientc
42、arboy, and reactor top; recommended diameter 37 mm.6.23 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 distilled water or water of equal purity.7.2 Culture Media:7.2.1 Bacterial Liquid Growth B
43、rothTryptic Soy Broth(TSB) 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 forthe continuous flow reactor operation.7.2.2 Bacterial Plating MediumR2A Agar is recom-mended.7.3 Buffered Water0.0425 g/
44、LKH2PO4distilled water,filter sterilized, and 0.405 g/LMgCl6H2O distilled water, filtersterilized (prepared according to Method 9050 C.1.a(6).FIG. 1 Expanded Schematic of Reactor TopFIG. 2 Expanded Schematic of Rod and CouponsFIG. 3 Expanded Schematic of Baffled Stir BarE2562 1738. Culture Preparati
45、on8.1 Pseudomonas aeruginosaATCC 700888 is the organismused in this test.Aseptically remove an isolated colony from anR2A plate and place into 100 mL of sterile TSB (300 mg/L).Incubate bacterial suspension in an environmental shaker at 366 2C for 22 6 2 h. Viable bacterial density should equal 108CF
46、U/mL, and may be checked 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 a
47、ccumulated debrisbefore each use by screening under a dissecting microscope at amagnification of at least 20. Discard those with visible damage to surfacetopography.9.1.1 Sonicate coupons for 30 s in a 1+99 dilution oflaboratory soap and tap water. The soapy water must com-pletely cover the coupons.
48、9.1.2 Rinse coupons with reagent grade water and sonicatefor 30 s in reagent grade water.9.1.3 Repeat rinsing and sonication with reagent gradewater until no soap is left on the coupons. Once the couponsare clean, care must be taken to prevent oils and other residuefrom contaminating the surface.NOT
49、E 6Coupons may be made out of alternative materials such asglass or stainless steel. The user should adjust the cleaning procedure sothat it is appropriate for the coupon material being used.9.1.4 Place a coupon into each hole in the reactor rods,leaving the top of the coupon flush with the inside rod surface.Tighten set screw.9.1.5 Place rods into reactor top loosely (not yet fitted intonotches).9.2 Preparation of Reactor Top:9.2.1 Invert the reactor top and place baffle onto glass rodpositioned in the center of
