ASTM_E_2647_-_13.pdf

Designation:E264713Standard Test Method forQuantification of Pseudomonas aeruginosa Biofilm GrownUsing 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 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.Scope1.1 This test method specifies the operational parametersrequired 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 fluidshear rather than representative of one particular environment.1.2 This test method uses the drip flow reactor.The dripflow reactor(DFR)is a plug flow reactor with laminar flowresulting in low fluid shear.The reactor is versatile and mayalso be used for growing and/or characterizing biofilms ofdifferent species,although this will require changing theoperational parameters to optimize the method based upon thegrowth requirements of the new organism.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 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-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.Referenced Documents2.1 ASTM Standards:3D5465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating Methods2.2 Other Standard:Method 9050 C.1.a Buffered Dilution Water Preparation,according to Eaton et al43.Terminology3.1 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 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 density,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.3.1.3 chamber,nreactor base containing four rectangularwells or channels.3.1.4 channel,none of four rectangular wells in reactorchamber(base)where coupon is placed.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides,Antimicrobials,and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1,2013.Published May 2013.Originallyapproved in 2008.Last previous edition approved in 2008 as E2647 08.DOI:10.1520/E2647-13.2Ellison,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,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 Water,19th Edition,American Public HealthAssociation,American Water Works Association,Water Environment Federation,Washington,DC,1995.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 4.Summary of Test Method4.1 This test method is used for growing a repeatable P.aeruginosa biofilm in a drip flow reactor.The biofilm isestablished by operating the reactor in batch mode(no flow ofnutrients)for 6 h.A mature biofilm forms while the reactoroperates for an additional 48 h with a continuous flow ofnutrients.During continuous flow,the biofilm experiences verylow shear caused by the gravity flow of media dripping onto asurface set at a 10 angle.At the end of the 54 h,biofilmaccumulation is quantified by removing coupons from thereactor channels,rinsing the coupons to remove the planktoniccells,scraping the biofilm from the coupon surface,disaggre-gating the clumps,then diluting and plating for viable cellenumeration.5.Si