ASTM_E_2562_-_12.pdf

Designation:E256212Standard 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 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 reproducible(1)2Pseudomonas aeruginosabiofilm under high shear.The resulting biofilm is representa-tive of generalized situations where biofilm exists under highshear rather than being representative of one particular envi-ronment.1.2 This test method uses the Centers for Disease Controland 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 forgrowing 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 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 Standards:Method 9050 C.1.a Buffered Dilution Water Preparationaccording to Eaton 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 exhibiting 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 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.4.Summary of Test Method4.1 This test method is used for growing a reproduciblePseudomonas 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 residencetime of the nutrients in the reactor is set to select for biofilm1This 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 1,2012.Published June 2012.Originallyapproved 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 ofthis standard.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.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 growth,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 the 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,harvesting the biofilm from the couponsurface,disaggregating the clumps,and diluting and plating forviable 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 biofilms are engineeredin growth reactors designed to produce a specific biofilm type.Altering system parameters wil