1、Designation:E219612Standard Test Method forQuantification of Pseudomonas aeruginosa Biofilm Grownwith Medium Shear and Continuous Flow Using RotatingDisk Reactor1This standard is issued under the fixed designation E2196;the number immediately following the designation indicates the year oforiginal a
2、doption 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 is used for growing a reproducible(1)2Pseudomonas aeruginos
3、a biofilm in a continuously stirred tankreactor(CSTR)under medium shear conditions.In addition,the test method describes how to sample and analyze biofilmfor viable cells.1.2 Although this test method was created to mimic condi-tions within a toilet bowl,it can be adapted for the growth andcharacter
4、ization of varying species of biofilm(rotating diskreactorrepeatability and relevance(2).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(rotating diskreactorefficacy test method(3).1.
5、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
6、 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 Platin
7、g Methods2.2 Other Standards:Method 9050 C.1.a Buffered Dilution Water Preparation(4)3.Terminology3.1 biofilm,n microorganisms living in a self-organizedcommunity attached to surfaces,interfaces,or each other,embedded in a matrix of extracellular polymeric substances ofmicrobial origin,while exhibit
8、ing altered phenotypes withrespect to growth rate and gene transcription.3.1.1 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,taxo
9、nomic diversity,thickness,chemical gradients,chemicalcomposition,consistency,and other materials in the matrix thatare not produced by the biofilm microorganisms,are controlledby the physiochemical environment in which it exists.3.2 coupon,nbiofilm sample surface.4.Summary of Test Method4.1 This tes
10、t method is used for growing a reproduciblePseudomonas aeruginosa biofilm in a rotating disk reactor.The biofilm is established by operating the reactor in batchmode(no flow)for 24 h.Steady state growth(attachment isequal to detachment)is reached while the reactor operates foran additional 24 h with
11、 continuous flow of the nutrients.Theresidence time of the nutrients in the reactor is set to select forbiofilm growth,and is species and reactor parameter specific.During the entire 48 h,the biofilm is exposed to continuousfluid shear from the rotation of the disk.At the end of the 48 h,biofilm acc
12、umulation is quantified by removing coupons fromthe disk,harvesting the biofilm from the coupon surface,disaggregating the clumps,then diluting and plating for viablecell enumeration.5.Significance and Use5.1 Bacteria that exist in a biofilm are phenotypicallydifferent from suspended cells of the sa
13、me genotype.The studyof biofilm in the laboratory requires protocols that account forthis difference.Laboratory biofilms are engineered in growth1This test method is under the jurisdiction of ASTM Committee E35 onPesticides,Antimicrobials,and Alternative Control Agents and is the directresponsibilit
14、y of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1,2012.Published June 2012.Originallyapproved in 2002.Last previous edition approved in 2007 as E2196 07.DOI:10.1520/E2196-12.2The boldface numbers in parentheses refer to a list of references at the end ofthis standard.
15、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,Wes
16、t Conshohocken,PA 19428-2959.United States1 reactors designed to produce a specific biofilm type.Alteringsystem parameters will correspondingly result in a change inthe biofilm.The purpose of this method is to direct a user in thelaboratory study of biofilms by clearly defining each systemparameter.This method will enable a person to grow,sample,and analyze a laboratory biofilm.6.Apparatus6.1 Wooden Applicator Sticks,sterile.6.2 Inoculating Loop.6.3 Petri Dish,100-by 15-mm,plastic,sterile and em
