ASTM_E_3180_-_18.pdf

Designation:E318018Standard Test Method forQuantification of a Bacillus subtilis Biofilm Comprised ofVegetative Cells and Spores Grown Using the ColonyBiofilm Model1This standard is issued under the fixed designation E3180;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 and quantify a Bacillus subtilis biofilmcomprised of vegetative cells and endospores(spores)usingthe colony biofilm method(CBM).2,3The resulting biofilm isrepresentative of static environments that can develop a spo-rulating biofilm rather than being representative of one particu-lar environment.1.2 This test method utilizes a modified CBM to grow thebiofilm.The CBM uses a semipermeable membrane on an agarplate as the biofilm growth surface and nutrient source.2,3Inthis test method,membranes are inoculated and incubated fora total of 8 days to promote sporulation within the biofilm.1.3 This test method describes how to sample and analyzethe biofilm for vegetative cells and spores.Biofilm populationis expressed as total colony forming units(CFU)and spores permembrane.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,health,and environmental practices and deter-mine the applicability 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 International Standards,Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade(TBT)Committee.2.Referenced Documents2.1 ASTM Standards:4E2756 Terminology Relating to Antimicrobial and AntiviralAgents3.Terminology3.1 DefinitionsFor definitions of terms used in this TestMethod see Terminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 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.3.2.2 spore,na dormant,robust,and non-metabolicallyactive structure produced by certain bacteria enabling pro-longed survival and greater resistance to deleterious environ-mental factors.4.Summary of Test Method4.1 This test method utilizes a modified CBM to grow thebiofilm.The CBM uses an inoculated semipermeable mem-brane on an agar plate as the biofilm growth surface andnutrient source.2In the published method,the inoculatedmembranes are transferred to fresh agar plates regularly(usually every 8-24 h)to provide new nutrients to themembrane-grown cells and are incubated for a total of approxi-mately 48 h before sampling or subjecting the biofilm to furthertests.3In this test method,membranes are inoculated andincubated for 24 h before transferring the membrane to a newmedia plate.This initial growth step is similar to the publishedCBM and allows the colony biofilm to grow and spread across1This 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 Oct.1,2018.Published January 2019.DOI:10.1520/E3180182Anderl,J.N.,Franklin,M.J.,&Stewart,P.S.(2000).Role of AntibioticPenetration Limitation in Klebsiella pneumoniae Biofilm Resistance to Ampicillinand Ciprofloxacin.Antimicrobial Agents and Chemotherapy,44(7)18181824.3Merritt,J.H.,Kadouri,D.E.,and OToole,G.A.(2011).Growing and analyzingstatic biofilms.Current Protocols in Microbiology.22:B:1B.1:1B.1.11B.1.184For 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 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 Trade Organization Technical Barriers to Trade(TBT)Committee.1 the membrane.After the transfer,the colony biofilms areincubated for an additional