ASTM_D_5465_-_16_2020.pdf

Designation:D546516(Reapproved 2020)Standard Practices forDetermining Microbial Colony Counts from Waters Analyzedby Plating Methods1This standard is issued under the fixed designation D5465;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 These practices cover recommended procedures forcounting colonies and reporting colony-forming units(CFU)on membrane filters(MF)and standard pour and spread plates.1.2 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard.1.3 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.1.4 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:2D1129 Terminology Relating to WaterD5259 Test Method for Isolation and Enumeration of En-terococci from Water by the Membrane Filter ProcedureD5392 Test Method for Isolation and Enumeration of Es-cherichia coli in Water by the Two-Step Membrane FilterProcedureD6161 Terminology Used for Microfiltration,Ultrafiltration,Nanofiltration,and Reverse Osmosis Membrane ProcessesD6974 Practice for Enumeration of Viable Bacteria andFungi in Liquid FuelsFiltration and Culture ProceduresE2563 Practice for Enumeration of Non-Tuberculosis Myco-bacteria in Aqueous Metalworking Fluids by Plate CountMethod2.2 Other Standards:APHA 9215 Heterotrophic Plate Count33.Terminology3.1 Definitions:3.1.1 For definitions of terms used in this standard,refer toTerminologies D1129 and D6161.3.2 Definitions of Terms Specific to This Standard:3.2.1 colony forming unit(CFU),nin microbiology,avisible mass of cells(algae,bacteria,or fungi)originating fromeither an individual cell or cluster of cells that have been placedonto or dispersed into a solid or semi-solid nutrient mediumand subsequently incubated under prescribed conditions.3.2.1.1 DiscussionPrescribed growth conditions caninclude,but are not limited to:growth medium pH and nutrientcomposition,incubation temperature,incubation environment(for example:gas mixture,pressure and relative humidity),andincubation interval.Any given set of growth conditions willselect for the culture recovery of a fraction of a samplesmicrobiome and against the culture recovery of the balance ofthat microbiome.3.2.1.2 DiscussionRecognizing that all culture test meth-ods are selective,CFU data invariably underestimate thepopulation densities of viable microbes in samples tested bythose methods.Moreover,incomplete disaggregation ofmasses of microbial cells during sample preparation contrib-utes to decreasing the ratio of CFU to total viable microbes inthe sample.3.2.1.3 DiscussionColonies normally become visible tothe naked eye only after approximately 1 billion cells haveamassed.Assuming that the colony derived from a single cell,it requires approximately 30 generations for a single cell toproliferate to a mass of 1 billion cells.Consequently,the timelapse between inoculation and detection of a CFU is directlydependent on the generation time(s)of taxa present in sample.Moreover,because colony diameter increases as the cells1These practices are under the jurisdiction of ASTM Committee D19 on Waterand are the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved July 1,2020.Published July 2020.Originally approvedin 1993.Last previous edition approved in 2016 as D6465 16.DOI:10.1520/D5465-16R20.2For 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.3Available from American Public Health Association(APHA),800 I St.,NW,Washington,DC 20001,http:/www.apha.org.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.within the colony continue proliferate,in samples containingmicrobes with different generation times,colonies of microb