ASTM D5465-16(2020)
(Practice)Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
SIGNIFICANCE AND USE
4.1 Numerous ASTM test methods and practices (for example: Test Methods D5259 and D5392, and Practices D6974 and E2563) report colony counts as their measured parameter.
4.2 These practices provide a uniform set of counting, calculating, and reporting procedures for ASTM test methods in microbiology.
Section
A—Counting Colonies on Membrane Filters
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B—Counting Colonies on Pour Plates
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C—Counting Colonies on Spread Plates
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4.3 The counting rules provide a best attainable estimate of microorganisms in the sample, since the samples cannot be held and reanalyzed at a later date.
SCOPE
1.1 These practices cover recommended procedures for counting 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 as standard. No other units of measurement are included in this standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
General Information
Relations
Standards Content (Sample)
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D5465 − 16 (Reapproved 2020)
Standard Practices for
Determining Microbial Colony Counts from Waters Analyzed
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by Plating Methods
This standard is issued under the fixed designation D5465; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope E2563PracticeforEnumerationofNon-Tuberculosis Myco-
bacteria inAqueous Metalworking Fluids by Plate Count
1.1 These practices cover recommended procedures for
Method
counting colonies and reporting colony-forming units (CFU)
2.2 Other Standards:
onmembranefilters(MF)andstandardpourandspreadplates.
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APHA 9215Heterotrophic Plate Count
1.2 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
3. Terminology
standard.
3.1 Definitions:
1.3 This standard does not purport to address all of the
3.1.1 For definitions of terms used in this standard, refer to
safety concerns, if any, associated with its use. It is the
Terminologies D1129 and D6161.
responsibility of the user of this standard to establish appro-
3.2 Definitions of Terms Specific to This Standard:
priate safety, health, and environmental practices and deter-
3.2.1 colony forming unit (CFU), n—in microbiology, a
mine the applicability of regulatory limitations prior to use.
visiblemassofcells(algae,bacteria,orfungi)originatingfrom
1.4 This international standard was developed in accor-
eitheranindividualcellorclusterofcellsthathavebeenplaced
dance with internationally recognized principles on standard-
onto or dispersed into a solid or semi-solid nutrient medium
ization established in the Decision on Principles for the
and subsequently incubated under prescribed conditions.
Development of International Standards, Guides and Recom-
3.2.1.1 Discussion—Prescribed growth conditions can
mendations issued by the World Trade Organization Technical
include,butarenotlimitedto:growthmediumpHandnutrient
Barriers to Trade (TBT) Committee.
composition, incubation temperature, incubation environment
(forexample:gasmixture,pressureandrelativehumidity),and
2. Referenced Documents
incubation interval. Any given set of growth conditions will
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2.1 ASTM Standards:
select for the culture recovery of a fraction of a sample’s
D1129Terminology Relating to Water
microbiome and against the culture recovery of the balance of
D5259Test Method for Isolation and Enumeration of En-
that microbiome.
terococci from Water by the Membrane Filter Procedure
3.2.1.2 Discussion—Recognizing that all culture test meth-
D5392Test Method for Isolation and Enumeration of Es-
ods are selective, CFU data invariably underestimate the
cherichia coli inWater by theTwo-Step Membrane Filter
population densities of viable microbes in samples tested by
Procedure
those methods. Moreover, incomplete disaggregation of
D6161TerminologyUsedforMicrofiltration,Ultrafiltration,
masses of microbial cells during sample preparation contrib-
Nanofiltration,andReverseOsmosisMembraneProcesses
utes to decreasing the ratio of CFU to total viable microbes in
D6974Practice for Enumeration of Viable Bacteria and
the sample.
Fungi in Liquid Fuels—Filtration and Culture Procedures
3.2.1.3 Discussion—Colonies normally become visible to
the naked eye only after approximately 1 billion cells have
amassed.Assuming that the colony derived from a single cell,
it requires approximately 30 generations for a single cell to
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proliferate to a mass of 1 billion cells. Consequently, the time
These practices are under the jurisdiction ofASTM Committee D19 on Water
and are the direct responsibility of Subcommittee D19.24 on Water Microbiology.
lapse between inoculation and detection of a CFU is directly
CurrenteditionapprovedJuly1,2020.PublishedJuly2020.Originallyapproved
dependent on the generation time(s) of taxa present in sample.
in 1993. Last previous edition approved in 2016 as D6465–16. DOI: 10.1520/
Moreover, because colony diameter increases as the cells
D5465-16R20.
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For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
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Standards volume information, refer to the standard’s Document Summary page on Available from American Public Health Association (APHA), 800 I St., NW,
the ASTM website. Washington, DC 20001, http://www.apha.org.
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