ASTM D5465-16
(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
6
B—Counting Colonies on Pour Plates
7
C—Counting Colonies on Spread Plates
8
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 and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: D5465 − 16
Standard Practices for
Determining Microbial Colony Counts from Waters Analyzed
1
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* 2.2 Other Standards:
3
9215Heterotrophic Plate Count
1.1 These practices cover recommended procedures for
counting colonies and reporting colony-forming units (CFU)
3. Terminology
onmembranefilters(MF)andstandardpourandspreadplates.
3.1 Definitions:
1.2 The values stated in SI units are to be regarded as
3.1.1 For definitions of terms use in this standard, see
standard. No other units of measurement are included in this Terminologies D1129 and D6161.
standard.
3.1.2 colony forming unit (CFU), n—in microbiology, a
visible mass of cells (algae, bacteria or fungi) originating from
1.3 This standard does not purport to address all of the
eitheranindividualcellorclusterofcellsthathavebeenplaced
safety concerns, if any, associated with its use. It is the
onto or dispersed into a solid or semi-solid nutrient medium
responsibility of the user of this standard to establish appro-
and subsequently incubated under prescribed conditions.
priate safety and health practices and determine the applica-
3.1.2.1 Discussion—Prescribed growth conditions can
bility of regulatory limitations prior to use.
include,butarenotlimitedto:growthmediumpHandnutrient
composition, incubation temperature, incubation environment
2. Referenced Documents
(forexample:gasmixture,pressureandrelativehumidity),and
2
2.1 ASTM Standards:
incubation interval. Any given set of growth conditions will
D1129Terminology Relating to Water
select for the culture recovery of a fraction of a sample’s
D5259Test Method for Isolation and Enumeration of En-
microbiome and against the culture recovery of the balance of
terococci from Water by the Membrane Filter Procedure
that microbiome.
D5392Test Method for Isolation and Enumeration of Es-
3.1.2.2 Discussion—Recognizing that all culture test meth-
cherichia ColiinWaterbytheTwo-StepMembraneFilter
ods are selective, CFU data invariably underestimate the
Procedure
population densities of viable microbes in samples tested by
D6161TerminologyUsedforMicrofiltration,Ultrafiltration,
those methods. Moreover, incomplete disaggregation of
Nanofiltration and Reverse Osmosis Membrane Processes
masses of microbial cells during sample preparation contrib-
D6974Practice for Enumeration of Viable Bacteria and
utes to decreasing the ratio of CFU to total viable microbes in
Fungi in Liquid Fuels—Filtration and Culture Procedures
the sample.
E2563PracticeforEnumerationofNon-Tuberculosis Myco-
3.1.2.3 Discussion—Colonies normally become visible to
bacteria inAqueous Metalworking Fluids by Plate Count
the naked eye only after approximately 1 billion cells have
Method
amassed.Assuming that the colony derived from a single cell,
it requires approximately 30 generations for a single cell to
proliferate to a mass of 1 billion cells. Consequently, the time
1
lapse between inoculation and detection of a CFU is directly
These practices are under the jurisdiction ofASTM Committee D19 on Water
and are the direct responsibility of Subcommittee D19.24 on Water Microbiology.
dependent on the generation time(s) of taxa present in sample.
Current edition approved June 1, 2016. Published June 2016. Originally
Moreover, because colony diameter increases as the cells
approved in 1993. Last previous edition approved in 2012 as D6465–93 (2012).
DOI: 10.1520/D5465-16.
2 3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Available online from http://standardmethods.org/store/
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM ProductView.cfm?ProductID=102, or from American Public Health Association
Standards volume information, refer to the standard’s Document Summary page on (APHA), Standard Methods for the Examination of Water and Wastewater, 800 I
the ASTM website. Street, NW Washington, DC 20001, http://www.apha.org.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
D5465 − 16
within the colony continue proliferate, in samples containing lines. A suggested procedure for reducing error in counting is
microbes with different generation times, colonies of microbes showninFig.2.Countthecoloniesinthesquaresindicatedby
with longer generation times are likely to be eclipsed (and
the ar
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D5465 − 93 (Reapproved 2012) D5465 − 16
Standard PracticePractices for
Determining Microbial Colony Counts from Waters Analyzed
1
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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 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 and health practices and determine the applicability of regulatory
limitations prior to use.
2. Referenced Documents
2
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D5259 Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
D5392 Test Method for Isolation and Enumeration of Escherichia Coli in Water by the Two-Step Membrane Filter Procedure
D6161 Terminology Used for Microfiltration, Ultrafiltration, Nanofiltration and Reverse Osmosis Membrane Processes
D6974 Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
E2563 Practice for Enumeration of Non-Tuberculosis Mycobacteria in Aqueous Metalworking Fluids by Plate Count Method
2.2 Other Standards:
3
9215 Heterotrophic Plate Count
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms use in this standard, see Terminologies D1129 and D6161.
3.1.2 colony forming unit (CFU), n—in microbiology, a visible mass of cells (algae, bacteria or fungi) originating from either
an individual cell or cluster of cells that have been placed onto or dispersed into a solid or semi-solid nutrient medium and
subsequently incubated under prescribed conditions.
1
These practices are under the jurisdiction of ASTM Committee D19 on Water and are the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved June 1, 2012June 1, 2016. Published August 2012June 2016. Originally approved in 1993. Last previous edition approved in 20042012 as
D6465 – 93 (2004).(2012). DOI: 10.1520/D5465-93R12.10.1520/D5465-16.
2
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 Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
3
Available online from http://standardmethods.org/store/ProductView.cfm?ProductID=102, or from American Public Health Association (APHA), Standard Methods for
the Examination of Water and Wastewater, 800 I Street, NW Washington, DC 20001, http://www.apha.org.
3.1.2.1 Discussion—
Prescribed growth conditions can include, but are not limited to: growth medium pH and nutrient composition, incubation
temperature, incubation environment (for example: gas mixture, pressure and relative humidity), and incubation interval. Any
given set of growth conditions will select for the culture recovery of a fraction of a sample’s microbiome and against the culture
recovery of the balance of that microbiome.
3.1.2.2 Discussion—
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
D5465 − 16
Recognizing that all culture test methods are selective, CFU data invariably underestimate the population densities of viable
microbes in samples tested by those methods. Moreover, incomplete disaggregation of masses of microbial cells during sample
preparation contributes to decreasing the ratio of CFU to total viable microbes in the sample.
3.1.2.3 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 proliferate to a mass of 1 billion cells.
Consequently, the time lapse between inoculation and detection of a CFU is dire
...
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