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ASTM D8516-23

(Test Method)

Standard Test Method for Quantification of Culturable Waterborne Bacteria Using a Defined Culture Medium Coated Plate

Standard Test Method for Quantification of Culturable Waterborne Bacteria Using a Defined Culture Medium Coated Plate

SIGNIFICANCE AND USE
5.1 This plate format is useful for the routine monitoring of culturable, waterborne bacteria in potable and non-potable waters. The significance of finding these bacteria can help with identifying water quality or water system problems or evaluate compliance with maintenance protocols. This test method uses small volumes of water, or dilutions thereof, and provides an easy and reliable method that eliminates media preparation and reduces laboratory waste.
SCOPE
1.1 This test method describes a simple procedure for the quantification of culturable, waterborne bacteria in potable water (drinking water, bottled water, and dental water, for example) and non-potable waters (cooling towers, for example).  
1.1.1 The EasyDisc2, 3 plate format is designed to test 1 mL of a water sample on a 47 mm gridded plate containing a growth reagent embedded to the plate’s inner surface.  
1.1.2 Detection is based on colorimetric technology in which viable, aerobic, heterotrophic, waterborne bacteria grow when present in the water sample, displaying a color reaction which allows for a simplified visualization of colony growth.  
1.2 Each plate can accurately detect up to 300 colony forming units per 1 mL (CFU/1 mL) of sample. To increase the quantification range, a sample dilution can be used. Adjust the CFU/mL result to reflect dilutions.  
1.3 This test method can be used for potable (for example, drinking, bottled, and dental) waters and non-potable waters such as cooling tower waters. It is the user’s responsibility to adhere to all requirements by local regulations and ensure the validity of this test method for waters other than those tested as part of the Interlaboratory Study (ILS).  
1.4 The values stated in SI units are to be regarded as the standard. No other units of measurement are included in this standard.  
1.5 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.  
1.6 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

Status
Published
Publication Date
30-Jun-2023
ICS
07.100.20 - Microbiology of water
Technical Committee
D19 - Water
Drafting Committee
D19.24 - Water Microbiology
Current Stage
Ref Project

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ASTM D8516-23 - Standard Test Method for Quantification of Culturable Waterborne Bacteria Using a Defined Culture Medium Coated PlateASTM D8516-23 - Standard Test Method for Quantification of Culturable Waterborne Bacteria Using a Defined Culture Medium Coated Plate
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ASTM D8516-23 - Standard Test Method for Quantification of Culturable Waterborne Bacteria Using a Defined Culture Medium Coated Plate
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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: D8516 − 23
Standard Test Method for
Quantification of Culturable Waterborne Bacteria Using a
1
Defined Culture Medium Coated Plate
This standard is issued under the fixed designation D8516; 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 1.6 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.1 This test method describes a simple procedure for the
ization established in the Decision on Principles for the
quantification of culturable, waterborne bacteria in potable
Development of International Standards, Guides and Recom-
water (drinking water, bottled water, and dental water, for
mendations issued by the World Trade Organization Technical
example) and non-potable waters (cooling towers, for ex-
Barriers to Trade (TBT) Committee.
ample).
2,3
1.1.1 The EasyDisc plate format is designed to test 1 mL
2. Referenced Documents
of a water sample on a 47 mm gridded plate containing a
4
2.1 ASTM Standards:
growth reagent embedded to the plate’s inner surface.
D1129 Terminology Relating to Water
1.1.2 Detection is based on colorimetric technology in
D1193 Specification for Reagent Water
which viable, aerobic, heterotrophic, waterborne bacteria grow
D2777 Practice for Determination of Precision and Bias of
when present in the water sample, displaying a color reaction
Applicable Test Methods of Committee D19 on Water
which allows for a simplified visualization of colony growth.
D3370 Practices for Sampling Water from Flowing Process
1.2 Each plate can accurately detect up to 300 colony
Streams
forming units per 1 mL (CFU/1 mL) of sample. To increase the
5
2.2 ISO Standard:
quantification range, a sample dilution can be used. Adjust the
ISO 6222 Water quality—Enumeration of culturable micro-
CFU/mL result to reflect dilutions.
organisms—Colony count by inoculation in a nutrient
1.3 This test method can be used for potable (for example,
agar culture medium
drinking, bottled, and dental) waters and non-potable waters
2.3 Standard Methods for the Examination of Water and
6
such as cooling tower waters. It is the user’s responsibility to
Wastewater:
adhere to all requirements by local regulations and ensure the
9060 Samples
validity of this test method for waters other than those tested as
9215 Heterotrophic Plate Count
part of the Interlaboratory Study (ILS).
3. Terminology
1.4 The values stated in SI units are to be regarded as the
standard. No other units of measurement are included in this 3.1 Definitions:
standard. 3.1.1 For definitions of terms used in this test method, refer
to Terminology D1129.
1.5 This standard does not purport to address all of the
3.1.2 ambient temperature, n—temperature of the
safety concerns, if any, associated with its use. It is the
surroundings, generally assumed to be 20 °C to 25 °C.
responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter- 3.1.3 colony forming unit, CFU, n—in microbiology, a
mine the applicability of regulatory limitations prior to use. 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
1 and subsequently incubated under prescribed conditions.
This test method is under the jurisdiction of ASTM Committee D19 on Water
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved July 1, 2023. Published August 2023. DOI: 10.1520/
4
D8516-23. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
2
EasyDisc is a registered trademark of IDEXX Laboratories, Inc. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
3
The sole source of supply of the apparatus known to the committee at this time Standards volume information, refer to the standard’s Document Summary page on
is IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, ME, 04092, USA. If the ASTM website.
5
you are aware of alternative suppliers, please provide this information to ASTM Available from American National Standards Institute (ANSI), 25 W. 43rd St.,
International Headquarters. Your comments will receive careful consideration at a 4th Floor, New York, NY 10036, http://www.ansi.org.
1 6
meeting of the responsible technical committee, which you may attend. Available online from https:/
...

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5.1 Adverse effects on natural populations of aquatic organisms and their uses have demonstrated the need to assess the hazards of many new, and some presently used, materials. The process described herein will help producers, users, regulatory agencies, and others to efficiently and adequately compare alternative materials, completely assess a final candidate material, or reassess the hazard of a material already in use.  
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5.1 Polychaetes are an important component of the benthic community, in which they generally comprise 30 to 50 % of the macroinvertebrate population. They are preyed upon by many species of fish, birds, and larger invertebrate species. Larger polychaetes feed on small invertebrates, larval stages of invertebrates, and algae. Polychaetes are especially sensitive to inorganic toxicants and, to a lesser extent, to organic toxicants (1).4 The ecological importance of polychaetes and their wide geographical distribution, ability to be cultured in the laboratory, and sensitivity to contaminants make them appropriate acute and chronic toxicity test organisms. Their relatively short life cycle enables the investigator to measure the effect of contaminants on reproduction.  
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SIGNIFICANCE AND USE
5.1 Microbiological water testing procedures using membrane filtration are based on the premise that all bacteria within a specific size range will be retained by the membrane filter used. If the membrane filter does not retain these bacteria, false negative results or lowered density estimates may occur that could have serious repercussions due to the presence of unrecognized potential health hazards in the water being tested, especially in drinking water.  
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SCOPE
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SIGNIFICANCE AND USE
5.1 Tests with algae provide information on the toxicity of test materials to an important component of the aquatic biota and might indicate whether additional testing (2) is desirable. Specific testing procedures under various regulatory jurisdictions follow procedures similar to those described in this Guide (3, 4). Users should consult with any specific regulatory requirements to determine the applicability and consistency of this standard with such requirements.  
5.2 Algae are ubiquitous in aquatic ecosystems, where they incorporate solar energy into biomass, produce oxygen, function in nutrient cycling and serve as food for animals. Because of their ecological importance, sensitivity to many toxicants, ready availability, ease of culture, and fast growth rates (rendering it possible to conduct a multi-generation test in a short period of time), algae are often used in toxicity testing.  
5.3 Results of algal toxicity tests might be used to compare the sensitivities of different species of algae and the toxicities of different materials to algae and to study the effects of various environmental factors on results of such tests.  
5.4 Results of algal toxicity tests might be an important consideration when assessing the hazards of materials to aquatic organisms (See Guide E1023) or deriving water quality criteria for aquatic organisms (5).  
5.5 Results of algal toxicity tests might be useful for studying biological availability of, and structure-activity relationships between, test materials.  
5.6 Results of algal toxicity tests will depend on the temperature, composition of the growth medium, and other factors. These tests are conducted in solutions that contain concentrations of salts, minerals, and nutrients that greatly exceed those in most surface waters. These conditions may over- or under-estimate the effects of the test material if discharged to surface waters.
SCOPE
1.1 This guide covers procedures for obtaining laboratory data concerning the adverse effects of a test material added to growth medium on growth of certain species of freshwater and saltwater microalgae during a static exposure. These procedures will probably be useful for conducting short-term toxicity tests with other species of algae, although modifications might be necessary. Although the test duration is comparable to an acute toxicity test with aquatic animals, an algal toxicity test of short duration (72, 96 or 120 h) allows for examination of effects upon multiple generations of an algal population and thus should not be viewed as an acute toxicity test.  
1.2 Other modifications of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting toxicity tests with microalgae.  
1.3 These procedures are applicable to many chemicals, either individually or in formulations, commercial products, or known mixtures. With appropriate modifications, these procedures can be used to conduct tests on temperature, and pH and on such materials as aqueous effluents (see Guide E1192), leachates, oils, particulate matter, sediments, and surface waters. Static tests might not be applicable to materials that are highly volatile, are rapidly biologically or chemically transformed in aqueous solutions, or are removed from test solutions in substantial quantities by the test vessels or organisms during the test. (1)3 However, practical flow-through test procedures with microalgae have not been developed.  
1.4 Results of tests using microalgae should usually be reported in terms of the 96-h (or other time period) IC50 (see 3.2.5) b...

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