ASTM D8516-23

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
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
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
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
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/
D8516-23. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
EasyDisc is a registered trademark of IDEXX Laboratories, Inc. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
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.
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://www.standardmethods.org/doi.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D8516 − 23
3.2 Definitions of Terms Specific to This Standard: 4.2.2 EasyDisc R2A plates are incubated, white lid side up,
3.2.1 confluent growth, n—in this test method, plated results at 20 °C to 28 °C for five to seven days for potable and
observed do not contain isolated bacterial colonies but are non-potable water samples.
observed as continuous bacterial growth covering the entire 4.2.3 EasyDisc YEA plates are incubated, white lid side up,
area of the plate’s printed grid. in a 22 °C 6 2 °C incubator for 68 h 6 4 h or a 36 °C 6 2 °C
incubator for 44 h 6 4 h for potable and non-potable water
3.2.2 cooling tower water, n—water collected from a cool-
samples.
ing tower.
4.3 Culturable, waterborne bacteria are detected if any
3.2.3 culturable, adj—in this test method, any aerobic bac-
colony growth is observed.
teria able to form colonies on solid media.
4.4 Test results are reported as CFU/1 mL.
3.2.4 dental water, n—water that comes from dental devices
or waterlines of a dental unit providing a water source for
NOTE 2—Colonies may be counted with or without the aid of a manual
dental procedures.
colony counter with magnification under a uniform light.
3.2.5 drinking water, n—water that meets regulatory re-
5. Significance and Use
quirements or standards for human consumption.
5.1 This plate format is useful for the routine monitoring of
3.2.6 EasyDisc PCA, n—method used for the quantification
culturable, waterborne bacteria in potable and non-potable
of culturable, waterborne bacteria in drinking water, bottled
waters. The significance of finding these bacteria can help with
2,3
water, and cooling tower water.
identifying water quality or water system problems or evaluate
3.2.6.1 Discussion—The CFU data generated by this test
compliance with maintenance protocols. This test method uses
method correlate with the pour plate method using plate count
small volumes of water, or dilutions thereof, and provides an
agar (PCA) incubated at 35 °C for 48 h as described in Method
easy and reliable method that eliminates media preparation and
9215 (see 2.3).
reduces laboratory waste.
3.2.7 EasyDisc R2A, n—method used for the quantification
6. Interferences
of culturable, waterborne bacteria in drinking water, bottled
2,3
water, and dental water.
6.1 Do not use automated colony counters as the printed
3.2.7.1 Discussion—The CFU data generated by this
grid on the plate can interfere with results from an automatic
method correlate with the pour plate method using Reasoners
counter.
2 Agar (R2A) incubated at 20 °C to 28 °C for five to seven
6.2 Buffers containing phosphate should be avoided for
days as described in Method 9215 (see 2.3).
quality control samples or dilutions as they can interfere with
3.2.8 EasyDisc YEA, n—method used for the quantification
colony visualization.
2,3
of culturable, waterborne bacteria in drinking water.
6.3 Confluent growth can interfere with the colony count on
3.2.8.1 Discussion—The CFU data generated by this
the plates. It is recommended that any plates with confluent
method correlate with the pour plate method using yeast extract
growth are reanalyzed with dilutions of the original sample.
agar incubated at 22 °C for 68 h and 36 °C for 44 h as
described in ISO 6222.
7. Apparatus
3.2.9 plate, n—in this test method, a 47 mm sample plate
7.1 Colony counter, manual magnification (optional).
with a printed grid to aid in counting colonies, which contains
7.2 Equipment for Sample Collection and Transport:
a growth reagent for testing a 1 mL 6 0.1 mL specimen.
7.2.1 Sterile 100 mL vessels with or without sodium thio-
3.2.10 specimen, n—in this test method, a 1 mL portion of a
sulfate.
collected water sample.
7.2.1.1 Preparation of Vessels with Sodium Thiosulfate—
Vessels can be purchased containing sodium thiosulfate or
4. Summary of Test Method
0.1 mL of a 10 % solution of sodium thiosulfate can be added
4.1 Bacteria are grown on a defined culture medium coated
to a 120 mL vessel, to neutralize up to 15 mg/L of residual
on a plate.
chlorine as described in Method 9060 (see 2.3).
4.2 From a well-mixed sample, a 1 mL specimen is added to
NOTE 3—Sodium thiosulfate is required for any samples containing an
a plate, gently swirled, the white plate lid is reattached, and
oxidizing agent such as chlorine.
then the plate is incubated. NOTE 4—Dental waters may contain specific disinfectant agents (for
example, hydrogen peroxide), therefore the addition of an appropriate
NOTE 1—Incubation temperature and interval depends on the plate type
neutralizer may be required.
selected. Follow local regulatory requirements for plate selection.
7.2.2 Ice chest.
4.2.1 EasyDisc PCA plates are incubated, white lid side up,
7.2.3 Ice packs.
at 35 °C 6 2 °C for 48 h 6 3 h for potable and non-potable
7.3 Incubator, air microbiological type to maintain a tem-
water samples.
perature of the selected plate(s) (4.2.1 – 4.2.3).
7.4 Loop, inoculating sterile loop, 10 μL capacity or equiva-
Reasoner, D. J. and Geldreich, E. E., “A New Medium for the Enumeration and
lent.
Subculture of Bacteria from Potable Water,” Applied and Environmental
Microbiology, Vol 49, No. 1, January 1985, pp. 1-7. 7.5 Pipettor, capable of pipetting 1 mL.
D8516 − 23
7.6 Pipettor tips, disposable, 1 mL, sterile. storage temperatures. Take care that samples containers are not
totally immersed in water during transit.
8. Reagents and Materials
10.3 Handling Time Limitations—Adhere to all require-
8.1 Plates—Plates are commercially available in different
ments by local regulations, or it is recommended to examine
premade formats (3.2.6 – 3.2.8) and each plate is suitable for samples as soon as possible after collection.
single samples.
11. Quality Control
8.2 Purity of Water—Unless otherwise indicated, references
to water shall be understood to mean reagent water conforming
11.1 Observe and record incubator temperatures at least
to Specification D1193, Type IV. Sterilize water by either twice per day when in use. Individual observations shall be
autoclaving or sterile filtration (water filtered by an 0.22 μm
≥4 h apart to ensure temperature is within stated limits.
filter).
11.2 Quality control should be conducted at least once on
8.3 Purity of Reagents—Reagent-grade chemicals shall be
each new lot of plates. Perform the positive control procedure
used in all tests. Unless otherwise indicated, it is intended that (11.3) and the negative control procedure (11.4).
all reagents conform to the specifications of the Committee on
11.3 Preparation of Positive Control:
Analytical Reagents of the American Chemical Society, where
11.3.1 Use the quality control kit (8.4.1) and perform all
such specifications are available. Other grades may be used,
steps at ambient temperature.
provided it is first ascertained that the reagent is of sufficiently
11.3.2 Remove vial(s) from freezer. Equilibrate at ambient
high purity to permit its use without lessening the accuracy of
temperature for 15 min.
the determination.
11.3.3 Open a vial and aseptically transfer the pellet to an
8.4 Quality Control Strains: appropriately labeled vessel of 100 mL of sterile, nonbuffered,
8.4.1 IDEXX-QC HPC/TVC, or
oxidant-free water in a sterile vessel without sodium thiosul-
8.4.2 At least one target organism: fate.
8.4.2.1 Enterococcus faecalis, American Type Culture Col-
11.3.4 Swirl the sample and allow to stand for 5 min to
lection (ATCC) 29212/World Data Centre for Microorganisms dissolve the pellet completely.
(WDCM) 00087; or
11.3.5 After the pellet has dissolved, mix by inverting the
8.4.2.2 Escherichia coli, ATCC 11775/WDCM 00090.
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