ASTM D5246-24
(Test Method)Standard Test Method for Isolation and Enumeration of Pseudomonas aeruginosa from Water
Standard Test Method for Isolation and Enumeration of <emph type="ital">Pseudomonas aeruginosa</emph> from Water
SIGNIFICANCE AND USE
5.1 P. aeruginosa is an opportunistic pathogen and has been linked as the causative agent of numerous infections that may be transmitted through a contaminated water supply to a susceptible host.
Note 1: Fecal waste is >95 % E. coli which is found in humans and warm blooded animals.
5.2 The membrane filtration procedure described is a rapid and reliable test method of detecting P. aeruginosa in water.
SCOPE
1.1 The test method covers the isolation and enumeration of Pseudomonas aeruginosa. Testing was performed on spiked samples using reagent grade water as the diluent from surface waters; recreational waters; ground water, water supplies; especially rural nonchlorinated sources; waste water; and saline waters. The detection limit of this test method is one microorganism per 100 mL.
1.2 This test method was used successfully with reagent water. It is the user's responsibility to ensure the validity of this test method for surface waters, recreational waters, ground water, rural nonchlorinated sources; waste water; and saline waters.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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. Specific hazard statements are given in Section 10.
1.5 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.
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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: D5246 − 24
Standard Test Method for
Isolation and Enumeration of Pseudomonas aeruginosa from
1
Water
This standard is issued under the fixed designation D5246; 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 D1193 Specification for Reagent Water
D2777 Practice for Determination of Precision and Bias of
1.1 The test method covers the isolation and enumeration of
Applicable Test Methods of Committee D19 on Water
Pseudomonas aeruginosa. Testing was performed on spiked
D3370 Practices for Sampling Water from Flowing Process
samples using reagent grade water as the diluent from surface
Streams
waters; recreational waters; ground water, water supplies;
especially rural nonchlorinated sources; waste water; and
3. Terminology
saline waters. The detection limit of this test method is one
microorganism per 100 mL. 3.1 Definitions:
3.1.1 For definitions of terms used in this standard, refer to
1.2 This test method was used successfully with reagent
Terminology D1129.
water. It is the user’s responsibility to ensure the validity of this
3.2 Definitions of Terms Specific to This Standard:
test method for surface waters, recreational waters, ground
3.2.1 Pseudomonas aeruginosa, n—an aerobic, motile,
water, rural nonchlorinated sources; waste water; and saline
gram negative rod that produces fluorescent pigments and
waters.
pyocyanin.
1.3 The values stated in SI units are to be regarded as
3.2.1.1 Discussion—It is oxidase and caseinase positive, is
standard. No other units of measurement are included in this
able to grow at 42 °C, is relatively resistant to many antibiotics,
standard.
and may utilize acetamide.
1.4 This standard does not purport to address all of the
3.2.2 refrigeration, n—storage at 2 °C to 8 °C.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
4. Summary of Test Method
priate safety, health, and environmental practices and deter-
4.1 A water sample is passed through a 0.45 mm or
mine the applicability of regulatory limitations prior to use.
equivalent membrane filter. The filter carrying the retained
Specific hazard statements are given in Section 10.
3
organisms is placed on a selective medium (M-PA-C) and is
1.5 This international standard was developed in accor-
incubated at 41.5 °C 6 0.5 °C for 72 h. The resulting pink-
dance with internationally recognized principles on standard-
brown to black colonies of P. aeruginosa are counted and
ization established in the Decision on Principles for the
reported per 100 mL of the sample. Colonies may be verified
Development of International Standards, Guides and Recom-
on skim milk agar.
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
5. Significance and Use
2. Referenced Documents
5.1 P. aeruginosa is an opportunistic pathogen and has been
2
linked as the causative agent of numerous infections that may
2.1 ASTM Standards:
be transmitted through a contaminated water supply to a
D1129 Terminology Relating to Water
susceptible host.
NOTE 1—Fecal waste is >95 % E. coli which is found in humans and
1
warm blooded animals.
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.
5.2 The membrane filtration procedure described is a rapid
Current edition approved April 1, 2024. Published April 2024. Originally
and reliable test method of detecting P. aeruginosa in water.
approved in 1992. Last previous edition approved in 2019 as D5246 – 19. DOI:
10.1520/D5246-24.
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
3
Standards volume information, refer to the standard’s Document Summary page on Available from BBL Microbiological Systems, Division of Becton Dickinson
the ASTM website. and Co., Cockeysville, MD 21030. Other suppliers may be utilized if equivalent.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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D5246 − 24
6. Interferences 7.10 Equipment for membrane filter procedure:
7.10.1 Petri dishes, sterile, plastic, 9 mm × 50 mm, with
6.1 For certain samples, bacterial cells may have been
tight-fitting lids; or 15 mm × 60 mm, glass or plastic, with
exposed
...
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: D5246 − 19 D5246 − 24
Standard Test Method for
Isolation and Enumeration of Pseudomonas aeruginosa from
1
Water
This standard is issued under the fixed designation D5246; 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 The test method covers the isolation and enumeration of Pseudomonas aeruginosa. Testing was performed on spiked samples
using reagent grade water as the diluent from surface waters; recreational waters; ground water, water supplies; especially rural
nonchlorinated sources; waste water; and saline waters. The detection limit of this test method is one microorganism per 100 mL.
1.2 This test method was used successfully with reagent water. It is the user’s responsibility to ensure the validity of this test
method for surface waters, recreational waters, ground water, rural nonchlorinated sources; waste water; and saline waters.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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. Specific hazard statements are given in Section 10.
1.5 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.
2. Referenced Documents
2
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D2777 Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
D3370 Practices for Sampling Water from Flowing Process Streams
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this standard, refer to Terminology D1129.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 Pseudomonas aeruginosa, n—an aerobic, motile, gram negative rod that produces fluorescent pigments and pyocyanin.
1
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 Dec. 1, 2019April 1, 2024. Published December 2019April 2024. Originally approved in 1992. Last previous edition approved in 20152019 as
D5246 – 15.D5246 – 19. DOI: 10.1520/D5246-19.10.1520/D5246-24.
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.
*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 ----------------------
D5246 − 24
3.2.1.1 Discussion—
It is oxidase and caseinase positive, is able to grow at 42°C,42 °C, is relatively resistant to many antibiotics, and may utilize
acetamide.
3.2.2 refrigeration, n—storage at 2 to 8°C.2 °C to 8 °C.
4. Summary of Test Method
4.1 A water sample is passed through a 0.45 mm or equivalent membrane filter. The filter carrying the retained organisms is placed
3
on a selective medium (M-PA-C) and is incubated at 41.5 6 0.5°C41.5 °C 6 0.5 °C for 72 h. The resulting pink-brown to black
colonies of P. aeruginosa are counted and reported per 100 mL of the sample. Colonies may be verified on skim milk agar.
5. Significance and Use
5.1 P. aeruginosa is an opportunistic pathogen and has been linked as the causative agent of numerous infections that may be
transmitted through a contaminated water supply to a susceptible host.
NOTE 1—Fecal waste is >95 % E. coli which is found in humans and warm bloodiedblooded animals.
5.2 The membrane filtration procedure described is a rapid and reliable test method of detecting P. aeruginosa in water.
6. Interferences
6.1 For certain samples, bacterial cells may have been exposed to adverse environmental factors
...
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