ASTM D5246-13
(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 Pseudomonas 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 bloodied 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 reagent grade water samples.
1.2 It is the user’s responsibility to ensure the validity of this method for surface waters, ground waters, recreational waters fresh and marine), wastewaters.
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 and health practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 10.
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Designation: D5246 − 13
StandardTest 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.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope nin. It is oxidase and caseinase positive, is able to grow at
42°C,isrelativelyresistanttomanyantibiotics,andmayutilize
1.1 Thetestmethodcoverstheisolationandenumerationof
acetamide.
Pseudomonas aeruginosa. Testing was performed on spiked
3.2.2 refrigeration—storage at 2 to 8°C.
reagent grade water samples.
1.2 Itistheuser’sresponsibilitytoensurethevalidityofthis
4. Summary of Test Method
method for surface waters, ground waters, recreational waters
4.1 A water sample is passed through a 0.45 mm or
fresh and marine), wastewaters.
equivalent membrane filter. The filter carrying the retained
3
1.3 The values stated in SI units are to be regarded as
organisms is placed on a selective medium (M-PA-C) and is
standard. No other units of measurement are included in this
incubated at 41.5 6 0.5°C for 72 h. The resulting pink-brown
standard.
to black colonies of Pseudomonas aeruginosa are counted and
reported per 100 mL of the sample. Colonies may be verified
1.4 This standard does not purport to address all of the
on skim milk agar.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
5. Significance and Use
priate safety and health practices and determine the applica-
5.1 Pseudomonas aeruginosa is an opportunistic pathogen,
bility of regulatory limitations prior to use. Specific hazard
and has been linked as the causative agent of numerous
statements are given in Section 10.
infections that may be transmitted through a contaminated
water supply to a susceptible host.
2. Referenced Documents
2
NOTE 1—Fecal waste is >95 % E. coli which is found in humans and
2.1 ASTM Standards:
warm bloodied animals.
D1129Terminology Relating to Water
D1193Specification for Reagent Water 5.2 The membrane filtration procedure described is a rapid
D2777Practice for Determination of Precision and Bias of and reliable test method of detecting P. aeruginosa in water.
Applicable Test Methods of Committee D19 on Water
6. Interferences
D3370Practices for Sampling Water from Closed Conduits
6.1 For certain samples, bacterial cells may have been
3. Terminology exposed to adverse environmental factors that lower their
probability for survival and growth on a membrane filter
3.1 Definitions:
medium.Thiseffectmaybepronouncedinthistestmethoddue
3.1.1 For definitions of terms used in this test method, refer
to the presence of antibiotics and the elevated incubation
to Terminology D1129.
temperature.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 Pseudomonas aeruginosa—an aerobic, motile, gram
6.2 The selection of an appropriate dilution volume is
negative rod that produces fluorescent pigments and pyocya- essential.Toosmalladilutionvolumemayfailtodetectany P.
aeruginosa organisms, while too large a volume may cause an
overabundance of colonies that would interfere with an accu-
1
This test method is under the jurisdiction ofASTM Committee D19 on Water
rate count.
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
6.3 Chemicals or a combination of chemicals in certain
CurrenteditionapprovedMay1,2013.PublishedJuly2013.Originallyapproved
in 1992. Last previous edition approved in 2004 as D5246–92 (2004). DOI:
samples can have a toxic effect upon P. aeruginosa when
10.1520/D5246-13.
concentrated.
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
1
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D5246 − 13
6.4 Turbidity in samples may clog filter or effect color 7.11.6 Filter flask, vacuum, usually 1 L, with appropriate
detection of organisms that develop on the filter. tubing.
7.11.7 Flask for safety trap placed between the filter flask
6.5 Water samples containing residual chlorine can be
and the vacuum source.
detrimental to P. aeruginosa. Utilize the procedure defined in
7.11.8 Membrane filters, sterile, white, grid marked, 47 mm
Practices D3370 to address chlor
...
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 − 92 (Reapproved 2004) D5246 − 13
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
1.1 ThisThe test method covers the isolation and enumeration of Pseudomonas aeruginosaaeruginosa. (TestingP. aeruginosa
was) 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. performed on spiked reagent grade
water samples.
1.2 This test method was used successfully with reagent water and it is the user’sIt is the user’s responsibility to ensure the
validity of this test method for waters of untested matrices.surface waters, ground waters, recreational waters fresh and marine),
wastewaters.
1.3 The values stated in SI units are to be regarded as the 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 and health practices and determine the applicability of regulatory
limitations prior to use. Specific hazard statements are given in Section 10.
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 Closed Conduits
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this test method, refer to Terminology D1129.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 Pseudomonas aeruginosa—an aerobic, motile, gram negative rod that produces fluorescent pigments and pyocyanin. It is
oxidase and caseinase positive, is able to grow at 42°C, is relatively resistant to many antibiotics, and may utilize acetamide.
3.2.2 refrigeration—storage at 2 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
3
placed on a selective medium (M-PA-C) and is incubated at 41.5 6 0.5°C for 48 to 72 h. The resulting pink-brown to black
colonies of Pseudomonas 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 Pseudomonas 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. In addition to its direct pathogenicity, the
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 June 1, 2004May 1, 2013. Published June 2004July 2013. Originally approved in 1992. Last previous edition approved in 19982004 as
D5246 – 92 (1998).(2004). DOI: 10.1520/D5246-92R04.10.1520/D5246-13.
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 from BBL Microbiological Systems, Division of Becton Dickinson 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
1
---------------------- Page: 1 ----------------------
D5246 − 13
association of P. aeruginosa with human fecal waste indicates that where elevated levels of P. aeruginosa are found, a serious health
hazard may exist due to the presence of other pathogens.
NOTE 1—Fecal waste is >95 % E. coli which is found in humans and warm bloodied 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 that lower their pro
...
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