ASTM D5246-24

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.

General Information

• Status: Published
• Publication Date: 31-Mar-2024
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: D19 - Water
• Drafting Committee: D19.24 - Water Microbiology

Relations

• Replaces: ASTM D5246-19 - Standard Test Method for Isolation and Enumeration of <emph type="ital">Pseudomonas aeruginosa</emph> from Water
• Effective Date: 01-Apr-2024

Overview

ASTM D5246-24 is the internationally recognized standard test method for the isolation and enumeration of Pseudomonas aeruginosa from water. Developed by ASTM International’s Committee D19 on Water, this standard outlines a reliable and rapid membrane filtration technique to detect P. aeruginosa-an opportunistic pathogen-in water samples. Ensuring water quality is essential, given that P. aeruginosa can cause infections, especially in immunocompromised individuals, and is transmitted via contaminated water supplies.

Key Topics

• Pathogen Detection: Focused on identifying and quantifying Pseudomonas aeruginosa, an aerobic, motile, gram-negative bacterium that produces distinctive pigments and exhibits resistance to many antibiotics.
• Sample Types: Applicable to a broad range of water sources, including:
  • Surface waters
  • Recreational waters
  • Ground water
  • Rural nonchlorinated water supplies
  • Wastewater
  • Saline waters
• Detection Limit: As low as one microorganism per 100 mL, ensuring sensitivity for public health and environmental protection.
• Membrane Filtration Method: Utilizes a 0.45 μm membrane filter and selective media for efficient colony isolation and counting.
• Quality Assurance: Includes requirements for positive and negative controls, sterility checks for apparatus and media, and standardized reporting of results.

Applications

ASTM D5246-24 serves as an essential tool for environmental laboratories, water utilities, public health agencies, and industries requiring water quality monitoring. Key practical uses include:

• Municipal and Rural Water Supply Monitoring: Routine surveillance for P. aeruginosa in both chlorinated and nonchlorinated systems, with emphasis on rural, small-scale, or private supplies.
• Regulatory Compliance: Supports water quality standards and helps facilities meet health and environmental regulations linked to microbial contaminants.
• Industrial Water Testing: Applied in processes where P. aeruginosa presence can compromise product safety or system integrity, such as in cooling towers, pharmaceutical manufacturing, and food processing.
• Recreational Water Safety: Validates the biological safety of pools, spas, and natural bathing areas by monitoring for pathogen levels that pose health risks.
• Wastewater Treatment Validation: Supports assessment of treatment efficacy and monitoring of effluent to prevent environmental contamination.

Related Standards

For comprehensive water quality monitoring and laboratory practice, refer to these related ASTM standards:

• ASTM D1129 - Terminology Relating to Water
• ASTM D1193 - Specification for Reagent Water
• ASTM D2777 - Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
• ASTM D3370 - Practices for Sampling Water from Flowing Process Streams

These standards provide foundational terminology, sampling techniques, and technical requirements to ensure the accuracy and reliability of water testing results.

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Keywords: Pseudomonas aeruginosa, water testing, membrane filtration, ASTM D5246, water microbiology, pathogen detection, water quality standards, environmental monitoring, public health.

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
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.
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
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
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.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
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
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 to adverse environmental factors that lower their
loose-fitting lids; or 15 mm × 100 mm.
probability for survival and growth on a membrane filter
7.10.2 Membrane filtration units (filter base and funnel),
medium. This effect may be pronounced in this test method due
glass, plastic or stainless steel, wrapped with aluminum foil or
to the presence of antibiotics and the elevated incubation
kraft paper and sterilized. Purchased disposable plastic sterile
temperature.
filters can also be used.
6.2 The selection of an appropriate dilution volume is
7.10.3 Membrane filters, sterile, white, grid marked, 47 mm
essential. Too small a dilution volume may fail to detect any P.
diameter, with 0.45 μm 6 0.02 μm pore size.
aeruginosa organisms, while too large a volume may cause an
7.10.4 Ultraviolet unit (254 nm), for sanitization of the filter
overabundance of colonies that would interfere with an accu-
funnel between filtrations (optional).
rate count.
7.10.5 Line vacuum, electric vacuum pump, or aspirator, for
6.3 Chemicals or a combination of chemicals in certain use as a vacuum source. In an emergency or in the field, a hand
samples can have a toxic effect upon P. aeruginosa when pump or a syringe equipped with a check valve to prevent the
return flow of air, can be used.
concentrated.
7.10.6 Filter flask, vacuum, usually 1 L, with appropriate
6.4 Turbidity in samples may clog filter or effect color
tubing.
detection of organisms that develop on the filter.
7.10.7 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
7.10.8 Membrane filters, sterile, white, grid marked, 47 mm
Practices D3370 to address chlorinated water samples.
diameter, with 0.45 μm 6 0.02 μm pore size.
7.10.9 Flame or electric incinerator for sterilizing metal
7. Apparatus
inoculating loops and forceps.
7.1 Equipment for collection and transport of samples to
7.11 Forceps, straight or curved, with smooth tips to handle
laboratory:
filters.
7.1.1 Autoclavable Sample Container—Use sterile, non-
7.12 Incubator, hot air or water-jacketed microbiological
toxic, glass or plastic containers with a leak-proof lid. Ensure
type to maintain a temperature of 41.5 °C 6 0.5 °C and
that the sample container is capable of holding a 1 L sample
35.0 °C 6 0.5 °C.
with ample headspace to facilitate mixing of sample by shaking
prior to analysis. 7.13 Magnification of 10× to 15× with a cool white
7.1.2 Ice Chest. fluorescent light or optional stereoscopic microscope.
7.1.3 Ice Packs.
7.14 Colony counting device, mechanical, electric or hand
tally (optional).
7.2 Rinse water bottles, sterile, polypropylene or glass.
7.3 Pipettes, sterile, plastic or autoclavable glass pipettes 7.15 UV lamp, 6 W, 365 nm.
with a 2.5 % tolerance (Class A) with pipette bulbs or
8. Reagents and Materials
automatic pipette. Automatic pipettors and associated sterile
pipette tips capable of delivering up to 10 mL and 1 mL 8.1 Purity of Reagents—Reagent grade chemicals shall be
volumes (optional).
used in all tests. Unless otherwise indicated, it is intended that
all reagents shall conform to the specifications of the Commit-
7.4 Pipette container, autoclavable stainless steel, aluminum
tee on Analytical Reagents of the American Chemical Society,
or borosilicate glass (if using glass pipettes).
where such specifications are available. Other grades may be
7.5 Inoculation loops, at least 3 mm diameter, and needles,
used, provided it is first ascertained that the reagent is of
nichrome or platinum wire, 26 B&S gauge, in suitable holders.
sufficiently high purity to permit its use without lessening the
Sterile disposable applicator sticks or plastic loops are alterna-
accuracy of the determination.
tives to inoculation loops.
8.2 Purity of Water—Unless otherwise indicated, references
7.6 Graduated cylinders, from 100 mL to 1 L, sterile,
to water shall be understood to mean reagent water conforming
polypropylene or glass covered with aluminum foil or kraft
to Type II of Specification D1193.
paper.
8.3 Buffered Water—Dispense 1.25 mL of buffered water
7.7 Temperature monitoring device, glass, dial, or elec-
stock solution (see 8.4) and 5.0 mL magnesium chloride
tronic thermometer, graduated in 0.5 °C increments, checked
solution (see 8.5) and dilute to 1 L with water. Dispense in
against a National Institute of Standards and Technology
(NIST) certified thermometer or one traceable to an NIST
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
thermometer.
Standard-Grade Reference Materials, American Chemical Society, Washington,
7.8 Balance, with a readability of 0.1 g. DC. For suggestions on the testing of reagents not listed by the American Chemical
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
7.9 pH Meter, with accuracy 60.1 units and equipped with
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
surface electrode. copeial Convention, Inc. (USPC), Rockville, MD.
D5246 − 24
amount to provide 99 mL after sterilization. This can be 9.3 Soybean Casein Digest Agar —Formula per litre of
purchased or prepared in the laboratory. Add after stock water (it is recommended this should be purchased and not
prepared from individual ingredients):
solution (see 8.4). Add after 99 mL or volume as required
based on dilution.
Pancreatic digest of casein 15.0 g
Papaic digest of soybean meal 5.0 g
NOTE 2—Sterile buffered water can be purchased or made in the Sodium chloride 5.0 g
Agar 15.0 g
laboratory.
9.3.1 Soybean Casein Digest Agar—Prepare the media ac-
8.4 Buffered Water Stock—Dissolve 34.0 g potassium dihy-
cording to manufacturer’s instructions and dispense it asepti-
drogen phosphate (KH PO ) in 500 mL water, adjust to pH 7.2
2 4
cally into sterile petri dishes. (It is recommended this should be
with KOH solution (5.6 g/L) and dilute to 1 L with water.
purchased and not prepared from individual ingredients.)
8.5 Magnesium Chloride Solution (81.1 g/L)—Dissolve
81.1 g magne
...

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
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
2.1 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.
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.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. 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
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
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 that lower their probability for
survival and growth on a membrane filter medium. This effect may be pronounced in this test method due to the presence of
antibiotics and the elevated incubation temperature.
6.2 The selection of an appropriate dilution volume is essential. Too small a dilution volume may fail to detect any P. aeruginosa
organisms, while too large a volume may cause an overabundance of colonies that would interfere with an accurate count.
6.3 Chemicals or a combination of chemicals in certain samples can have a toxic effect upon P. aeruginosa when concentrated.
6.4 Turbidity in samples may clog filter or effect color detection of organisms that develop on the filter.
6.5 Water samples containing residual chlorine can be detrimental to P. aeruginosa. Utilize the procedure defined in Practices
D3370 to address chlorinated water samples.
7. Apparatus
7.1 Equipment for collection and transport of samples to laboratory:
7.1.1 Autoclavable Sample Container—Use sterile, non-toxic, glass or plastic containers with a leak-proof lid. Ensure that the
sample container is capable of holding a 1-L1 L sample with ample headspace to facilitate mixing of sample by shaking prior to
analysis.
7.1.2 Ice Chest.
7.1.3 Ice Packs.
7.2 Rinse water bottles, Rinse water bottles, sterile, polypropylene or glass.
7.3 Pipettes, Pipettes, sterile, plastic or autoclavable glass pipettes with a 2.5 % tolerance (Class A) with pipette bulbs or automatic
pipette. Automatic pipettors and associated sterile pipette tips capable of delivering up to 10 mL and 1 mL 1 mL volumes
(optional).
Available from BBL Microbiological Systems, Division of Becton Dickinson and Co., Cockeysville, MD 21030. Other suppliers may be utilized if equivalent.
D5246 − 24
7.4 Pipette container, Pipette container, autoclavable stainless steel, aluminum or borosilicate glass (if using glass pipettes).
7.5 Inoculation loops, Inoculation loops, at least 3 mm diameter, and needles, nichrome or platinum wire, 26 B&S gauge, in
suitable holders. Sterile disposable applicator sticks or plastic loops are alternatives to inoculation loops.
7.6 Graduated cylinders, Graduated cylinders from 100 mL to 1 L, sterile, polypropylene or glass covered with aluminum foil or
kraft paper.
7.7 Temperature monitoring device, glass, dial, or electronic thermometer, Temperature monitoring device, glass, dial, or
electronic thermometer graduated in 0.5°Cgraduated in 0.5 °C increments, checked against a National Institute of Standards and
Technology (NIST) certified thermometer or one traceable to an NIST thermometer.
7.8 Balance, Balance, with a readability of 0.1 g.
7.9 pH Meter, pH Meter, with accuracy 60.1 units and equipped with surface electrode.
7.10 Equipment for membrane filter procedure:
7.10.1 Petri dishes, Petri dishes, sterile, plastic, 99 mm × 50 mm, with tight-fitting lids; or 1515 mm × 60 mm, glass or plastic,
with loose-fitting lids; or 1515 mm × 100 mm.
7.10.2 Membrane filtration units (filter base and funnel), Membrane filtration units (filter base and funnel), glass, plastic or
stainless steel, wrapped with aluminum foil or kraft paper and sterilized. Purchased disposable plastic sterile filters can also be
used.
7.10.3 Membrane filters, Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45 6 0.02 μm 0.45 μm 6 0.02 μm
pore size.
7.10.4 Ultraviolet unit (254 nm), Ultraviolet unit for sanitization of the filter funnel between filtrations (optional).
7.10.5 Line vacuum, electric vacuum pump, or aspirator, Line vacuum, electric vacuum pump, or aspirator for use as a vacuum
source. In an emergency or in the field, a hand pump or a syringe equipped with a check valve to prevent the return flow of air,
can be used.
7.10.6 Filter flask, Filter flask, vacuum, usually 1 L, with appropriate tubing.
7.10.7 Flask for safety trap, Flask for safety trap placed between the filter flask and the vacuum source.
7.10.8 Membrane filters, Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45 6 0.02 μm 0.45 μm 6 0.02 μm
pore size.
7.10.9 Flame or electric incinerator Flame or electric incinerator for sterilizing metal inoculating loops and forceps.
7.11 Forceps, Forceps, straight or curved, with smooth tips to handle filters.
7.12 Incubator, Incubator, hot air or water-jacketed microbiological type to maintain a temperature of 41.5 6 0.5°C and 35.0 6
0.5°C.41.5 °C 6 0.5 °C and 35.0 °C 6 0.5 °C.
7.13 Magnification of 10× to 15× Magnification of 10–15× with a cool white fluorescent light or optional stereoscopic
microscope.
7.14 Colony counting device, Colony counting device, mechanical, electric or hand tally (optional).
D5246 − 24
7.15 UV lamp, 365-nm UV lamp.6 W, 365 nm.
8. Reagents and Materials
8.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where
such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high
purity to permit its use without lessening the accuracy of the determination.
8.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water conforming to
Type II of Specification D1193.
8.3 Buffered Water—Dispense 1.25 mL of buffered water stock solution (see 8.4) and 5.0 mL magnesium chloride solution (see
8.5) and dilute to 1 L with water. Dispense in amount to provide 99 mL after sterilization. This can be purchased or prepared in
the laboratory. Add after stock solution (see 8.4). Add after 99 mL or volume as required based on dilution.
NOTE 2—Sterile buffered water can be purchased or made in the laboratory.
8.4 Buffered Water Stock—Dissolve 34.0 g potassium dihydrogen phosphate (KH PO ) in 500 mL water, adjust to pH 7.2 with
2 4
KOH solution (5.6 g/L) and dilute to 1 L with water.
8.5 Magnesium Chloride Solution (81.1 g/L)—Dissolve 81.1 g magnesium chloride (MgCl 6H O) in water and dilute to 1 L with
2 2
water.
8.6 Potassium Hydroxide Solution (5.6 g/L)—Dissolve 5.6 g of potassium hydroxide (KOH) in water and dilute to 1 L with water.
9. Media Preparation
9.1 M-PA-C Medium —Formula per litre of water:
L-lysine 5.0 g
Sodium chloride 5.0 g
Yeast extract 2.0 g
Xylose 1.25 g
Sucrose 1.25 g
Lactose 1.25 g
Phenol red 0.08 g
Ferric ammonium citrate, anhydrous 0.80 g
Sodium thiosulfate, anhydrous 5.0 g
Agar 12.0 g
Magnesium sulfate, anhydrous 1.5 g
Kanamycin 0.008 g
Nalidixic acid 0.037 g
9.1.1 M-PA-C Medium or Equivalent—Dissolve mixture of above items into 1 L of water, boiling for 1 min to solubilize the
chemicals. Cool to 45 to 50°C45 °C to 50 °C before dispensing. Pour one plate of medium a
...