ASTM D5392-24

Standard Test Method for Isolation and Enumeration of <emph type="bdit">Escherichia coli</emph> in Water by the Two-Step Membrane Filter Procedure

SIGNIFICANCE AND USE
5.1 This test method is useful for measuring recreational water quality and chlorinated wastewaters, although it can be used for any water suspected of contamination by fecal wastes of warm-blooded animals. The significance of finding E. coli in recreational water samples, especially samples obtained from fresh recreational waters, is that there is a risk of gastrointestinal illness, directly related to the E. coli density, associated with swimming.5  
5.2 Since small or large volumes of water or dilutions thereof can be analyzed by the MF technique, a wider range of levels of E. coli in water can be detected and enumerated than with other methods.
SCOPE
1.1 This test method describes a membrane filter (MF) procedure for the detection and enumeration of Escherichia coli, a bacterium found exclusively in the feces of humans and other warm-blooded animals. The presence of these microorganisms in water is an indication of fecal pollution and the possible presence of enteric pathogens. These bacteria are found in water and wastewater in a wide range of densities. The detection limit of this procedure is one colony forming unit (CFU) per volume filtered.  
1.2 This test method has been used successfully with temperate fresh and marine ambient waters, and wastewaters. It is the user’s responsibility to ensure the validity of this test method for waters of other types.  
1.3 The values stated in SI units are to be regarded as 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. For specific hazard statements, see Section 9.  
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 D5392-19 - Standard Test Method for Isolation and Enumeration of <emph type="bdit">Escherichia coli</emph> in Water by the Two-Step Membrane Filter Procedure
• Effective Date: 01-Apr-2024
• Referred By: ASTM D5465-16(2020) - Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
• Effective Date: 01-Apr-2024
• Referred By: ASTM D8506-23 - Standard Guide for Microbial Contamination and Biodeterioration in Turbine Oils and Turbine Oil Systems
• Effective Date: 01-Apr-2024

Overview

ASTM D5392-24 is the Standard Test Method for Isolation and Enumeration of Escherichia coli (E. coli) in Water by the Two-Step Membrane Filter Procedure, developed by ASTM International. This method is widely used to detect and quantify E. coli-a key indicator of fecal contamination-in various water types, including recreational waters, wastewaters, and ambient surface waters. Detection of E. coli is crucial for assessing water safety, particularly due to its direct association with gastrointestinal health risks in humans following exposure to contaminated water during activities like swimming.

This membrane filter (MF) method allows for sensitive detection, with a lower limit of one colony-forming unit (CFU) per volume filtered, making it a valuable tool for both low- and high-contamination scenarios.

Key Topics

• Purpose & Significance

  • Detects E. coli, indicating recent fecal pollution and possible presence of pathogens.
  • Enables health risk assessment in recreational and potable waters.
  • Facilitates regulatory compliance and monitoring for public health protection.

• Test Method Overview

  • Utilizes a two-step incubation process on selective mTEC agar.
  • Suitable for a variety of water matrices, including fresh and marine waters, and chlorinated wastewaters.
  • Applicable for a broad concentration range due to adaptability in sample volume and dilution.
  • Achieves enumeration by direct count of bacterial colonies observable on the membrane.

• Quality & Limitations

  • Offers high specificity and acceptable levels of precision and bias.
  • Potential interference from high turbidity or chemicals toxic to bacteria.
  • Verification steps support accurate identification of E. coli colonies.

• Health & Safety Considerations

  • Good laboratory practices and safety protocols are essential.
  • Sample handling, incubator calibration, and equipment maintenance are critical for valid results.

Applications

• Recreational Water Quality

  • Helps public health officials evaluate swimming lakes, beaches, and pools for microbial safety.
  • Informs timely issuance of advisories or closures in case of contamination.

• Wastewater Monitoring

  • Used in wastewater treatment plants to assess effluent quality and regulatory compliance.
  • Monitors disinfection effectiveness in treated wastewater before environmental discharge.

• Environmental Water Surveillance

  • Assesses contamination in rivers, reservoirs, and estuaries.
  • Supports watershed management and pollution source tracking.

• Research and Regulatory Compliance

  • Used in scientific studies investigating microbial water quality.
  • Meets requirements for water quality monitoring programs and standards under various national and international regulations.

Related Standards

The following ASTM standards are referenced or relevant to ASTM D5392-24:

• ASTM D1129 – Terminology Relating to Water: Provides definitions critical for understanding water-related test methods.
• ASTM D1193 – Specification for Reagent Water: Describes the quality and purity required for water used in analytical testing.
• ASTM D3370 – Practices for Sampling Water from Flowing Process Streams: Details best practices for water sample collection.
• ASTM D5465 – Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods: Offers guidelines for accurate enumeration of microbial colonies.

Practical Value

Implementing ASTM D5392-24 ensures reliable detection and enumeration of E. coli in water, supporting proactive water quality management, protection of public health, and adherence to environmental standards. By enabling laboratories, utilities, and regulatory agencies to effectively monitor and respond to contamination events, this standard underpins both routine water assessments and investigations of waterborne disease outbreaks.

Keywords:E. coli, water quality, membrane filter, recreational water, wastewater, fecal pollution, ASTM D5392-24, mTEC agar, enumeration, environmental monitoring.

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: D5392 − 24
Standard Test Method for
Isolation and Enumeration of Escherichia coli in Water by
the Two-Step Membrane Filter Procedure
This standard is issued under the fixed designation D5392; 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
D3370 Practices for Sampling Water from Flowing Process
1.1 This test method describes a membrane filter (MF)
Streams
procedure for the detection and enumeration of Escherichia
D3870 Practice for Establishing Performance Characteristics
coli, a bacterium found exclusively in the feces of humans and
for Colony Counting Methods in Microbiology (With-
other warm-blooded animals. The presence of these microor-
drawn 2000)
ganisms in water is an indication of fecal pollution and the
D5465 Practices for Determining Microbial Colony Counts
possible presence of enteric pathogens. These bacteria are
from Waters Analyzed by Plating Methods
found in water and wastewater in a wide range of densities. The
detection limit of this procedure is one colony forming unit
3. Terminology
(CFU) per volume filtered.
3.1 Definitions:
1.2 This test method has been used successfully with
3.1.1 For definitions of terms used in this standard, refer to
temperate fresh and marine ambient waters, and wastewaters. It
Terminology D1129.
is the user’s responsibility to ensure the validity of this test
method for waters of other types.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 Escherichia coli (E. coli), n—a species of bacteria that
1.3 The values stated in SI units are to be regarded as
is a member of the total coliform group and known to originate
standard.
in the feces of warm-blooded animals.
1.4 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the 3.3 Performance Characteristics (Practice D3870):
responsibility of the user of this standard to establish appro-
3.3.1 accuracy, n—the proportion of the observed count to
priate safety, health, and environmental practices and deter-
the true density of a sample.
mine the applicability of regulatory limitations prior to use.
3.3.2 bias, n—the persistent positive or negative deviation
For specific hazard statements, see Section 9.
of the average value of the test method from the assumed or
1.5 This international standard was developed in accor-
accepted true value.
dance with internationally recognized principles on standard-
3.3.3 precision, n—the degree of agreement of repeated
ization established in the Decision on Principles for the
measurements of the same parameter expressed quantitatively
Development of International Standards, Guides and Recom-
as the standard deviation or as the 95 % confidence limits of
mendations issued by the World Trade Organization Technical
the mean computed from the results of a series of controlled
Barriers to Trade (TBT) Committee.
determinations.
2. Referenced Documents
3.3.4 specificity, n—the ability of a test method to select or
distinguish, or both, the target bacteria in the same water
2.1 ASTM Standards:
sample; the specificity characteristic of the method is usually
D1129 Terminology Relating to Water
reported as the percent of false positive and false negative
results.
This test method is under the jurisdiction of ASTM Committee D19 on Water
3.3.5 upper counting limit (UCL), n—that colony count
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
above which there is an unacceptable counting error; the error
Current edition approved April 1, 2024. Published April 2024. Originally
may be due to overcrowding or antibiosis.
approved in 1993. Last previous edition approved in 2019 as D5392 – 19. DOI:
10.1520/D5392-24.
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 last approved version of this historical standard is referenced on
the ASTM website. www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5392 − 24
4. Summary of Test Method 7.2 Microscope Lamp, producing diffuse light from a cool,
4 white fluorescent lamp adjusted to give maximum visibility.
4.1 This two-step test method provides a direct count of
bacterial colonies developing on the surface of the filter when 7.3 Counting Device, hand tally or electronic.
placed on a selective nutrient medium. The water sample is
7.4 Pipet Container, stainless steel, aluminum, or borosili-
passed through a membrane filter that retains the bacteria. After
cate glass, for glass pipets.
filtration, the membrane filter containing the bacterial cells is
7.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or
placed on a selective, differential medium, mTEC. The mem-
plastic, of appropriate volume.
brane on the medium is first incubated at 35 °C for 2 h so that
injured or stressed bacteria can be resuscitated and then the
7.6 Graduated Cylinders, 100 mL to 1000 mL, covered with
medium is incubated at 44.5 °C for 22 h. Following incubation, aluminum foil or kraft paper and sterile.
the filter is transferred to a filter pad saturated with urea
7.7 Membrane Filtration Units (filter base and funnel),
substrate. After 15 min all yellow or yellow-brown colonies are
glass, plastic, or stainless steel, wrapped in aluminum foil or
counted with the aid of 10× to 15× magnifier and a fluorescent
kraft paper and sterilized or disposable sterile units.
lamp.
7.8 Ultraviolet Unit (254 nm), for sterilizing the filtration
unit (optional).
5. Significance and Use
7.9 Line Vacuum, Electric Vacuum Pump, or Aspirator, for
5.1 This test method is useful for measuring recreational
use as a vacuum source. In an emergency or in the field, a hand
water quality and chlorinated wastewaters, although it can be
pump or a syringe equipped with a check valve to prevent the
used for any water suspected of contamination by fecal wastes
return flow of air, can be used.
of warm-blooded animals. The significance of finding E. coli in
recreational water samples, especially samples obtained from
7.10 Flask, filter, vacuum, usually 1 L, with appropriate
fresh recreational waters, is that there is a risk of gastrointes-
tubing. A filter manifold to hold a number of filter bases is
tinal illness, directly related to the E. coli density, associated
optional.
with swimming.
7.11 Forceps, straight or curved, with smooth tips to handle
5.2 Since small or large volumes of water or dilutions
filters without damage.
thereof can be analyzed by the MF technique, a wider range of
7.12 Thermometers, checked against a National Institute of
levels of E. coli in water can be detected and enumerated than
Standards and Technology (NIST) certified thermometer, or
with other methods.
one traceable to a NIST thermometer.
6. Interferences
7.13 Petri Dishes, sterile, plastic, 50 mm by 12 mm, with
tight-fitting lids.
6.1 Water with high levels of colloidal or suspended mate-
rials can clog the membrane filter pores and prevent filtration.
7.14 Bottles, milk dilution, borosilicate glass, screw-cap
Also, suspended materials cause spreading colonies that could
with neoprene liners, marked at 99 mL for 1 to 100 dilutions.
interfere with target colonies and thereby prevent accurate
Dilution bottles marked at 90 mL or tubes marked at 9 mL may
counting.
be used for 1 to 10 dilutions.
6.2 Smaller sample size or sample dilution can be used to
7.15 Inoculation Loops, at least 3 mm diameter, and
minimize the interference of turbidity or high background
needles, nichrome or platinum wire, 26 B & S gauge, in
(nontarget) bacterial densities. Replicates of sample volumes
suitable holders.
or dilutions of sample may be filtered and the results combined.
7.16 Incubator, air, maintained at 35 °C 6 0.5 °C.
However, the membrane filter techniques may not be appli-
7.17 Incubator, Waterbath, maintained at 44 °C 6 0.2 °C.
cable to high turbid waters with low bacterial densities.
7.18 Test Tubes, 150 mm by 20 mm, borosilicate glass or
6.3 In some samples, chemicals may have toxic effects on
plastic.
the target organism.
7.19 Test Tubes, 75 mm by 10 mm, borosilicate glass.
7. Apparatus
7.20 Caps, aluminum or autoclavable plastic, for 20 mm
7.1 Stereoscopic Microscope, wide-field type with magnifi-
diameter test tubes.
cation of 10× to 15×.
7.21 Test Tubes, screw-cap, borosilicate glass, 125 mm by
16 mm or other appropriate size.
Dufour, A., Strickland, E., and Cabelli, V., “Membrane Filter Method for
8. Reagents and Materials
Enumerating Escherichi coli,” Applied and Environmental Microbiology, Vol 41,
1981, pp. 1152–1158.
8.1 Purity of Reagents—Reagent grade chemicals shall be
Cabelli, V. J., Dufour, A. P., Levin, M. A., McCabe, L. J., and Haberman, P. W.,
used in all tests. Unless otherwise indicated, it is intended that
“Relationship of Microbial Indicators to Health Effects at Marine Bathing Beaches,”
American Journal of Public Health, Vol 69, 1979, pp. 690–696. all reagents conform to the specifications of the Committee on
D5392 − 24
Analytical Reagents of the American Chemical Society where 8.7.2 Dissolve the ingredients and bring the pH of the
such specifications are available. Other grades may be used, solution to between 4.5 and 5.2. The substrate should be a
provided it is first ascertained that the reagent is of sufficiently straw yellow color at this pH range.
high purity to permit its use without lessening the accuracy of
8.8 Cytochrome Oxidase Reagent (1 g/L)—Dissolve 1 g of
the determination. The agar used in preparation of culture
N,N,N',N'-tetramethyl-p-phenylenediamine-dihydrochloride in
media must be of microbiological grade. Whenever possible,
water and dilute to 100 mL.
use commercial culture media and reagents as means of quality
8.9 Kovac’s Indole Reagent (acid/alcohol solution)—
control.
Dissolve 10 g p-dimethylaminobenzaldehyde in 150 mL of
8.2 Purity of Water—Unless otherwise indicated, references
amyl or isoamyl alcohol, then slowly add 50 mL of concen-
to water shall be understood to mean reagent water as defined
trated hydrochloric acid and mix. The indole reagent is also
by Type III of Specification D1193.
commercially available. (Warning—This is highly flammable
8.3 Ethanol, Methanol, or Isopropanol, denatured, in a
and corrosive; take precaution when handling.)
small, wide-mouth container, for flame-sterilization or pipets.
8.10 Tryptic Soy Broth (TSB):
8.4 Membrane Filters, sterile, white, grid marked, 47 mm
8.10.1 Composition per Litre:
diameter, with 0.45 μm 6 0.02 μm pore size or other pore sizes
Casein Peptone (pancreatic) 17.0 g
for which the manufacturer provides data demonstrating
Dipotassium Hydrogen Phosphate 2.5 g
Dextrose (glucose) 2.5 g
equivalency.
Sodium Chloride 5.0 g
Soy Peptone 3.0 g
8.5 Buffered Dilution Water/Buffered Rinse Water:
8.5.1 Composition per Litre:
8.10.2 Procedure—Follow vendor recommended procedure.
Sodium Dihydrogen Phosphate (NaH PO ) 0.58 g
2 4 8.10.2.1 Add 30 g of TSB in 1 L of reagent water and mix
Sodium Monohydrogen Phosphate (Na HPO ) 2.50 g
2 4
to dissolve. If necessary, warm slightly to dissolve.
Sodium Chloride 8.50 g
8.10.2.2 Dispense as required, such as 5 mL to 10 mL into
8.5.2 Preparation—Dissolve the ingredients in 1 L of water
tubes.
in a flask and dispense in appropriate amounts for dilutions in
8.10.2.3 Sterilize in the autoclave at 121 °C for 15 min to
screw-cap bottles or culture tubes or into containers for use as
20 min at 15 lb pressure.
rinse water, or both. Autoclave after preparation at 121 °C for
8.10.2.4 Final pH should be 7.3 6 0.2 at 25 °C.
15 min. The final pH is 7.4 6 0.2.
8.11 Simmon’s Citrate Agar:
8.6 mTEC Agar:
8.11.1 Composition per Litre:
8.6.1 Composition per Litre:
Magnesium Sulfate (7H O) 0.2 g
Proteose Peptone 5.0 g
Ammonium Dihydrogen Phosphate 1.0 g
Yeast Extract 3.0 g
Dipotassium Hydrogen Phosphate 1.0 g
Lactose 10.0 g
Sodium Citrate (2H O) 2.0 g
Sodium Chloride 7.5 g
Sodium Chloride 5.0 g
Dipotassium Phosphate 3.3 g
Agar 15.0 g
Monopotassium Phosphate 1.0 g
Bromothymol Blue 0.08 g
Sodium Lauryl Sulfate 0.2 g
Sodium Desoxycholate 0.1 g
8.11.2 Add 24.2 g in 1 L of reagent water and heat to boiling
Brom Cresol Purple 0.08 g
to dissolve completely while mixing.
Brom Phenol Red 0.08 g
Agar 15.0 g
8.11.3 Dispense in
...

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: D5392 − 19 D5392 − 24
Standard Test Method for
Isolation and Enumeration of Escherichia coli in Water by
the Two-Step Membrane Filter Procedure
This standard is issued under the fixed designation D5392; 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 This test method describes a membrane filter (MF) procedure for the detection and enumeration of Escherichia coli, a
bacterium found exclusively in the feces of humans and other warm-blooded animals. The presence of these microorganisms in
water is an indication of fecal pollution and the possible presence of enteric pathogens. These bacteria are found in water and
wastewater in a wide range of densities. The detection limit of this procedure is one colony forming unit (CFU) per volume filtered.
1.2 This test method has been used successfully with temperate fresh and marine ambient waters, and wastewaters. It is the user’s
responsibility to ensure the validity of this test method for waters of other types.
1.3 The values stated in SI units are to be regarded as 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. For specific hazard statements, see Section 9.
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
D3370 Practices for Sampling Water from Flowing Process Streams
D3870 Practice for Establishing Performance Characteristics for Colony Counting Methods in Microbiology (Withdrawn 2000)
D5465 Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
3. Terminology
3.1 Definitions:
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 April 1, 2019April 1, 2024. Published April 2019April 2024. Originally approved in 1993. Last previous edition approved in 20142019 as
D5392 – 14.D5392 – 19. DOI: 10.1520/D5392-19.10.1520/D5392-24.
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.
The last approved version of this historical standard is referenced on www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5392 − 24
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 Escherichia coli (E. coli), n—a species of bacteria that is a member of the total coliform group and known to originate in
the feces of warm-blooded animals.
3.3 Performance Characteristics (Practice D3870):
3.3.1 accuracy, n—the proportion of the observed count to the true density of a sample.
3.3.2 bias, n—the persistent positive or negative deviation of the average value of the test method from the assumed or accepted
true value.
3.3.3 precision, n—the degree of agreement of repeated measurements of the same parameter expressed quantitatively as the
standard deviation or as the 95 % confidence limits of the mean computed from the results of a series of controlled determinations.
3.3.4 specificity, n—the ability of a test method to select or distinguish, or both, the target bacteria in the same water sample; the
specificity characteristic of the method is usually reported as the percent of false positive and false negative results.
3.3.5 upper counting limit (UCL), n—that colony count above which there is an unacceptable counting error; the error may be due
to overcrowding or antibiosis.
4. Summary of Test Method
4.1 This two-step test method provides a direct count of bacterial colonies developing on the surface of the filter when placed
on a selective nutrient medium. The water sample is passed through a membrane filter that retains the bacteria. After filtration, the
membrane filter containing the bacterial cells is placed on a selective, differential medium, mTEC. The membrane on the medium
is first incubated at 35°C for 2 h 35 °C for 2 h so that injured or stressed bacteria can be resuscitated and then the medium is
incubated at 44.5°C for 22 h. 44.5 °C for 22 h. Following incubation, the filter is transferred to a filter pad saturated with urea
substrate. After 15 min 15 min all yellow or yellow-brown colonies are counted with the aid of 1010× to 15× magnifier and a
fluorescent lamp.
5. Significance and Use
5.1 This test method is useful for measuring recreational water quality and chlorinated wastewaters, although it can be used for
any water suspected of contamination by fecal wastes of warm-blooded animals. The significance of finding E. coli in recreational
water samples, especially samples obtained from fresh recreational waters, is that there is a risk of gastrointestinal illness, directly
related to the E. coli density, associated with swimming.
5.2 Since small or large volumes of water or dilutions thereof can be analyzed by the MF technique, a wider range of levels of
E. coli in water can be detected and enumerated than with other methods.
6. Interferences
6.1 Water with high levels of colloidal or suspended materials can clog the membrane filter pores and prevent filtration. Also,
suspended materials cause spreading colonies that could interfere with target colonies and thereby prevent accurate counting.
6.2 Smaller sample size or sample dilution can be used to minimize the interference of turbidity or high background (nontarget)
bacterial densities. Replicates of sample volumes or dilutions of sample may be filtered and the results combined. However, the
membrane filter techniques may not be applicable to high turbid waters with low bacterial densities.
Dufour, A., Strickland, E., and Cabelli, V., “Membrane Filter Method for Enumerating Escherichi coli,” Applied and Environmental Microbiology, Vol 41, 1981, pp.
1152–1158.
Cabelli, V. J., Dufour, A. P., Levin, M. A., McCabe, L. J., and Haberman, P. W., “Relationship of Microbial Indicators to Health Effects at Marine Bathing Beaches,”
American Journal of Public Health, Vol 69, 1979, pp. 690–696.
D5392 − 24
6.3 In some samples, chemicals may have toxic effects on the target organism.
7. Apparatus
7.1 Stereoscopic Microscope, wide-field type with magnification of 1010× to 15×.
7.2 Microscope Lamp, producing diffuse light from a cool, white fluorescent lamp adjusted to give maximum visibility.
7.3 Counting Device, hand tally or electronic.
7.4 Pipet Container, stainless steel, aluminum, or borosilicate glass, for glass pipets.
7.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, of appropriate volume.
7.6 Graduated Cylinders, 100 to 1000 mL, 100 mL to 1000 mL, covered with aluminum foil or kraft paper and sterile.
7.7 Membrane Filtration Units (filter base and funnel), glass, plastic, or stainless steel, wrapped in aluminum foil or kraft paper
and sterilized.sterilized or disposable sterile units.
7.8 Ultraviolet Unit, Unit (254 nm), for sterilizing the filtration unit (optional).
7.9 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 Flask, filter, vacuum, usually 1 L, 1 L, with appropriate tubing. A filter manifold to hold a number of filter bases is optional.
7.11 Forceps, straight or curved, with smooth tips to handle filters without damage.
7.12 Thermometers, checked against a National Institute of Standards and Technology (NIST) certified thermometer, or one
traceable to a NIST thermometer.
7.13 Petri Dishes, sterile, plastic, 50 by 12 mm, 50 mm by 12 mm, with tight-fitting lids.
7.14 Bottles, milk dilution, borosilicate glass, screw-cap with neoprene liners, marked at 99 mL for 1 to 100 dilutions. Dilution
bottles marked at 90 mL or tubes marked at 9 mL may be used for 1 to 10 dilutions.
7.15 Inoculation Loops, at least 3-mm3 mm diameter, and needles, nichrome or platinum wire, 26 B & S gage,gauge, in suitable
holders.
7.16 Incubator, air, maintained at 35 6 0.5°C.35 °C 6 0.5 °C.
7.17 Incubator, Waterbath, maintained at 44 6 0.2°C.44 °C 6 0.2 °C.
7.18 Test Tubes, 150 by 20 mm, 150 mm by 20 mm, borosilicate glass or plastic.
7.19 Test Tubes, 75 by 10 mm, 75 mm by 10 mm, borosilicate glass.
7.20 Caps, aluminum or autoclavable plastic, for 20-mm20 mm diameter test tubes.
7.21 Test Tubes, screw-cap, borosilicate glass, 125 by 16 mm 125 mm by 16 mm or other appropriate size.
D5392 − 24
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 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. The agar used in preparation of culture media must be of
microbiological grade. Whenever possible, use commercial culture media and reagents as means of quality control.
8.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water as defined by Type
III of Specification D1193.
8.3 Ethanol, Methanol, or Isopropanol, denatured, in a small, wide-mouth container, for flame-sterilization or pipets.
8.4 Membrane Filters, sterile, white, grid marked, 47-mm47 mm diameter, with 0.450.45 μm 6 0.02 μm pore size or other pore
sizes for which the manufacturer provides data demonstrating equivalency.
8.5 Buffered Dilution Water/Buffered Rinse Water:
8.5.1 Composition per Litre:
Sodium Dihydrogen Phosphate (NaH PO ) 0.58 g
2 4
Sodium Monohydrogen Phosphate (Na HPO ) 2.50 g
2 4
Sodium Chloride 8.50 g
Sodium Dihydrogen Phosphate (NaH PO ) 0.58 g
2 4
Sodium Monohydrogen Phosphate (Na HPO ) 2.50 g
2 4
Sodium Chloride 8.50 g
8.5.2 Preparation—Dissolve the ingredients in 1 L of water in a flask and dispense in appropriate amounts for dilutions in
screw-cap bottles or culture tubes, and/ortubes or into containers for use as rinse water. water, or both. Autoclave after preparation
at 121°C for 15 min. 121 °C for 15 min. The final pH is 7.4 6 0.2.
8.6 mTEC Agar:
8.6.1 Composition per Litre:
Proteose Peptone 5.0 g
Yeast Extract 3.0 g
Lactose 10.0 g
Sodium Chloride 7.5 g
Dipotassium Phosphate 3.3 g
Monopotassium Phosphate 1.0 g
Sodium Lauryl Sulfate 0.2 g
Sodium Desoxycholate 0.1 g
Brom Cresol Purple 0.08 g
Brom Phenol Red 0.08 g
Agar 15.0 g
Proteose Peptone 5.0 g
Yeast Extract 3.0 g
Lactose 10.0 g
Sodium Chloride 7.5 g
Dipotassium Phosphate 3.3 g
Monopotassium Phosphate 1.0 g
Sodium Lauryl Sulfate 0.2 g
Sodium Desoxycholate 0.1 g
Reagent Chemicals, American Chemical Society Specifications,ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference
Materials, American Chemical Society, Washington, 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, U.K., and the United States Pharmacopeia and National Formulary, U.S. PharmaceuticalPharmacopeial Convention, Inc.
(USPC), Rockville, MD.
This is available commercially and is recommended to purchase and not prepare from individual components.
D5392 − 24
Brom Cresol Purple 0.08 g
Brom Phenol Red 0.08 g
Agar 15.0 g
8.6.2 Add 45.26 g of dehydrated mTEC medium to 1 L of water in a flask and heat to boiling, until ingredients dissolve. Autoclave
at 121°C121 °C for 15 min and pour into 1010 mm by 47 mm plates (4 mL/plate) after cooling. The pH of the medium is 7.3.7.3.
8.7 Urea Substrate Medium:
8.7.1 Composition per Litre:
Ingredients Grams/100 mL
Urea 2.01 g
Urea 2.01 g
Phenol Red 0.01 g
Phenol Red 0.01 g
8.7.2 Dissolve the ingredients and bring the pH of the solution to between 4.5 and 5.2.5.2. The substrate should be a straw yellow
color at this pH range.
8.8 Cytochrome Oxidase Reagent (1 g/L)—Dissolve 1 g of N,N,N',N'-tetramethyl-p-phenylenediamine-dihydrochloride in water
and dilute to 100 mL. The cytochrome oxidase reagent is also commercially available.
8.9 Kovac’s Indole Reagent (acid/alcohol solution)—Dissolve 10 g 10 g p-dimethylaminobenzaldehyde in 150 mL of amyl or
isoamyl alcohol, then slowly add 50 mL of concentrated hydrochloric acid and mix. The indole reagent is also commercially
available. (Warning—This is highly flammable and corrosive; take precaution when handling.)
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