United States " Office Of Water EPA/600/4-91/016
Environmental Protection (WH-550) JULY 1991
Agency
Research And Development Cincinnati. OH
vvEPA Test Methods For
Escherichia Coll In
Drinking Water
EC Medium With Mug Tube
Procedure
Nutrient Agar With Mug
Membrane Filter Procedure
Printed on Recycled Paper
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DISCLAIMER
This document has been reviewed in accordance with U, S, Environmental
Protection Agency policy and approved for publication. Mention of trade names
or commercial products does not constitute endorsement or recommendation for
use.
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FOREWORD
Environmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. The Environmental
Monitoring Systems Laboratory - Cincinnati (EMSL-Cincinnati) conducts research
to:
o Develop and evaluate analytical methods to identify and measure
the concentration of chemical pollutants in drinking waters,
surface waters, groundwaters, wastewaters, sediments, sludges, and
solid wastes.
o Investigate methods for the identification and measurement of
viruses, bacteria and other microbiological organisms in aqueous
samples and to determine the responses of aquatic organisms to
water quality.
o Develop and operate a quality assurance program to support the
achievement of data quality objectives in measurements of
pollutants in drinking water, surface water, groundwater,
wastewater, sediment and solid waste.
The two analytical methods described in this report can be used to
measure the bacteriological quality of potable water. They are approved for
determining compliance of public water systems with the National Primary
Drinking Water Regulations. The presence of Escherichia coli in drinking
water is an indication of fecal pollution and the possible presence of enteric
pathogens.
Thomas A. Clark, Director
Environmental Monitoring Systems
Laboratory - Cincinnati
ill
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Test Method 1104
Detection of Escherichia Coli in Drinking Water by the
EC Medium with Mug T\ibe Procedure
1. Scope and Application
1.1 This method describes a
procedure for thedetection and
enumeration of Escherichia
coli (E. coin in drinking water
by transfer from total colif orm-
positive presumptive tests to
EC + MUG medium. Because
this species is a natural
inhabitant only of the intestinal
tractofwarm-bloodedanimals, ;
its presence in water samples
is an indication of fecal'
pollution and the possible
presence of enteric pathogens.
1.2 This method can be applied to
positive presumptive results
from the multiple tube
fermentation (MTF), presence-
absence (P-A), and membrane
filter (MF) procedures for total
coliforms.
13 The detection limit of this
procedure is one micro-
organism per 100 mL.
1.4 The Total Coliform Rule1
requires that all total coliform-
positive cultures be tested for
either fecal coliforms or £. coli.
This method for £. coli is
approved in the National
Primary Drinking Water
Regulations; Analytical
Techniques; Coliform
Bacteria2.
2. Summary
2.1 Culture from total coliform-
positive tubes or bottles of
lauryl tryptose, lactose or P-A
medium, or from total coli form
MF colonies or entire surface
growth in the presumptive
phase of these procedures for
total coliforms is inoculated
into EC broth containing 4-
methylumbelliferyl-6-D-
glucuronide (EC + MUG) and
incubated at 44.5 ± 0.2°C for
24 hours3. Observance of
bright blue fluorescence when
subjected to long-wave (366
nm) ultraviolet (UV) light
indicates a positive test for £.
coli.
3. Definition
,3.1
In this method £. coli are
''.:,;\ definedasthosebacteriawhich
)• ', produce bright blue
5 fluorescence in EC + MUG
; medium after initial culture in .
lauryl tryptose, lactose or P-A
broth or on Endo MF plates.
4. Interferences
4.1 Certain brands of test tubes
fluoresce under long-wave UV
light and may interfere with
test results. Tubes should be
examined before use.
4.2 Do not use an inverted vial; gas
production is not relevant to
the test and observation for this
reaction may cause confusion
in test interpretation.
5. Safety Precautions
5.1 The analyst/technician must
know and observe the normal
safety procedures required in a
microbiology laboratory while
preparing, using, and disposing
of cultures, reagents and
materials and while operating
sterilization equipment.
6. Apparatus and Equipment
6.1 Water bath, gable-covered
incubator that maintains a44.5
+ 0.2°C temperature.
6.2 Lamp, ultraviolet, long-wave,
366 nm, preferably with a 6-
watt bulb.
6.3 Inoculation loop, 3 mm
diameterorneedleofnichrome
wire, 26 B &S gauge, in suitable
holder. Sterileapplicatorsticks
are a suitable alternative to
inoculation loops or needles.
Sterile cotton-tipped applicator
sticks are used for the transfer
of growth from the entire MF
surface by the swab technique.
6.4 Bunsen or Fisher-type burner
or electric incinerator unit.
6.5 Thermometer, glass/mercury
or dial calibrated in 0.2°C
increments or less, checked
against a National Bureau of
Standards (NBS) certified
thermometer, or one traceable
to an NBS thermometer.
6.6 Ethanol, methanol or
isopropanol in small, wide-
mouth container, for flame-
sterilizing forceps.
6.7 Pyrex test tubes, 150 x 20 mm.
6.8 Culture tube racks to hold 20
mm diameter tubes.
6.9 Flasks, borosilicate glass,
screw-cap, 250 - 1000 mL
volume.
7. Media and Reagents
7.1 Purity of Reagents: Reagent
grade chemicals shall be used
in all tests. Unless otherwise
indicated, reagents shall
conform to the specifications
of the Committee on Analytical
Reagents of the American
Chemical Society. The agar
used in preparation of culture
media must be of
microbiological grade.
7.2 Whenever possible, use
commercial culture media as a
means of quality control.
73 EC Medium with MUG (Difco
0022-17), EC Broth with MUG
(BBL12332) or equivalent
Composition:
Tryptose or Pancreatic Digest
of Casein Peptone 20.0 g
Lactose 5.0 g
Bile Salts No. 3 or
Bile Salts Mixture 1.5 g
Dipotassium Phosphate 4.0 g
Monopotassium Phosphate 1.5 g
Sodium Chloride 5.0 g
4-methyl-B-D-umbelliferyl
glucuronide (MUG) 0.05 g
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Reagent Grade Water
Final pH: 6.9 ±0.2
1L
Preparation: Add 37.1 grams
(Difco) or 37.0 grams (BBL)
of EC with MUG medium or
equivalent to 1 liter of water
and warm slightly to dissolve
completely. Dispense into
tubes 050x20 mm). Sterilize
for 12-15 minutes at 121°C (15
Ibs. pressure).
Alternatively, O.OSg MUG per
liter may be added to EC
Medium (Difco 0314.02), EC
Broth (BBL 11187) or equivalent
before autoclaving.
7.4 Check test tubes before use
with a 366 nm UV light to
ensure they do not fluoresce.
7.5 For quality control of the
medium, include a MUG-
positive (E. coin and MUG-
negatitfe (e.g., uninoculated)
control for each analysis or set
of analyses. Check medium
before use with a 366 nm UV
light to ensure it does not
fluoresce.
7.6 Incubate positive and negative
control cultures at 35 ± 0.5°C
for 24 hours in lauryl tryptose
broth. Transferaloopful toEC
medium with MUG and
incubateat44.5 ± 0.2°C for 24
hours. Read and record results.
7.7 Use prepared medium in tubes
with loose-fitting closures
within one week. Store sterile
refrigerated medium for up to
three months in screw-cap
tubes/containers, and incubate
stored medium overnight at
35°C before use; discard tubes
with growth.
8. Sample Collection, Preserv-
ation and Holding Time
8.1 This test method is a transfer
procedure from a proceeding
sample analysis; therefore, it
doesnotinvolvedirect analysis
of the water sample.
Consequently, sample coll-
ection, preservation and
holdingtimearenotprocedures
specifically applicable to this
method. However, adherence
to sample collection and
preservation procedures and
holding time limits for the
original water sample is critical
to the production of valid data.
9. Calibration and Standard-
ization
9.1 Check temperatures in
incubators daily to ensure
operation within stated limits.
9.2 Check thermometers at least
. annually against an NBS
certified thermometer or one
traceable to NBS. Check
mercury columns for breaks.
10. Quality Control
10.1 Verify at least 5% of both
MUG-positive results and
turbid total coliform-positive,
MUG-negative results.
Verification of a pure culture
may be performed by the use
of API 20 E or an equivalent
bacterial identification system;
standard biochemical tests (e.g.
citrate,indoleandurease tests);
serotyping after biochemical
identification if desired; or the
indole testat44°C and growth
in citrate.
10.2 See recommendations on
quality control for
microbiological analyses in
Standard Methods for the
Examination of Water and
Wastewater.4
11. Procedure
11.1 Gently swirl the presumptive
total coliform tube or bottle.
Using a sterile inoculating loop
or wooden applicator, transfer
inocula from total coliform-
positive presumptive phase
tubes or bottles at 24 hours (or
48 hours if needed)to EC +
MUG tubes.
Transfer inocula from total
coliform MF colonies with a
sterile needle or wooden
applicator stick. Alternatively,
use a sterilecotton-tipped swab
to transfer the entire surface
growth from a total coliform-
positiveMF plate to EC+MUG
tubes. Do not leave the cotton
swab in the medium. Gently
swirl inoculated EC + MUG
tubes to ensure mixing of
inoculum with medium.
11.2 Incubate inoculated EC +
MUG tubes at 44.5+0.2°C for
24 + 2 hours. Tubes must be
placed in the incubator within
30 minutes after inoculation.
The water depth in the water
bath incubator must come to
the top level of the culture
medium in the tube.
11.3 Detect fluorescence using an
ultraviolet lamp (366 nm),
preferably with a 6-watt bulb.
Ensure that weak
autofluorescence of medium,
if present, is not misinterpreted
as positive forE. coli. A MUG-
positive (E- coli) and MUG-
negative (e.g., uninoculated)
control are necessary for each
analysis. The observation of
bright blue fluorescence in the
EC + MUG tubes after 24 + 2
hours constitutes apositive test
for £. coli.
12. Reporting
12.1 Reportthepresenceorabsence
of E. coli.
References
1. Drinking Water; National Primary
Drinking Water Regulations; Total
Coliforms (Including Fecal
Coliforms and £. Coli): Final Rule.
40 Code of Federal Regulations
(CFR) Parts 141 and 142. Federal
Register 54: p. 27544, June 29,1989.
2. National Primary Drinking Water
Regulations; Analytical Techniques;
Coliform Bacteria. 40 Code of
FederalRegulations (CFR),Part 141,
Federal Register 56, p. 636, January
8,1991.
3. Rippey, S.R..L. A. Chandler and W.
D. Watkins. 1987. Fluorometric
Method for Enumeration of
Escherichia coli in Molluscan
Shellfish. I. Food Protection
50:685-690.
4. American Public Health Association.
1985. Standard Methods for the
Examination of Water and
Wastewater. 16th edition. American
Public Health Association,
Washington, D.C.
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Test Method 1105
Detection of Escherichia Coli in Drinking Water by the
Nutrient Agar with Mug Membrane Filter Procedure
1. Scope and Application
1.1 This method describes a
membrane filter (MF)
procedure for the detection and
enumeration of Escherichia
coli (£. coin in drinking water
by transfer of a total coliform-
positive membrane containing
sheen colonies from m-Endo
medium to another substrate
selective for E. coli. Because
this species is a natural
inhabitant only of the intestinal
tractofwarm-bloodedanimals,
its presence in water samples
is an indication of fecal
pollution and the possible
presence of enteric pathogens.
1.2 The detection limit of this
procedure is one£. coli per 100
mL.
1.3 The Total Coliform Rule1
requires that all total coliform-
positive cultures be tested for
either fecal coliforms orE_. coli.
This method for g.. coli
detection is approved in the
National Primary Drinking
Water Regulations; Analytical
Techniques; Coliform
Bacteria2.
2. Summary
2.1 The MFmethod determines the
presence or absenceorprovides
a direct count of £. goJi in water
based on the development of
fluorescent colonies on the
surfaceoftheMF". AlOOmL
water sample is filtered through
the membrane which retains
the bacteria. After filtration,
the membrane containing the
bacterial cells is placed on
Endo-type medium and
incubated at 35 ± 0.5°C for 24
+ 2 hours. Following
incubation, the total coliform-
positive filter is transferred to
a nutrient agar substrate
containing 4-methylum-
belliferyl-B-D-glucuronide
(NA + MUG). Production of
bright blue fluorescence around
the sheen colonies after an
additional 4 hours incubation
at 35°C indicates £. coli.
Fluorescent halo colonies are
observed and/or counted with
a long-wave (366 nm)
ultraviolet (UV) lamp.
3. Definition
3.1 In this method. E. coli are those
bacteria which produce bright
blue fluorescence around the
periphery of total coliform
colonies.
4. Interferences
4.1 Dull fluorescence on the entire
surface of some colonies or
green fluorescence produced
by some pseudomonads
capable of growing on Endo
media may be mistaken for the
brightblue fluorescence typical
of E. coli.
5. Safety Precautions
5.1 The analyst/technician must
know and observe the normal
safety procedures required in a
microbiology laboratory while
preparing, using, and disposing
of cultures, reagents and
materials and while operating
sterilization equipment.
6. Apparatus and Equipment
6.1 Stereoscopic microscope,
wide-field type with
magnification of 10-15X.
6.2 Microscope lamp producing
diffuse light from a cool, white
fluorescent tube and diffuser.
63 UV lamp, long-wave, 366 nm,
preferably with a 6-watt bulb.
6.4 Forceps, straight or curved,
with smooth tips to handle
filters without damage.
6.5 Ethanol, methanol or
isopropanol in a small, wide-
mouth container, for flame-
sterilizing forceps.
6.6 Bumer.BunsenorFishertype,
or electric incinerator unit for
sterilizing inoculation needles.
6.7 Thermometer, glass/mercury
or dial calibrated in 0.5°C
increments or less, checked
against a National Bureau of
Standards (NBS) certified
thermometer, or one traceable
to an NBS thermometer.
6.8 Flask, borosilicate glass,
screw-cap, 250-1000 mL
volume.
6.9 Needles, nichrome wire, 26 B
& S gauge, in suitable holders.
Disposable applicatorsticksare
alternatives.
6.10 Incubator maintained at 35 +
0.5°C.
6.11 Petri dishes, plastic, sterile,
with tight-fitting lids. 50 X 12
mm.
7. Media and Reagents
7.1 Purity of Reagents: Reagent
grade chemicals shall be used
in all tests. Unless otherwise
indicated, reagents shall
conform to the specifications
of theCommitteeon Analytical
Reagents of the American
Chemical Society. The agar
used in preparation of culture
media must be of
microbiological grade.
7.2 Whenever possible, use
commercial culture media as
assurance of quality control.
7.3 Nutrient Agar with MUG (NA
+ MUG) (Difco 0023-15-6)or
equivalent, or
NutrientAgarfDifcoOOOl-02.
BBL 11471)orequivalentwith
MUG added separately.
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Composition:
Peptone 5.0 g
Beef Extract 3.0 g
Agar 15.0 g
4-Mcthyl-B-d-umbelliferyl
glucuronide (MUG) 0.1 g
Reagent Grade Water 1L
Final pH: 6.8 ±0.1
Preparation: Add 23 grams of
NA + MUG agar per liter of
reagent grade water and mix
well. Heat in boiling water
bath to dissolve the agar
completely. Dispense in bottles
or flasks and sterilize for 15
minutes at 121°C (15 Ibs.
pressure). Pour into 50 mm
petri plates (4 mL per plate).
7.4 If medium in petri dishes is not
used immediately, store
refrigerated for up to two
weeks. Incubate stored
medium overnight at 35°C
before use; inspect and discard
plates with growth.
7.5 For quality control of the
medium spot inoculate control
culture(s) onto a MF placed on
tn-Endo agar and incubate at
35 ± 0.5°C for 18-24 hours.
Then transfer the MF to NA+
MUG and incubate at 35°C for
4 hours. Readforfluorescence
around periphery of growth
and record results. Fluores-
cence over the entire colony or
growth area usually does not
confirm as £. Coli. Test each
commercial lot or laboratory
batch prepared from basic
ingredients.
8. Sample Collection, Preserv-
ation and Holding Time
8.1 This test method is a transfer
procedure from a proceeding
sample analysis; therefore, it
doesnotinvolvedirect analysis
of the water sample. Con-
sequently, sample collection,
preservation and holding time
are not procedures specifically
applicable to this method.
However, adherence to sample
collection and preservation
procedures and holding time
limits for the original water
sample is critical to the
production of valid data.
9. Calibration and Standard-
ization
9.1 Check temperatures in
incubators daily to insure
operation within stated limits.
9.2 Check thermometers at least
annually against an NBS
certified thermometer or one
traceable to NBS. Check
mercury columns for breaks.
10. Quality Control
10.1 Verify at least 5% of both
MUG-positive results and
MUG-negative, total coliform-
positive results. Also verify
any non-sheen colonies that
fluoresce. Verification of a
pure culture may be performed
by the use of API 20 E or an
equivalent bacterial identifi-
cation system; standard
biochemical tests (e.g. citrate,
indole and urease tests);
serotyping after biochemical
identification if desired; or the
indole testat44 °C and growth
in citrate.
10.2 See recommendations on
quality control for
microbiological analyses in
Standard Methods for the
Examination of Water and
Wastewater.4
11. Procedure
11.1 Record the total coliform-
positive MF results, and count
and record the sheen colonies
on Endo medium, if desired.
11.2 Prior to transfer of the
membrane, transfer a small
portion of each sheen colony
to theappropriate total coliform
verification media using a
sterile needle. Alternatively,
transfer a small portion of the
sheen fluorescent colonies or
swab the surface growth on the
entire membrane after transfer
of the membrane and
incubation on NA + MUG.
11-3 Use sterile forceps to transfer
the MF from a total coliform
positive Endo plate to the NA
+ MUG medium. Mark each
sheen colony withafine-tipped
marker or by puncturing a hole
in the MF adjacent to the colony
with a needle.
11.4 Incubate at 35 ± 0.5°C for 4
hours.
11.5 Observeindividualcoloniesfor
fluorescence using a long wave
(366 nm) UV light source,
preferably with a 6-watt bulb.
Any amount of bright blue
fluorescence on the periphery
(outer edge) of the colony is
considered positive for E . coli.
Dull fluorescent sheen on the
entire colony is not typical of
£. coli. Read fluorescence on
the membrane surface, not from
the bottom of the plate; the
fluorescent halo cannot always
be read clearly from the
underside. The analyst must
distinguish the blue
fluorescence of a MUG-
positive reaction from the green
fluorescence caused by some
Pseudomonas species. The
number of E. coli colonies
with fluorescent halo may be
counted if desired.
12. Reporting
12.1 Reportthepresenceorabsence
References
1. Drinking Water; National Primary
Drinking Water Regulations; Total
Coliforms (Including Fecal
Coliforms and 1. Coli): Final Rule.
40 Code of Federal Regulations
(CFR) Parts 141 and 142. Federal
Busier 54:p.27544,June29, 1989.
2. National Primary Drinking- Water
Regulations; Analytical Techniques;
Coliform Bacteria. 40 Code of
FederalRegulations (CFR), Part 14 1 ,
Federal Register 56, p. 636, January
8, 1991.
3. Mates, A. and M. Shaffer. 1989.
Membrane Filtration Differentiation
of £. coli from Coli forms in the
Examination of Water. Jour. Appl.
Bacteriol. 67, 343-346.
4. American Public Health Association.
1985. Standard Methods for the
Examination of Water and
Wastewater. 16th edition. American
Public Health Association,
Washington, D.C.
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