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EPA
United States
Environmental Protection
Agency
Office of Research
and Development
Cincinnati OH 45268
EPA 600-R-00-013
February 2000
Membrane Filter Method for
the Simultaneous Detection
of Total Coliforms and
Escherichia coli in
Drinking Water
1
MEMBRANE FILTER METHOD FOR THE SIMULTANEOUS DETECTION OF
TOTAL COLIFORMS AND ESCHERICHIA COLI IN DRINKING WATER
1. Scope and Application
1.1 This test method describes a membrane filter (MF)
medium, MI Agar, for the simultaneous detection and
enumeration of both total coliforms (TC) and Escherichia
coli in water samples in 24 hours or less on the
basis of their specific enzyme activities. Two enzyme
substrates, the fluorogen 4-Methylumbelliferyl-$-Dgalactopyranoside
(MUGal) and a chromogen Indoxyl-$-Dglucuronide
(IBDG), are included in the medium to
detect the enzymes $-galactosidase and $-glucuronidase,
respectively, produced by TCs and E. coli, respectively.
1.2 Total Coliforms include species that may inhabit the
intestines of warm-blooded animals or occur naturally
in soil, vegetation, and water. They are usually found
in fecally-polluted water and are often associated with
disease outbreaks. Although they are not usually pathogenic
themselves, their presence in drinking water
indicates the possible presence of pathogens. E. coli,
one species of the coliform group, is always found in
feces and is, therefore, a more direct indicator of
fecal contamination and the possible presence of
enteric pathogens. In addition, some strains of E.
coli are pathogenic (12).
1.3 This method, which has been validated for use with
drinking water in single-lab and multi-lab studies
(8-10), will be used primarily by certified drinking
water laboratories for microbial analysis of potable
water. Other uses include recreational, surface, or
marine water, bottled water, groundwater, well water,
treatment plant effluents, water from drinking water
distribution lines, drinking water source water, and
possibly foods, pharmaceuticals, clinical specimens
(human or veterinary), other environmental samples
(e.g., aerosols, soil, runoff, or sludge) and/or
isolation and separation of transformants through the
use of E. coli lac Z or gus A/uid reporter genes (11).
1.4 Since a wide range of sample volumes or dilutions can
be analyzed by the MF technique, a wide range of E.
coli and TC levels in water can be detected and enumerated.
2
2. Summary of the Method
An appropriate volume of a water sample (100 ml for drinking
water) is filtered through a 47-mm, 0.45-µm pore size cellulose
ester membrane filter that retains the bacteria present in the
sample. The filter is placed on a 5-ml plate of MI agar, a
selective and differential medium, and the plate is incubated at
35EC for up to 24 hours. The bacterial colonies that grow on the
plate are inspected for the presence of blue color from the
breakdown of IBDG by the E. coli enzyme $-glucuronidase and
fluorescence under longwave ultraviolet light (366 nm) from the
breakdown of MUGal by the TC enzyme $-galactosidase (8).
3. Definitions
3.1 Total Coliforms (TC) - In this method, TCs are those
bacteria that produce fluorescent colonies upon exposure
to longwave ultraviolet light (366 nm) after primary
culturing on MI agar (See Figure 1.). The
fluorescent colonies can be completely blue-white (TCs
other than E. coli) or blue-green (E. coli) in color or
fluorescent halos may be observed around the edges of
the blue-green E. coli colonies. In addition, nonfluorescent
blue colonies, which rarely occur, are
added to the total count because the fluorescence is
masked by the blue color from the breakdown of IBDG
(8).
3.2 Escherichia coli - In this method, the E. coli are
those bacteria that produce blue colonies under ambient
light after primary culturing on MI agar (See Figures 1
and 2.). These colonies can be fluorescent or nonfluorescent
under longwave ultraviolet light (366 nm)
(8).
4. Interferences
4.1 Water samples containing colloidal or suspended particulate
material can clog the membrane filter, thereby
preventing filtration, or cause spreading of bacterial
colonies which could interfere with identification of
target colonies. However, the blue E. coli colonies
can often be counted on plates with heavy particulates
or high concentrations of total bacteria (See Figures 2
and 3.)(8).
4.2 The presence of some lateral diffusion of blue color
away from the target E. coli colonies can affect
enumeration and colony picking on plates with high
concentrations of E. coli. This problem should not
3
affect filters with low counts, such as those obtained
with drinking water or properly diluted samples (8).
4.3 Tiny, flat or peaked pinpoint blue colonies (< 0.5 mm
in diameter on filters containing < 200 colonies) may
be due to species other than E. coli. These colonies
occur occasionally in low numbers and should be
excluded from the count of the E. coli colonies, which
are usually much larger in size (1-3 mm in diameter).
The small colonies have never been observed in the
absence of typical E. coli, but, if such should occur,
the sample should not be considered E. coli-positive
unless at least one colony has been verified by another
method [e.g., EC medium with 4-Methylumbelliferyl-$-Dglucuronide
(MUG) or API 20E strips] (8).
4.4 Bright green, fluorescent, non-blue colonies, observed
along with the typical blue/white or blue-green fluorescent
TC colonies, may be species other than coliforms.
These colonies, which generally occur in low
numbers (< 5%) and can usually be distinguished from
the TCs, should be eliminated from the TC count. An
increase in the number of bright green colonies may
indicate an unusual sample population or a breakdown of
the cefsulodin in the medium (8).
5. Safety and Health
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.
5.2 Mouth-pipetting is prohibited.
5.3 Avoid prolonged exposure to longwave or germicidal
ultraviolet light.
5.4 Autoclave all contaminated plates and materials at the
end of the analysis.
6. Apparatus, Equipment, and Supplies
6.1 Incubator set at 35E ± 0.5EC, with approximately 90%
humidity if loose-lidded petri dishes are used.
6.2 Stereoscopic microscope, with magnification of 10-15X,
wide-field type.
4
6.3 A microscope lamp producing diffuse light from cool,
white fluorescent lamps adjusted to give maximum color.
6.4 Hand tally.
6.5 Pipet container of stainless steel, aluminum, or pyrex
glass, for pipets.
6.6 Graduated cylinders (100-ml for drinking water),
covered with aluminum foil or kraft paper and
sterilized.
6.7 Membrane filtration units (filter base and funnel),
glass, plastic or stainless steel. These are wrapped
with aluminum foil or kraft paper and sterilized.
6.8 Germicidal ultraviolet (254 nm) light box for
sanitizing the filter funnels is desirable, but
optional.
6.9 Line vacuum, electric vacuum pump, or aspirator is used
as a vacuum source. In an emergency, a hand pump or a
syringe can be used. Such vacuum-producing devices
should be equipped with a check valve to prevent the
return flow of air.
6.10 Vacuum filter flask, usually 1-liter, with appropriate
tubing. Filter manifolds to hold a number of filter
bases are desirable, but optional.
6.11 Safety trap flask, placed between the filter flask and
the vacuum source.
6.12 Forceps, straight (preferred) or curved, with smooth
tips to permit easy handling of filters without damage.
6.13 Alcohol, 95% ethanol, in small wide-mouthed vials, for
sterilizing forceps.
6.14 Bunsen or Fisher-type burner or electric incinerator
unit.
6.15 Sterile T.D. (To Deliver) bacteriological or Mohr
pipets, glass or plastic (1-ml and 10-ml volumes).
6.16 Membrane Filters (MF), white, grid-marked, cellulose
ester, 47-mm diameter, 0.45 µm ± 0.02-µm pore size,
presterile or sterilized for 10 min at 121EC (15 lb
pressure).
5
6.17 Longwave ultraviolet lamp (366 nm), handheld 4-watt
(preferred) or 6-watt, or microscope attachment.
6.18 Dilution Water: Sterile phosphate-buffered dilution
water, prepared in large volumes (e.g., 1 liter) for
wetting membranes before addition of the sample and for
rinsing the funnel after sample filtration or in 99-ml
dilution blanks [Section 9050C in Standard Methods
(2)].
6.19 Indelible ink marker for labeling plates.
6.20 Thermometer, checked against a National Institute of
Science and Technology (NIST)-certified thermometer, or
one traceable to an NIST thermometer.
6.21 Petri dishes, sterile, plastic, 9 x 50 mm, with tightfitting
lids, or 15 x 60 mm, glass or plastic, with
loose-fitting lids. 15 x 100 mm dishes may also be
used.
6.22 Bottles, milk dilution, borosilicate glass, screw-cap
with neoprene liners, marked at 99 ml for 1:100 dilutions
(if needed). Dilution bottles marked at 90 ml,
or tubes marked at 9 ml may be used for 1:10 dilutions.
6.23 Flasks, borosilicate glass, screw-cap, 250- to 2000-ml
volume, for agar preparation.
6.24 Waterbath maintained at 50EC for tempering agar.
6.25 Syringe filter, sterile, disposable, 25-mm diameter,
0.22-µm pore size, to filter cefsulodin for MI agar.
6.26 Syringe, sterile, plastic, disposable, 20-cc capacity.
Autoclaved glass syringes are also acceptable.
6.27 Test tubes, sterile, screw-cap, 20 x 150 mm, borosilicate
glass or plastic, with lids.
6.28 Sterilization filter units, presterile, disposable,
500- or 1000-ml capacity, 0.2-µm pore size, to filter
stock buffer solutions.
7. Reagents and Materials
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
6
Chemical Society (1). 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.
7.3 Purity of Water: Reagent-grade distilled water
conforming to Specification D1193, Type II water or
better, ASTM Annual Book of Standards (3).
7.4 Buffered Dilution Water (2)
7.4.1 Stock Phosphate Buffer Solution (2):
Potassium Dihydrogen
Phosphate (KH2PO4) 34.0 g
Reagent-Grade Distilled Water 500 ml
7.4.2 Preparation of Stock Buffer Solution:
Adjust the pH of the solution to 7.2 with 1 N
NaOH, and bring volume to 1000 ml with reagentgrade
distilled water. Sterilize by filtration
or autoclave for 15 minutes at 121EC (15 lbs
pressure).
7.4.3 MgCl2Solution (2): Dissolve 38 g anhydrous
MgCl2 (or 81.1 g MgCl2@6H2O) in one liter of
reagent-grade distilled water. Sterilize by
filtration or autoclave for 15 min at 121EC (15
lb pressure).
7.4.4 Storage of Stock Buffer and MgCl2Solutions:
After sterilization of the stock solutions,
store in the refrigerator until used. Handle
aseptically. If evidence of mold or other
contamination appears in either stock, the
solution should be discarded, and a fresh
solution should be prepared.
7.4.5 Working Solution (Final pH 7.0 ± 0.2): Add
1.25 ml phosphate buffer stock and 5 ml MgCl2
stock for each liter of reagent-grade distilled
water prepared. Mix well, and dispense in
appropriate amounts for dilutions in screw-cap
dilution bottles or culture tubes, and/or into
larger containers for use as rinse water.
Autoclave at 121EC (15 lb pressure) for 15 min.
Longer sterilization times may be needed
depending on the container and load size and the
7
amount of time needed for the liquid to reach
121EC.
7.5 MI Agar (8)
7.5.1 Composition:
Proteose Peptone #3 5.0 g
Yeast Extract 3.0 g
$-D-Lactose 1.0 g
4-Methylumbelliferyl-$-DGalactopyranoside
(MUGal)
(final concentration 100 µg/ml) 0.1 g
Indoxyl-$-D-Glucuronide (IBDG)
(final concentration 320 µg/ml) 0.32 g
NaCl 7.5 g
K2HPO4 3.3 g
KH2PO4 1.0 g
Sodium Lauryl Sulfate 0.2 g
Sodium Desoxycholate 0.1 g
Agar 15.0 g
Reagent-Grade Distilled Water 1000 ml
7.5.2 Cefsulodin Solution (1 mg/1 ml): Add 0.02 g
of cefsulodin to 20 ml reagent-grade distilled
water, sterilize using a 0.22-µm syringe filter,
and store in a sterile tube at 4EC until needed.
Prepare fresh solution each time. Do not save
the unused portion.
7.5.3 Preparation: Autoclave the medium for 15 minutes
at 121EC (15 lb pressure), and add 5 ml
of the freshly-prepared solution of Cefsulodin
(5 µg/ml final concentration) per liter of
tempered agar medium. Pipet the medium into
9 x 50 mm Petri dishes (5 ml/plate). Store
plates at 4EC for up to 2 weeks. The final pH
should be 6.95 + 0.2.
7.6 Tryptic Soy Agar/Trypticase Soy Agar (Difco 0369-17-6,
BD 4311043, Oxoid CM 0129B) (TSA)
7.6.1 Composition:
Tryptone 17 g
Soytone 3 g
Dextrose 2.5 g
NaCl 5 g
K2HPO4 2.5 g
8
7.6.2 Preparation: Add the dry ingredients listed
above to 1000 ml of reagent-grade distilled
water, and heat to boiling to dissolve the agar
completely. Autoclave at 121EC (15 lb pressure)
for 15 min. Dispense the agar into 9 x 50 mm
petri dishes (5 ml/plate). Incubate the plates
for 24-48 hr at 35EC to check for contamination.
Discard any plates with growth. If $ 5% of the
plates show contamination, discard all plates,
and make new medium. Store at 4EC until needed.
The final pH should be 7.3 ± 0.2.
8. Sample Collection, Preservation and Holding Times
8.1 Water samples are collected in sterile polypropylene
sample containers with leakproof lids.
8.2 Sampling procedures are described in detail in Sections
9060A and 9060B of the 18th edition of Standard Methods
for the Examination of Water and Wastewater (2) or in
the USEPA Microbiology Methods Manual, Section II, A
(6). Residual chlorine in drinking water (or
chlorinated effluent) samples should be neutralized
with sodium thiosulfate (1 ml of a 10% solution per
liter of water) at the time of collection. Adherence
to sample preservation procedures and holding time
limits is critical to the production of valid data.
Samples not collected according to these rules should
not be analyzed.
8.2.1 Storage Temperature and Handling Conditions:
Ice or refrigerate water samples at a temperature
of 1-4EC during transit to the laboratory.
Use insulated containers to assure proper maintenance
of storage temperature. Take care that
sample bottles are not totally immersed in water
from melted ice during transit or storage.
8.2.2 Holding Time Limitations: Analyze samples as
soon as possible after collection. Drinking
water samples should be analyzed within 30 h of
collection (13). Do not hold source water
samples longer than 6 h between collection and
initiation of analyses, and the analyses should
be complete within 8 h of sample collection.
9. Calibration and Standardization
9.1 Check temperatures in incubators twice daily to insure
operation within stated limits (14).
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9.2 Check thermometers at least annually against an NISTcertified
thermometer or one traceable to NIST. Check
mercury columns for breaks.
10. Quality Control (QC)
10.1 Pretest each batch of MI agar for performance (i.e.,
correct enzyme reactions) with known cultures (E. coli,
TC, and a non-coliform).
10.2 Test new lots of membrane filters against an acceptable
reference lot using the method of Brenner and Rankin
(7).
10.3 Perform specific filtration control tests each time
samples are analyzed, and record the results.
10.3.1 Filter Control: Place one or more membrane
filters on TSA plates, and incubate the plates
for 24 hours at 35EC. Absence of growth
indicates sterility of the filter(s).
10.3.2 Phosphate-Buffered Dilution Water Controls:
Filter a 50-ml volume of sterile dilution
water before beginning the sample filtrations
and a 50-ml volume of dilution water after
completing the filtrations. Place the filters
on TSA plates, and incubate the plates for 24
hours at 35EC. Absence of growth indicates
sterility of the dilution water.
10.3.3 Agar Controls: Place one or more TSA plates and
one or more MI agar plates in the incubator for
24 hours at 35o C. Absence of growth indicates
sterility of the plates.
10.4 See recommendations on quality control for microbiological
analyses in the "Manual for the Certification
of Laboratories Analyzing Drinking Water: Criteria and
Procedures; Quality Assurance" (14) and the USEPA
Microbiology Methods Manual, Part IV, C (6).
11. Procedure
11.1 Prepare MI Agar and TSA as described in 7.5 and 7.6,
respectively. If plates are made ahead of time and
stored in the refrigerator, remove them and allow
them to warm to room temperature. The crystals that
form on MI Agar after refrigeration will disappear as
the plates warm up (8).
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11.2 Label the bottom of the MI Agar plates with the sample
number/identification and the volume of sample to
be analyzed. Label QC TSA plates and the MI agar
sterility control plate(s).
11.3 Using a flamed forceps, place a membrane filter,
grid-side up, on the porous plate of the filter base.
If you have difficulties in removing the separation
papers from the filters due to static electricity,
place a filter with the paper on top on the funnel
base and turn on the vacuum. The separation paper
will curl up, allowing easier removal.
11.4 Attach the funnel to the base of the filter unit,
taking care not to damage or dislodge the filter.
The membrane filter is now located between the funnel
and the base.
11.5 Put about 30 ml of sterile dilution water in the
bottom of the funnel.
11.6 Shake the sample container vigorously 25 times.
11.7 Measure an appropriate volume (100 ml for drinking
water) or dilution of the sample with a sterile
pipette or graduated cylinder, and pour it into the
funnel. Turn on the vacuum, and leave it on while
rinsing the funnel twice with about 30 ml sterile
dilution water.
11.8 Remove the funnel from the base of the filter unit.
A germicidal ultraviolet (254 nm)light box can be
used to hold and sanitize the funnel between
filtrations. At least 2 minutes of exposure time is
required for funnel decontamination. Protect eyes
from UV irradiation with glasses, goggles, or an
enclosed UV chamber.
11.9 Holding the membrane filter at its edge with a flamed
forceps, gently lift and place the filter grid-side
up on the MI agar plate. Slide the filter onto the
agar, using a rolling action to avoid trapping air
bubbles between the membrane filter and the
underlying agar. Run the tip of the forceps around
the outside edge of the filter to be sure the filter
makes contact with the agar. Reseat the membrane if
non-wetted areas occur due to air bubbles.
11.10 Invert the petri dish, and incubate the plate at 35EC
for 24 hours.
11
11.11 Count all blue colonies on each MI agar plate under
normal/ambient light, and record the results (See
Figures 1 and 2.). This is the E. coli count.
11.12 Expose each MI agar plate to longwave ultraviolet
light (366 nm), and count all fluorescent colonies
[blue/green fluorescent E. coli, blue/white fluorescent
TCs other than E. coli, and blue/green with
fluorescent edges (also E. coli)] (See Figure 1.).
Record the data.
11.13 Add any blue, non-florescent colonies (if any) found
on the same plate to the TC count (8).
12. Calculation of Results
12.1 Use the following general rules to calculate the E.
coli or TC per 100 ml of sample:
12.1.1 Select and count filters with < 200 total
colonies per plate.
12.1.2 Select and count filters with < 100 target
colonies (ideally, 20-80).
12.1.3 If the total number of colonies or TC on a
filter are too-numerous-to-count or confluent,
record the results as "TC+ (TNTC)"
and count the number of E. coli. If both
target organisms are > 200, record the
results as "TC+ EC+ (TNTC)".
12.1.4 Calculate the final values using the
formulae:
E. coli/100 ml = Number of blue colonies x (100)
Volume of sample filtered (ml)
Number of fluorescent colonies +
number of blue, non-fluorescent
TC/100 ml = colonies (if any) x (100)
Volume of sample filtered (ml)
12.2 See the USEPA Microbiology Manual, Part II, Section
C, 3.5, for general counting rules (6).
12.3 Report results as E. coli or TC per 100 ml of drinking
water.
13. Performance Characteristics
13.1 The detection limits of this method are one E. coli
12
and/or one total coliform per sample volume or dilution
tested (8).
13.2 The false-positive and false-negative rates for E.
coli are both reported to be 4.3% (8).
13.3 The single lab recovery of E. coli is reported (8)
to be 97.9% of the Heterotrophic Plate Count (pour
plate) (2) and 115% of the R2A spread plate (2).
For Klebsiella pneumoniae and Enterobacter aerogenes,
two total coliforms, the recoveries are 87.5% and
85.7% of the HPC (8), respectively, and 89.3% and
85.8% of the R2A spread plate, respectively.
13.4 The specificities for E. coli and total coliforms are
reported to be 95.7% and 93.1% (8), respectively.
13.5 The single lab coefficients of variation for E. coli
and total coliforms are reported to be 25.1% and
17.6% (8), respectively, for a variety of water
types.
13.6 In a collaborative study (4,5,9), 19 laboratories
concurrently analyzed six wastewater-spiked
Cincinnati tap water samples, containing 3 different
concentrations of E. coli (< 10, 11-30, and > 30 per
100 ml).
13.6.1 The single laboratory precision (coefficient
of variation), a measure of the repeatability
ranged from 3.3% to 27.3% for E. coli and
from 2.5% to 5.1% for TC for the six samples
tested, while the overall precision
(coefficient of variation), a measure of
reproducibility, ranged from 8.6% to 40.5%
and from 6.9% to 27.7%, respectively. These
values are based on log10-transformed data
(5).
13.6.2 Table 1 contains the statistical summary of
the collaborative study (9) results.
14. Pollution Prevention
14.1 Pollution prevention is any technique that reduces or
eliminates the quantity or toxicity of waste at the
point of generation. It is the environmental management
tool preferred over waste disposal or recycling.
When feasible, laboratory staff should use a pollution
prevention technique, such as preparation of the
13
smallest practical volumes of reagents, standards,
and media or downsizing of the test units in a
method.
14.2 The laboratory staff should also review the procurement
and use of equipment and supplies for other ways
to reduce waste and prevent pollution. Recycling
should be considered whenever practical.
15. Waste Management:
The Environmental Protection Agency requires that laboratory
waste management practices be consistent with all applicable
rules and regulations. The Agency urges laboratories to protect
the air, water, and land by minimizing and controlling releases
from hoods and bench operations, complying with the letter and
spirit of sewer discharge permits and regulations and by complying
with solid and hazardous waste regulations, particularly the
hazardous waste identification rules and land disposal restrictions.
All infectious wastes should be autoclaved before
disposal.
16. References
1. American Chemical Society. 1981. Reagent Chemicals.
In American Chemical Society Specifications, 6th
edition. American Chemical Society, Washington, D.C.
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.
2. American Public Health Association. 1992. Standard
Methods for the Examination of Water and Wastewater,
18th edition. American Public Health Association,
Washington, D.C.
3. American Society for Testing and Materials. 1993.
Standard Specification for Reagent Water, designation
D1193-91, p. 45-47. In 1993 Annual Book of ASTM
Standards: Water and Environmental Technology, volume
11.01. American Society for Testing and Materials,
Philadelphia, Pennsylvania.
14
4. American Society for Testing and Materials. 1994.
Standard practice for determination of precision and
bias of applicable methods of committee D-19 on water,
designation D 2777-86, p. 31-44. In 1994 Annual book
of ASTM standards, section 11: water and environmental
technology, vol. 11.01. American Society for Testing
and Materials, Philadelphia, Pennsylvania.
5. Association of Official Analytical Chemists. 1989.
Guidelines for Collaborative Study Procedure to
Validate Characteristics of a Method of Analysis.
J. Assoc. Off. Anal. Chem. 72(4):694-704.
6. Bordner, R., J. Winter, and P. Scarpino (ed). 1978.
Microbiological methods for monitoring the environment:
water and wastes. EPA-600/8-78-017, Environmental
Monitoring and Support Laboratory, U.S. Environmental
Protection Agency, Cincinnati.
7. Brenner, K.P., and C.C. Rankin. 1990. New screening
test to determine the acceptability of 0.45-µm membrane
filters for analysis of water. Appl. Environ. Microbiol.
56:54-64.
8. Brenner, K.P., C.C. Rankin, Y.R. Roybal, G.N. Stelma,
Jr., P.V. Scarpino, and A.P. Dufour. 1993. New medium
for the simultaneous detection of total coliforms and
Escherichia coli in water. Appl. Environ. Microbiol.
59:3534-3544.
9. Brenner, K.P., C.C. Rankin, and M. Sivaganesan. 1996.
Interlaboratory Evaluation of MI Agar and the U.S.
Environmental Protection Agency-Approved Membrane
Filter Method for the Recovery of Total Coliforms and
Escherichia coli from Drinking Water. J. Microbiol.
Methods 27:111-119.
10. Brenner, K.P., C.C. Rankin, M. Sivaganesan, and P.V.
Scarpino. 1996. Comparison of the Recoveries of
Escherichia coli and Total Coliforms from Drinking
Water by the MI Agar Method and the U.S. Environmental
Protection Agency-Approved Membrane Filter Method.
Appl. Environ. Microbiol. 62(1):203-208.
11. Buntel, C.J. 1995. E. coli $-glucuronidase (GUS) as a
marker for recombinant vaccinia viruses. BioTechniques
19(3):352-353.
15
12. Federal Register. 1985. National primary drinking
water regulations; synthetic organic chemicals,
inorganic chemicals and microorganisms; proposed rule.
Fed. Regist. 50:46936-47022.
13. Federal Register. 1994. National primary and
secondary drinking water regulations: analytical
methods for regulated drinking water contaminants;
final rule. Fed. Regist. 59:62456-62471.
14. U.S. Environmental Protection Agency. 1992. Manual
for the certification of laboratories analyzing drinking
water: criteria and procedures, quality assurance,
third edition. EPA-814B-92-002, Office of Ground
Water and Drinking Water, Technical Support Division,
U.S. Environmental Protection Agency, Cincinnati, Ohio.
16
TABLE 1. Statistical Summary of the Collaborative Study Results1
____________________________________________________________________________________________________________
E. Coli
Count RSDR
Target Sample Category Initial Final RSDr
6 RSDR
9 RSDr
Organism Number (Range)2 n3 n4 Sr
5 (%) X7 SR
8 (%) Ratio
____________________________________________________________________________________________________________
Escherichia 1 Low (< 10) 63 63 0.17 27.3 0.64 0.26 40.5 1.49
coli
2 63 63 0.21 25.0 0.84 0.33 39.0 1.56
3 Medium 63 63 0.10 7.9 1.27 0.15 12.1 1.52
(11-30)
4 63 60 0.07 5.6 1.32 0.12 9.2 1.65
5 High (> 30) 63 60 0.06 3.3 1.87 0.16 8.6 2.62
6 63 63 0.09 4.3 1.99 0.25 12.6 2.91
____________________________________________________________________________________________________________
Total 1 Low (< 10) 63 63 0.10 4.3 2.35 0.62 26.4 6.11
Coliforms
2 63 63 0.09 3.8 2.31 0.64 27.7 7.25
3 Medium 63 63 0.11 5.1 2.17 0.47 21.8 4.28
(11-30)
4 63 57 0.10 3.3 3.07 0.21 6.9 2.08
5 High (> 30) 63 63 0.15 4.8 3.10 0.43 14.0 2.96
6 63 63 0.08 2.5 3.14 0.46 14.7 5.97
____________________________________________________________________________________________________________
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Table 1. (cont’d)
1 The values are based on log10 transformed data (5).
2 The samples were grouped by their E. coli count on MI agar into the
following categories:
Low (< 10 E. coli/100 ml, samples 1 and 2), Medium (11-30 E. coli/100 ml,
samples 3 and 4),and High (> 30 E. coli/100 ml, samples 5 and 6).
3 These values are based on triplicate analyses by each laboratory. The
reference laboratory analyzed three sets of samples (the initial and final
samples prepared and a sample shipped along with the other 18 lab samples.
4 These values were obtained after removing outliers by the AOAC procedure(5).
5 sr, Single Operator Standard Deviation, a measure of repeatability.
6 RSDr, Single Operator Relative Standard Deviation (Coefficient of Variance),
a measure of repeatability.
7 The mean of the replicate analyses for all laboratories.
8 sR, Overall Standard Deviation, a measure of reproducibility.
9 RSDR, Overall Relative Standard Deviation (Coefficient of Variation), a
measure of reproducibility.
18
FIGURE 1. This photograph shows Escherichia coli (blue/green
fluorescence) and total coliforms other than E. coli
(blue/white fluorescence) on MI agar under longwave UV
light (366 nm). The sample used was a wastewater-spiked
Cincinnati, Ohio tap water.
19
FIGURE 2. These photographs show Escherichia coli and total coliforms from cistern water on
MI agar. The confluent plate was photographed under different lighting: ambient
light on the left, and longwave UV light (366 nm) on the right. Under ambient
light, E. coli are blue, and total coliforms other than E. coli and non-coliforms
are their natural color. Under longwave UV light, all total coliforms, including
E. coli, are fluorescent, and non-coliforms are non-fluorescent (i.e., they are not
visible).
20
FIGURE 3. This photograph shows that Escherichia coli (blue/green
fluorescence) and total coliforms other than E. coli
(blue/white fluorescence) can easily be detected on MI
agar plates from samples with high turbidity levels. The
sample used was a surface water-spiked Cincinnati, Ohio
tap water.
