Method
1603:
Escherichia
coli
(
E.
coli)
in
Water
by
Membrane
Filtration
Using
Modified
membrane­
Thermotolerant
Escherichia
coli
Agar
(
Modified
mTEC)

September
2002
U.
S.
Environmental
Protection
Agency
Office
of
Water
(
4303T)
1200
Pennsylvania
Avenue,
NW
Washington,
DC
20460
EPA­
821­
R­
02­
023
Disclaimer
The
Engineering
and
Analysis
Division,
of
the
Office
of
Science
and
Technology,
has
reviewed
and
approved
this
report
for
publication.
The
Office
of
Science
and
Technology
directed,
managed,
and
reviewed
the
work
of
DynCorp
in
preparing
this
report.
Neither
the
United
States
Government
nor
any
of
its
employees,
contractors,
or
their
employees
make
any
warranty,
expressed
or
implied,
or
assumes
any
legal
liability
or
responsibility
for
any
third
party s
use
of
or
the
results
of
such
use
of
any
information,
apparatus,
product,
or
process
discussed
in
this
report,
or
represents
that
its
use
by
such
party
would
not
infringe
on
privately
owned
rights.
This
document
combines
the
information
previously
published
in
Test
Methods
for
Escherichia
coli
and
Enterococci
in
Water
by
the
Membrane
Filter
Procedure
(
EPA­
600/
4­
85­
076)
(
Reference
18.6
and
Improved
Enumeration
Methods
for
the
Recreational
Water
Quality
Indicators:
Enterococci
and
Escherichia
coli
(
EPA/
821/
R­
97/
004)
(
Reference
18.4.
Mention
of
trade
names
or
commercial
products
does
not
constitute
endorsement
or
recommendation
for
use.

Questions
concerning
this
method
or
its
application
should
be
addressed
to:

Robin
K.
Oshiro
Engineering
and
Analysis
Division
(
4303T)
U.
S.
EPA
Office
of
Water,
Office
of
Science
and
Technology
1200
Pennsylvania
Avenue,
NW
Washington,
DC
20460
oshiro.
robin@
epa.
gov
202­
566­
1075
202­
566­
1053
(
facsimile)
Table
of
Contents
1.0
Scope
and
Application
.........................................................
1
2.0
Summary
of
Method
...........................................................
1
3.0
Definitions...................................................................
1
4.0
Interferences
and
Contamination
.................................................
1
5.0
Safety
......................................................................
1
6.0
Equipment
and
Supplies
........................................................
2
7.0
Reagents
and
Standards
........................................................
3
8.0
Sample
Collection,
Preservation,
and
Storage
.......................................
6
9.0
QualityControl
...............................................................
6
10.0
Calibration
and
Standardization
..................................................
6
11.0
Procedure
...................................................................
6
12.0
Data
Analysis
and
Calculations
..................................................
7
13.0
Method
Performance...........................................................
7
14.0
Reporting
Results
.............................................................
7
15.0
VerificationProcedure
.........................................................
7
16.0
PollutionPrevention
...........................................................
8
17.0
WasteManagement............................................................
9
18.0
References...................................................................
9
Method
1603:
Escherichia
coli
(
E.
coli)
in
Water
by
Membrane
Filtration
Using
Modified
membrane­
Thermotolerant
Escherichia
coli
Agar
(
modified
mTEC)

September
2002
1.0
Scope
and
Application
1.1
This
method
describes
a
revised
Escherichia
coli
membrane
filter
(
MF)
procedure,
a
single­
step
method
that
uses
one
medium,
modified
mTEC
Agar,
and
does
not
require
the
transfer
of
the
membrane
filter
to
another
medium
or
other
substrate.
The
modified
medium
contains
a
chromogen
(
5­
bromo­
6­
chloro­
3­
indolyl­$­
D­
glucuronide),
which
is
catabolized
to
glucuronic
acid
and
a
red­
or
magenta­
colored
compound
by
E.
coli
that
produce
the
enzyme
$­
Dglucuronidase
The
apparatus
and
equipment,
and
sampling,
filtration,
and
verification
procedures
for
this
modified
mTEC
method
are
identical
to
those
of
the
original
mTEC
method.
Because
the
bacterium
is
a
natural
inhabitant
only
of
the
intestinal
tract
of
warm­
blooded
animals,
its
presence
in
water
samples
is
an
indication
of
fecal
pollution
and
the
possible
presence
of
enteric
pathogens.

1.2
The
E.
coli
test
is
recommended
as
a
measure
of
recreational
fresh
water
quality.
Epidemiological
studies
have
led
to
the
development
of
criteria
which
can
be
used
to
promulgate
recreational
water
standards
based
on
established
relationships
between
health
effects
and
water
quality.
The
significance
of
finding
E.
coli
in
recreational
fresh
water
samples
is
the
direct
relationship
between
the
density
of
E.
coli
and
the
risk
of
gastrointestinal
illness
associated
with
swimming
in
the
water
(
Reference
18.3.

1.3
The
test
for
E.
coli
can
be
applied
to
fresh,
estuarine,
and
marine
waters.

1.4
Since
a
wide
range
of
sample
volumes
or
dilutions
can
be
analyzed
by
the
MF
technique,
a
wide
range
of
E.
coli
levels
in
water
can
be
detected
and
enumerated.

2.0
Summary
of
Method
2.1
The
MF
method
provides
a
direct
count
of
E.
coli
in
water
based
on
the
development
of
colonies
that
grow
on
the
surface
of
a
membrane
filter.
A
water
sample
is
filtered
through
the
membrane,
which
retains
the
bacteria.
After
filtration,
the
membrane
containing
the
bacteria
is
placed
on
a
selective
and
differential
medium,
modified
mTEC
Agar,
incubated
at
35
±
0.5
°
C
for
2
h
to
resuscitate
the
injured
or
stressed
bacteria,
and
then
incubated
at
44.5
±
0.2
°
C
for
22
h.
The
modified
method
eliminates
the
transfer
of
the
membrane
filter
to
another
substrate.
The
target
colonies
on
modified
mTEC
agar
are
red
or
magenta
in
color
after
the
incubation
period.

3.0
Definitions
3.1
In
this
method,
E.
coli
are
those
bacteria
which
produce
red
or
magenta
colonies
on
the
modified
mTEC
agar.

4.0
Interferences
and
Contamination
4.1
Water
samples
containing
colloidal
or
suspended
particulate
material
can
clog
the
membrane
filter
and
prevent
filtration,
or
cause
spreading
of
bacterial
colonies
which
could
interfere
with
identification
of
target
colonies.

5.0
Safety
September
2002
Method
1603
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.

6.0
Equipment
and
Supplies
6.1
Glass
lens
with
magnification
of
2­
5x,
or
stereoscopic
microscope.

6.2
Lamp,
with
a
cool,
white
fluorescent
tube.

6.3
Hand
tally
or
electronic
counting
device.

6.4
Pipet
container,
stainless
steel,
aluminum
or
borosilicate
glass,
for
glass
pipets.

6.5
Pipets,
sterile,
T.
D.
bacteriological
or
Mohr,
glass
or
plastic,
of
appropriate
volume.

6.6
Graduated
cylinders,
100­
1000
mL,
covered
with
aluminum
foil
or
kraft
paper
and
sterile.

6.7
Membrane
filtration
units
(
filter
base
and
funnel),
glass,
plastic
or
stainless
steel,
wrapped
with
aluminum
foil
or
kraft
paper
and
sterilized.

6.8
Ultraviolet
unit
for
sanitization
of
the
filter
funnel
between
filtrations
(
optional).

6.9
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.

6.10
Filter
flask,
vacuum,
usually
1
L,
with
appropriate
tubing.
A
filter
manifold
to
hold
a
number
of
filter
bases
is
optional.

6.11
Flask
for
safety
trap
placed
between
the
filter
flask
and
the
vacuum
source.

6.12
Forceps,
straight
or
curved,
with
smooth
tips
to
handle
filters
without
damage.

6.13
Ethanol,
methanol
or
isopropanol
in
a
small,
wide­
mouth
container,
for
flame­
sterilizing
forceps.

6.14
Burner,
Bunsen
or
Fisher
type,
or
electric
incinerator
unit
for
sterilizing
loops
and
needles.

6.15
Thermometer,
checked
against
a
National
Institute
of
Standards
and
Technology
(
NIST)
certified
thermometer,
or
one
that
meets
the
requirements
of
NIST
Monograph
SP
250­
23.

6.16
Petri
dishes,
sterile,
plastic,
9
x
50
mm,
with
tight­
fitting
lids;
or
15
x
60
mm,
glass
or
plastic,
with
loose­
fitting
lids;
or
15
x
100
mm.

6.17
Bottles,
milk
dilution,
borosilicate
glass,
screw­
cap
with
neoprene
liners,
marked
at
99
mL
for
1:
100
dilutions.
Dilution
bottles
marked
at
90
mL
or
tubes
marked
at
9
mL
may
be
used
for
1:
10
dilutions.

6.18
Flasks,
borosilicate
glass,
screw­
cap,
250­
2000
mL
volume.

6.19
Membrane
filters,
sterile,
white,
grid
marked,
47
mm
diameter,
with
0.45
±
0.02
µ
m
pore
size.

September
2002
2
Method
1603
6.20
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.

Note:
A
platinum
loop
is
required
for
the
cytochrome
oxidase
test
in
the
verification
procedure.

6.21
Incubator
maintained
at
35
±
0.5
°
C,
with
approximately
90%
humidity
if
loose­
lidded
petri
dishes
are
used.

6.22
Waterbath
maintained
at
44.5
±
0.5
°
C.

6.23
Waterbath
maintained
at
50
°
C
for
tempering
agar.

6.24
Test
tubes,
20
x
150
mm,
borosilicate
glass
or
plastic.

6.25
Test
tubes,
10
x
75
mm,
borosilicate
glass
6.26
Caps,
aluminum
or
autoclavable
plastic,
for
20
mm
diameter
test
tubes.

6.27
Test
tubes
screw­
cap,
borosilicate
glass,
16
x
125
mm
or
other
appropriate
size.

6.28
Filter
Paper.

6.29
Whirl­
Pak
®
bags.

7.0
Reagents
and
Standards
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
(
Reference
18.5).
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
water
conforming
to
Specification
D1193,
reagent
water
conforming
Type
II,
Annual
Book
of
ASTM
Standards
(
Reference
18.1).

7.4
Phosphate
Buffered
Saline
7.4.1
Composition:

Sodium
Dihydrogen
Phosphate
0.58
g
Sodium
Monohydrogen
Phosphate
2.5
g
Sodium
Chloride
8.5
g
Reagent­
Grade
Distilled
Water
1.0
L
7.4.2
Preparation:
Dissolve
the
ingredients
in
1
L
of
reagent­
grade
distilled
water
in
a
flask
,
and
dispense
in
appropriate
amounts
for
dilutions
in
screw­
cap
bottles
or
culture
tubes,
and/
or
into
containers
for
use
as
rinse
water.
Autoclave
at
121
°
C
(
15
lb
pressure)
for
15
min.
Final
pH
should
be
7.4
+
0.2.

7.5
Phosphate
Buffered
Dilution
Water
(
References
18.2)

3
September
2002
Method
1603
7.5.1
Composition
of
Stock
Phosphate
Buffer
Solution:

Phosphate
Dihydrogen
Phosphate
34.0
g
Reagent­
Grade
Distilled
Water
500.0
mL
Preparation:
Adjust
the
pH
of
the
solution
to
7.2
with
1
N
NaOH,
and
bring
the
volume
to
1
L
with
reagent­
grade
distilled
water.
Sterilize
by
filtration
or
autoclave
at
121
°
C
(
15
lb
pressure)
for
15
min.

7.5.2
Preparation
of
Stock
Magnesium
Chloride
Solution:
Add
38
g
anhydrous
MgCl2
or
81.1
g
MgCl2
C6H2
O
to
1
L
reagent­
grade
distilled
water.
Sterilize
by
filtration
or
autoclave
at
121
°
C
(
15
lb
pressure)
for
15
min.

7.5.3
Storage
of
Stock
Solutions:
After
sterilization,
store
the
stock
solutions
in
the
refrigerator
until
used.
Handle
aseptically.
If
evidence
of
mold
or
other
contamination
appears,
the
affected
stock
solution
should
be
discarded
and
a
fresh
solution
should
be
prepared.

7.5.4
Working
Phosphate
Buffered
Dilution
Water:
Mix
1.25
mL
of
the
stock
phosphate
buffer
and
5
mL
of
the
MgCl2
stock
per
liter
of
reagent­
grade
distilled
water.
Dispense
in
appropriate
amounts
for
dilutions
in
screwcap
bottles
or
culture
tubes,
and/
or
into
containers
for
use
as
rinse
water.
Autoclave
at
121
°
C
(
15
lb
pressure)
for
15
min.
Final
pH
should
be
7.0
±
0.2.

7.6
Modified
mTEC
Agar
7.6.1
Composition:

Protease
Peptone
#
3
Yeast
Extract
Lactose
NaCl
Dipotassium
Phosphate
Monopotassium
Phosphate
Sodium
Lauryl
Sulfate
Sodium
Desoxycholate
Chromogen
(
5­
bromo­
6­
chloro­
3­
indolyl­
$­
D­
glucuronide)
Agar
Reagent­
Grade
Distilled
Water
5.0
g
3.0
g
10.0
g
7.5
g
3.3
g
1.0
g
0.2
g
0.1
g
0.5
g
15.0
g
1.0
L
7.6.2
Preparation:
Add
dry
ingredients
to
1
L
of
reagent­
grade
distilled
water
in
a
flask,
and
heat
to
boiling
until
the
ingredients
dissolve.
Autoclave
at
121
°
C
(
15
lb
pressure)
for
15
min,
and
cool
in
a
50
°
C
waterbath.
Pour
the
medium
into
each
9
x
50
mm
culture
dish
to
a
4­
5
mm
depth
(
approximately
4­
6
mL),
and
allow
to
solidify.
Final
pH
should
be
7.3
±
0.2.
Store
in
a
refrigerator.

7.7
Nutrient
Agar
7.7.1
Composition:

Peptone
5.0
g
Beef
Extract
3.0
g
Agar
15.0
g
Reagent­
Grade
Distilled
Water
1.0
L
September
2002
4
Method
1603
7.7.2
Preparation:
Add
dry
ingredients
to
1
L
of
reagent­
grade
distilled
water,
and
mix
well.
Heat
to
boiling
to
dissolve
the
agar
completely.
Dispense
in
screwcap
tubes,
and
autoclave
at
121
°
C
(
15
lb
pressure)
for
15
min.
Remove
the
tubes
and
slant.
Final
pH
should
be
6.8
±
0.2.

7.8
Tryptic
Soy
Broth;
Trypticase
Soy
Broth
7.8.1
Composition:

Tryptone
or
Trypticase
Soytone
or
Phytone
Sodium
Chloride
Dextrose
Dipotassium
Phosphate
Reagent­
Grade
Distilled
Water
17.0
g
3.0
g
5.0
g
2.5
g
2.5
g
1.0
L
7.8.2
Preparation:
Add
dry
ingredients
to
1
L
of
reagent­
grade
distilled
water.
Warm
the
broth,
and
mix
gently
to
dissolve
the
medium
completely.
Dispense
in
screw­
cap
tubes,
and
autoclave
at
121
°
C
(
15
lb
pressure)
for
15
min.
Final
pH
should
be
7.3
±
0.2.

7.9
Simmons
Citrate
Agar
7.9.1
Composition:

Magnesium
Sulfate
Monoammonium
Phosphate
Dipotassium
Phosphate
Sodium
Citrate
Sodium
Chloride
Brom
Thymol
Blue
Agar
Reagent­
Grade
Distilled
Water
0.2
g
1.0
g
1.0
g
2.0
g
5.0
g
0.08
g
15.0
g
1.0
L
7.9.2
Preparation:
Add
dry
ingredients
to
1
L
of
reagent­
grade
distilled
water.
Heat
to
boiling
to
dissolve
completely.
Dispense
into
screwcap
tubes,
and
autoclave
at
121
°
C
(
15
lb
pressure)
for
15
min.
Cool
the
tubes
and
slant.
Final
pH
should
be
6.8
±
0.2.

7.10
Tryptone
1%;
Tryptophane
Broth
7.10.1
Composition:

Tryptone
or
Trypticase
Peptone
10.0
g
Reagent­
Grade
Distilled
Water
1.0
L
7.10.2
Preparation:
Add
the
tryptone
or
trypticase
peptone
to
1
L
or
reagent­
grade
distilled
water,
and
heat,
mixing
until
dissolved.
Dispense
in
5­
mL
volumes
into
tubes,
and
autoclave
121
°
C
(
15
lb
pressure)
for
15
min.
Final
pH
should
be
7.2
±
0.2.

7.11
EC
Broth
7.11.1
Composition:

Tryptose
or
Trypticase
Peptone
Lactose
Bile
Salts
no.
3
or
Bile
Salts
Mixture
Dipotassium
Phosphate
Monopotassium
Phosphate
Sodium
Chloride
Reagent­
Grade
Distilled
Water
20.0
g
5.0
g
1.5
g
4.0
g
1.5
g
5.0
g
1.0
L
5
September
2002
Method
1603
7.11.2
Preparation:
Add
dry
ingredients
to
1
L
of
reagent­
grade
distilled
water,
and
warm
to
dissolve
completely.
Dispense
into
fermentation
tubes
(
20
x
150
mm
tubes
containing
inverted
10
x
75
mm
vials).
Autoclave
at
121
°
C
(
15
lb
pressure)
for
15
min.
Final
pH
should
be
6.9
±
0.2.

7.12
Oxidase
Reagent
7.12.1
Composition:

N,
N,
N ,
N ­
tetramethyl­
D­
phenylenediamine
dihydrochloride,
1%
aqueous
solution
(
1
g
per
100
mL
sterile
reagent­
grade
distilled
water).

7.13
Kovacs
Indole
Reagent
7.13.1
Composition:

D­
dimethylaminobenzaldehyde
10.0
g
Amyl
or
Isoamyl
Alcohol
150.0
mL
Concentrated
(
12
M)
Hydrochloric
Acid
50.0
mL
7.13.2
Preparation:
Dissolve
D­
dimethylaminobenzaldehyde
in
alcohol,
slowly
add
hydrochloric
acid,
and
mix.

8.0
Sample
Collection,
Preservation,
and
Storage
8.1
Sampling
procedures
are
described
in
detail
in
the
USEPA
microbiology
methods
manual,
Section
II,
A
(
Reference
18.2).
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.

8.1.1
Storage
Temperature
and
Handling
Conditions
Ice
or
refrigerate
water
samples
at
a
temperature
of
1­
4
°
C
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
during
transit
or
storage.

8.1.2
Holding
Time
Limitations
Examine
samples
as
soon
as
possible
after
collection.
Do
not
hold
samples
longer
than
6
h
between
collection
and
initiation
of
analyses.

9.0
Quality
Control
9.1
See
recommendations
on
quality
control
for
microbiological
analyses
in
the
USEPA
microbiology
methods
manual,
Part
IV,
C
(
Reference
18.2).

10.0
Calibration
and
Standardization
10.1
Check
temperatures
in
incubators
daily
to
ensure
operation
within
stated
limits.

10.2
Check
thermometers
at
least
annually
against
a
NIST
certified
thermometer
or
one
that
meets
the
requirements
of
NIST
Monograph
SP
250­
23.
Check
mercury
columns
for
breaks.

11.0
Procedure
11.1
Prepare
the
modified
mTEC
Agar
as
directed
in
section
7.6.

September
2002
6
Method
1603
11.2
Mark
the
petri
dish
and
report
form
with
the
sample
identification
and
volume.

11.3
Place
a
sterile
membrane
filter
on
the
filter
base,
grid
side
up,
and
attach
the
funnel
to
the
base
so
that
the
membrane
filter
is
held
between
the
funnel
and
the
base.

11.4
Shake
the
sample
bottle
vigorously
at
least
25
times
to
distribute
the
bacteria
uniformly,
and
measure
the
desired
volume
of
sample
or
dilution
into
the
funnel.

11.5
Select
sample
volumes
based
on
previous
knowledge
of
the
pollution
level,
to
produce
20­
80
E.
coli
colonies
on
the
membranes.
Sample
volumes
of
1­
100
mL
are
normally
tested
at
half­
log
intervals
(
e.
g.,
100,
30,
10,
3
mL).

11.6
Smaller
sample
sizes
or
sample
dilutions
can
be
used
to
minimize
the
interference
of
turbidity
or
for
high
bacterial
densities.
Multiple
volumes
of
the
same
sample
or
sample
dilutions
may
be
filtered,
and
the
results
may
be
combined.

11.7
Filter
the
sample,
and
rinse
the
sides
of
the
funnel
at
least
twice
with
20­
30
mL
of
sterile
buffered
rinse
water.
Turn
off
the
vacuum,
and
remove
the
funnel
from
the
filter
base.

11.8
Use
sterile
forceps
to
aseptically
remove
the
membrane
filter
from
the
filter
base,
and
roll
it
onto
the
modified
mTEC
Agar
to
avoid
the
formation
of
bubbles
between
the
membrane
and
the
agar
surface.
Reseat
the
membrane
if
bubbles
occur.
Run
the
forceps
around
the
edge
of
the
filter
to
be
sure
that
the
filter
is
properly
seated
on
the
agar.
Close
the
dish,
invert,
and
incubate
35
±
0.5
°
C
for
2
h.

11.9
After
a
2
h
incubation
at
35
±
0.5
°
C,
transfer
the
plate
to
a
Whirl­
Pak
®
bag,
seal
the
bag,
place
the
bag
with
the
plate
inverted
in
a
test­
tube
rack,
and
put
the
rack
in
a
44.5
±
0.2
°
C
waterbath
for
22­
24
h.

11.10
After
22­
24
h,
remove
the
plate
from
the
waterbath,
count
and
record
the
number
of
red
or
magenta
colonies
with
the
aid
of
an
illuminated
lens
with
a
2­
5x
magnification
or
a
stereoscopic
microscope
(
See
Photo
1).

12.0
Data
Analysis
and
Calculations
Use
the
following
general
rules
to
calculate
the
E.
coli
count
per
100
mL
of
sample:

12.1
Select
the
membrane
filter
with
an
acceptable
number
of
magenta
or
red
colonies
(
20­
80),
and
calculate
the
number
of
E.
coli
per
100
mL
according
to
the
following
general
formula:

Number
of
E.
coli
colonies
E.
coli/
100
mL
=
X
100
Volume
of
sample
filtered
(
mL)

12.2
See
the
USEPA
microbiology
methods
manual,
Part
II,
Section
C,
3.5,
for
general
counting
rules
(
Reference
18.2).

12.3
Reporting
Results
12.3.1
There
should
be
at
least
three
volumes
filtered
per
sample.
Report
the
results
as
E.
coli
per
100
mL
of
sample.

13.0
Method
Performance
13.1
The
false­
positive
and
false­
negative
rates,
reported
for
various
environmental
water
samples,
were
<
1%
and
4%,
respectively.

14.0
Reporting
Results
7
September
2002
Method
1603
14.1
There
should
be
at
least
three
volumes
filtered
per
sample.
Report
the
results
as
E.
coli
per
100
mL
of
sample.

15.0
Verification
Procedure
15.1
Red
or
magenta
colonies
can
be
verified
as
E.
coli.
Verification
of
colonies
may
be
required
in
evidence
gathering
and
is
also
recommended
as
a
means
of
quality
control
for
the
initial
use
of
the
test
and
for
changes
in
sample
sites,
lots
of
commercial
media,
or
major
ingredients
in
media
compounded
in
the
laboratory.
The
verification
procedure
follows.

Photo
1.
Escherichia
coli
colonies
on
modified
mTEC
agar.
E.
coli
colonies
are
red
to
magenta.

15.2
Using
a
sterile
inoculation
loop,
transfer
growth
from
the
centers
of
at
least
10
well­
isolated
colonies
to
Nutrient
Agar
plates
or
slants
and
to
Trypticase
Soy
Broth.
Incubate
the
agar
and
broth
cultures
for
24
h
at
35
±
0.5
°
C.

15.3
After
incubation,
remove
a
loopful
of
growth
from
the
Nutrient
Agar
slant
with
a
platinum
loop,
and
deposit
it
on
the
surface
of
a
piece
of
filter
paper
that
has
been
saturated
with
freshly
prepared
Cytochrome
Oxidase
Reagent.
If
the
spot
where
the
bacteria
were
deposited
turns
deep
purple
within
15
seconds,
the
test
is
positive.

15.4
Transfer
growth
from
the
Trypticase
Soy
Broth
tube
to
Simmons
Citrate
Agar,
Tryptone
Broth,
and
an
EC
Broth
fermentation
tube.

15.4.1
Incubate
the
Simmons
Citrate
Agar
and
Tryptone
Broth
for
48
h
at
35
±
0.5
°
C.

15.4.2
Incubate
the
EC
Broth
at
44.5
±
0.2
°
C
in
a
waterbath
for
24
h.
The
water
level
must
be
above
the
level
of
the
EC
Broth
in
the
tube.

15.4.3
Add
0.5
mL
of
Kovacs
Indole
Reagent
to
the
48
h
Tryptone
Broth
culture,
and
shake
the
tube
gently.
A
positive
test
for
indole
is
indicated
by
a
deep
red
color
which
develops
in
the
alcohol
layer
on
top
of
the
broth.

15.4.4
E.
coli
is
EC
gas­
positive,
indole­
positive,
and
oxidase­
negative,
and
does
not
utilize
citrate
(
i.
e.,
the
medium
remains
green).

15.5
Alternately,
commercially
available
multi­
test
identification
systems
may
be
used
to
verify
colonies.
Inoculate
the
colonies
into
an
identification
system
for
Enterobacteriaceae
that
includes
lactose
fermentation,
F­
nitrophenyl­$­
D­
galactopyranoside
(
ONPG),
and
cytochrome
oxidase
test
reactions.

September
2002
8
Method
1603
16.0
Pollution
Prevention
16.1
The
solutions
and
reagents
used
in
this
method
pose
little
threat
to
the
environment
when
recycled
and
managed
properly.

16.2
Solutions
and
reagents
should
be
prepared
in
volumes
consistent
with
laboratory
use
to
minimize
the
volume
of
expired
materials
to
be
disposed.

17.0
Waste
Management
17.1
It
is
the
laboratory's
responsibility
to
comply
with
all
federal,
state,
and
local
regulations
governing
waste
management,
particularly
the
biohazard
and
hazardous
waste
identification
rules
and
land
disposal
restrictions,
and
to
protect
the
air,
water,
and
land
by
minimizing
and
controlling
all
releases
from
fume
hoods
and
bench
operations.
Compliance
with
all
sewage
discharge
permits
and
regulations
is
also
required.

17.2
Samples,
reference
materials,
and
equipment
known
or
suspected
to
have
viable
E.
coli
attached
or
contained
must
be
sterilized
prior
to
disposal.

17.3
Samples
preserved
with
HCl
to
pH
<
2
are
hazardous
and
must
be
neutralized
before
being
disposed,
or
must
be
handled
as
hazardous
waste.

17.4
For
further
information
on
waste
management,
consult
 
The
Waste
Management
Manual
for
Laboratory
Personnel 
and
 
Less
Is
Better:
Laboratory
Chemical
Management
for
Waste
Reduction, 
both
available
from
the
American
Chemical
Society's
Department
of
Government
Relations
and
Science
Policy,
1155
16th
Street
NW,
Washington,
DC
20036.

18.0
References
18.1
Annual
Book
of
ASTM
Standards,
Vol.
11.01,
American
Society
for
Testing
and
Materials,
Philadelphia,
PA
19103.

18.2
Bordner
R.,
J.
A.
Winter,
P.
V.
Scarpino
(
eds.).
1978.
Microbiological
methods
for
Monitoring
the
Environment:
Water
and
Wastes,
EPA­
600/
8­
78­
017.
Office
of
Research
and
Development,
USEPA.

18.3
Cabelli,
V.
J.,
A.
P.
Dufour,
M.
A.
Levin,
L.
J.
McCabe,
and
P.
W.
Haberman.
1979.
Relationship
of
Microbial
Indicators
to
Health
Effects
at
Marine
Bathing
Beaches.
Am.
J.
Public
Health.
69:
690­
696.

18.4
Improved
Enumeration
Methods
for
the
Recreational
Water
Quality
Indicators:
Enterococci
and
Escherichia
coli.
2000.
EPA/
821/
R­
97/
004.
Office
of
Science
and
Technology,
Washington
D.
C.

18.5
Reagent
Chemicals,
American
Chemical
Society
Specifications,
American
Chemical
Society,
Washington,
DC.
For
suggestions
of
the
testing
of
reagents
not
listed
by
the
American
Chemical
Society,
see
Analar
Standards
for
Laboratory
Chemicals,
BDH
Ltd.,
Poole,
Dorset,
UK
and
the
United
States
Pharmacopeia.

18.6
Test
methods
for
Escherichia
coli
and
Enterococci
in
Water
by
the
Membrane
Filter
Procedure.
1985.
EPA­
600/
4­
85/
076.
Environmental
Monitoring
and
Support
Laboratory,
Cincinnati,
USEPA.

9
September
2002
