Document ID: EPA-HQ-OW-2002-0039-0087
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-07-09T04:00Z

IDEXX
COLILERT
®
TEST
METHOD
FOR
THE
SIMULTANEOUS
DETECTION
OF
TOTAL
COLIFORMS
AND
E.
COLI
IN
WATER
JUNE
2003
DRAFT
Draft
1
June
2003
IDEXX
COLILERT
®
TEST
METHOD
FOR
THE
SIMULTANEOUS
DETECTION
OF
TOTAL
COLIFORMS
AND
E.
COLI
IN
WATER
1.0
Scope
and
Application
1.1
This
method
is
intended
for
use
in
the
simultaneous
detection
and
confirmation
of
total
coliforms
and
E.
coli
in
water.
Any
positive
sample
for
total
coliforms
is
an
indication
of
contamination.
Any
positive
sample
for
both
total
coliforms
and
E.
coli
is
an
acute
violation.

1.2
The
minimum,
non­
zero
number
of
bacterial
counts
detectable
with
this
method
is
a
function
of
the
dilution
scheme
used
when
processing
the
sample.

1.3
Colilert
®
method
can
be
applied
to
fresh
waters,
drinking
waters
and
waste
waters.
It
can
be
used
as
a
Presence/
Absence
test
or
quantification
with
either
5,
10,
or
15
tube
serial
dilution
MPN
tubes
or
with
the
Quanti­
Tray
 
system
(
see
package
insert)
(
1).

1.4
Since
there
can
be
a
wide
range
of
coliform
levels
in
surface
waters
and
wastewaters,
dilutions
can
be
used
with
this
method
for
detecting
and
enumerating
the
actual
level.

1.5
Colilert
®
is
not
for
intended
for
use
with
marine
waters.

2.0
Summary
of
Method
2.1
This
method
is
based
on
Defined
Substrate
Technology
®
.
This
product
utilizes
nutrient
indicators
that
produce
color/
fluorescence
when
metabolized
by
total
coliforms
and
E.
coli.
When
the
reagent
is
added
to
the
sample
and
incubated,
it
can
detect
these
bacteria
at
1
CFU/
100
mL
within
24
hours
with
as
many
as
2
million
heterotrophic
bacteria/
100
mL
present.
This
test
is
not
intended
for
marine
waters.

3.0
Definitions
3.1
In
this
method,
coliform
bacteria
are
those
bacteria
which
produce
a
yellow
color,
and
for
E.
coli,
also
produce
a
fluorescent
signal
under
a
6­
watt,
365­
nm
UV
light
after
incubation
at
35
°
C
±
0.5
°
C
for
24
hours.

4.0
Interferences
4.1
Heterotrophic
bacteria
greater
than
2,000,000/
100
mL
can
yield
a
positive
reaction
for
coliforms.
Some
water
samples
containing
humic
material
may
have
an
innate
color
and
a
control
blank
of
the
same
water
sample
may
be
may
be
required
for
comparison
to
the
inoculated
sample.

5.0
Safety
5.1
The
analyst/
technician
must
know
and
observe
the
normal
safety
procedures
required
in
a
microbiology
laboratory
preparing
using
and
disposing
of
samples,
reagents
and
materials,
and
while
operating
sterilizing
equipment.

5.2
Mouth­
pipetting
is
prohibited.
Draft
2
June
2003
6.0
Equipment
and
Supplies
6.1
Pipettes,
sterile,
T.
D.
bacteriological
or
Mohr,
glass
or
plastic
of
appropriate
volume.

6.2
Sterile
vessels,
glass
or
plastic
(
free
from
fluorescence),
100­
to
200­
mL
volume.

6.3
Incubator
maintained
at
35
°
C
±
0.5
°
C.

6.4
6­
watt,
365­
nm,
UV
light.

7.0
Reagents
7.1
Sterile
deionized
or
distilled
water:
Water
conforming
to
specification
D1193,
reagent
water
conforming
Type
II,
Annual
Book
of
ASTM
Standards
(
2).
Autoclave
at
121
°
C
(
15­
lb
pressure)
for
15
minutes
or
sterile
filter
using
a
0.22
micron
filter
into
a
sterile
container.

7.2
Sodium
thiosulfate
is
used
to
dechlorinate
drinking
water
samples.

7.3
Store
Colilert
®
at
4
°
C
­
30
°
C
away
from
light.
The
expiration
date
is
indicated
on
the
package
(
12
months
from
the
date
of
manufacture).

8.0
Sample
Collection,
Preservation
and
Storage
8.1
Sampling
procedures
as
described
in
detail
in
the
USEPA
microbiology
methods
manual,
Section
II,
A
(
3)
and
in
Standard
Methods
for
the
Examination
of
Water
and
Wastewater
(
4).

8.1.1
Storage
Temperature
and
Handling
Conditions:
Ice
or
refrigerate
bacteriological
samples
at
a
temperature
less
than
10
°
C
during
transit
to
the
laboratory.
Use
insulated
containers
to
assure
proper
maintenance
of
storage
temperature.
Take
care
that
sample
vessels
are
not
totally
immersed
in
water
during
transit.

8.1.2
Holding
Time
Limitations:
Examine
samples
as
soon
as
possible
after
collection.
For
drinking
water
samples
do
not
exceed
30
hours
holding
time
from
collection
to
analysis.
For
non­
potable
water
for
compliance,
do
not
exceed
6
hours
holding
time
and
process
within
2
hours.

9.0
Quality
Control
9.1
Quality
control
should
be
conducted
on
each
lot
of
Colilert
®
or
more
often
as
regulations
required.
Inoculate
100
mL
of
sterile
water
with
Quanti­
Cult
 
or
American
Type
Culture
Collection
(
ATCC)
listed
below.
Follow
the
procedure
in
Section
11.

Quanti­
Cult
 
Organism
ATCC
#
Expected
Result
E.
coli
25922
or
11775
Yellow,
fluorescent
Klebsiella
pnuemoniae
31488
Yellow
Pseudomonas
aeruginosa
10145
or
27853
Colorless,
no
fluorescence
Draft
3
June
2003
10.0
Calibration
and
Standardization
10.1
Check
temperatures
in
incubators
daily
to
insure
operation
within
stated
limits.

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

11.0
Procedure
11.1
Presence/
Absence
11.1.1
Carefully
separate
one
Snap
Pack
from
the
strip
taking
care
not
to
accidentally
open
adjacent
pack.

11.1.2
Tap
the
Snap
Pack
to
ensure
that
all
of
the
Colilert
®
powder
is
in
the
bottom
of
the
pack.

11.1.3
Open
one
pack
by
snapping
back
the
top
at
the
scoreline.

11.1.4
Add
the
reagent
to
the
100­
mL
water
sample
contained
in
a
sterile,
non­
fluorescent
vessel.

11.1.5
Aseptically
cap
and
seal
the
vessel.

11.1.6
Shake
until
dissolved.

11.1.7
Incubate
for
24
hours
at
35
°
C
±
0.5
°
C
11.1.8
Read
the
results
at
24
hours.
Compare
each
result
against
the
comparator
dispensed
into
an
identical
vessel.

11.1.9
If
no
yellow
color
is
observed,
the
test
is
negative.

11.1.10
If
the
sample
has
a
yellow
color
equal
to
or
greater
than
the
comparator,
the
presence
of
total
coliforms
is
confirmed.
If
color
is
not
uniform,
mix
by
inversion,
then
recheck.

11.1.11
If
the
sample
is
yellow,
but
lighter
than
the
comparator,
it
may
be
incubated
an
additional
4
hours
(
but
no
more
than
28
hours
total).
If
the
sample
is
total
coliform
positive,
the
color
will
intensify.
If
it
does
not
intensify,
the
sample
is
negative.

11.1.12
If
yellow
is
observed,
check
vessel
for
fluorescent
by
placing
a
6­
watt,
365­
nm
UV
light
within
five
inches
of
the
sample
in
a
dark
environment.
Be
sure
the
light
is
facing
away
from
your
eyes
and
towards
the
vessel.
If
the
fluorescence
is
greater
or
equal
to
the
fluorescence
of
the
comparator,
the
presence
of
E.
coli
is
confirmed.

11.2
Quantification
11.2.1
Colilert
®
can
be
used
for
multiple
tube
MPN
analysis
(
e.
g.,
5­
tube,
10­
tube,
or
15­
tube
serial
dilutions).
Consult
Standard
Methods
for
appropriate
MPN
tables.
For
accuracy
and
counting
range,
use
the
IDEXX
Quanti­
Tray
 
or
Quanti­
Tray
2000
 
and
follow
the
above
Presence/
Absence
Sections
11.1.1
through
11.1.5.

11.2.2
If
a
dilution
is
required,
use
sterile
water,
not
buffered
water
for
making
dilutions.
Colilert
®
is
already
buffered.
Always
add
Colilert
®
to
the
proper
volume
of
diluted
sample
after
making
dilutions.
Draft
4
June
2003
11.2.3
Follow
the
package
insert
for
Quanti­
Tray
 
(
5).
Remove
a
sterile
tray
from
the
plastic
bag
and
squeeze
the
tray
at
the
top
to
open.
Pour
the
sample
reagent
mixture
from
step
11.1.5
above
directly
into
the
tray
avoiding
contact
with
the
foil
tab,
and
then
seal
the
tray
with
the
Quanti­
Tray
 
sealer.

11.2.4
Incubate
at
24
hours
at
35
°
C
±
0.5
°
C.

12.0
Data
Analysis
and
Calculations
12.1
Quanti­
Tray
 
12.1.1
Follow
the
same
interpretation
directions
from
Section
11.1.8
above
to
count
the
number
of
positive
wells.
Refer
to
the
MPN
table
provided
with
the
Quanti­
Tray
 
to
determine
the
Most
Probable
Number
(
MPN)
of
total
coliforms
(
yellow
wells)
and
E.
coli
(
yellow/
fluorescent
wells)
in
the
sample.
The
color
and
fluorescent
intensity
of
positive
wells
may
vary.

12.1.2
Record
the
results
as
the
Most
Probable
Number/
100
mL.
If
any
dilutions
were
made,
multiply
the
MPN/
mL
by
the
dilution
factor
to
obtain
the
final
MPN/
100
value.

13.0
Method
Performance
13.1
Colilert
®
found
to
be
equally
sensitive
to
LTB,
EC+
MUG
(
6).

13.2
Correlation
of
0.905
found
between
Colilert
®
and
m­
Tec
for
E.
coli
(
6).

14.0
Reporting
Results
14.1
Report
results
as
Presence
or
Absence
for
total
coliforms
and
for
E.
coli.
For
quantification,
report
results
as
MPN/
100
mL
for
total
coliforms
and
E.
coli.

15.0
Verification
Procedure
15.1
Not
applicable.

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
identification
rules
and
land
disposal
restrictions.
Compliance
with
all
sewage
discharge
permits
and
regulations
is
also
required.

17.2
Samples,
reference
materials
and
equipment
known
or
suspected
to
have
viable
bacteria
attached
or
contained
must
be
sterilized
prior
to
disposal.
Draft
5
June
2003
18.0
References
18.1
Colilert
®
Package
Insert
from
IDEXX.

18.2
Annual
Book
of
ASTM
Standards,
Vol.
11.01,
American
Society
for
Testing
Materials,
Philadelphia,
PA
19103.

18.3
Bordner,
R.,
J.
A.
Winter
and
P.
V.
Scarpino
(
eds.).
Microbiological
Methods
for
Monitoring
the
Environment,
Water
and
Wastes,
EPA­
600/
8­
78­
017.
Office
of
Research
and
Development,
USEPA.

18.4
Clesceri,
L.
S.,
A.
E.
Greenberg,
A.
D.
Eaton
(
eds.).
1998.
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
20th
Edition,
American
Public
Health
Association,
Washington,
DC.

18.5
Quanti­
Tray
 
Package
Insert
from
IDEXX.

18.6
Federal
Register
/
Vol.
66,
No.
169
/
Thursday,
August
30,
2001,
page
45818.