TOTAL
COLIFORMS
AND
E.
COLI
MEMBRANE
FILTRATION
METHOD
JUNE
2003
DRAFT
Draft
1
June
2003
TOTAL
COLIFORMS
AND
E.
COLI
MEMBRANE
FILTRATION
METHOD
Sensitivity:
1
CFU/
100
mL
m­
ColiBlue24
®
Broth
U.
S.
Environmental
Protection
Agency
(
USEPA)
Approved
for
Drinking
Water
Method
No.
10029
Revision
3,
2/
13/
02
1.0
Scope
and
Application
1.1
This
method
determines
the
presence
or
absence
of
total
coliforms
and
E.
coli
in
finished
potable
water
using
a
selective
and
differential
membrane
filtration
(
MF)
medium,
m­
ColiBlue24
Broth.

1.2
This
method
can
detect
the
presence
or
absence
of
both
total
coliforms
and
E.
coli
simultaneously
within
24
hours
and
without
the
need
for
a
confirmation
step.

1.3
The
detection
limit
of
the
method
is
one
colony
forming
unit
(
CFU)
of
coliform
bacteria
per
100
mL
of
sample.
See
Attachment
1.3.

2.0
Summary
of
Method
2.1
Coliform
bacteria
are
identified
in
water
either
as
total
coliforms
or
E.
coli.
Total
coliforms
can
be
present
in
water
without
E.
coli
being
present;
E.
coli
cannot
be
present
in
water
without
total
coliforms
also
being
present.

2.2
A
100­
mL
volume
of
sample
is
filtered
through
a
47­
mm
membrane
filter
using
standard
techniques.
The
filter
is
then
transferred
to
a
50­
mm
petri
plate
containing
an
absorbent
pad
saturated
with
m­
ColiBlue24
Broth.
The
filter
is
then
incubated
at
35
°
C
±
0.5
°
C
for
24
hours.
If
coliform
bacteria
are
present
in
the
sample,
both
red
and
blue
colonies
may
appear;
the
blue
colonies
are
specific
to
the
presence
of
E.
coli.

2.3
m­
ColiBlue24
Broth
is
a
nutritive,
lactose­
based
medium,
containing
inhibitors
to
selectively
eliminate
growth
of
non­
coliforms.
It
is
analogous
to
an
improved
version
of
m­
Endo.
Total
coliform
colonies
growing
on
the
medium
are
highlighted
by
a
non­
selective
dye,
2,3,5­
Triphenoltetrazolium
Chloride
(
TTC),
which
produces
red
colored
colonies.
Among
the
total
coliform
colonies,
which
grow
up
on
the
medium,
any
E.
coli
colonies
are
distinguishable
by
a
selective
blue
color,
resulting
from
the
action
of
b­
glucuronidase
enzyme
on
5­
Bromo­
4­
Chloro­
3­
Indolyl­
Beta­
D­
glucuronide
(
BCIG).

3.0
Definitions
3.1
Material
Safety
Data
Sheet
(
MSDS)
­
Written
information
provided
for
each
chemical
reagent
or
standard
about
a
chemical's
toxicity,
health
hazards,
physical
properties,
flammability,
and
reactivity.
It
also
includes
storage,
spill,
and
handling
precautions.
Draft
2
June
2003
3.2
Total
Coliform
Bacteria
­
Bacteria
belonging
to
the
genera
Klebsiella
sp.,
Enterobacter
sp.,
Citrobacter
sp.,
or
Escherichia
sp.

3.3
Coliform
Positive
Colony
­
A
red
or
blue
colony.

3.4
Coliform
Negative
Colony
­
A
clear
or
white
colony.

3.5
Escherichia
coli
or
E.
coli
Bacteria
­
A
genus
within
the
total
coliform
group
typified
by
possession
of
the
enzyme
b­
Glucuronidase,
ability
to
grow
at
44.5
°
C,
and
form
indole
from
tryptophan.

3.6
E.
coli
Positive
Colony
­
A
blue
colony.

3.7
E.
coli
Negative
Colony
­
A
non­
blue
colony.

4.0
Interferences
4.1
No
interferences
to
the
colony
color
development
have
been
found
in
finished
potable
water
samples.
Similarly,
particulates
in
water
samples
do
not
alter
the
efficacy
of
the
medium,
although
excess
particulates
may
cause
colonies
to
grow
together
on
crowded
filters
or
slow
the
sample
filtration
process.

5.0
Safety
5.1
Standard
safety
practices
appropriate
to
microbiology
laboratories
should
be
followed.
Follow
the
test
procedure
carefully
and
observe
all
precautionary
measures.

5.2
Solid
and
liquid
waste
materials
containing
or
suspected
to
contain
viable
bacteria
should
be
decontaminated
using
an
autoclave
or
by
using
an
appropriate
disinfectant
before
discarding.

5.3
Refer
to
the
appropriate
Material
Safety
Data
Sheets
supplied
for
each
reagent
for
comprehensive
safety
data
essential
to
proper
use.

6.0
Equipment
and
Supplies
6.1
Equipment
6.1.1
Air
Incubator
­
Capable
of
operating
at
35
°
C
±
0.5
°
C.

6.1.2
Vacuum
pump.

6.1.3
Membrane
filtration
funnel
unit
and
flask.

6.1.4
Dissecting
microscope,
capable
of
10
 
15X
magnification.
The
microscope
should
be
equipped
with
a
fluorescent
illuminator.
Draft
3
June
2003
6.2
Supplies/
Glassware
­
Cleanse
all
glassware
thoroughly
with
a
suitable
detergent
and
hot
water,
rinse
with
hot
water
to
remove
traces
of
detergent
residual,
and
rinse
again
with
laboratory­
pure
water.
Sterilize
all
glassware
by
autoclaving
15
min.
at
121
°
C
or
by
heating
in
an
oven
for
at
least
1
hour
at
170
°
C.

6.2.1
Presterilized
50­
mm
MF
petri
plates
with
pads.

6.2.2
45­
mm
presterilized
membrane
filters.

6.2.3
Sterile
Forceps.

6.2.4
Sterile
glass
or
plastic
sample
collection
containers.

6.2.5
Sterile
graduated
cylinders.

6.2.6
Sterile
pipets.

6.2.7
Sterile
MF
filtration
unit.

6.2.8
Side­
arm
flask.

6.2.9
Biohazard
bag.

6.2.10150
mL
of
sterile
rinse
water.

7.0
Reagents
and
Standards
7.1
Growth
Medium
7.1.1
The
membrane
filtration
method
for
total
coliforms
and
E.
coli
utilizes
m­
ColiBlue24
Broth,
Hach
product
26084­
20,
26084­
42,
or
26084­
50.
The
broth
is
provided
in
ready
to
use
2­
mL
ampules
or
100­
mL
glass
bottles.
Under
proper
storage
conditions
(
2
 
8
°
C)
the
broth
has
a
shelf
life
of
one
year.
The
broth
contains
a
nutritive
medium
and
colorimetric
indicators.

7.2
Dechlorinating
Reagent
7.2.1
Hach
decholorinating
reagent
Powder
Pillow
(
14363­
69)
contains
sodium
thiosulfate
(
Na2S2O3
)
and
sodium
sulfate
(
Na2SO4).

7.2.2
To
make
a
3%
sodium
thiosulfate
(
Na2S2O3
)
solution,
add
48.18
g
of
sodium
thiosulfate
pentahydrate
to
approximately
500
mL
of
deionized
water,
then
dilute
to
1
L
with
deionized
water.
*
Whirl­
Pak
®
is
a
registered
trademark
and
patented
product
of
Nasco;
U.
S.
Patent
2,973,131.

Draft
4
June
2003
8.0
Sample
Collection,
Dechlorination,
Preservation,
Shipment
and
Storage
8.1
Water
Sample
Collection
8.1.1
Sample
Collection
Containers
­
Samples
should
be
collected
in
sterile,
clean
glass
or
heat­
resistant
bottles.
Presterilized
Whirl­
Pak
®
1
Bags
may
also
be
used.

8.1.2
Sampling
Procedure
­
Potable
water
samples
are
taken
by
first
flushing
the
tap
2
 
3
minutes
to
clear
service
line.
Collect
samples
using
aseptic
techniques
to
avoid
contamination.
For
other
water
samples,
aseptically
collect
water
representative
of
the
source.

8.2
Dechlorination
­
Water
containing
chlorine
or
other
halogens
must
be
treated
with
sodium
thiosulfate
to
allow
accurate
evaluation
of
microbial
content.
Add
one
decholorinating
reagent
Powder
Pillow
(
14363­
69)
by
aseptically
cutting
off
the
tip
of
an
alcohol­
rinsed
pillow
and
pouring
the
contents
into
100
mL
of
chlorinated
water
sample.
Alternatively,
pipet
0.1
mL
of
a
3%
sodium
thiosulfate
(
Na2S2O3)
solution
into
100
mL
of
a
chlorinated
water
sample.
Presterilized
Whirl­
Pak
®
Bags
contain
sufficient
sodium
thiosulfate
powder
to
neutralize
a
100­
mL
chlorinated
water
sample.

8.3
Preservation,
Shipment,
Storage
­
Samples
should
be
tested
as
soon
as
possible.
If
analysis
cannot
be
done
within
1
hr
of
collection,
potable
water
samples
should
be
held
on
ice
or
at
2
 
8
°
C
in
a
refrigerator
for
a
maximum
holding
time
of
30
hours
(
16.1).
Non­
potable
samples
should
be
held
at
<
10
°
C
for
a
maximum
period
of
8
hours
from
sampling
to
analysis.

9.0
Quality
Control
9.1
m­
ColiBlue24
Broth
undergoes
quality
control
(
QC)
testing
at
the
time
of
manufacture.
A
Certificate
of
Analysis
is
included
with
every
m­
ColiBlue24
product,
stating
that
the
m­
ColiBlue24
Broth,
as
received
by
the
analyst,
is
ready
for
use
in
analyzing
water
samples
by
the
membrane
filtration
procedure.
Each
laboratory
should
test
quality
control
using
the
following
procedure.
Three
ampules
will
be
required,
as
well
as
membrane
filtration
equipment.
The
three
ampules
will
be
used
as
an
uninoculated
blank,
a
positive
control
for
both
total
coliforms
and
E.
coli,
and
a
negative
control.

9.2
The
day
before
the
QC
procedure,
inoculate
three
10­
mL
Tryptic
Soy
Broth
tubes
with
Psuedomonas
aeruginosa
(
ATCC
27853).
Incubate
at
35
°
C
±
0.5
°
C
for
24
±
4
hours.

9.3
Aseptically
open
three
ampules
of
m­
ColiBlue24
Broth
and
saturate
the
pads
in
three
MF
petri
dishes.

9.4
Filter
100
mL
of
sterile
water
and
place
filter
on
the
first
pad.
This
is
the
uninoculated
blank.
Draft
5
June
2003
9.5
Prepare
serial
dilutions
as
described
in
Standard
Methods
9222B
(
16.2).
Prepare
a
serial
dilution
of
the
24
hr.
P.
aeruginosa
culture
so
that
filtration
of
a
100­
mL
volume
would
theoretically
yield
20
 
80
colonies.
Filter
the
bacteria­
containing
diluent.
After
filtration,
rinse
the
funnel
with
several
20
 
30­
mL
volumes
of
sterile
rinse
water.
Place
membrane
filter
into
the
second
plate
of
m­
ColiBlue24
Broth;
this
is
the
negative
control.
Prepare
appropriate
serial
dilutions
of
the
E.
coli
culture
and
filter.
Rinse
the
funnel
as
previously
indicated.
Using
this
same
membrane
filter,
serially
dilute
the
E.
cloacae
culture,
filter
and
rinse.
This
will
introduce
both
E.
coli
cells
and
E.
cloacae
cells
onto
the
same
filter
for
a
dual
positive
control.
Incubate
all
three
MF
petri
plates
for
24
hours
at
35
°
C
±
0.5
°
C.
The
uninoculated
control
should
remain
blank.
The
P.
aeruginosa
control
should
have
no
colonies.
The
positive
control
should
have
both
blue
(
E.
coli)
and
red
(
E.
cloacae)
colonies.

9.6
Colonies
may
be
picked
from
membrane
filters
and
inoculated
into
Lauryl
Tryptose
Broth
(
LTB),
Brilliant
Green
Lactose
Bile
(
BGLB),
EC+
MUG,
or
other
media
for
further
QC
testing
if
desired.

10.0
Calibration
and
Standardization
10.1
No
calibration
or
standardization
of
m­
ColiBlue24
Broth
is
required.

10.2
All
equipment
used
in
Section
6
should
be
calibrated
and
maintained
according
to
manufacturer's
instructions.

11.0
Procedure
11.1
Test
Procedure
11.1.1
Aseptically
open
an
ampule
containing
m­
ColiBlue24
Broth
and
pour
the
broth
onto
the
pad
in
a
50­
mm
MF
petri
plate.

11.1.2
Place
a
sterile
filter
onto
a
sterile
filter
holder.
Using
sterile
graduated
cylinders
and
pipets,
measure
an
appropriate
sample
aliquot.
Pour
water
sample
into
reservoir
funnel
on
filter
holder
and
draw
the
water
through
the
filter
using
a
vacuum
pump.
Rinse
the
funnel
with
several
20
 
30­
mL
volumes
of
sterile
rinse
water.
With
sterile
forceps,
transfer
the
filter
to
a
petri
plate
containing
the
pad
saturated
with
m­
ColiBlue24.
Invert
plate
and
incubate
at
35
°
C
±
0.5
°
C
for
24
hours.

11.2
Interpretation
11.2.1
Examine
filters
for
colony
growth.
Colonies
are
typically
readily
visible,
but
a
dissecting
microscope
may
prove
useful.
Draft
6
June
2003
11.2.2
A
red
or
blue
colony
is
a
Total
Coliform
Positive
result.
A
clear
or
white
colony
is
a
Total
Coliform
Negative
result.
A
blue
colony
is
specifically
an
E.
coli
Positive
result.
A
non­
blue
colony
is
an
E.
coli
Negative
result:

Positive
Result
Negative
Result
Total
Coliform
Red
or
Blue
Colony
Clear
or
White
Colony
E.
coli
Blue
Colony
only
Non­
Blue
Colony
11.2.3
The
incubation
time
is
24
hours.
If
no
colonies
are
visible
after
24
hours,
the
sample
is
free
of
total
coliforms
and
E.
coli.

12.0
Data
Analysis,
Calculations,
Interpretation
and
Reporting
12.1
Presence/
Absence
12.1.1
The
presence
of
at
least
one
red
or
blue
colony
indicates
the
sample
is
total
coliform
positive.
The
presence
of
at
least
one
blue
colony
also
indicates
the
sample
contains
E.
coli.
Absence
of
red
or
blue
colonies
indicates
the
sample
contains
no
total
coliforms
or
E.
coli.
No
further
analysis
or
calculation
is
required.

12.2
Quantitation
12.2.1
This
method
is
approved
by
the
USEPA
for
the
purposes
of
determining
the
presence
or
absence
of
total
coliforms
and
E.
coli
in
drinking
water.
Since
a
protocol
for
evaluating
the
use
of
a
new
method
for
the
enumeration
of
total
coliforms
and/
or
E.
coli
in
drinking
water
does
not
exist,
new
methods,
such
as
m­
ColiBlue24,
cannot
be
approved
by
the
USEPA
as
quantitative
methods
for
USEPA
reporting
purposes.
However,
we
have
included
the
enumeration
protocol
for
other
(
non­
USEPA
reporting)
purposes.

12.2.2
Coliform
density
determinations
can
often
provide
additional
data
for
problem
solving
within
the
water
plant
or
for
analysis
of
source
water
samples.
m­
Coliblue24
Broth
can
be
used
to
make
density
determinations.

12.2.3
If
enumeration
of
total
coliform
and/
or
E.
coli
populations
is
desired,
refer
to
Standard
Methods
9222B
for
appropriate
dilutions
of
the
sample
to
filter
so
that
20
 
80
coliform
colonies
are
present
after
incubation.
For
analysis
of
potable
water
samples,
a
standard
sample
of
100
mL
is
always
required.
Calculate
the
number
of
microorganisms
per
100­
mL
sample
also
as
described
in
Standard
Methods
9222B.
Draft
7
June
2003
13.0
Method
Performance
Characteristics
13.1
Specificity:
The
specificity
of
m­
ColiBlue24
Broth
for
recovery
of
total
coliforms
and
E.
coli
was
evaluated.
These
experiments
were
conducted
according
to
the
EPA
protocol
of
June
30,
1992
(
16.3).
For
E.
coli,
the
false
positive
error
was
2.5%
and
the
undetected
target
error
was
0%
(
Section
17.0,
Table
17.1).
Overall
agreement
between
m­
ColiBlue24
and
E.
coli
reference
methods
was
98.8%.
For
total
coliforms,
the
false
positive
error
was
26.8%
and
the
undetected
target
error
was
1.6%
(
Table
17.2).
Using
m­
Endo
as
a
comparison
for
total
coliform
recovery
to
m­
ColiBlue24,
m­
Endo
total
coliform
false
positive
error
was
29.6%
and
the
undetected
target
error
was
3.4
%
(
Table
17.3).
Overall
agreement
for
total
coliform
recovery
was
86.2%
for
m­
ColiBlue24,
and
85.7%
with
m­
Endo.
Although
the
total
coliform
false
positive
errors
seemed
large
initially,
review
of
the
literature
revealed
that
many
existing
media
for
total
coliform
recovery
have
similar
false
positive
error
rates
(
16.4
­
16.10).

13.2
Precision
and
Bias:
Not
applicable,
as
this
protocol
was
conducted
on
a
presence/
absence
basis.
If
individuals
wish
to
use
this
medium
for
quantitative
determinations
they
may
use
precision
and
bias
calculations
specified
by
ASTM
(
16.11).

14.0
Pollution
Prevention
14.1
Base
the
quantity
of
chemicals
purchased
on
expected
usage
during
its
shelf
life
and
disposal
cost
of
unused
material.
Actual
reagent
preparation
volume
should
reflect
anticipated
usage
and
reagent
stability.

14.2
For
information
about
pollution
prevention
that
may
be
applicable
to
laboratories
and
research
institutions,
consult
Less
is
better:
Laboratory
Chemical
Management
for
Waste
Reduction,
available
from
the
American
Chemical
Society's
department
of
Government
Regulations
and
Science
Policy,
1155
16th
Street
N.
W.,
Washington
D.
C.,
20036.
Phone:
(
202)
872­
4477.

15.0
Waste
Management
15.1
It
is
the
laboratory's
responsibility
to
comply
with
all
federal,
state,
and
local
regulations
governing
waste
management,
particularly
the
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.

15.2
See
the
MSDS
for
product
composition
information
and
further
guidance
on
waste
disposal.

15.3
For
more
information
on
laboratory
waste
management,
consult
Waste
Management
Manual
for
Laboratory
Personnel,
available
from
the
American
Chemical
Society's
department
of
Government
Regulations
and
Science
Policy,
1155
16th
Street
N.
W.,
Washington
D.
C.,
20036.
Phone:
(
202)
872­
4477.
Draft
8
June
2003
16.0
References
16.1
Federal
Register,
Title
40,
Part
141,
Section
141.21
Coliform
sampling.
December
5,
1994.

16.2
"
American
Public
Health
Association,
American
Water
Works
Association,
Water
Environment
Federation.
Microbiological
Examination,
Part
9000"
in:
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
18th
ed.,
A.
E.
Greenberg,
L.
S.
Clesceri,
A.
D.
Eaton,
eds.
Washington,
D.
C.,
American
Public
Health
Association,
1993.

16.3
U.
S.
Environmental
Protection
Agency.
Requirements
for
the
nationwide
approval
of
new
or
optionally
revised
methods
for
total
coliforms,
fecal
coliforms,
and/
or
E.
coli,
in
national
drinking
water
monitoring.
Revision
1.2,
June
30,
1992.
Environmental
Monitoring
Systems
Laboratory,
Cincinnati,
OH.

16.4
Brenner,
K.
P.,
C.
C.
Rankin,
Y.
R.
Roybal,
G.
N.
Stelma,
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.

16.5
Cenci,
G.,
A.
De
Bartolomeo,
and
G.
Caldini.
1993.
Comparison
of
fluorogenic
and
conventional
membrane
filter
media
for
enumerating
coliform
bacteria.
Microbios
76:
47
 
54.

16.6
Covert
T.
C.,
L.
C.
Shadix,
E.
W.
Rice,
J.
R.
Haines,
and
R.
W.
Freyberg.
1989.
Evaluation
of
the
autoanalysis
colilert
test
for
detection
and
enumeration
of
total
coliforms.
Appl.
Environ.
Microbiol.
55:
2443
 
2447.

16.7
Edberg,
S.
C.,
M.
J.
Allen,
D.
B.
Smith,
and
the
national
collaborative
study.
1988.
National
field
evaluation
of
a
defined
substrate
method
for
the
simultaneous
enumeration
of
total
coliforms
and
Escherichia
coli
from
drinking
water:
comparison
with
the
standard
multiple
tube
fermentation
method.
Appl.
Environ.
Microbiol.
54:
1595
 
1601.

16.8
Jacobs,
N.
J.,
W.
L.
Zeigler,
F.
C.
Reed,
T.
A.
Stukel,
and
E.
W.
Rice.
1986.
Comparison
of
membrane
filter,
multiple­
fermentation­
tube,
and
presence­
absence
techniques
for
detecting
total
coliforms
in
small
community
water
systems.
Appl.
Environ.
Microbiol.
51:
1007
 
1012.
16.9
Lupo,
L.,
E.
Stickland,
A.
Dufour,
and
V.
Cabelli.
11977.
The
effect
of
oxidase
positive
bacteria
on
total
coliform
density
estimates.
Health
Lab.
Sci.
14:
117­
121.

16.10
Sartory,
D.
P.
and
L.
Howard.
11992.
A
medium
detecting
b­
glucuronidase
for
the
simultaneous
membrane
filtration
enumeration
of
Escherichia
coli
and
coliforms
from
drinking
water.
Lett.
Appl.
Microbiol.
15:
273­
276.

16.11
American
Society
for
Testing
and
Materials.
Determination
of
precision
and
bias
applicable
methods
of
committee
D­
19
on
water.
D
2777­
86.
Annual
Book
of
ASTM
Standards,
Vol.
11.01.
ASTM,
Philadelphia,
1993.
Draft
9
June
2003
17.0
Tables
17.1
E.
coli
recovery
on
m­
ColiBlue24
Reference
Positive
Negative
Total
Positive
234
6
240
Negative
0
250
250
Total
234
256
490
Sensitivity
100.0%
Specificity
97.7%
False
Positive
Error
2.5%
Undetected
Target
Error
0%
Overall
Agreement
98.8%

17.2
Coliform
recovery
on
m­
ColiBlue24
Reference
Positive
Negative
Total
Positive
183
67
250
Negative
2
248
250
Total
185
315
500
Sensitivity
96.8%
Specificity
80.1%
False
Positive
Error
29.0%
Undetected
Target
Error
3.2%
Overall
Agreement
85.7%

Reference
Positive
Negative
Total
Positive
149
61
210
Negative
5
245
250
Total
154
306
460
Sensitivity
96.8%
Specificity
80.1%
False
Positive
Error
29.0%
Undetected
Target
Error
3.2%
Overall
Agreement
85.7%
