Document ID: EPA-HQ-OW-2002-0039-0134
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-07-09T04:00Z

STANDARD
METHODS
9222
B.
STANDARD
TOTAL
COLIFORM
MEMBRANE
FILTER
PROCEDURE,

STANDARD
METHODS
9222
D.
FECAL
COLIFORM
MEMBRANE
FILTER
PROCEDURE,
AND
STANDARD
METHODS
9222G:
MF
PARTITION
PROCEDURES
ESCHERICHIA
COLI
PARTITION
METHODS
JUNE
2003
DRAFT
Draft
1
June
2003
STANDARD
METHODS
9222
B.
STANDARD
TOTAL
COLIFORM
MEMBRANE
FILTER
PROCEDURE
Reprinted
by
Permission
from
the
20th
Edition
1.
Laboratory
Apparatus
For
MF
analyses
use
glassware
and
other
apparatus
composed
of
material
free
from
agents
that
may
affect
bacterial
growth.

a.
Sample
bottles:
See
Section
9030B.
18.

b.
Dilution
bottles:
See
Section
9030B.
13.

c.
Pipets
and
graduated
cylinders:
See
Section
9030B.
9.
Before
sterilization,
loosely
cover
opening
of
graduated
cylinders
with
metal
foil
or
a
suitable
heavy
wrapping­
paper
substitute.
Immediately
after
sterilization
secure
cover
to
prevent
contamination.

d.
Containers
for
culture
medium:
Use
clean
borosilicate
glass
flasks.
Any
size
or
shape
of
flask
may
be
used,
but
erlenmeyer
flasks
with
metal
caps,
metal
foil
covers,
or
screw
caps
provide
for
adequate
mixing
of
the
medium
contained
and
are
convenient
for
storage.

e.
Culture
dishes:
Use
sterile
borosilicate
glass
or
disposable,
presterilized
plastic
petri
dishes,
60
×
15
mm,
50
×
9
mm,
or
other
appropriate
size.
Wrap
convenient
numbers
of
clean,
glass
culture
dishes
in
metal
foil
if
sterilized
by
dry
heat,
or
suitable
heavy
wrapping
paper
when
autoclaved.
Incubate
loose­
lidded
glass
and
disposable
plastic
culture
dishes
in
tightly
closed
containers
with
wet
paper
or
cloth
to
prevent
moisture
evaporation
with
resultant
drying
of
medium
and
to
maintain
a
humid
environment
for
optimum
colony
development.
Presterilized
disposable
plastic
dishes
with
tight­
fitting
lids
that
meet
the
specifications
above
are
available
commercially
and
are
used
widely.
Reseal
opened
packages
of
disposable
dish
supplies
for
storage.

f.
Filtration
units:
The
filter­
holding
assembly
(
constructed
of
glass,
autoclavable
plastic,
porcelain,
or
stainless
steel)
consists
of
a
seamless
funnel
fastened
to
a
base
by
a
locking
device
or
by
magnetic
force.
The
design
should
permit
the
membrane
filter
to
be
held
securely
on
the
porous
plate
of
the
receptacle
without
mechanical
damage
and
allow
all
fluid
to
pass
through
the
membrane
during
filtration.
Discard
plastic
funnels
with
deep
scratches
on
inner
surface
or
glass
funnels
with
chipped
surfaces.

Wrap
the
assembly
(
as
a
whole
or
separate
parts)
in
heavy
wrapping
paper
or
aluminum
foil,
sterilize
by
autoclaving,
and
store
until
use.
Alternatively
expose
all
surfaces
of
the
previously
cleaned
assembly
to
ultraviolet
radiation
(
2
min
exposure)
for
the
initial
sanitization
before
use
in
the
test
procedure,
or
before
reusing
units
between
successive
filtration
series.
Field
units
may
be
sanitized
by
dipping
or
spraying
with
alcohol
and
then
igniting
or
immersing
in
boiling
water
for
2
min.
After
submerging
unit
in
boiling
water,
cool
it
to
room
temperature
before
reuse.
Do
not
ignite
plastic
parts.
Sterile,
disposable
field
units
may
be
used.

For
filtration,
mount
receptacle
of
filter­
holding
assembly
on
a
1­
L
filtering
flask
with
a
side
tube
or
other
suitable
device
(
manifold
to
hold
three
to
six
filter
assemblies)
such
that
a
pressure
differential
(
34
to
51
kPa)
can
be
exerted
on
the
filter
membrane.
Connect
flask
to
a
vacuum
line,
an
electric
vacuum
pump,
a
filter
pump
operating
on
water
pressure,
a
hand
aspirator,
or
other
means
of
securing
a
pressure
differential
(
138
to
207
kPa).
Connect
a
flask
of
approximately
the
same
capacity
between
filtering
flask
and
vacuum
source
to
trap
carry­
over
water.
Draft
2
June
2003
g.
Membrane
filter:
Use
membrane
filters
(
for
additional
specifications,
see
Section
9020)
with
a
rated
pore
diameter
such
that
there
is
complete
retention
of
coliform
bacteria.
Use
only
those
filter
membranes
that
have
been
found,
through
adequate
quality
control
testing
and
certification
by
the
manufacturer,
to
exhibit:
full
retention
of
the
organisms
to
be
cultivated,
stability
in
use,
freedom
from
chemical
extractables
that
may
inhibit
bacterial
growth
and
development,
a
satisfactory
speed
of
filtration
(
within
5
min),
no
significant
influence
on
medium
pH
(
beyond
±
0.2
units),
and
no
increase
in
number
of
confluent
colonies
or
spreaders
compared
to
control
membrane
filters.
Use
membranes
grid­
marked
in
such
a
manner
that
bacterial
growth
is
neither
inhibited
nor
stimulated
along
the
grid
lines
when
the
membranes
with
entrapped
bacteria
are
incubated
on
a
suitable
medium.
Preferably
use
fresh
stocks
of
membrane
filters
and
if
necessary
store
them
in
an
environment
without
extremes
of
temperature
and
humidity.
Obtain
no
more
than
a
year's
supply
at
any
one
time.

Preferably
use
presterilized
membrane
filters
for
which
the
manufacturer
has
certified
that
the
sterilization
technique
has
neither
induced
toxicity
nor
altered
the
chemical
or
physical
properties
of
the
membrane.
If
membranes
are
sterilized
in
the
laboratory,
autoclave
for
10
min
at
121
°
C.
At
the
end
of
the
sterilization
period,
let
the
steam
escape
rapidly
to
minimize
accumulation
of
water
of
condensation
on
filters.

h.
Absorbent
pads
consist
of
disks
of
filter
paper
or
other
material
certified
for
each
lot
by
the
manufacturer
to
be
of
high
quality
and
free
of
sulfites
or
other
substances
of
a
concentration
that
could
inhibit
bacterial
growth.
Use
pads
approximately
48
mm
in
diameter
and
of
sufficient
thickness
to
absorb
1.8
to
2.2
mL
of
medium.
Presterilized
absorbent
pads
or
pads
subsequently
sterilized
in
the
laboratory
should
release
less
than
1
mg
total
acidity
(
calculated
as
CaCO3)
when
titrated
to
the
phenolphthalein
end
point,
pH
8.3,
using
0.02N
NaOH
and
produce
pH
levels
of
7
±
0.2.
Sterilize
pads
simultaneously
with
membrane
filters
available
in
resealable
kraft
envelopes,
or
separately
in
other
suitable
containers.
Dry
pads
so
they
are
free
of
visible
moisture
before
use.
See
sterilization
procedure
described
for
membrane
filters
above
and
Section
9020
for
additional
specifications
on
absorbent
pads.

i.
Forceps:
Smooth
flat
forceps,
without
corrugations
on
the
inner
sides
of
the
tips.
Sterilize
before
use
by
dipping
in
95%
ethyl
or
absolute
methyl
alcohol
and
flaming.

j.
Incubators:
Use
incubators
to
provide
a
temperature
of
35
±
0.5
°
C
and
to
maintain
a
humid
environment
(
60%
relative
humidity).

k.
Microscope
and
light
source:
To
determine
colony
counts
on
membrane
filters,
use
a
magnification
of
10
to
15
diameters
and
a
cool
white
fluorescent
light
source
adjusted
to
give
maximum
sheen
discernment.
Optimally
use
a
binocular
wide­
field
dissecting
microscope.
Do
not
use
a
microscope
illuminator
with
optical
system
for
light
concentration
from
an
incandescent
light
source
for
discerning
coliform
colonies
on
Endo­
type
media.
1
Dehydrated
Difco
M­
Endo
Agar
LES
(
No.
0736),
dehydrated
BBL
M­
Endo
Agar
LES
(
No.
11203),
or
equivalent.

Draft
3
June
2003
2.
Materials
and
Culture
Media
The
need
for
uniformity
dictates
the
use
of
commercial
dehydrated
media.
Never
prepare
media
from
basic
ingredients
when
suitable
dehydrated
media
are
available.
Follow
manufacturer's
directions
for
rehydration.
Store
opened
supplies
of
dehydrated
media
in
a
desiccator.
Commercially
prepared
media
in
liquid
form
(
sterile
ampule
or
other)
may
be
used
if
known
to
give
equivalent
results.
See
Section
9020
for
media
quality
control
specifications.

Test
each
new
medium
lot
against
a
previously
acceptable
lot
for
satisfactory
performance
as
described
in
Section
9020B.
With
each
new
lot
of
Endo­
type
medium,
verify
a
minimum
10%
of
coliform
colonies,
obtained
from
natural
samples
or
samples
with
known
additions,
to
establish
the
comparative
recovery
of
the
medium
lot.

Before
use,
test
each
batch
of
laboratory­
prepared
MF
medium
for
performance
with
positive
and
negative
culture
controls.
Check
for
coliform
contamination
at
the
beginning
and
end
of
each
filtration
series
by
filtering
20
to
30
mL
of
dilution
or
rinse
water
through
the
filter.
If
controls
indicate
contamination,
reject
all
data
from
affected
samples
and
request
resample.

a.
LES
Endo
agar:
1
Yeast
extract......................................................................
1.2
g
Casitone
or
trypticase.........................................................
3.7
g
Thiopeptone
or
thiotone......................................................
3.7
g
Tryptose.............................................................................
7.5
g
Lactose..............................................................................
9.4
g
Dipotassium
hydrogen
phosphate,
K2HPO4........................
3.3
g
Potassium
dihydrogen
phosphate,
KH2PO4.........................
1.0
g
Sodium
chloride,
NaCl......................................................
.3.7
g
Sodium
desoxycholate........................................................
0.1
g
Sodium
lauryl
sulfate........................................................
0.05
g
Sodium
sulfite,
Na2SO3......................................................
1.6
g
Basic
fuchsin....................................................................
0.8
g
Agar...............................................................................
15.0
g
Reagent­
grade
water........................................................
.1.0
L
Rehydrate
product
in
1
L
water
containing
20
mL
95%
ethanol.
Do
not
use
denatured
ethanol,
which
reduces
background
growth
and
coliform
colony
size.
Bring
to
a
near
boil
to
dissolve
agar,
then
promptly
remove
from
heat
and
cool
to
45
to
50
°
C.
Do
not
sterilize
by
autoclaving.
Final
pH
7.2
±
0.2.
Dispense
in
5­
to
7­
mL
quantities
into
lower
section
of
60­
mm
glass
or
plastic
petri
dishes.
If
dishes
of
any
other
size
are
used,
adjust
quantity
to
give
an
equivalent
depth
of
4
to
5
m.
Do
not
expose
poured
plates
to
direct
sunlight;
refrigerate
in
the
dark,
preferably
in
sealed
plastic
bags
or
other
containers
to
reduce
moisture
loss.
Discard
unused
medium
after
2
weeks
or
sooner
if
there
is
evidence
of
moisture
loss,
medium
contamination,
or
medium
deterioration
(
darkening
of
the
medium).
2
Dehydrated
Difco
M­
Endo
Broth
MF
(
No.
0749),
dehydrated
BBL
m­
Endo
Broth
(
No.
11119),
or
equivalent
may
be
used
if
absorbent
pads
are
used.

Draft
4
June
2003
b.
M­
Endo
medium:
2
Tryptose
or
polypeptone.....................................................
10.0
g
Thiopeptone
or
thiotone........................................................
5.0
g
Casitone
or
trypticase..........................................................
5.0
g
Yeast
extract......................................................................
1.5
g
Lactose............................................................................
12.5
g
Sodium
chloride,
NaCl........................................................
5.0
g
Dipotassium
hydrogen
phosphate,
K2HPO4.....................
4.375
g
Potassium
dihydrogen
phosphate,
KH2PO4.....................
1.375
g
Sodium
lauryl
sulfate......................................................
..
0.05
g
Sodium
desoxycholate.......................................................
0.10
g
Sodium
sulfite,
Na2SO3.....................................................
2.10
g
Basic
fuchsin.................................................................
..
1.05
g
Agar
(
optional)...............................................................
.15.0
g
Reagent­
grade
water........................................................
.1.0
L
1)
Agar
preparation
 
Rehydrate
product
in
1
L
water
containing
20
mL
95%
ethanol.
Heat
to
near
boiling
to
dissolve
agar,
then
promptly
remove
from
heat
and
cool
to
between
45
and
50
°
C.
Dispense
5­
to
7­
mL
quantities
into
60­
mm
sterile
glass
or
plastic
petri
dishes.
If
dishes
of
any
other
size
are
used,
adjust
quantity
to
give
an
equivalent
depth.
Do
not
sterilize
by
autoclaving.
Final
pH
should
be
7.2
±
0.2.
A
precipitate
is
normal
in
Endo­
type
media.
Refrigerate
finished
medium
in
the
dark
and
discard
unused
agar
after
2
weeks.

2)
Broth
preparation
 
Prepare
as
above,
omitting
agar.
Dispense
liquid
medium
(
at
least
2.0
mL
per
plate)
onto
absorbent
pads
(
see
absorbent
pad
specifications,
Section
9222B.
1)
and
carefully
remove
excess
medium
by
decanting
the
plate.
The
broth
may
have
a
precipitate
but
this
does
not
interfere
with
medium
performance
if
pads
are
certified
free
of
sulfite
or
other
toxic
agents
at
a
concentration
that
could
inhibit
bacterial
growth.
Refrigerated
broth
may
be
stored
for
up
to
4
d.

c.
Buffered
dilution
rinse
water:
See
Section
9050C.
1.

4.
Bibliography
Fifield,
C.
W.
&
C.
P.
Schaufus.
1958.
Improved
membrane
filter
medium
for
the
detection
of
coliform
organisms.
J.
Amer.
Water
Works
Assoc.
50:
193.

McCarthy,
J.
A.
&
J.
E.
Delaney.
1958.
Membrane
filter
media
studies.
Water
Sewage
Works
105:
292.

Rhines,
C.
E.
&
W.
P.
Cheevers.
1965.
Decontamination
of
membrane
filter
holders
by
ultraviolet
light.
J.
Amer.
Water
Works
Assoc.
57:
500.

Geldreich,
E.
E.,
H.
L.
Jeter
&
J.
A.
Winter.
1967.
Technical
considerations
in
applying
the
membrane
filter
procedure.
Health
Lab.
Sci.
4:
113.

Watling,
H.
R.
&
R.
J.
Watling.
1975.
Note
on
the
trace
metal
content
of
membrane
filters.
Water
SA
1:
28.
Draft
5
June
2003
Lin,
S.
D.
1976.
Evaluation
of
Millipore
HA
and
HC
membrane
filters
for
the
enumeration
of
indicator
bacteria.
Appl.
Environ.
Microbiol.
32:
300.

Standridge,
J.
H.
1976.
Comparison
of
surface
pore
morphology
of
two
brands
of
membrane
filters.
Appl.
Environ.
Microbiol.
31:
316.

Geldreich,
E.
E.
1976.
Performance
variability
of
membrane
filter
procedure.
Pub.
Health
Lab.
34:
100.

Grabow,
W.
O.
K.
&
M.
Du
Preez.
1979.
Comparison
of
m­
Endo
LES,
MacConkey
and
Teepol
media
for
membrane
filtration
counting
of
total
coliform
bacteria
in
water.
Appl.
Environ.
Microbiol.
38:
351.

Dutka,
B.
D.,
ed.
1981.
Membrane
Filtration
Applications,
Techniques
and
Problems.
Marcel
Dekker,
Inc.,
New
York,
N.
Y.

Evans,
T.
M.,
R.
J.
Seidler
&
M.
W.
LeChevallier.
1981.
Impact
of
verification
media
and
resuscitation
on
accuracy
of
the
membrane
filter
total
coliform
enumeration
technique.
Appl.
Environ.
Microbiol.
41:
1144.

Franzblau,
S.
G.,
B.
J.
Hinnebusch,
T.
M.
Kelley
&
N.
A.
Sinclair.
1984.
Effect
of
noncoliforms
on
coliform
detection
in
potable
groundwater:
improved
recovery
with
an
anaerobic
membrane
filter
technique.
Appl.
Environ.
Microbiol.
48:
142.

McFeters,
G.
A.,
J.
S.
Kippin
&
M.
W.
LeChevallier.
1986.
Injured
coliforms
in
drinking
water.
Appl.
Environ.
Microbiol.
51:
1.
3
Rosolic
acid
reagent
will
decompose
if
sterilized
by
autoclaving.
Refrigerate
stock
solution
in
the
dark
and
discard
after
2
weeks
or
sooner
if
its
color
changes
from
dark
red
to
muddy
brown.

Draft
6
June
2003
9222
D.
FECAL
COLIFORM
MEMBRANE
FILTER
PROCEDURE
Fecal
coliform
bacterial
densities
may
be
determined
either
by
the
multiple­
tube
procedure
or
by
the
MF
technique.
See
Section
9225
for
differentiation
of
Escherichia
coli,
the
predominant
fecal
coliform.
If
the
MF
procedure
is
used
for
chlorinated
effluents,
demonstrate
that
it
gives
comparable
information
to
that
obtainable
by
the
multiple­
tube
test
before
accepting
it
as
an
alternative.
The
fecal
coliform
MF
procedure
uses
an
enriched
lactose
medium
and
incubation
temperature
of
44.5
±
0.2
°
C
for
selectivity.
Because
incubation
temperature
is
critical,
submerge
waterproofed
(
plastic
bag
enclosures)
MF
cultures
in
a
water
bath
for
incubation
at
the
elevated
temperature
or
use
an
appropriate
solid
heat
sink
incubator
or
other
incubator
that
is
documented
to
hold
the
44.5
°
C
temperature
within
0.2
°
C
throughout
the
chamber,
over
a
24­
h
period.
Areas
of
application
for
the
fecal
coliform
method
in
general
are
stated
in
the
introduction
to
the
multiple­
tube
fecal
coliform
procedures,
Section
9221E.

1.
Materials
and
Culture
Medium
a.
M­
FC
medium:
The
need
for
uniformity
dictates
the
use
of
dehydrated
media.
Never
prepare
media
from
basic
ingredients
when
suitable
dehydrated
media
are
available.
Follow
manufacturer's
directions
for
rehydration.
Commercially
prepared
media
in
liquid
form
(
sterile
ampule
or
other)
also
may
be
used
if
known
to
give
equivalent
results.
See
Section
9020
for
quality
control
specifications.

M­
FC
medium:

Tryptose
or
biosate............................................
10.0
g
Proteose
peptone
No.
3
or
polypeptone..............
5.0
g
Yeast
extract....................................................
..
3.0
g
Sodium
chloride,
NaCl........................................
5.0
g
Lactose.............................................................
12.5
g
Bile
salts
No.
3
or
bile
salts
mixture...................
1.5
g
Aniline
blue.........................................................
0.1
g
Agar
(
optional)..................................................
15.0
g
Reagent­
grade
water.........................................
..
1.0
L
Rehydrate
product
in
1
L
water
containing
10
mL
1%
rosolic
acid
in
0.2N
NaOH.
3
Heat
to
near
boiling,
promptly
remove
from
heat,
and
cool
to
below
50
°
C.
Do
not
sterilize
by
autoclaving.
If
agar
is
used,
dispense
5­
to
7­
mL
quantities
to
50­
×
12­
mm
petri
plates
and
let
solidify.
Final
pH
should
be
7.4
±
0.2.
Refrigerate
finished
medium,
preferably
in
sealed
plastic
bags
or
other
containers
to
reduce
moisture
loss,
and
discard
unused
broth
after
96
h
or
unused
agar
after
2
weeks.
Test
each
medium
lot
against
a
previously
acceptable
lot
for
satisfactory
performance
as
described
in
Section
9020B,
by
making
dilutions
of
a
culture
of
E.
coli
(
Section
9020)
and
filtering
appropriate
volumes
to
give
20
to
60
colonies
per
filter.
With
each
new
lot
of
medium
verify
10
or
more
colonies
obtained
from
several
natural
samples,
to
establish
the
absence
of
false
positives.
For
most
samples
M­
FC
medium
may
be
used
without
the
1%
rosolic
acid
addition,
provided
there
is
no
interference
with
background
growth.
Such
interference
may
be
expected
in
stormwater
samples
collected
during
the
first
runoff
(
initial
flushing)
after
a
long
dry
period.
Before
use,
test
each
batch
of
laboratory­
prepared
MF
medium
for
performance
with
positive
and
Draft
7
June
2003
negative
culture
controls.
Check
for
coliform
contamination
at
the
beginning
and
end
of
each
filtration
series
by
filtering
20
to
30
mL
of
dilution
or
rinse
water
through
filter.
If
controls
indicate
contamination,
reject
all
data
from
affected
samples
and
request
resample.

b.
Culture
dishes:
Tight­
fitting
plastic
dishes
are
preferred
because
the
membrane
filter
cultures
are
submerged
in
a
water
bath
during
incubation.
Place
fecal
coliform
cultures
in
plastic
bags
or
seal
individual
dishes
with
waterproof
(
freezer)
tape
to
prevent
leakage
during
submersion.
Specifications
for
plastic
culture
dishes
are
given
in
Section
9222B.
1e.

c.
Incubator:
The
specificity
of
the
fecal
coliform
test
is
related
directly
to
the
incubation
temperature.
Static
air
incubation
may
be
a
problem
in
some
types
of
incubators
because
of
potential
heat
layering
within
the
chamber,
slower
heat
transfer
from
air
to
the
medium,
and
the
slow
recovery
of
temperature
each
time
the
incubator
is
opened
during
daily
operations.
To
meet
the
need
for
greater
temperature
control
use
a
water
bath,
a
heat­
sink
incubator,
or
a
properly
designed
and
constructed
incubator
shown
to
give
equivalent
results.
A
temperature
tolerance
of
44.5
±
0.2
°
C
can
be
obtained
with
most
types
of
water
baths
that
also
are
equipped
with
a
gable
top
for
the
reduction
of
water
and
heat
losses.

2.
Procedure
a.
Selection
of
sample
size:
Select
volume
of
water
sample
to
be
examined
in
accordance
with
the
information
in
Table
9222:
III.
Use
sample
volumes
that
will
yield
counts
between
20
and
60
fecal
coliform
colonies
per
membrane.
When
the
bacterial
density
of
the
sample
is
unknown,
filter
several
volumes
or
dilutions
to
achieve
a
countable
density.
Estimate
volume
and/
or
dilution
expected
to
yield
a
countable
membrane
and
select
two
additional
quantities
representing
onetenth
and
ten
times
this
volume,
respectively.

b.
Filtration
of
sample:
Follow
the
same
procedure
and
precautions
as
prescribed
under
Section
9222B.
5b
above.

c.
Preparation
of
culture
dish:
Place
a
sterile
absorbent
pad
in
each
culture
dish
and
pipet
at
least
2.0­
mL
M­
FC
medium,
prepared
as
directed
above,
to
saturate
pad.
Carefully
remove
any
excess
liquid
from
culture
dish
by
decanting
the
plate.
Aseptically,
place
prepared
filter
on
mediumimpregnated
pad
as
described
in
Section
9222B
above.
As
a
substrate
substitution
for
the
nutrient­
saturated
absorbent
pad,
add
1.5%
agar
to
M­
FC
broth
as
described
in
Section
9222B
above.

d.
Incubation:
Place
prepared
dishes
in
waterproof
plastic
bags
or
seal,
invert,
and
submerge
petri
dishes
in
water
bath,
and
incubate
for
24
±
2
h
at
44.5
±
0.2
°
C.
Anchor
dishes
below
water
surface
to
maintain
critical
temperature
requirements.
Place
all
prepared
cultures
in
the
water
bath
within
30
min
after
filtration.
Alternatively,
use
an
appropriate,
accurate
solid
heat
sink
or
equivalent
incubator.

e.
Counting:
Colonies
produced
by
fecal
coliform
bacteria
on
M­
FC
medium
are
various
shades
of
blue.
Nonfecal
coliform
colonies
are
gray
to
cream­
colored.
Normally,
few
nonfecal
coliform
colonies
will
be
observed
on
M­
FC
medium
because
of
selective
action
of
the
elevated
temperature
and
addition
of
rosolic
acid
salt
reagent.
Count
colonies
with
a
low­
power
(
10
to
15
magnifications)
binocular
wide­
field
dissecting
microscope
or
other
optical
device.
Draft
8
June
2003
f.
Verification:
Verify
typical
blue
colonies
and
any
atypical
grey
to
green
colonies
as
described
in
Section
9020
for
fecal
coliform
analysis.
Simultaneous
inoculation
at
both
temperatures
is
acceptable.
Draft
9
June
2003
3.
Bibliography
Geldreich,
E.
E.,
H.
F.
Clark,
C.
B.
Huff
&
L.
C.
Best.
1965.
Fecal­
coliform­
organism
medium
for
the
membrane
filter
technique.
J.
Amer.
Water
Works
Assoc.
57:
208.

Rose,
R.
E.,
E.
E.
Geldreich
&
W.
Litsky.
1975.
Improved
membrane
filter
method
for
fecal
coliform
analysis.
Appl.
Microbiol.
29:
532.

Lin,
S.
D.
1976.
Membrane
filter
method
for
recovery
of
fecal
coliforms
in
chlorinated
sewage
effluents.
Appl.
Environ.
Microbiol.
32:
547.

Presswood,
W.
G.
&
D.
K.
Strong.
1978.
Modification
of
M­
FC
medium
by
eliminating
rosolic
acid.
Appl.
Environ.
Microbiol.
36:
90.

Green,
B.
L.,
W.
Litsky
&
K.
J.
Sladek.
1980.
Evaluation
of
membrane
filter
methods
for
enumeration
of
faecal
coliforms
from
marine
waters.
Mar.
Environ.
Res.
67:
267.

Sartory,
D.
P.
1980.
Membrane
filtration
faecal
coliform
determinations
with
unmodified
and
modified
MFC
medium.
Water
SA
6:
113.

Grabow,
W.
O.
K.,
C.
A.
Hilner
&
P.
Coubrough.
1981.
Evaluation
of
standard
and
modified
M­
FC,
MacConkey,
and
Teepol
media
for
membrane
filter
counting
of
fecal
coliform
in
water.
Appl.
Environ.
Microbiol.
42:
192.

Rychert,
R.
C.
&
G.
R.
Stephenson.
1981.
Atypical
Escherichia
coli
in
streams.
Appl.
Environ.
Microbiol.
41:
1276.

Pagel,
J.
E.,
A.
A.
Qureshi,
D.
M.
Young
&
L.
T.
Vlassoff.
1982.
Comparison
of
four
membrane
filter
methods
for
fecal
coliform
enumeration.
Appl.
Environ.
Microbiol.
43:
787.

U.
S.
Environmental
Protection
Agency.
1992.
Environmental
Regulations
and
Technology.
Control
of
Pathogens
and
Vector
Attraction
in
Sewage
Sludge.
EPA­
626/
R­
92­
013,
Washington,
D.
C.

U.
S.
Environmental
Protection
Agency.
1993.
Standards
for
the
Use
or
Disposal
of
Sewage
Sludge:
Final
Rule.
40
CFR
Part
257;
Federal
Register
58:
9248,
Feb.
19,
1993.
Draft
10
June
2003
9222
G.
MF
PARTITION
PROCEDURES
ESCHERICHIA
COLI
PARTITION
METHODS
Escherichia
coli
is
a
member
of
the
fecal
coliform
group
of
bacteria;
its
presence
is
indicative
of
fecal
contamination.
Rapid
quantitation
and
verification
may
be
achieved
with
the
MF
procedure
by
transferring
the
membrane
from
a
total­
coliform­
or
fecal­
coliform­
positive
sample
to
a
nutrient
agar
substrate
containing
4­
methylumbelliferyl­$­
D­
glucuronide
(
MUG).
In
this
method
E.
coli
is
defined
as
any
coliform
that
produces
the
enzyme
$­
glucuronidase
and
hydrolyzes
the
MUG
substrate
to
produce
a
blue
fluorescence
around
the
periphery
of
the
colony.
In
the
examination
of
drinking
water
samples,
use
this
method
to
verify
the
presence
of
E.
coli
from
a
total­
coliform­
positive
MF
on
Endo­
type
media.
In
the
examination
of
wastewater
and
other
nonpotable
water
samples,
use
this
procedure
to
verify
positive
filters
from
mFC
medium
used
in
the
fecal
coliform
MF
procedure.

1.
Laboratory
Apparatus
a.
Culture
dishes:
See
Section
9222B.
1e.

b.
Filtration
units:
See
Section
9222B.
1f.

c.
Forceps:
See
Section
9222B.
1i.

d.
Incubator:
See
Section
9222B.
1j.

e.
Ultraviolet
lamp,
long
wave
(
366
nm),
preferably
6
W.

f.
Microscope
and
light
source:
See
Section
9222B.
1
k.

2.
Materials
and
Culture
Media
a.
Nutrient
agar
with
MUG
(
NA­
MUG):
Peptone................................................................
5.0
g
Beef
extract..........................................................
3.0
g
Agar...................................................................
15.0
g
4­
methylumbelliferyl­$­
D­
glucuronide...............
0.1
g
Reagent­
grade
water...............................................
1
L
Add
dehydrated
ingredients
to
reagent­
grade
water,
mix
thoroughly,
and
heat
to
dissolve.
Sterilize
by
autoclaving
for
15
min
at
121
°
C.
Dispense
aseptically
into
50­
mm
plastic
culture
plates.
The
final
pH
should
be
6.8
±
0.2.
Refrigerated
prepared
medium
may
be
held
for
2
weeks.

b.
EC
broth
with
MUG
(
EC­
MUG):
Tryptose
or
trypticase........................................
20.0
g
Lactose..............................................................
.5.0
g
Bile
salts
mixture
or
bile
salts
No.......................
31.5
g
Dipotassium
hydrogen
phosphate,
K2HPO4.....
....
4.0
g
Potassium
dihydrogen
phosphate,
KH2PO4...........
1.4
g
Sodium
chloride,
NaCl........................................
.5.0
g
4­
methylumbelliferyl­$­
D­
glucuronide.................
0.1
g
Reagent­
grade
water...........................................
..
1
L
Draft
11
June
2003
Add
dehydrated
ingredients
to
reagent­
grade
water,
mix
thoroughly
and
heat
to
dissolve.
pH
should
be
6.9
±
0.2
after
sterilization.
Before
sterilization,
dispense
into
culture
tubes
and
cap
with
metal
or
heat­
resistant
plastic
caps.

3.
Procedures
a.
Selection
of
sample
size:
See
Section
9222B.
5
for
selection
of
sample
size
and
filtration
procedure.

For
drinking
water
samples
using
Endo­
type
medium,
count
and
record
the
metallic
golden
sheen
colonies.
Before
transfer
of
the
membrane,
transfer
a
small
portion
of
each
target
colony
to
the
appropriate
total
coliform
verification
medium,
using
a
sterile
needle.
See
Section
9222B.
5
for
total
coliform
verification
procedures.

b.
Alternative
coliform
verifications:
After
transfer
and
incubation
on
NA­
MUG,
swab
the
surface
growth
on
the
filter
and
transfer
to
the
appropriate
total
coliform
verification
medium.
Aseptically
transfer
the
membrane
from
the
Endo­
type
medium
to
NA­
MUG
or
EC­
MUG
medium.
If
differentiation
of
the
total
coliforms
is
desired
using
NA­
MUG
medium,
mark
each
sheen
colony
with
a
fine­
tipped
marker
or
by
puncturing
a
hole
in
the
membrane
adjacent
to
the
colony
with
a
sterile
needle.
Incubate
NA­
MUG
at
35
±
0.5
°
C
for
4
h
or
EC­
MUG
at
44.5
±
0.2
for
24
±
2
h.
Observe
individual
colonies
or
tubes
using
a
long­
wave­
length
(
366­
nm)
ultraviolet
light
source,
preferably
containing
a
6­
W
bulb.
The
presence
of
a
blue
fluorescence
in
the
tube,
on
the
periphery
(
outer
edge)
of
a
colony,
or
observed
from
the
back
of
the
plate
is
considered
a
positive
response
for
E.
coli.
Count
and
record
the
number
of
target
colonies,
if
quantification
is
desired,
or
just
record
presence
or
absence
of
fluorescence.

For
nonpotable
water
samples,
use
mFC
medium
for
initial
isolation
before
transfer
to
NA­
MUG
or
EC­
MUG
medium.
The
procedure
is
the
same
as
the
above,
with
the
exception
of
the
total
coliform
verification
process.

For
the
EC­
MUG
method,
a
positive
control
consisting
of
a
known
E.
coli
(
MUG­
positive)
culture,
a
negative
control
consisting
of
a
thermotolerant
Klebsiella
pneumoniae
(
MUG­
negative)
culture,
and
an
uninoculated
medium
control
may
be
necessary
to
interpret
the
results
and
to
avoid
confusion
of
weak
autofluorescence
of
the
medium
as
a
positive
response.
See
Section
9221F.
Draft
12
June
2003
4.
Bibliography
U.
S.
Environmental
Protection
Agency.
1989.
Drinking
Water;
National
Primary
Drinking
Water
Regulations;
Total
Coliforms
(
Including
Fecal
Coliforms
and
E.
coli);
Final
Rule.
40
CFR
Parts
141
and
142.
Federal
Register
54:
27544.
June
29,
1989.

Mates,
A.
&
M.
Schaffer.
1989.
Membrane
Filtration
Differentiation
of
E.
coli
from
Coliforms
in
the
Examination
of
Water.
J.
Appl.
Bacteriol.
67:
343.

U.
S.
Environmental
Protection
Agency.
1991.
National
Primary
Drinking
Water
Regulations;
Analytical
Techniques;
Coliform
Bacteria.
40
CFR
Part
141,
Federal
Register
56:
636.
Jan
8,
1991.

Mates,
A.
&
M.
Schaffer.
1992.
Quantitative
Determination
of
Escherichia
coli
from
Coliforms
and
Fecal
Coliforms
in
Sea
Water.
Microbios.
71:
27.

Sartory,
D.
&
L.
Howard.
1992.
A
Medium
Detecting
beta­
glucuronidase
for
the
Simultaneous
Membrane
Filtration
enumeration
of
Escherichia
coli
and
Coliforms
from
Drinking
Water.
Lett.
Appl.
Microbiol.
15:
273.

Shadix,
L.
C.,
M.
E.
Dunnigan
&
E.
W.
Rice.
1993.
Detection
of
Escherichia
coli
by
the
Nutrient
Agar
plus
4­
methylumbelliferyl­
D­
glucuronide
(
MUG)
Membrane
Filter
Method.
Can.
J.
Microbiol.
39:
1066.