Document ID: EPA-HQ-OW-2002-0039-0166
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-07-09T04:00Z

STANDARD
METHODS
9221
B.
STANDARD
TOTAL
COLIFORM
FERMENTATION
TECHNIQUE
JUNE
2003
DRAFT
Draft
1
June
2003
STANDARD
METHODS
9221
B.
STANDARD
TOTAL
COLIFORM
FERMENTATION
TECHNIQUE
Reprinted
by
Permission
from
the
20th
Edition
1.
Presumptive
Phase
Use
lauryl
tryptose
broth
in
the
presumptive
portion
of
the
multiple­
tube
test.
If
the
medium
has
been
refrigerated
after
sterilization,
incubate
overnight
at
room
temperature
(
20/
C)
before
use.
Discard
tubes
showing
growth
and/
or
bubbles.

a.
Reagents
and
culture
medium:

1)
Lauryl
tryptose
broth:

Tryptose
................................................................................
20.0
g
Lactose
...............................................................................
.....
5.0
g
Dipotassium
hydrogen
phosphate.
K2HPO4
...........................
2.75
g
Potassium
dihydrogen
phosphate.
KH2PO4
...........................
2.75
g
Sodium
chloride.
NaC1
...........................................................
5.0
g
Sodium
lauryl
sulfate
..............................................................
0.1
g
Reagent­
grade
water
..............................................................
..
1
L
Add
dehydrated
ingredients
to
water,
mix
thoroughly,
and
heat
to
dissolve.
pH
should
be
6.8
±
0.2
after
sterilization.
Before
sterilization,
dispense
sufficient
medium,
in
fermentation
tubes
with
an
inverted
vial,
to
cover
inverted
vial
at
least
one­
half
to
two­
thirds
after
sterilization.
Alternatively,
omit
inverted
vial
and
add
0.01
g/
L
bromcresol
purple
to
presumptive
medium
to
determine
acid
production.
the
indicator
of
a
positive
result
in
this
part
of
the
coliform
test.
Close
tubes
with
metal
or
heat­
resistant
plastic
caps.

Make
lauryl
tryptose
broth
of
such
strength
that
adding
100­
mL,
20­
mL,
or
l0­
mL
portions
of
sample
to
medium
will
not
reduce
ingredient
concentrations
below
those
of
the
standard
medium.
Prepare
in
accordance
with
Table
9221:
1.

b.
Procedure:

1)
Arrange
fermentation
tubes
in
rows
of
five
or
ten
tubes
each
in
a
test
tube
rack.
The
number
of
rows
and
the
sample
volumes
selected
depend
upon
the
quality
and
character
of
the
water
to
be
examined.
For
potable
water
use
five
20­
mL
portions,
ten
10­
mL
portions,
or
a
single
bottle
of
100
mL
portion;
for
nonpotable
water
use
five
tubes
per
dilution
(
of
10,
1,
0.1
mL,
etc.).

In
making
dilutions
and
measuring
diluted
sample
volumes,
follow
the
precautions
given
in
Section
9215B.
2.
Use
Figure
9215:
1
as
a
guide
to
preparing
dilutions.
Shake
sample
and
dilutions
vigorously
about
25
times.
Inoculate
each
tube
in
a
set
of
five
with
replicate
sample
volumes
(
in
increasing
decimal
dilutions,
if
decimal
quantities
of
the
sample
are
used).
Mix
test
portions
in
the
medium
by
gentle
agitation.
Draft
2
June
2003
2)
Incubate
inoculated
tubes
or
bottles
at
35
±
0.5C.
After
24
±
2
h
swirl
each
tube
or
bottle
gently
and
examine
it
for
growth,
gas,
and
acidic
reaction
(
shades
of
yellow
color)
and,
if
no
gas
or
acidic
reaction
is
evident,
reincubate
and
reexamine
at
the
end
of
48
±
3
h.
Record
presence
or
absence
of
growth,
gas,
and
acid
production.
If
the
inner
vial
is
omitted,
growth
with
acidity
signifies
a
positive
presumptive
reaction.

c.
Interpretation:
Production
of
an
acidic
reaction
or
gas
in
the
tubes
or
bottles
within
48
±
3
h
constitutes
a
positive
presumptive
reaction.
Submit
tubes
with
a
positive
presumptive
reaction
to
the
confirmed
phase
(
9221B.
2).

9221:
I.
Preparation
of
Lauryl
Tryptose
Broth
Inoculum
mL
Amount
of
Medium
in
Tube
mL
Volume
of
Medium
+
Inoculum
mL
Dehydrated
Lauryl
Tryptose
Broth
Required
g/
L
1
10
or
more
11
or
more
35.6
10
10
20
71.2
10
20
30
53.4
20
10
30
106.8
100
50
150
106.8
100
35
135
137.1
100
20
120
213.6
The
absence
of
acidic
reaction
or
gas
formation
at
the
end
of
48
±
3
h
of
incubation
constitutes
a
negative
test.
Submit
drinking
water
samples
demonstrating
growth
without
a
positive
gas
or
acid
reaction
to
the
confirmed
phase
(
9221B.
2).
An
arbitrary
48­
h
limit
for
observation
doubtless
excludes
occasional
members
of
the
coliform
group
that
grow
very
slowly
(
see
Section
9212).

2.
Confirmed
Phase
a.
Culture
medium:
Use
brilliant
green
lactose
bile
broth
fermentation
tubes
for
the
confirmed
phase.

Brilliant
green
lactose
bile
broth:

Peptone
...................................................................................
10.0
g
Lactose
...................................................................................
10.0
g
Oxgall
.....................................................................................
20.0
g
Brilliant
green
....................................................................
0.0133
g
Reagent­
grade
water
................................................................
1.0
L
Add
dehydrated
ingredients
to
water,
mix
thoroughly,
and
heat
to
dissolve.
pH
should
be
7.2
±
0.2
after
sterilization.
Before
sterilization,
dispense,
in
fermentation
tubes
with
an
inverted
vial,
sufficient
medium
to
cover
inverted
vial
at
least
one­
half
to
two­
thirds
after
sterilization.
Close
tubes
with
metal
or
heat­
resistant
plastic
caps.
Draft
3
June
2003
b.
Procedure:
Submit
all
presumptive
tubes
or
bottles
showing
growth,
any
amount
of
gas,
or
acidic
reaction
within
24
±
2
h
of
incubation
to
the
confirmed
phase.
If
active
fermentation
or
acidic
reaction
appears
in
the
presumptive
tube
earlier
than
24
±
2
h,
transfer
to
the
confirmatory
medium;
preferably
examine
tubes
at
18
±
1
h.
If
additional
presumptive
tubes
or
bottles
show
active
fermentation
or
acidic
reaction
at
the
end
of
a
48
±
3
h
incubation
period,
submit
these
to
the
confirmed
phase.

Gently
shake
or
rotate
presumptive
tubes
or
bottles
showing
gas
or
acidic
growth
to
resuspend
the
organisms.
With
a
sterile
loop
3.0
to
3.5
mm
in
diameter,
transfer
one
or
more
loopfuls
of
culture
to
a
fermentation
tube
containing
brilliant
green
lactose
bile
broth
or
insert
a
sterile
wooden
applicator
at
least
2.5
cm
into
the
culture,
promptly
remove,
and
plunge
applicator
to
bottom
of
fermentation
tube
containing
brilliant
green
lactose
bile
broth.
Remove
and
discard
applicator.
Repeat
for
all
other
positive
presumptive
tubes.

Incubate
the
inoculated
brilliant
green
lactose
bile
broth
tube
at
35
±
0.5/
C.
Formation
of
gas
in
any
amount
in
the
inverted
vial
of
the
brilliant
green
lactose
bile
broth
fermentation
tube
at
any
time
(
e.
g.,
6
±
1
h,
24
±
2
h)
within
48
±
3
h
constitutes
a
positive
confirmed
phase.
Calculate
the
MPN
value
from
the
number
of
positive
brilliant
green
lactose
bile
tubes
as
described
in
Section
9221C.

c.
Alternative
procedure:
Use
this
alternative
only
for
polluted
water
or
wastewater
known
to
produce
positive
results
consistently.

If
all
presumptive
tubes
are
positive
in
two
or
more
consecutive
dilutions
within
24
h,
submit
to
the
confirmed
phase
only
the
tubes
of
the
highest
dilution
(
smallest
sample
inoculum)
in
which
all
tubes
are
positive
and
any
positive
tubes
in
still
higher
dilutions.
Submit
to
the
confirmed
phase
all
tubes
in
which
gas
or
acidic
growth
is
produced
only
after
48
h.

3.
Completed
Phase
To
establish
the
presence
of
coliform
bacteria
and
to
provide
quality
control
data,
use
the
completed
test
on
at
least
10%
of
positive
confirmed
tubes
(
see
Figure
9221:
1).
Simultaneous
inoculation
into
brilliant
green
lactose
bile
broth
for
total
coliforms
and
EC
broth
for
fecal
coliforms
(
see
Section
9221E
below)
or
EC­
MUG
broth
for
Escherichia
coli
may
be
used.
Consider
positive
EC
and
EC­
MUG
broths
elevated
temperature
(
44.5/
C)
results
as
a
positive
completed
test
response.
Parallel
positive
brilliant
green
lactose
bile
broth
cultures
with
negative
EC
or
EC­
MUG
broth
cultures
indicate
the
presence
of
nonfecal
coliforms.
Draft
4
June
2003
a.
Culture
media
and
reagents:

1)
LES
Endo
agar:
See
Section
9222B.
Use
100­
x
15­
mm
petri
plates.

2)
MacConkey
agar:

Peptone
................................................................................
17.0
g
Proteose
peptone
..............................................................
....
3.0
g
Lactose
................................................................................
10.0
g
Bile
salts
..............................................................................
.1.5
g
Sodium
chloride.
NaC1
......................................................
..
5.0
g
Agar
..................................................................................
..
13.5
g
Neutral
red
...........................................................................
0.03
g
Crystal
violet
......................................................................
0.001
g
Reagent­
grade
water
...............................................................
1.0
L
Add
ingredients
to
water,
mix
thoroughly,
and
heat
to
boiling
to
dissolve.
Sterilize
by
autoclaving
for
15
min
at
121/
C.
Temper
agar
after
sterilization
and
pour
into
petri
plates
(
100
X
15
mm).
pH
should
be
7.1
±
0.2
after
sterilization.

3)
Nutrient
agar:

Peptone
................................................................................
..
5.0
g
Beef
extract
............................................................................
3.0
g
Agar
......................................................................................
15.0
g
Reagent­
grade
water
...............................................................
1.0
L
Add
ingredients
to
water,
mix
thoroughly,
and
heat
to
dissolve.
pH
should
be
6.8
±
0.2
after
sterilization.
Before
sterilization.
dispense
in
screw­
capped
tubes.
After
sterilization,
immediately
place
tubes
in
an
inclined
position
so
that
the
agar
will
solidify
with
a
sloped
surface.
Tighten
screw
caps
after
cooling
and
store
in
a
protected,
cool
storage
area.

4)
Gram­
stain
reagents:

a)
Ammonium
oxalate­
crystal
violet
(
Hucker
*
s):
Dissolve
2
g
crystal
violet
(
90%
dye
content)
in
20
mL
95%
ethyl
alcohol;
dissolve
0.8
g
(
NH4)
2C2O4H2O
in
80
mL
reagentgrade
water;
mix
the
two
solutions
and
age
for
24
h
before
use;
filter
through
paper
into
a
staining
bottle.

b)
Lugol
*
s
solution,
Gram's
modification:
Grind
1
g
iodine
crystals
and
2
g
K1
in
a
mortar.
Add
reagent­
grade
water,
a
few
milliliters
at
a
time,
and
grind
thoroughly
after
each
addition
until
solution
is
complete.
Rinse
solution
into
an
amber
glass
bottle
with
the
remaining
water
(
using
a
total
of
300
mL).

c)
Counterstain:
Dissolve
2.5
g
safranin
dye
in
100
mL
95%
ethyl
alcohol.
Add
10
mL
to
100
mL
reagent­
grade
water.

d)
Acetone
alcohol:
Mix
equal
volumes
of
ethyl
alcohol
(
95%)
with
acetone.
Draft
5
June
2003
b.
Procedure:

1)
Using
aseptic
technique,
streak
one
LES
Endo
agar
(
Section
9222B.
2)
or
MacConkey
agar
plate
from
each
tube
of
brilliant
green
lactose
bile
broth
showing
gas,
as
soon
as
possible
after
the
observation
of
gas.
Streak
plates
in
a
manner
to
insure
presence
of
some
discrete
colonies
separated
by
at
least
0.5
cm.
Observe
the
following
precautions
when
streaking
plates
to
obtain
a
high
proportion
of
successful
isolations
if
coliform
organisms
are
present:
(
a)
Use
a
sterile
3­
mm­
diam
loop
or
an
inoculating
needle
slightly
curved
at
the
tip;
(
b)
tap
and
incline
the
fermentation
tube
to
avoid
picking
up
any
membrane
or
scum
on
the
needle;
(
c)
insert
end
of
loop
or
needle
into
the
liquid
in
the
tube
to
a
depth
of
approximately
0.5
cm;
and
(
d)
streak
plate
for
isolation
with
curved
section
of
the
needle
in
contact
with
the
agar
to
avoid
a
scratched
or
torn
surface.
Flame
loop
between
second
and
third
quadrants
to
improve
colony
isolation.
Incubate
plates
(
inverted)
at
35
±
0.5/
C
for
24
±
2
h.

2)
The
colonies
developing
on
LES
Endo
agar
are
defined
as
typical
(
pink
to
dark
red
with
a
green
metallic
surface
sheen)
or
atypical
(
pink,
red,
white,
or
colorless
colonies
without
sheen)
after
24
h
incubation.
Typical
lactose­
fermenting
colonies
developing
on
MacConkey
agar
are
red
and
may
be
surrounded
by
an
opaque
zone
of
precipitated
bile.
From
each
plate
pick
one
or
more
typical,
well­
isolated
coliform
colonies
or,
if
no
typical
colonies
are
present,
pick
two
or
more
colonies
considered
most
likely
to
consist
of
organisms
of
the
coliform
group,
and
transfer
growth
from
each
isolate
to
a
single­
strength
lauryl
tryptose
broth
fermentation
tube
and
onto
a
nutrient
agar
slant.
(
The
latter
is
unnecessary
for
drinking
water
samples.)

If
needed,
use
a
colony
magnifying
device
to
provide
optimum
magnification
when
colonies
are
picked
from
the
LES
Endo
or
MacConkey
agar
plates.
When
transferring
colonies,
choose
well­
isolated
ones
and
barely
touch
the
surface
of
the
colony
with
a
flame­
sterilized,
aircooled
transfer
needle
to
minimize
the
danger
of
transferring
a
mixed
culture.

Incubate
secondary
broth
tubes
(
lauryl
tryptose
broth
with
inverted
fermentation
vials
inserted)
at
35
±
0.5/
C
for
24
±
2
h;
if
gas
is
not
produced
within
24
±
2
h
reincubate
and
examine
again
at
48
±
3
h.
Microscopically
examine
Gram­
stained
preparations
from
those
24­
h
nutrient
agar
slant
cultures
corresponding
to
the
secondary
tubes
that
show
gas.

3)
Gram­
stain
technique
 
The
Gram
stain
may
be
omitted
from
the
completed
test
for
potable
water
samples
only
because
the
occurrences
of
gram­
positive
bacteria
and
spore­
forming
organisms
surviving
this
selective
screening
procedure
are
infrequent
in
drinking
water.

Various
modifications
of
the
Gram
stain
technique
exist.
Use
the
following
modification
by
Hucker
for
staining
smears
of
pure
culture:
include
a
gram­
positive
and
a
gram­
negative
culture
as
controls.

Prepare
separate
light
emulsions
of
the
test
bacterial
growth
and
positive
and
negative
control
cultures
on
the
same
slide
using
drops
of
distilled
water
on
the
slide.
Air­
dry
and
fix
by
passing
slide
through
a
flame
and
stain
for
1
min
with
ammonium
oxalate­
crystal
violet
solution.
Rinse
slide
in
tap
water
and
drain
off
excess;
apply
Lugol*
s
solution
for
1
min.
Draft
6
June
2003
Rinse
stained
slide
in
tap
water.
Decolorize
for
approximately
15
to
30
s
with
acetone
alcohol
by
holding
slide
between
the
fingers
and
letting
acetone
alcohol
flow
across
the
stained
smear
until
the
solvent
flows
colorlessly
from
the
slide.
Do
not
overdecolorize.
Counterstain
with
safranin
for
15
s,
rinse
with
tap
water,
blot
dry
with
absorbent
paper
or
air
dry,
and
examine
microscopically.
Gram­
positive
organisms
are
blue;
gram­
negative
organisms
are
red.
Results
are
acceptable
only
when
controls
have
given
proper
reactions.

c.
Interpretation:
Formation
of
gas
in
the
secondary
tube
of
lauryl
tryptose
broth
within
48
±
3
h
and
demonstration
of
gram­
negative,
nonspore­
forming,
rod­
shaped
bacteria
from
the
agar
culture
constitute
a
positive
result
for
the
completed
test,
demonstrating
the
presence
of
a
member
of
the
coliform
group.
Draft
7
June
2003
4.
Bibliography
Meyer,
E.
M.
1918.
An
aerobic
spore­
forming
bacillus
giving
gas
in
lactose
broth
isolated
in
routine
water
examination.
J.
Bacteriol.
3:
9.

Hucker,
G.
J.
&
H.
J.
Conn.
1923.
Methods
of
Gram
Staining.
N.
Y.
State
Agr.
Exp.
Sm.
Tech.
Bull.
No.
93.

Norton,
J.
F.
&
J.
J.
Weight.
1924.
Aerobic
spore­
forming
lactose
fermenting
organisms
and
their
significance
in
water
analysis.
Amer.
J.
Pub.
Health
14:
1019.

Hucker,
G.
J.
&
H.
J.
Conn.
1927.
Further
Studies
on
the
Methods
of
Gram
Staining.
N.
Y.
State
Agr.
Exp.
Sta.
Tech.
Bull.
No.
128.

Porter,
R,
C.
S.
McCleskey
&
M.
Levine.
1937.
The
facultative
sporulating
bacteria
producing
gas
from
lactose.
J.
Bacteriol.
33:
163.

Cowles,
P.
B.
1939.
A
modified
fermentation
tube.
J.
Bacteriol.
38:
677.

Sherman,
V.
B.
D.
1967.
A
Guide
to
the
Identification
of
the
Genera
of
Bacteria.
Williams
&
Wilkins.
Baltimore,
Md.

Geldreich,
E.
E.
1975.
Handbook
for
Evaluating
Water
Bacteriological
Laboratories.
2nd
ed.
EPA­
670/
9­
75­
006.
U.
S.
Environmental
Protection
Agency,
Cincinnati,
Ohio.

Evans,
T.
M..,
C.
E.
Waarvick,
R.
J.
Seidler
&
M.
W.
LeChevallier,
1981.
Failure
of
the
most­
probable
number
technique
to
detect
coliforms
in
drinking
water
and
raw
water
supplies.
Appl.
Environ.
Microbiol.
41:
130.

Seidler,
R.
J.,
T.
M.
Evans,
J.
R.
Kaufman,
C.
E.
Waarvick
&
M.
W.
LeChevallier.
1981.
Limitations
of
standard
coliform
enumeration
techniques.
J.
Amer.
Water
Works
Assoc.
73:
538.

Gerhards,
P.,
ed.
1981.
Manual
of
Methods
for
General
Bacteriology.
American
Soc.
Microbiology.
Washington,
D.
C.

Krieg,
N.
R.
&
J.
G.
Holt,
eds.
1984.
Bergey's
Manual
of
Systematic
Bacteriology,
Vol
1.
Williams
&
Wilkins.
Baltimore,
Md.

Greenberg,
A.
E.
&
D.
A.
Hunt,
eds.
1985.
Laboratory
Procedures
for
the
Examination
of
Seawater
and
Shellfish.
5th
ed.
American
Public
Health
Assoc.
Washington,
D.
C.

U.
S.
Environmental
Protection
Agency.
1989.
National
primary
drinking
water
regulations;
analytical
techniques;
coliform
bacteria;
final
rule.
Federal
Register
54(
135):
29998
(
July
17,
1989).
Draft
8
June
2003
STANDARD
METHODS
9221
C.
ESTIMATION
OF
BACTERIAL
DENSITY
Reprinted
by
Permission
from
Supplement
to
the
20th
Edition
1.
Precision
of
Fermentation
Tube
Test
Unless
a
large
number
of
sample
portions
is
examined,
the
precision
of
the
fermentation
tube
test
is
rather
low.
For
example,
Table
9221:
IV
shows
that
the
95%
confidence
limits
are
often
one­
third
times
and
three
times
the
estimate.
Consequently,
use
caution
when
interpreting
the
sanitary
significance
of
any
single
coliform
result.
When
several
samples
from
a
given
sampling
point
are
estimated
separately
and
the
results
combined
in
their
geometric
mean,
the
precision
is
greatly
improved
2.
Table
Reading
and
Recording
of
MPN
Record
coliform
concentration
as
the
Most
Probable
Number
(
MPN)/
100
mL.
The
MPN
values,
for
a
variety
of
positive
and
negative
combinations,
are
given
in
Tables
9221:
II,
III,
and
IV.
The
sample
volumes
indicated
in
Tables
9221:
II
and
III
are
chosen
especially
for
examining
finished
waters.
Table
9221:
IV
illustrates
MPN
values
for
combinations
of
positive
and
negative
results
when
five
10­
mL,
five
1.0­
mL,
and
five
0.1­
mL
sample
portion
volumes
are
tested.
If
the
sample
portion
volumes
used
are
those
found
in
the
tables,
report
the
value
corresponding
to
the
number
of
positive
and
negative
results
in
the
series
as
the
MPN/
100
mL,
or,
when
appropriate,
as
presence
or
absence.
When
the
series
of
decimal
dilutions
is
different
from
that
in
the
table,
select
the
MPN
value
from
Table
9221:
IV
for
the
combination
of
positive
results
and
calculate
according
to
the
following
formulas:

MPN/
l00
mL
=
MPN
value
(
from
table)
×
10/
V
where:

V
=
volume
of
one
sample
portion
at
the
lowest
selected
dilution.

When
more
than
three
dilutions
are
used
in
a
decimal
series
of
dilutions,
select
the
three
most
appropriate
dilutions
and
refer
to
Table
9221:
IV.
Several
examples
are
shown
in
the
following
table.

Volume
mL
Example
10
mL
1
mL
0.1
mL
0.01
mL
0.001
mL
Combination
of
positives
MPN
Index
No./
100
mL
a
5
5
1
0
0
5­
1­
0
330
b
4
5
1
0
0
4­
5­
1
48
c
0
0
1
0
0
0­
0­
1
1.8
d
5
4
4
1
0
4­
4­
1
400
e
5
4
4
0
1
4­
4­
1
400
f
5
5
5
5
2
5­
5­
2
54,000
Draft
9
June
2003
Select
highest
dilution
that
gives
positive
results
in
all
five
portions
tested
(
no
lower
dilution
giving
any
negative
results)
and
the
two
next
succeeding
higher
dilutions
(
Example
a).
If
the
lowest
dilution
tested
has
less
than
five
portions
with
positive
results,
select
it
and
the
two
next
succeeding
higher
dilutions
(
Examples
b,
c).

When
a
positive
result
occurs
in
a
dilution
higher
than
the
three
selected
according
to
the
fore­
going
rules,
change
the
selection
to
the
lowest
dilution
that
has
less
than
five
positive
results
and
the
next
two
higher
dilutions
(
Example
d).
When
all
the
foregoing
selection
rules
have
left
unselected
any
higher
dilutions
with
positive
results,
add
those
higher­
dilution
positive
results
to
the
tested
to
select
three
dilutions,
then
select
the
next
lower
dilution
(
Example
f).

When
it
is
desired
to
summarize
with
a
single
MPN
value
the
results
from
several
samples,
use
the
geometric
mean
or
the
median.
The
geometric
mean
is
computed
by
averaging
the
logarithmic
values;
e.
g.,
the
geometric
mean
of
A,
B,
and
C
is
10L
where
L
=
(
log10A
+
log10B
+
log10C)/
3
Mean
values
are
reported
as
the
antilog
of
L.

Table
9221:
IV
shows
all
but
the
very
improbable
positive
tube
combinations
for
a
three­
dilution
series.
In
testing
10
different
samples,
there
is
a
99%
chance
of
finding
all
the
results
among
these
95
outcomes.
If
untabulated
combinations
occur
with
a
frequency
greater
than
0.1%,
it
indicates
that
the
technique
is
faulty
or
that
the
statistical
assumptions
underlying
the
MPN
estimate
are
not
being
fulfilled
(
e.
g.,
growth
inhibition
at
low
dilutions).

The
MPN
for
combinations
not
appearing
in
the
table,
or
for
other
combinations
of
tubes
or
dilutions,
may
be
estimated
as
follows:
First
select
the
lowest
dilution
that
does
not
have
all
positive
results.
Second,
select
the
highest
dilution
with
at
least
one
positive
result.
Finally,
select
all
the
dilutions
between
them.
For
example,
from
(
5/
5,
10/
10,
4/
10,
1/
10,
0/
5)
use
only
(­,
­,
4/
10,
1/
10,
­),
4/
10
@
0.1
mL
sample/
tube
and
1/
10
@
0.01
mL
sample/
tube;
from
(
5/
5,
10/
10,
10/
10,
0/
10,
0/
5)
select
only
(­,­,
10/
10,
0/
10,
­),
10/
10
@
0.1
mL
sample/
tube
and
0/
10
@
0.01
mL
sample/
tube.
Use
only
the
selected
dilutions
in
the
following
formula
of
Thomas1
:

MPN/
100
mL
(
approx.)
=
100
×
P/(
N
×
T)
1/
2
where:

P
=
number
of
positive
results,
N
=
volume
of
sample
in
all
the
negative
portions
combined,
mL,
and
T
=
total
volume
of
sample
in
the
selected
dilutions,
mL.

In
the
first
example
above,

MPN/
100
mL
(
approx.)
=
100
×
5/
(
0.69
×
1.1)
1/
2
=
500/
0.87
=
570/
100
mL
In
the
second
example
above,

MPN/
100
mL
(
approx)
=
100
×
10/(
0.1
×
1.1)
1/
2
=
1000/
0.332
=
3000/
100
mL
Draft
10
June
2003
The
two
examples
compare
well
with
the
true
MPNs,
590/
100
mL
and
2400/
100
mL,
respectively.
The
second
example
is
a
special
case
for
which
an
exact
solution
can
be
calculated
directly
for
the
two
selected
dilutions.

Although
MPN
tables
and
calculations
are
described
for
use
in
the
coliform
test,
they
also
determine
the
MPN
of
any
other
organisms
provided
that
suitable
test
media
are
available.

When
all
the
results
at
the
lower
dilutions
are
positive
and
all
the
results
at
higher
dilutions
are
negative,
it
is
possible
to
calculate
an
exact
MPN
for
two
selected
dilutions
as
follows:
When
V
is
the
volume
of
each
individual
sample
portion
at
the
highest
dilution
with
all
positive
portions,

MPN/
100
mL
=
(
1/
V)
[
230.3
log10
(
T/
N)]

where
T
and
N
are
defined
as
for
Thomas's
formula.
The
last
example
discussed
above
was
(
5/
5,
10/
10,
10/
10,
0/
10,
0/
5),
with
portions
10,
1,
0.1,
0.01,
and
0.001.
The
third
dilution
is
the
highest
with
positive
portions,
so
V
=
0.1.
The
MPN
for
the
third
and
fourth
dilution
would
be
exactly
MPN/
100
mL
=
(
1/
0.1)
×
[
230.3
log10
(
1.1/
0.1)]

=
2400/
100
mL
3.
Reference
Thomas,
H.
A.,
JR.
1942.
Bacterial
densities
from
fermentation
tube
tests.
J.
Amer.
Water
Works
Assoc.
34:
572.

4.
Bibliography
McCrady,
M.
H.
1915.
The
numerical
interpretation
of
fermentation
tube
results.
J.
Infect.
Dis.
12:
183.

McCrady,
M.
H.
1918.
Tables
for
rapid
interpretation
of
fermentation­
tube
results.
Pub.
Health
J.
9:
201.

Hoskins,
J.
K.
1933.
The
most
probable
numbers
of
B.
coli
in
water
analysis.
J.
Amer.
Water
Works
Assoc.
25:
867.

Hoskins,
J.
K.
1934.
Most
Probable
Numbers
for
evaluation
of
coli­
aerogenes
tests
by
fermentation
tube
method.
Pub.
Health
Rep.
49:
393.

Halvorson,
H.
O.
&
N.
R.
Ziegler.
1933­
35.
Application
of
statistics
to
problems
in
bacteriology.
J.
Bacteriol.
25:
101:
26:
331.559;
29:
609.

Eisenhart,
C.
&
P.
W.
Wilson.
1943.
Statistical
methods
and
control
in
bacteriology.
Bacteriol.
Rev.
7:
57.

Cochran,
W.
G.
1950.
Estimationof
bacterial
densities
by
means
of
the
"
Most
Probable
Number."
Biometrics
6:
105.

Woodward,
R.
L.
1957.
How
probable
is
the
Most
Probable
Number?
J.
Amer.
Water
Works
Assoc.
49:
1060.

DeMan,
J.
C.
1983.
MPN
tables,
corrected.
Eur.
J.
Appl.
Biotechnol.
17:
301.

Garthright,
W.
E.
1998.
Appendix
2.
Most
probable
number
form
serial
dilutions.
FDA
Bacteriological
Analytical
Manual,
8th
ed.,
Rev.
A.
AOAC
International,
Gaithersburg,
Md.
Draft
11
June
2003
Table
9221:
II.
MPN
Index
and
95%
Confidence
Limits
for
Various
Combinations
of
Positive
and
Negative
Results
When
Five
20­
mL
Portions
Are
Used
No.
of
Tubes
Giving
Positive
Reaction
Out
of
5
(
20mLEach)
MPN
Index/
I00
mL
95%
Confidence
Limits
(
Exact)

Lower
Upper
0
<
1.1
 
3.5
I
1.1
0.051
5.4
2
2.6
0.4
8.4
3
4.6
1.0
13
4
8.0
2.1
23
5
>
8.0
3.4
 
Table
9221.
III.
MPN
Index
and
95%
Confidence
Limits
for
Various
Combinations
of
Positive
and
Negative
Results
When
Ten
10­
mL
Portions
Are
Used
No.
of
Tubes
Giving
Positive
Reaction
Out
of
10
(
10
mL
Each)
MPN
Index/
100
mL
95%
Confidence
Limits
(
Exact)

Lower
Upper
0
<
1.1
 
3.4
1
1.1
0.051
5.9
2
2.2
0.37
8.2
3
3.6
0.91
9.7
4
5.1
1.6
13
5
6.9
2.5
15
6
9.2
3.3
19
7
12
4.8
24
8
16
5.8
34
9
23
8.1
53
10
>
23
13
 
Draft
12
June
2003
Table
9221.
IV.
MPN
Index
and
95%
Confidence
Limits
for
Various
Combinations
of
Positive
Results
When
Five
Tubes
Are
Used
per
Dilution
(
10
mL,
1.0
mL,
0.1
mL)*

Combination
of
Positives
MPN
Index/
100
mL
Confidence
Limits
Combination
of
Positives
MPN
Index/
100
mL
Confidence
Limits
Low
High
Low
High
0­
0­
0
0­
0­
1
0­
1­
0
0­
1­
1
0­
2­
0
0­
2­
1
0­
3­
0
1­
0­
0
1­
0­
1
1­
0­
2
1­
1­
0
1­
1­
1
1­
1­
2
1­
2­
0
1­
2­
1
1­
3­
0
1­
3­
1
1­
4­
0
2­
0­
0
2­
0­
1
2­
0­
2
2­
1­
0
2­
1­
1
2­
1­
2
2­
2­
0
2­
2­
1
2­
2­
2
2­
3­
0
2­
3­
1
2­
4­
0
3­
0­
0
3­
0­
1
3­
0­
2
3­
1­
0
3­
1­
1
3­
1­
2
3­
2­
0
3­
2­
1
3­
2­
2
3­
3­
0
3­
3­
1
3­
3­
2
3­
4­
0
3­
4­
1
3­
5­
0
4­
0­
0
4­
0­
1
4­
0­
2
<
1.8
1.8
1.8
3.6
3.7
5.5
5.6
2.0
4.0
6.0
4.0
6.1
8.1
6.1
8.2
8.3
10
10
4.5
6.8
9.1
6.8
9.2
12
9.3
12
14
12
14
15
7.8
11
13
11
14
17
14
17
20
17
21
24
21
24
25
13
17
21
 
0.090
0.090
0.70
0.70
1.8
1.8
0.10
0.70
1.8
0.71
1.8
3.4
1.8
3.4
3.4
3.5
3.5
0.79
1.8
3.4
1.8
3.4
4.1
3.4
4.1
5.9
4.1
5.9
5.9
2.1
3.5
5.6
3.5
5.6
6.0
5.7
6.8
6.8
6.8
6.8
9.8
6.8
9.8
9.8
4.1
5.9
6.8
6.8
6.8
6.9
10
10
15
15
10
10
15
12
15
22
15
22
22
22
22
15
15
22
17
22
26
22
26
36
26
36
36
22
23
35
26
36
36
36
40
40
40
40
70
40
70
70
35
36
40
4­
0­
3
4­
1­
0
4­
1­
1
4­
1­
2
4­
1­
3
4­
2­
0
4­
2­
1
4­
2­
2
4­
2­
3
4­
3­
0
4­
3­
1
4­
3­
2
4­
4­
0
4­
4­
1
4­
4­
2
4­
5­
0
4­
5­
1
5­
0­
0
5­
0­
1
5­
0­
2
5­
0­
3
5­
1­
0
5­
1­
1
5­
1­
2
5­
1­
3
5­
2­
0
5­
2­
1
5­
2­
2
5­
2­
3
5­
2­
4
5­
3­
0
5­
3­
1
5­
3­
2
5­
3­
3
5­
3­
4
5­
4­
0
5­
4­
1
5­
4­
2
5­
4­
3
5­
4­
4
5­
4­
5
5­
5­
0
5­
5­
1
5­
5­
2
5­
5­
3
5­
5­
4
5­
5­
5
25
17
21
26
31
22
26
32
38
27
33
39
34
40
47
41
48
23
31
43
58
33
46
63
84
49
70
94
120
150
79
110
140
170
210
130
170
220
280
350
430
240
350
540
920
1600
>
1600
9.8
6.0
6.8
9.8
10
6.8
9.8
10
14
9.9
10
14
14
14
15
14
15
6.8
10
14
22
10
14
22
34
15
22
34
36
58
22
34
52
70
70
36
58
70
100
100
150
70
100
150
220
400
700
70
40
42
70
70
50
70
70
100
70
70
100
100
100
120
100
120
70
70
100
150
100
120
150
220
150
170
230
250
400
220
250
400
400
400
400
400
440
710
710
1100
710
1100
1700
2600
4600
 
*
Results
to
two
significant
figures
Draft
13
June
2003
STANDARD
METHODS
9221
E.
FECAL
COLIFORM
PROCEDURE
Reprinted
by
Permission
from
the
20th
Edition
Elevated­
temperature
tests
for
distinguishing
organisms
of
the
total
coliform
group
that
also
belong
to
the
fecal
coliform
group
are
described
herein.
Modifications
in
technical
procedures.
standardization
of
methods,
and
detailed
studies
of
the
fecal
coliform
group
have
established
the
value
of
this
procedure.
The
test
can
be
performed
by
one
of
the
multiple­
tube
procedures
described
here
or
by
membrane
filter
methods
as
described
in
Section
9222.
The
procedure
using
A­
l
broth
is
a
single­
step
method.

The
fecal
coliform
test
(
using
EC
medium)
is
applicable
to
investigations
of
drinking
water,
stream
pollution,
raw
water
sources,
wastewater
treatment
systems,
bathing
waters,
seawaters,
and
general
waterquality
monitoring.
Prior
enrichment
in
presumptive
media
is
required
for
optimum
recovery
of
fecal
coliforms
when
using
EC
medium.
The
test
using
A­
1
medium
is
applicable
to
source
water,
seawater,
and
treated
wastewater.

1.
Fecal
Coliform
Test
(
EC
Medium)

The
fecal
coliform
test
is
used
to
distinguish
those
total
coliform
organisms
that
are
fecal
coliforms.
Use
EC
medium
or,
for
a
more
rapid
test
of
the
quality
of
shellfish
waters,
treated
wastewaters,
or
source
waters,
use
A­
1
medium
in
a
direct
test.

a.
EC
medium:

Tryptose
or
trypticase
..............................................................
20.0
g
Lactose
.......................................................................................
5.0
g
Bile
salts
mixture
or
bile
salts
No.
..........................................
3
1.5
g
Dipotassium
hydrogen
phosphate.
K2HPO4
...............................
4.0
g
Potassium
dihydrogen
phosphate.
KH2PO4
................................
1.5
g
Sodium
chloride.
NaCl
...............................................................
5.0
g
Reagent­
grade
water
...................................................................
1.0
L
Add
dehydrated
ingredients
to
water,
mix
thoroughly,
and
heat
to
dissolve.
pH
should
be
6.9
±
0.2
after
sterilization.
Before
sterilization,
dispense
in
fermentation
tubes,
each
with
an
inverted
vial,
sufficient
medium
to
cover
the
inverted
vial
at
least
partially
after
sterilization.
Close
tubes
with
metal
or
heat­
resistant
plastic
caps.

b.
Procedure:
Submit
all
presumptive
fermentation
tubes
or
bottles
showing
any
amount
of
gas,
growth,
or
acidity
within
48
h
of
incubation
to
the
fecal
coliform
test.

1)
Gently
shake
or
rotate
presumptive
fermentation
tubes
or
bottles
showing
gas,
growth,
or
acidity.
Using
a
sterile
3­
or
3.5­
mm­
diam
loop
or
sterile
wooden
applicator
stick,
transfer
growth
from
each
presumptive
fermentation
tube
or
bottle
to
EC
broth
(
see
Section
9221B.
2).

2)
Incubate
inoculated
EC
broth
tubes
in
a
water
bath
at
44.5
±
0.2/
C
for
24
±
2
h.

Place
all
EC
tubes
in
water
bath
within
30
min
after
inoculation.
Maintain
a
sufficient
water
depth
in
water
bath
incubator
to
immerse
tubes
to
upper
level
of
the
medium.
Draft
14
June
2003
c.
Interpretation:
Gas
production
with
growth
in
an
EC
broth
culture
within
24
±
2
h
or
less
is
considered
a
positive
fecal
coliform
reaction.
Failure
to
produce
gas
(
with
little
or
no
growth)
constitutes
a
negative
reaction.
If
multiple
tubes
are
used,
calculate
MPN
from
the
number
of
positive
EC
broth
tubes
as
described
in
Section
9221C.
When
using
only
one
tube
for
subculturing
from
a
single
presumptive
bottle,
report
as
presence
or
absence
of
fecal
coliforms.

2.
Fecal
Coliform
Direct
Test
(
A­
1
Medium)

a.
A­
1
broth:
This
medium
may
be
used
for
the
direct
isolation
of
fecal
coliforms
from
water.
Prior
enrichment
in
a
presumptive
medium
is
not
required.

Lactose
.....................................................................................
5.0
g
Tryptone..................................................................................
20.0
g
Sodium
chloride.
NaCl
.............................................................
5.0
g
Salicin
.......................................................................................
0.5
g
Polyethylene
glycol
p­
isooctylphenyl
ether*
.........................
1.0
mL
Reagent­
grade
water
..................................................................
1.0
L
*
Triton
X­
l00.
Rohm
and
Haas
Co.,
or
equivalent.

Heat
to
dissolve
solid
ingredients,
add
polyethylene
glycol
p­
isooctyiphenyl
ether,
and
adjust
to
pH
6.9
±
0.1.
Before
sterilization
dispense
in
fermentation
tubes
with
an
inverted
vial
sufficient
medium
to
cover
the
inverted
vial
at
least
partially
after
sterilization.
Close
with
metal
or
heat­
resistant
plastic
caps.
Sterilize
by
autoclaving
at
121/
C
for
10
min.
Store
in
dark
at
room
temperature
for
not
longer
than
7
d.
Ignore
formation
of
precipitate.

Make
A­
1
broth
of
such
strength
that
adding
l0­
mL
sample
portions
to
medium
will
not
reduce
ingredient
concentrations
below
those
of
the
standard
medium.
For
l0­
mL
samples
prepare
double­
strength
medium.

b.
Procedure:
Inoculate
tubes
of
A­
1
broth
as
directed
in
Section
9221B.
1b1).
Incubate
for
3
h
at
35
±
0.5/
C.
Transfer
tubes
to
a
water
bath
at
44.5
±
0.2/
C
and
incubate
for
an
additional
21
±
2
h.

c.
Interpretation:
Gas
production
in
any
A­
1
broth
culture
within
24
h
or
less
is
a
positive
reaction
indicating
the
presence
of
fecal
coliforrns.
Calculate
MPN
from
the
number
of
positive
A­
1
broth
tubes
as
described
in
Section
9221C.
Draft
15
June
2003
3.
Bibliography
Perry,
C.
A.
&
A.
A.
Haina.
1933.
A
modified
Eijkman
medium.
J.
Bacteriol.
26:
419.

Perry,
C.
A.
&
A.
A.
Haina.
1944.
Further
evaluation
of
EC
medium
for
the
isolation
of
coliform
bacteria
and
Eschenchia
coli.
Amer.
J.
Pub.
Health
34:
735.

Geldreich,
E.
E.,
H.
F.
Clark,
P.
W.,
Kabler,
C.
B.
Huff
&
R.
H.
Bordner.
1958.
The
coliform
group.
II.
Reactions
in
EC
medium
at
45/
C.
Appl.
Microbiol.
6:
347.

Geldreich,
E.
E.,
R.
H.
Bordner,
C.
B.
Huff,
H.
F.
Clark
&
P.
W.
Kabler.
1962.
Type
distribution
of
coliform
bacteria
in
the
feces
of
warmblooded
animals.
J.
Water
Pollut.
Control
Fed.
34:
295.

Geldreich,
E.
E.
1966.
Sanitary
significance
of
fecal
coliforms
in
the
environment.
FWPCA
Publ.
WP­
20­
3
(
Nov.).
U.
S.
Dep.
Interior,
Washington,
D.
C.

Andrews,
W.
H.
&
M.
W.
Presnell.
1972.
Rapid
recovery
of
Eschenchia
coli
from
estuarine
water.
Appl.
Microbiol.
23:
521.

Olson,
B.
H.
1978.
Enhanced
accuracy
of
coliform
testing
in
seawater
by
a
modification
of
the
mostprobable
number
method.
Appl.
Microbiol.
36:
438.

Strandridge,
J.
H.
&
J.
J.
Delfino.
1981.
A­
1
Medium:
Alternative
technique
for
fecal
coliform
organism
enumeration
in
chlorinated
wastewaters.
Appl.
Environ.
Microbiol.
42:
9
18.
Draft
16
June
2003
STANDARD
METHODS
9221
F.
ESCHERICHIA
COLI
PROCEDURE
Reprinted
by
Permission
from
the
Supplement
to
the
20th
Edition
­
Proposed
Escherichia
coli
is
a
member
of
the
fecal
coliform
group
of
bacteria.
This
organism
in
water
indicates
fecal
contamination.
Enzymatic
assays
have
been
developed
that
allow
for
the
identification
of
this
organism.
Assays
for
$­
glucuronidase
or
glutamate
decarboxylase
may
be
used
to
determine
the
presence
of
E.
coli.
In
method
9221F.
1,
E.
coli
are
defined
as
coliform
bacteria
that
possess
the
enzyme
$­
glucuronidase
and
are
capable
of
cleaving
the
fluorogenic
substrate
4­
methylumbel­
liferyl­$­
Dglucuronide
(
MUG)
with
the
corresponding
release
of
the
fluorogen
when
grown
in
EC­
MUG
medium
at
44.5/
C
within
24
±
2
h
or
less.
In
method
9221F.
2,
E.
coli
are
defined
as
coliform
bacteria
that
possess
the
enzyme
glutamate
decarboxylase
and
are
capable
of
producing
an
alkaline
reaction
within
4
h
in
a
reagent
containing
a
lytic
agent
and
glutamic
acid.
The
procedure
is
used
as
a
confirmatory
test
after
prior
enrichment
in
a
presumptive
medium
for
total
coliform
bacteria.
These
tests
are
performed
by
the
tube
procedure
described
here
or
by
the
membrane
filter
method
as
described
in
Section
9222.
The
chromogenic
substrate
procedure
(
Section
9223)
can
be
used
for
direct
detection
of
E.
coli.

Tests
for
E.
coli
are
applicable
for
the
analysis
of
drinking
water,
surface
and
ground
water,
and
wastewater.
E.
coli
is
a
member
of
the
indigenous
fecal
flora
of
warm­
blooded
animals.
The
occurrence
of
E.
coli
is
considered
a
specific
indicator
of
fecal
contamination
and
the
possible
presence
of
enteric
pathogens.

1.
Escherichia
coli
Test
(
EC­
MUG
medium)

Use
EC­
MUG
medium
for
the
confirmation
of
E.
coli.

a.
EC­
MUG
medium:

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
.................................
1.5
g
Sodium
chloride,
NaC1
.............................................................
..
5.0
g
4­
methylumbelliferyl­$­
D­
glucuronide
(
MUG)
.........................
0.05
g
Reagent­
grade
water
.....................................................................
1.0
L
Add
dehydrated
ingredients
to
water,
mix
thoroughly,
and
heat
to
dissolve.
pH
should
be
6.9
±
0.2
after
sterilization.
Before
sterilization,
dispense
in
tubes
that
do
not
fluoresce
under
long­
wavelength
(
366
nm)
ultraviolet
(
UV)
light.
An
inverted
tube
is
not
necessary.
Close
tubes
with
metal
or
heat­
resistant
plastic
caps.

b.
Procedure:
Submit
all
presumptive
fermentation
tubes
or
bottles
showing
growth,
gas,
or
acidity
within
48
±
3
h
of
incubation
to
the
E.
coli
test.

1)
Gently
shake
or
rotate
presumptive
fermentation
tubes
or
bottles
showing
growth.
gas,
or
acidity.
Using
a
sterile
3­
or
3.5­
mm­
diam
metal
loop
or
sterile
wooden
applicator
stick,
transfer
growth
from
presumptive
fermentation
tube
or
bottle
to
EC­
MUG
broth.
Draft
17
June
2003
2)
Incubate
inoculated
EC­
MUG
tubes
in
a
water
bath
or
incubator
maintained
at
44.5
±
0.2/
C
for
24
±
2
h.
Place
all
EC­
MUG
tubes
in
water
bath
within
30
min
after
inoculation.
Maintain
a
sufficient
water
depth
in
the
water­
bath
incubator
to
immerse
tubes
to
upper
level
of
medium.

c.
Interpretation:
Examine
all
tubes
exhibiting
growth
for
fluorescence
using
a
longwavelength
UV
lamp
(
preferably
6
W).
The
presence
of
bright
blue
fluorescence
is
considered
a
positive
response
for
E.
coli.
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
auto­
fluorescence
of
the
medium
as
a
positive
response.
If
multiple
tubes
are
used,
calculate
MPN
from
the
number
of
positive
ECMUG
broth
tubes
as
described
in
Section
9221C.
When
using
only
one
tube
or
subculturing
from
a
single
presumptive
bottle,
report
as
presence
or
absence
of
E.
coli.

2.
Escherichia
coli
Test
(
GAD
Procedure)

The
GAD
test
procedure
is
not
approved
for
use
in
LT2
monitoring.

3.
Bibliography
Feng,
P.
C.
S.
&
P.
A.
Hartman.
1982.
Fluorogenic
assays
for
immediate
confirmation
of
Escherichia
coli.
Appl.
Environ.
Microbiol.
43:
1320.

Hartman,
P.
A.
1989.
The
MUG
(
glucuronidase)
test
for
E.
coli
in
food
and
water.
In
A.
Balows
et
al.,
eds.,
Rapid
Methods
and
Automation
in
Microbiology
and
Immunology.
Proc.
5th
Intl.
Symp.
on
Rapid
Methods
and
Automation
in
Microbiology
&
Immunology,
Florence,
Italy,
Nov.
4­
6,
1987.

Fieldler,
J.
&
J.
Reiske.
1990.
Glutaminsauredecarboxylase­
schnelltest
zur
identifikation
von
Escherichia
coli.
Z.
Ges.
Hyg.
Grenzgeb.
36:
620.

Shadix,
L.
C.
&
E.
W.
Rice.
1991.
Evaluation
of
$­
glucuronidase
assay
for
the
detection
of
Escherichia
coli
from
environmental
waters.
Can.
J.
Microbiol.
37:
908.

Rice,
E.
W.,
C.
H.
Johnson,
M.
E.
Dunnigan
&
D.
J.
Reasoner.
1993.
Rapid
glutamate
decarboxylase
assay
for
the
detection
of
Escherichia
coli.
Appl.
Environ.
Microbiol.
59:
4937.
Errata.
1995.
Appl.
Environ.
Microbiol.
61:
847.

Rice,
E.
W.,
C.
H.
Johnson,
&
D.
J.
Reasoner.
1996.
Detection
of
Escherichia
coli
O157:
H7
in
water
from
coliform
enrichment
cultures.
Lett.
Appl.
Microbiol.
23:
179.