King
County
Environmental
Laboratory
Standard
Operating
Procedure
MICROBIOLOGY,
ENTEROCOCCI
IN
ENVIRONMENTAL
WATER
BY
MF
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SOP
#:
05­
04­
001­
000
STANDARD
OPERATING
PROCEDURE
for
Enterococci
in
Environmental
Water
by
Membrane
Filtration
SOP
#
05­
04­
001­
000
Date
of
Implementation:
3/
17/
03
Supersedes
SOP:
Microbiology
Manual
Section
6.3
Approved
by:

Supervisor:
_______________________________
Date:
________________

QA
Officer:
_______________________________
Date:
________________

King
County
Environmental
Laboratory
322
W.
Ewing
Street
Seattle
WA
98119­
1507
King
County
Environmental
Laboratory
Standard
Operating
Procedure
MICROBIOLOGY,
ENTEROCOCCI
IN
ENVIRONMENTAL
WATER
BY
MF
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SOP
#:
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CHRONOLOGY
OF
SOP
MODIFICATIONS
Revision
Number
Section
Revised
Date
Implemented
/
Initials
Type
of
Change
Rationale
King
County
Environmental
Laboratory
Standard
Operating
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MICROBIOLOGY,
ENTEROCOCCI
IN
ENVIRONMENTAL
WATER
BY
MF
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1.0
Scope
and
Application
Enterococci
are
a
subgroup
of
bacteria
of
the
fecal
streptococcus
group
that
includes
Enterococcus
faecalis,
E.
faecium,
E.
gallinarum
and
E.
avium.
Enterococci
are
an
indicator
of
human
and
animal
fecal
bacteria
contamination.
This
SOP
describes
the
analysis
of
environmental
water
samples
using
the
Membrane
Filtration
(
MF)
procedure
and
growth
on
mE
agar
followed
by
the
hydrolysis
of
esculin
iron
agar(
EIA).

2.0
Method
Summary
Appropriate
dilutions
of
the
sample,
if
necessary,
are
prepared
using
APHA
buffered
water
as
the
diluent.
The
sample
and
it's
dilutions
are
filtered
through
a
sterile
0.45
µ
m
nitrocellulose
membrane.
The
membrane
is
then
applied
to
mE
agar
plates.
After
incubation
on
the
mE
agar
plate
at
41
±
0.5
oC
for
48
±
3
hours,
the
filter
is
transferred
to
EIA
agar
and
incubated
for
an
additional
20­
30
minutes.
All
colonies
that
have
a
red
to
pink
color
with
a
black
or
reddish­
brown
precipitate
are
counted.
The
number
of
colonies
per
100
mLs
is
calculated
based
on
the
colonies
counted
and
the
dilutions
used
per
plate.

3.0
Definitions
Membrane
Filtration
(
MF):
The
technique
used
to
determine
the
number
of
colonies
in
a
sample
using
filtration
through
a
nitrocellulose
filter
of
nominal
0.45
µ
m
pore
size
and
incubation
on
an
agar
plate
or
a
liquid
substrate
pad.

Most
Probable
Number
(
MPN):
The
density
of
the
bacteria
in
a
sample
calculated
using
probability
formulas
and
the
observations
of
replicate
tubes
and
multiple
dilutions.

4.0
Interferences
Algae,
particulates,
colloids,
solids
or
other
material,
as
identified
by
sample
turbidity,
may
interfere
with
MF
analyses.
Samples
affected
by
these
interferences
should
be
tested
with
the
MPN
method.
Non­
sterile
equipment,
materials
or
work
environment
may
also
interfere
with
the
MF
technique.

5.0
Safety
and
Hazardous
Materials
Management
Each
sample
to
be
tested
for
Enterococci
should
be
considered
a
potential
biohazard
or
chemical
hazard.
Protective
equipment
such
as
lab
coats
should
be
worn
whenever
handling
samples.
Latex
or
nitrile
gloves
may
also
be
worn
as
a
protective
barrier.
Aerosols
may
also
be
generated
in
handling
the
samples;
exercise
protective
measures.
Used
plates
and
contaminated
disposable
supplies
must
be
stored
in
containers
clearly
labeled
as
biohazardous
until
sterilized
by
autoclaving.
Autoclaved
solid
waste
may
then
King
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#:
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be
discarded
in
a
solid
waste
container.
Liquids
not
identified
as
hazardous
waste
may
be
discarded
in
a
sanitary
drain.

6.0
Sample
Collection,
Containers,
Preservation
and
Storage
Polypropylene
(
PP)
or
high
density
polyethylene
(
HDPE)
bottles
must
be
autoclaved
and
identified
as
sterile
with
autoclave
tape
before
being
used
for
sample
collection.
Containers
must
be
filled
such
that
a
minimum
of
1
inch
of
headspace
is
present.
NPDES
water
samples
must
be
preserved
with
sterile
sodium
thiosulfate
solution
if
residual
chlorine
is
present
or
expected,
stored
at
4oC,
and
analyzed
within
6
hours
of
collection.
Non­
NPDES
water
samples
should
be
preserved
with
sterile
sodium
thiosulfate
solution
if
residual
chlorine
is
present
or
expected,
stored
at
4oC,
and
analyzed
within
24
hours
of
collection.

7.0
Apparatus
and
Equipment
41
±
0.5
oC
incubator
Sterile
100
ml
graduated
cylinders
Sterile
filter
funnels
Manifold
UV
sterilizer
for
filter
funnel
units
Bunsen
burner
or
other
flame
source
Vacuum
source
Pipetter
Wescodyne
or
other
disinfectant
disposal
bucket
Non­
serrated
forceps
Sterile
rinse
water
dispenser
8.0
Preventative
Maintenance
All
reusable
equipment
that
may
have
been
in
contact
with
the
samples
must
be
cleaned
and
sterilized
between
batches.

9.0
Reagents,
Media
and
Consumables
Sterile
APHA
phosphate­
buffered
dilution
water
Sterile
18mL
APHA
phosphate­
buffered
dilution
tubes
Sterile
1,
5,
10
and
25
ml
pipettes
or
pipette
tips
(<
2.5%
tolerance)
70%
alcohol
(
ethanol
or
isopropyl
alcohol)
Sterile
0.45
µ
m
membrane
filters,
nitrocellulose
with
gridlines,
47
mm
diameter
Sterile
mE
and
EIA
medium
in
sterile
Petri
dishes
(
See
SOP#
05­
01­
014­
000
for
media
preparation)
King
County
Environmental
Laboratory
Standard
Operating
Procedure
MICROBIOLOGY,
ENTEROCOCCI
IN
ENVIRONMENTAL
WATER
BY
MF
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10.0
Procedures
10.1
Preparation
Determine
what
volumes
need
to
be
run
for
the
type
of
water
being
analyzed.
Consult
Appendix
A
for
the
suggested
analyses
volumes
for
different
types
of
water.
The
samples
are
workgrouped
in
LIMS
such
that
the
lab
ID#
s
("
L"
numbers),
parameters,
dilutions,
and
associated
QC
samples
are
defined
for
a
particular
batch.
Once
workgrouped,
LIMS
can
generate
a
label
to
be
placed
on
each
plate
of
medium
for
each
specific
dilution.
The
plate
must
be
associated
with
a
sample
identifier,
the
volume
of
sample
tested,
the
date
tested,
and
the
type
of
test.
An
electronic
login
sheet
is
also
generated
by
LIMS,
which
records
the
sample,
product,
and
date
workgrouped.
The
login
sheet
is
used
to
record
the
date
and
time
a
sample
was
received
and
processed,
as
well
as
the
analysts
performing
these
procedures.
For
specific
information
on
the
use
of
LIMS
for
labeling
and
documenting
sample
preparation,
see
"
LIMS
user's
manual
for
the
microbiology
section"
(
SOP
#
05­
01­
001­
000).

If
needed,
prepare
dilutions
of
the
sample.
To
prepare
a
1:
10
dilution,
add
2
mL
of
the
sample
to
an
18
mL
sterile
APHA
buffered
water
dilution
tube.
Subsequent
dilutions
are
made
in
the
same
manner.
Alternate
volumes
may
be
used
to
accommodate
the
volume
of
sample
available
for
testing.

10.2
Filtration
Set
up
the
vacuum
manifold
with
an
inline
filter
between
the
vacuum
valve
and
the
collection
vessel.
Place
the
filtration
units
on
the
vacuum
manifold
and
close
all
manifold
valves.
Disinfect
the
non­
serrated
forceps
by
placing
the
tips
in
70%
alcohol
solution
for
90
seconds.
Remove
residual
alcohol
by
placing
the
tips
of
the
forceps
briefly
in
the
flame
of
the
Bunsen
burner.
With
the
sterile
forceps,
center
a
sterile,
0.45
µ
m
membrane
filter
on
the
base
of
each
filter
unit,
and
then
place
the
funnel
portion
of
the
unit
on
the
base.
Measure
the
amount
of
water
to
be
filtered
with
a
graduated
cylinder
if
the
volume
is
>
30
ml,
or
use
a
pipette
if
the
volume
is
 
30
ml.
If
the
volume
filtered
is
<
30
ml,
add
a
30­
50
ml
reservoir
of
APHA
buffered
water
to
the
filter
funnel
before
adding
the
sample.
If
the
volume
to
be
filtered
is
100mLs,
the
corresponding
marking
on
the
filter
funnel
may
be
used.
Add
the
sample
to
the
filter
funnel
and
turn
on
the
vacuum.
Once
the
sample
is
filtered
completely,
thoroughly
rinse
the
funnel
with
APHA
buffered
water,
making
sure
that
the
sides
of
the
funnel
are
washed
down.
Use
a
minimum
of
approximately
30
ml
for
each
rinse.
Once
all
the
rinse
water
is
filtered
through,
turn
off
the
vacuum
by
closing
the
manifold
valve.

Remove
the
membrane
filter
from
the
filtering
unit
by
grasping
the
edge
of
the
filter
with
the
sterile
forceps,
and
then
place
the
filter
onto
mE
agar
plates
previously
prepared.
Try
to
"
roll"
the
filter
onto
the
agar
such
that
the
filter
is
in
complete
contact
with
the
agar
surface.
Check
for
air
bubbles
underneath
the
filter;
if
there
are
air
bubbles,
try
lifting
the
filter
by
the
edge
and
placing
it
on
the
agar
again.
If
this
does
not
work,
then
touch
the
edge
of
the
filter
with
the
forceps
in
such
a
way
as
to
make
it
sit
in
contact
with
the
medium.
Do
not
touch
the
area
of
the
filter
that
the
sample
has
gone
through­­
only
handle
the
very
outside
edge.
Repeat
the
disinfection
and
flaming
of
the
forceps
between
each
King
County
Environmental
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ENTEROCOCCI
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WATER
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MF
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sample.
UV
sterilize
funnels
for
90
seconds
between
each
sample.

10.3
Incubation
Plates
should
be
transferred
to
the
incubator
within
30
minutes
of
filtration.
Place
the
plates,
inverted,
in
the
41
±
0.5
oC
incubator
for
48
±
3
hr.
After
the
48
hour
incubation,
transfer
the
filter
to
the
EIA
medium
and
incubate
at
41
±
0.5
oC
for
an
additional
20­
30
minutes.
Read
the
EIA
plates
immediately
after
removal.

10.4
Counting
Colonies
Count
with
the
aid
of
at
least
1­
2x
magnification
with
the
aid
of
a
fluorescent
lamp.
Enterococci
colonies
are
those
which
appear
pink
to
red
with
a
black
or
reddish­
brown
precipitate.
Size
of
the
colony
can
vary
from
1­
3
mm.
Monthly
confirmations
are
performed
on
samples
as
a
minimum
(
section
11.5).
All
questionable
colonies
should
have
verification
steps
performed.

10.5
Verification
Procedure
Questionable
colonies,
especially
colonies
from
a
sample
exhibiting
atypical
morphology,
should
be
verified.
Colonies
identified
as
enterococci
can
be
confirmed
by
incubation
in
Brain­
heart
infusion
(
BHI)
broth
tubes.
After
each
of
the
BHI
isolates
are
checked
for
purity,
they
are
then
characterized
by
lack
of
catalase
production,
tolerance
to
40%
Bile
agar,
successful
esculin
hydrolysis,
reduction
of
Methylene
Blue,
growth
in
BHI
containing
6.5%
NaCl,
growth
in
BHI
at
both
10oC
and
45oC,
and
growth
in
pH
9.6
BHI
broth.

10.6
Data
Entry
and
Qualifiers
(
see
LIMS
Data
Entry
SOP)
Manually
enter
into
LIMS
the
number
of
enterococci
colonies
counted
into
the
"
num"
value
field
for
each
sample
and
dilution
that
was
workgrouped.
A
zero
value
should
be
entered
into
the
"
num"
value
field
only
if
no
characteristic
colonies
are
observed.
During
manual
data
entry,
qualifiers
are
entered
into
the
qualifier
field
in
LIMS
according
to
the
following
conditions:

 
If
unable
to
count
the
colonies
due
to
confluent
growth,
use
a
C
and
do
not
enter
a
"
num"
value.
 
If
the
number
of
target
colonies
slightly
exceeds
60
without
confluent
growth,
or
there
is
background
or
confluent
growth
but
the
analyst
believes
colony
crowding
is
not
adversely
affecting
a
reliable
estimate,
enter
a
number
value
and
qualify
it
with
a
C.
 
If
the
plate
exceeds
the
maximum
countable
range,
an
estimate
can
be
taken.
For
estimates
of
plates
with
3
to
10
target
colonies
per
square,
count
10
squares
and
obtain
the
average
count
per
square.
For
plates
with
10
to
20
target
colonies
per
square,
count
5
squares
and
obtain
the
average
count
per
square.
Multiply
the
average
count
per
square
by
100
to
obtain
the
plate
count,
and
then
report
that
number
with
an
E
qualifier.
(
Standard
Methods,
9­
33).
 
If
the
number
of
target
colonies
exceeds
the
countable
range
and
no
reliable
estimate
is
available,
use
TNTC.
Once
the
data
is
approved,
TNTC
will
be
converted
to
>
###
(
the
maximum
number
possible
to
count,
given
the
volume
tested)
by
LIMS.
King
County
Environmental
Laboratory
Standard
Operating
Procedure
MICROBIOLOGY,
ENTEROCOCCI
IN
ENVIRONMENTAL
WATER
BY
MF
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#:
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000
 
Other
general­
use
qualifiers
may
also
be
used
for
microbiology
data
(
see
Appendix
B
of
KCEL
QA
Manual)

10.7
Calculating
Results
Results
are
entered
and
calculated
using
specific
programs
in
Seedpak
in
LIMS.
The
LIMS
program
will
calculate
the
colony
count
per
100
mL
for
each
sample
by
using
the
"
batching"
method.
This
method
calculates
by
summing
the
colony
counts
of
all
dilutions
where
a
"
num"
value
was
entered
and
dividing
by
the
corresponding
summed
total
volume
of
sample
filtered.
The
adjusted
count
is
calculated
by
multiplying
the
resultant
by
100.
See
Appendix
B.

11.0
QA/
QC
Requirements
Routine
QC
analyses
for
Microbiology
include
both
those
procedures
used
to
insure
the
quality
of
each
batch
of
media
and
containers
and
those
procedures
used
to
monitor
method
performance
of
each
sample
analysis
batch
or
analysis
session.
A
sample
analysis
batch
should
not
exceed
20
samples
of
the
same
matrix
which
are
all
prepared
and
analyzed
together,
using
the
same
reagents,
media,
and
equipment.
An
analysis
session
consists
of
one
or
more
batches
done
within
a
working
day.

Each
batch
of
media
must
be
tested
to
confirm
pH,
sterility,
and
positive
and
negative
performance
characteristics.
These
specific
QC
procedures
are
defined
in
SOP(
s)
that
describe
the
preparation
and
QC
checks
of
mE
and
EIA
media.
(
See
SOP#
05­
01­
014­
000
for
media
preparation).
After
washing
and
sterilization,
each
batch
of
containers
used
for
the
collection
of
the
samples
must
be
evaluated
for
appropriate
pH,
soap
residue,
and
sterility.

11.1
Lab
Duplicates
Lab
duplicates
are
prepared
for
each
matrix
type
at
a
frequency
of
1
per
batch
or
5%,
whichever
is
more
frequent.
For
MF,
the
acceptance
limits
are
based
on
the
precision
criterion
calculated
from
the
duplicates
of
the
previous
15
duplicate
pairs
(
see
Standard
Methods,
19th
Edition.
9­
9).
These
acceptance
limits
are
updated
automatically
by
LIMS
whenever
duplicate
sample
results
are
entered.
If
the
15
previous
duplicate
pairs
are
unavailable,
a
senior
analyst
and/
or
supervisor
must
evaluate
the
duplicate
value.

11.2
Negative
Control
A
negative
control
is
prepared
at
a
frequency
of
1
per
session.
The
negative
control
is
a
suspension
of
Staphylococcus
species,
coagulase
negative.
The
negative
control
should
show
an
appropriate
qualitative
response
on
the
media
for
the
test
organism
and
should
not
be
identified
as
containing
enterococci.

11.3
Positive
Control
A
positive
control
is
prepared
at
a
frequency
of
1
per
session.
The
positive
control
is
a
suspension
of
Enterococcus
faecalis
or
faecium.
The
positive
control
should
show
an
King
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appropriate
qualitative
response
on
the
media
for
the
test
organism
and
should
be
identified
as
containing
enterococci.

11.4
System
Controls
For
the
MF
procedure,
both
a
"
pre"
and
a
"
post"
filtration
blank
are
prepared
on
the
least
specific
media
being
used
that
day
at
a
frequency
of
1
set
per
session.
The
filtration
blanks
challenge
the
same
batch
of
APHA
water
(
100
mL)
used
to
dilute
and
analyze
the
samples.
These
system
controls
are
considered
acceptable
if
the
target
organism
for
the
media
is
not
detected.
If
the
"
pre"
and
"
post"
filtration
blanks
show
positive
results
by
the
least­
specific
method,
the
controls
must
be
evaluated
by
each
of
the
more­
specific
methods.

11.5
Verification
To
ensure
analysts'
accuracy
and
method
performance,
monthly
verification
tests
are
performed
on
10
colonies
picked
from
various
samples
being
processed
that
month.
Ten
colonies
identified
as
enterococci
are
confirmed
by
incubation
in
Brain­
heart
infusion
(
BHI)
broth
tubes.
Each
of
the
10
BHI
isolates
should
first
be
checked
for
purity
and
then
characterized
by
lack
of
catalase
production,
tolerance
to
40%
Bile
agar,
successful
esculin
hydrolysis,
reduction
of
Methylene
Blue,
growth
in
BHI
containing
6.5%
NaCl,
growth
in
BHI
at
both
10oC
and
45oC,
and
growth
in
pH
9.6
BHI
broth.
80%
of
these
isolates
must
be
positive
for
enterococcus.
Verification
results
are
archived
in
the
QC
data
book.

11.6
Corrective
Actions
Corrective
actions
must
be
taken
whenever
a
QC
failure
occurs.
Unacceptable
QC
results
within
a
batch
or
session
of
analyses
require
that
all
samples
associated
with
those
batches
and/
or
session
must
be
reviewed.
A
decision
should
be
made
as
to
if
and
how
the
samples
should
be
qualified.
A
Corrective
Action
form
should
be
prepared
to
document
the
failure
and
describe
how
it
was
resolved,
if
possible,
and
how
it
could
be
avoided
in
the
future.

12.0
Reporting,
Review
and
Documentation
LIMS
raw
data
sheets
are
attached
to
the
LIMS
hard
data
review
sheets
and
given
to
the
project
coordinator.
The
project
coordinator
reviews
the
results
for
acceptability
based
on
compliance
with
holding
times,
storage
and
testing
conditions,
positive
and
negative
controls,
filtration
blank
results,
and
conformance
with
both
historical
and
synoptic
data.
Appropriate
dilution
effects
and
consistency
with
other
parameters
are
also
evaluated.
Once
the
results
have
been
shown
to
meet
all
criteria,
the
project
coordinator
will
approve
and
post
the
data
to
EDS
tables
in
LIMS.
All
hardcopy
data
is
archived
by
year
and
project.

13.0
References
American
Public
Health
Association.
1998.
Method
9230
C.
Membrane
Filter
Techniques.
Clesceri,
L.
S.,
A.
D.
Eaton,
and
A.
E.
Greenberg
(
ed).
Standard
Methods
King
County
Environmental
Laboratory
Standard
Operating
Procedure
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ENTEROCOCCI
IN
ENVIRONMENTAL
WATER
BY
MF
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for
the
Examination
of
Water
and
Wastewater.
20th
Edition.
American
Public
Health
Association,
Washington,
DC.

14.0
Training
New
microbiologists
are
to
be
trained
at
this
procedure
by
first
reading
the
reference
method
and
this
SOP,
and
then
observing
the
method
as
performed
by
an
experienced
analyst.
While
being
directly
supervised,
the
trainee
will
then
perform
the
method
on
two
separate
sets
of
split
samples
also
performed
by
an
experienced
analyst.
Once
the
trainee
has
produced
acceptable
QC
results
and
the
results
of
the
two
sets
match
those
of
the
experienced
analyst,
the
trainee
will
be
approved
as
an
analyst.
King
County
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Standard
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Procedure
MICROBIOLOGY,
ENTEROCOCCI
IN
ENVIRONMENTAL
WATER
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MF
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Appendix
A
Suggested
Volumes
for
Enterococcus
Analyses
by
MF
Volume
Analyzed
(
mL)
102
101
100
10­
1
10­
2
10­
3
10­
4
10­
5
10­
6
Project
Streams
(
routine)
x
x
x
Streams
(
storms)
x
x
x
x
Lake
Wash.
x
x
Lake
Samm.
x
x
Lake
Union
x
x
Beaches
(
Lakes)
x
x
x
Sewage
(
raw)
x
x
x
x
x
CSO
x
x
x
x
x
x
Trouble
Call
(
water)
x
x
x
x
x
x
Stormwater
x
x
x
x
x
x
WP
Offshore
x
Renton
Offshore
x
Beaches
*
(
Marine)
x
x
*
Volumes
routinely
analyzed
are
10,
30
and
100
mL.
King
County
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Operating
Procedure
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MF
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Appendix
B
Calculation
of
Final
MF
Results
"
Batching"
Method
This
process
takes
the
colonies
counted
for
the
sample
and
the
volume
filtered
from
each
of
the
selected
dilutions
and
performs
the
following
calculations:

1.
Sum
all
counted
colonies.
2.
Sum
all
filtered
volumes
(
only
use
the
volumes
of
dilutions
which
produce
a
numeric
value).
Remember
to
also
include
the
volumes
of
duplicated
dilutions
if
the
sample
was
run
in
duplicate.
3.
Multiply
the
sum
of
the
counted
colonies
(#
1
above)
by
100
and
divide
by
the
sum
of
the
filtered
volumes
(#
2
above).

(
All
Counted
Colonies)
X
100
(
Total
Volume
Filtered)

This
is
the
adjusted
reported
value.

The
method
detection
limit
is
found
by
following
the
instructions
under
special
conditions,
number
two.
Special
Conditions:

1.
If
all
dilutions
resulted
in
a
count
of
TNTC:

Divide
the
maximum
countable
number
for
the
specific
procedure
(
see
chart
below)
by
the
smallest
volume
filtered.
Multiply
this
result
by
100.
Place
this
result
in
the
Qualifier
Field
as
part
of
the
">
#####"
where
"####"
is
the
result.
The
Value
Field
is
left
blank.

Procedure
Maximum
Countable
Number
Fecal
Coliform
60
Total
Coliform
80
E.
coli
80
Enterococci
60
Enterococci
(
modified
incubation)
60
Fecal
Streptococci
60
Heterotrophic
Plate
Count
200
2.
If
all
dilutions
resulted
in
zero
counts:

If
the
largest
volume
is
 
100,
divide
1
by
the
largest
volume
filtered,
and
then
multiply
the
resultant
by
100
to
obtain
the
count
per
100ml.
Enter
this
result
as
`<####'
in
the
qualifier
field.
King
County
Environmental
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If
the
largest
volume
is
<
100,
divide
100
by
the
largest
volume
filtered
to
obtain
the
count
per
100
ml.
Enter
this
result
as
`<####'
in
the
qualifier
field.

Significant
Digits:

1.
If
each
value
used
from
individual
dilutions
has
only
one
significant
figure
(
counts
<
10),
the
final
calculated
value
should
only
have
one
sig.
fig.
2.
If
any
value
from
individual
dilutions
has
2
or
3
sig.
figs,
the
final
calculated
value
should
have
no
more
than
the
smaller
of
the
number
of
sig
figs
(
i.
e.,
if
the
calculation
has
both
2
and
3
sig
fig
numbers,
the
result
should
be
reported
with
2
sig
figs).
3.
Sig.
figs
should
never
exceed
3.
