Document ID: EPA-HQ-OW-2002-0039-0084
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-07-09T04:00Z

Draft
June
2003
1
Checklist
for
the
Laboratory
Quality
Assurance
Evaluation
Program
for
Analysis
of
Cryptosporidium
under
the
Safe
Drinking
Water
Act
Part
A:
Personnel,
Facilities,
Equipment,
and
Quality
Assurance
Laboratory
Name
Date
of
Evaluation
Name
and
Affiliation
of
Evaluator
for
Part
A
Part
A:
Facilities,
Equipment,
and
Quality
Assurance
Item
to
be
Evaluated
Classification
Yes,
No,
Unknown*,
or
NA
1
Laboratory
Equipment
and
Supplies
1.1
Reagent­
grade
water
testing
1.1.1
Is
reagent
water
tested
monthly
for
these
minimum
parameters:
conductivity,
total
chlorine
residual;
and
annually
for
metals­
Pb,
Cd,
Cr,
Cu,
Ni,
Zn?
Requirement
1.1.2
Were
the
results
for
the
above
parameters
acceptable,
total
chlorine
residual
not
greater
than
0.1
mg/
L,
conductivity
not
greater
than
2
µ
mhos/
cm,
and
each
metal
not
greater
than
0.05
mg/
L
and
collectively
not
greater
than
0.1
mg/
L?
Requirement
1.1.3
Is
reagent
water
tested
monthly
for
heterotrophic
plate
count?
Requirement
1.1.4
Are
the
results
for
the
heterotrophic
plate
count
acceptable,
<
500/
mL?
Requirement
1.2
Laboratory
pH
meter:

1.2.1
Accuracy
±
0.1
units,
scale
graduations,
0.1
units?
Requirement
1.2.2
Is
a
record
maintained
for
pH
measurements
and
calibrations
used?
Requirement
1.2.3
Is
pH
meter
standardized
each
use
period
with
pH
7,
4
or
10
standard
buffers
(
selection
dependant
upon
desired
pH)?
Requirement
1.2.4
All
pH
buffers
are
dated
when
received
and
opened
and
are
discarded
before
expiration
date?
Requirement
1.3
Balances
(
top
loader
or
pan
balance):

1.3.1
Are
balances
calibrated
monthly
using
Class
S/
S­
1
weights,
or
weights
traceable
to
Class
S/
S­
1
weights?
Requirement
1.3.2
Is
correction
data
available
with
S/
S­
1
weights?
Requirement
1.3.3
Is
preventative
maintenance
conducted
yearly
at
a
minimum?
Recommendation
Item
to
be
Evaluated
Classification
Yes,
No,
Unknown*,
or
NA
Draft
June
2003
2
1.4
Autoclave:

1.4.1
Is
unit
equipped
with
a
temperature
gauge/
operational
safety
valve?
Requirement
1.4.2
Are
date,
contents,
sterilization
time
and
temperature
recorded
for
each
cycle?
Requirement
1.4.3
Is
a
maximum
registering
thermometer
or
continuous
monitoring
device
used
during
each
autoclave
cycle?
Requirement
1.4.4
Is
automatic
timing
mechanism
checked
with
stopwatch
quarterly?
Requirement
1.4.5
Are
spore
strips
or
ampules
used
monthly
to
confirm
sterilization?
Requirement
1.5
Refrigerator/
Freezer:

1.5.1
Is
refrigerator
able
to
maintain
temperature
of
1
°
C
to
5
°
C?
Requirement
1.5.2
Is
temperature
recorded
once
daily
for
days
in
use?
Requirement
1.6
Temperature
recording
device:

1.6.1
Are
calibration
of
glass/
mercury
thermometers
checked
annually
(
dial
thermometers
quarterly)
at
the
temperature
used
against
a
reference
NIST
thermometer
or
equivalent?
Requirement
1.7
Micropipetters:

1.7.
Have
micropipetters
been
calibrated
within
the
past
year?
[
Section
9.2.1]
Requirement
1.8
Centrifuge
1.8.1
Is
a
maintenance
contract
in
place,
or
internal
maintenance
protocol
available?
Requirement
1.8.2
Is
RPM
and
RCF
calibrated
yearly?
Requirement
1.9
General
1.9.1
Are
calibration
and
maintenance
records
complete
and
well
organized?
Recommendation
2
Quality
Assurance
2.1
Does
the
laboratory
have
a
formal
QA
laboratory
plan
prepared
and
ready
for
examination?
Requirement
2.2
Are
employee
resumes
present
and
complete?
Requirement
2.3
Is
a
training
protocol
for
new
employees
present?
Recommendation
2.4
Is
the
laboratory
performing
analyst
verification
of
examination
monthly
and
does
the
lab
have
corrective
action
procedures
in
place
if
criteria
are
not
met?
(
Section
10.5)
Requirement
2.5
Are
employee
training
records
available
and
up
to
date?
Requirement
2.5.1
Have
technicians/
analysts
analyzed
the
required
number
of
samples
using
Method
1622/
1623?
Requirement
2.6
Are
all
relevant
SOPs
present
and
current?
Requirement
2.7
Are
sampling
instructions
present
for
clients
collecting
and/
or
filtering
samples
in
the
field?
Requirement
Item
to
be
Evaluated
Classification
Yes,
No,
Unknown*,
or
NA
Draft
June
2003
3
2.8
Does
the
laboratory
have
criteria
for
sample
acceptance
and
corrective
action
procedures?
Requirement
2.9
Are
data
recording
procedures
present?
Requirement
2.9.1
Does
the
laboratory
have
an
SOP
for
checking
all
manual
calculations?
Requirement
2.10
Are
corrective
action
contingencies
present?
Requirement
2.10.1
For
OPR
failures?
[
Section
9.7.4]
Requirement
2.10.2
For
method
blank
contamination?
Requirement
2.10.3
For
positive/
negative
staining
control
failures?
Requirement
2.11
Does
the
quality
assurance
plan
specifically
address
requirements
for
protozoa
analysis
under
the
programs
for
which
the
laboratory
intends
to
analyze
samples?
Requirement
2.12
Is
a
laboratory
organization
chart
or
other
information
available
listing
staff
organization
and
responsibilities?
Does
it
identify
the
QA
manager?
Requirement
2.12.1
Is
the
QA
manager
separate
from
the
lab
manager?
Recommendation
2.13
Does
the
laboratory
have
a
list
of
preventative
maintenance
procedures
and
schedules?
Requirement
2.14
Date
range
covered
for
quality
control
(
QC)
sample
audit?

2.15
When
did
the
laboratory
begin
processing
samples
with
the
Envirochek
filter?
/
/

2.16
When
did
the
laboratory
begin
processing
samples
with
the
Filta­
Max
filter
(
if
applicable)?
/
/

2.17
When
did
the
laboratory
begin
processing
samples
with
the
CrypTest
filter
(
if
applicable)?
/
/

2.18
Approximately
how
many
field
samples
were
analyzed
using
methods
1622/
1623
since
the
lab
started
using
Method
1622/
1623?
Field
samples
___
MS_____

2.19
Have
acceptable
initial
precision
and
recovery
analyses
been
performed
for
each
version
of
the
method
the
laboratory
is
using?
Requirement
2.20
Were
method
blanks
run
once
per
week
or
per
20
samples
during
this
period?
[
Section
9.6.1]
Requirement
2.20.1
If
the
answer
to
2.20
is
no,
then
at
what
frequency
where
method
blanks
performed?

2.20.2
What
percentage
of
method
blanks
evaluated
were
without
contamination?

2.20.3
Was
an
acceptable
method
blank
associated
with
each
field
sample
examined?
Requirement
2.20.4
How
many
method
blanks
were
evaluated?

2.21
Were
ongoing
precision
and
recovery
(
OPR)
samples
run
once
per
week
or
per
20
samples
during
this
period?
[
Section
9.7]
Requirement
2.21.1
If
the
answer
to
2.21
is
no,
then
at
what
frequency
where
OPR
samples
performed?

2.21.2
What
percentage
of
OPR
samples
evaluated
met
the
recovery
criteria?
[
Table
3;
Section
9.7.3]
Item
to
be
Evaluated
Classification
Yes,
No,
Unknown*,
or
NA
Draft
June
2003
4
2.21.3
Does
the
laboratory
maintain
control
charts
of
OPR
results?
[
Section
9.7.5]
Requirement
2.21.4
Was
an
acceptable
OPR
associated
with
each
field
sample
examined?
Requirement
2.21.5
How
many
OPR
samples
were
evaluated?

2.21.6
How
many
OPR
samples
were
analyzed
during
the
past
six
months?

2.21.7
What
is
the
mean
and
relative
standard
deviation
of
the
recoveries
of
the
OPR
samples
analyzed
during
the
past
six
months?
Mean_______
RSD________

2.22
Were
matrix
spike
(
MS)
samples
analyzed
at
the
method
­
specified
frequency?
[
Section
9.1.8]
Requirement
2.22.1
If
the
answer
to
2.22
is
no,
then
at
what
frequency
were
MS
samples
analyzed?

2.22.2
How
many
MS
samples
were
evaluated?

2.22.3
How
many
MS
samples
were
analyzed
during
the
past
six
months?

2.22.4
What
is
the
mean
and
relative
standard
deviation
of
the
MS
samples
analyzed
during
the
past
six
months?
Mean_______
RSD________

2.23
Were
OPR
and
MS
samples
spiked
with
100
­
500
organisms?
[
Section
9.7]
Requirement
2.23.1
If
the
answer
to
2.23
is
no,
then
at
what
level
were
samples
spiked?

2.24
Are
the
laboratory
personnel
performing
the
QC
analyses
representative
of
the
personnel
seeking
approval
under
this
program?
Requirement
2.25
Does
the
laboratory
have
records
of
all
QC
checks
available
for
inspection?
Requirement
2.26
Does
the
laboratory
have
an
adequate
record
system
for
tracking
samples
from
collection
through
log­
in,
analysis,
and
data
reporting?
Requirement
2.27
Are
results
from
each
sample
maintained
electronically?

2.28
If
data
are
stored
electronically,
are
files
backed
up
on
more
than
one
disk
to
ensure
data
are
not
lost
in
the
eventuality
of
some
hardware
failure?
Requirement
2.29
If
data
is
stored
electronically,
does
the
laboratory
have
an
SOP
for
checking
the
accuracy
of
data
entry
into
an
electronic
system?
Requirement
2.30
Is
the
laboratory
using
the
April
2001
version
of
Method
1622/
1623?
Requirement
3
Data
Recording
Procedures
3.1
Is
shipping
information
complete,
including
the
time
and
date
of
sample
receipt,
sample
condition,

and
noting
any
discrepancies
between
samples
on
the
traffic
report
and
samples
received?
Requirement
3.2
Do
sample
numbers
on
the
shipping
forms
match
the
sample
numbers
on
the
report
forms?
Requirement
3.3
Are
current
Method
1622/
1623
bench
sheets
used
to
record
sample
processing
data?
Recommendation
3.4
Are
all
primary
measurements
during
each
step
recorded,
including
all
raw
data
used
in
calculations?
Requirement
Item
to
be
Evaluated
Classification
Yes,
No,
Unknown*,
or
NA
Draft
June
2003
5
3.5
Name
of
analyst
or
technician
performing
the
elution
is
recorded?
Requirement
3.6
Date
and
time
of
elution
is
recorded?
Requirement
3.7
Name
of
analyst
or
technician
performing
the
concentration
is
recorded?
Requirement
3.8
Date
and
time
of
concentration
is
recorded?
Requirement
3.9
Are
batch
and
lot
numbers
of
reagents
used
in
the
analysis
of
the
sample
recorded?
Requirement
3.10
Lot
number
for
the
IMS
kit
is
recorded?
Requirement
3.11
Are
Method
1622/
1623
Cryptosporidium
report
forms
used
to
record
sample
examination
results?
Requirement
3.12
Name
of
examining
analyst
is
recorded?
Requirement
3.13
Date
and
time
of
sample
examination
is
recorded?
Requirement
3.14
Are
calculations
of
final
concentrations
and
recoveries
complete
and
correct?
Requirement
3.15
Do
values
recorded
on
the
data
sheets
match
the
reported
values?
Requirement
3.16
Are
mistakes
on
all
forms
crossed
out
with
a
single
line,
initialed,
and
dated?
Requirement
3.17
Are
data
always
recorded
in
pen?
Requirement
3.18
Are
hardcopy
records
well
organized,
complete,
and
easily
accessible?
Requirement
3.19
Does
the
laboratory
include
a
disclaimer
on
the
report
to
the
client
if
method
QC
requirements
were
not
met?
Recommendation
3.20
Is
the
manually
recorded
data
legible?
Requirement
3.21
Do
records
demonstrate
each
analyst's
characterization
of
3
oocysts
and
3
cysts
from
positive
control
for
each
microscopy
session?
[
Section
15.2.1.1]
Requirement
3.22
Data
shows
that
no
more
than
0.5
mL
of
pellet
was
used
per
IMS?
[
Section
13.2.4]
Requirement
4
Holding
Times
4.1
Samples
analyzed
according
to
December
1999
version
of
Method
1622/
1623
4.1.1
Is
time
from
initiation
of
sample
collection
to
completion
of
concentration
72
hours
or
less?

[
Section
8.1]
Requirement
4.1.2
Concentrate
is
held
no
longer
than
24
hours
between
IMS
and
staining?
[
Section
8.2]
Requirement
4.1.3
Are
stained
slides
read
and
confirmed
within
72
hours
of
staining?
[
Section
8.4]
Requirement
4.2
Samples
analyzed
according
to
April
2001
version
of
Method
1622/
1623
4.2.1
Is
sample
elution
initiated
within
96
hours
of
sample
collection
or
field
filtration?
[
Section
8.2.1]
Requirement
4.2.2
Are
sample
elution,
concentration,
and
purification
steps
completed
in
one
work
day?

[
Section
8.2.2]
Requirement
Item
to
be
Evaluated
Classification
Yes,
No,
Unknown*,
or
NA
Draft
June
2003
6
4.2.3
Are
slides
stained
within
72
hours
of
application
of
the
purified
sample
to
the
slide?

[
Section
8.2.3]
Requirement
4.2.4
Are
stained
slides
read
and
confirmed
within
7
days
of
staining?
[
Section
8.2.4]
Requirement
5
Spike
enumeration
procedures
5.1
What
method
does
the
laboratory
currently
use
to
estimate
spike
doses:(
A)
flow­
sorted
spikes,
(
B)

well­
slide­
counted
spikes,
(
C)
hemacytometer­
counted
spikes,
or
(
D)
membrane­
filter­
counted
spikes
Circle
one:
A
B
C
D
5.1.1
If
flow­
sorted
spikes
are
used,
on
what
date
did
the
laboratory
begin
using
flow­
sorted
spikes?
/
/

5.1.2
If
counted
manually,
does
the
laboratory
follow
Method
1622/
1623
procedures
for
establishing
spike
level?
[
Section
11.3]
Requirement
5.1.3
What
were
the
relative
standard
deviations
of
the
last
four
spike
enumerations?
1.
2.
3.
4.

5.2
Source
of
oocysts
for
spikes
5.3
If
50­
L
samples
are
analyzed,
what
positive
control
procedure
does
the
laboratory
follow
for
OPR
and
MS
samples:
(
A)
spike
entire
50
L,
(
B)
spike
and
filter
10
L
before
filtering
40
L,
or
(
C)
filter
40
L
before
spiking
and
filtering
10
L.

*
Unknown
response
requires
an
explanation
Note:
All
section
references
in
[
]
refer
to
Method
1623
April
2001
Comments:
Draft
June
2003
7
Part
B:
Sample
Processing
and
Examination
Laboratory
Name
Date
of
Evaluation
Name
and
Affiliation
of
Evaluator
for
Part
B
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
6
Laboratory
Facilities
and
Laboratory
Safety
6.1
Are
laboratory
coats
and
gloves
worn
in
the
laboratory?
Requirement
6.2
No
other
safety
or
facility
issues
were
observed?

7
Sample
Spiking
Technician:

7.1
What
method
does
laboratory
currently
use
to
estimate
spike
doses:(
A)
flow­
sorted
spikes,
(
B)

wellslide
counted
spikes,
(
C)
hemacytometer­
counted
spikes,
or
(
D)
membrane­
filter­
counted
spikes
Circle
one:

A
B
C
D
7.2
With
what
filter
type
did
the
laboratory
demonstrate
their
spiking
procedure?

7.3
Is
the
carboy
used
for
negative
control
randomly
selected
from
carboy
stock
to
check
efficacy
of
cleaning
system?
Requirement
7.4
If
flow­
sorted
spikes
are
used,
was
suspension
vial
vortexed
for
two
minutes
or
per
manufacturers
instructions?
[
Section
11.4.3]
Method
Procedure
7.5
Was
the
suspension
vial
adequately
rinsed?
[
Section
11.4.3.1]
Method
Procedure
7.6
Does
the
laboratory
have
an
acceptable
SOP
for
sample
spiking?
Requirement
7.7
Other
than
the
issues
noted
for
items
7.2
through
7.6
(
if
any)
was
sample
spiking
demonstrated
successfully?

8
Envirochek
(
Complete
Sections
that
apply)

8.1
Envirochek
Filtration
Technician:

8.1.1
Are
all
components
required
for
sample
filtration
present
and
in
good
condition?
[
Section
6.2]
Requirement
8.1.2
Is
the
filter
assembly
set
up
correctly?
[
Figure
3,
pg
48]
Method
Procedure
8.1.3
Is
the
pump
adequate
for
needs?
[
Section
6.3.3]
Requirement
8.1.4
Is
the
appropriate
flow
rate
maintained
(
approximately
2L/
min)?
[
Section
12.2.1.2]
Method
Procedure
8.1.5
Is
the
volume
filtered
measured
using
a
flow
totalizer
or
calibrated
carboy?
[
Section
12.2.4.2]
Requirement
8.1.6
Is
the
system
well
maintained
and
cleaned
appropriately
following
use?
Requirement
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
Draft
June
2003
8
8.1.7
Is
the
system
able
to
maintain
seal
during
use
with
no
leaks?
Requirement
8.1.8
Does
the
laboratory
have
an
acceptable
SOP
for
Envirochek
filtration?
Requirement
8.1.9
Other
than
the
issues
noted
in
items
8.1.1
through
8.1.8,
was
Envirochek
filtration
demonstrated
successfully?

8.2
Envirochek
capsule
filter
elution
Technician:

8.2.1
Is
the
elution
buffer
prepared
as
per
Method
1622/
1623?
[
Section
7.4]
Method
Procedure
8.2.2
Is
the
wrist­
shaker
assembly
set
up
correctly?
[
Section
12.2.6.1.1]
Method
Procedure
8.2.3
Does
the
eluting
solution
cover
the
membrane?
[
Section
12.2.6.2.2]
Method
Procedure
8.2.4
Are
the
samples
shaken
at
an
appropriate
speed?
[
Section
12.2.6.2.3]
Method
Procedure
8.2.5
Are
the
samples
shaken
three
times
for
5
minutes
each
time,
and
each
in
a
different
orientation?
[
Section
12.2.6.2]
Method
Procedure
8.2.6
Does
the
laboratory
have
an
acceptable
SOP
for
Envirochek
capsule
filter
elution?
Requirement
8.2.7
Other
than
the
issues
noted
for
items
8.2.1
through
8.2.7
(
if
any)
was
Envirochek
filter
elution
demonstrated
successfully?

9
CrypTest
9.1
CrypTest
Filtration
Technician:

9.1.1
Are
all
components
required
for
sample
filtration
present
and
in
good
condition?
[
Section
6.2.3]
Requirement
9.1.2
Is
the
filter
assembly
set
up
correctly?
Method
Procedure
9.1.3
Is
the
pump
adequate
for
needs?
[
Section
6.3.3]
Requirement
9.1.4
Is
the
appropriate
flow
rate
maintained
(
approximately
2L/
min)?
Method
Procedure
9.1.5
Is
the
volume
filtered
measured
using
a
flow
meter
or
a
calibrated
carboy?
Requirement
9.1.6
Is
the
system
well
maintained
and
cleaned
appropriately
following
use?
Requirement
9.1.7
Is
the
system
able
to
maintain
seal
during
use
with
no
leaks?
Requirement
9.1.8
Does
the
laboratory
have
an
acceptable
SOP
for
CrypTest
Filtration?
Requirement
9.1.9
Other
than
the
issues
noted
in
items
9.1.3
through
9.1.10
(
if
any)
was
CrypTest
filtration
demonstrated
successfully?

9.2
CrypTest
cartridge
filter
elution
Technician:

9.2.1
Does
the
filter
seat
properly
in
the
filter
housing,
so
there
are
no
leaks?
Requirement
9.2.2
Is
the
elution
buffer
prepared
according
to
manufacturer's
instructions?
[
Section
7.4.2]
Method
Procedure
9.2.3
Is
an
appropriate
amount
of
elution
solution
backwashed
into
the
filter
housing?
(
approx.

150
mL)
Method
Procedure
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
Draft
June
2003
9
9.2.4
Is
the
assembly
well
sealed
(
no
leaks)?
Requirement
9.2.5
Is
sonication
performed
for
2
minutes?
Method
Procedure
9.2.6
Is
the
filter
elution
repeated,
according
to
the
manufacturer's
instructions?
Method
Procedure
9.2.7
Following
the
last
elution,
is
the
remaining
elution
buffer
driven
from
the
outlet
side
to
the
inlet
side
and
into
the
sample
bottle?
Requirement
9.2.7.1
Is
the
regulated
compressed
air
source
used,
sufficient
to
drive
the
eluting
buffer
from
the
filter?
Requirement
9.2.8
After
elution
is
complete,
is
the
filter
removed
from
the
housing
and
the
base,
lid,
and
lip
of
the
filter
housing
rinsed
using
eluting
solution
and
added
to
the
sample
bottle?
Requirement
9.2.9
Does
the
laboratory
have
an
acceptable
SOP
for
CrypTest
elution?
Requirement
9.2.10
Other
than
the
issues
noted
in
items
9.2.1
through
9.2.9
(
if
any)
was
CrypTest
filter
elution
demonstrated
successfully?

10
Filta­
Max
10.1
Filta­
Max
filtration
Technician:

10.1.1
Are
all
components
required
for
sample
filtration
present
and
in
good
condition?
[
Section
6.2.4]
Requirement
10.1.2
Is
the
filter
assembly
set
up
correctly?
Method
Procedure
10.1.3
Is
appropriate
flow
rate
maintained
of
<
4
L
per
minute?
Method
Procedure
10.1.4
Is
the
volume
filtered
measured
correctly
using
a
flow
meter
or
calibrated
carboy?
Requirement
10.1.5
Is
system
well
maintained
and
cleaned
appropriately
following
use?
Requirement
10.1.6
Is
system
able
to
maintain
seal
during
use
with
no
leaks?
Requirement
10.1.7
Does
the
laboratory
have
an
acceptable
SOP
for
Filta­
Max
filtration?
Requirement
10.1.8
Does
the
laboratory
indicate
on
the
filter
housing
the
correct
direction
of
flow?
Requirement
10.1.9
Other
than
the
issues
noted
in
items
10.1.1
through
10.1.8
(
if
any)
was
Filta­
Max
filtration
demonstrated
successfully?

10.2
Filta­
Max
filter
wash
station
elution
Technician:

10.2.1
Is
an
automatic
or
manual
wash
station
used?

10.2.2
Is
the
filter
wash
station
set
up
correctly?
Requirement
10.2.3
Is
PBST
used
to
elute
the
filter?
[
Section
7.4.2]
Method
Procedure
10.2.4
Is
an
appropriate
amount
of
PBST
used
for
each
wash?
(
approx.
600
mL)
Method
Procedure
10.2.5
During
the
first
wash,
is
the
plunger
moved
up
and
down
20
times?
Method
Procedure
10.2.6
Is
the
plunger
moved
up
and
down
gently
to
avoid
generating
excess
foam?
Method
Procedure
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
Draft
June
2003
10
10.2.7
During
the
second
wash,
is
the
plunger
moved
up
and
down
10
times?
Method
Procedure
10.2.8
If
the
automatic
washer
is
used,
is
the
machine
operating
properly?
Requirement
10.2.9
Is
the
wash
station
cleaned
adequately
between
samples?
Requirement
10.2.10
Does
the
laboratory
have
an
acceptable
SOP
for
Filta­
Max
elution
with
the
wash
station?
Requirement
10.2.11
Other
than
the
issues
noted
for
items
10.2.2
through
10.2.10
(
if
any)
was
elution
of
the
Filtamax
filter
using
the
wash
station
demonstrated
successfully?

10.3
Filta­
Max
filter
stomacher
elution
Technician
10.3.1
Is
PBST
used
to
elute
the
filter?
[
Section
7.4.3.4]
Method
Procedure
10.3.2
Is
an
appropriate
amount
of
PBST
used
for
each
wash?
(
approx.
600
mL)
Method
Procedure
10.3.3
Are
two
washes
performed
for
5
minutes
each?
Method
Procedure
10.3.4
Is
the
stomacher
in
good
condition
and
operating
properly?
Requirement
10.3.5
Does
the
laboratory
have
an
acceptable
SOP
for
Filta­
Max
elution
using
a
stomacher?
Requirement
10.3.6
Other
than
the
issues
noted
for
items
10.3.1
through
10.3.5
(
if
any)
was
elution
of
the
Filta­

Max
filter
using
the
stomacher
demonstrated
successfully?

10.4
Filta­
Max
filter
sample
concentration
(
as
an
alternative
to
Section
11)
Technician:

10.4.1
Is
concentrator
set
up
correctly?
Requirement
10.4.2
Is
the
force
of
the
vacuum
maintained
below
30
cm
Hg?
Method
Procedure
10.4.3
Is
concentration
performed
after
each
of
the
washes?
Method
Procedure
10.4.4
Is
the
concentrate
from
the
first
wash
added
to
the
600mL
of
eluate
from
the
second
wash?
Method
Procedure
10.4.5
Is
the
sample
concentrated
so
that
some
liquid
remains
above
the
filter
(
enough
to
cover
the
stirbar
about
half­
way)?
Method
Procedure
10.4.6
Is
the
stir
bar
and
concentration
tube
rinsed
after
each
concentration
and
the
liquid
added
to
the
concentrate?
Requirement
10.4.7
Was
the
filter
membrane
washed
twice?
Method
Procedure
10.4.8
Was
5
mL
of
PBST
used
each
time?
Method
Procedure
10.4.9
Is
the
membrane
adequately
washed
to
remove
oocysts
from
filter?
Method
Procedure
10.4.10
Is
the
pellet
volume
determined?
Requirement
10.4.11
Is
there
a
set
of
standards
for
comparison
of
pellet
size?
Recommendation
10.4.12
Does
the
laboratory
have
an
acceptable
SOP
for
concentration
using
the
Filta­
Max
concentrator?
Requirement
10.4.13
Other
than
the
issues
noted
in
items
10.4.1
through
10.4.12
(
if
any)
was
sample
concentration
using
the
Filta­
Max
concentrator
demonstrated
successfully?
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
Draft
June
2003
11
11
Concentration
11.1
Envirochek,
CrypTest,
and
Filta­
Max
filter
sample
centrifugation
Technician:

11.1.1
Is
the
sample
centrifuged
at
1500
x
G
using
a
swinging
bucket
rotor?
[
Section
13.2.1]
Method
Procedure
11.1.2
Are
the
centrifuge
tubes
properly
balanced
prior
to
centrifugation?
Requirement
11.1.3
Is
the
sample
centrifuged
for
15
minutes?
[
Section
13.2.1]
Method
Procedure
11.1.4
Is
the
centrifuge
slowly
decelerated
at
the
end
without
the
brake?
[
Section
13.2.1]
Method
Procedure
11.1.5
Is
the
pellet
volume
determined?
Requirement
11.1.6
Is
there
a
set
of
standards
for
comparison
of
pellet
size?
Recommendation
11.1.7
Does
the
laboratory
have
an
acceptable
SOP
for
sample
concentration?
Requirement
11.1.8
Is
residual
suspension
rinsed
from
all
containers
and
gloves?
Requirement
11.1.9
Other
than
the
issues
noted
in
items
11.1.1
through
11.1.8
(
if
any)
was
sample
concentration
demonstrated
successfully?

12
Reagents,
equipment
and
clean­
up
12.1
Source
for
reagent­
grade
water:

12.1.1
Is
still
or
DI
unit
maintained
according
to
manufacturer's
instructions?
Requirement
12.1.2
Is
reagent
grade
water
used
to
prepare
all
media
and
reagents?
[
Section
7.3]
Requirement
12.2
Centrifuge:

12.2.1
Does
centrifuge
have
a
swinging
bucket
rotor?
[
Section
6.8.1]
Requirement
12.2.2
Does
lab
have
easily
accessible
method
fro
determining
relative
centrifugal
force
of
centrifuges?
Requirement
12.3
SOP's
for
Reagents
12.3.1
Are
SOP's
available
for
the
preparation
of
all
essential
chemicals
and
reagents?
Requirement
12.3.2
Are
SOP's
posted
or
easily
accessible
at
the
bench?
Recommendation
12.3.3
Are
all
reagents
clearly
labeled
with
date
of
preparation,
technician
initials,
and
expiration
date?
Requirement
12.4
Clean­
up
12.4.1
Is
all
glassware
and
plasticware
washed
well
and
stored
appropriately
between
uses?
Requirement
12.4.2
Is
distilled
or
deionized
water
used
for
final
rinse?
Requirement
12.4.3
Is
an
SOP
available
for
glassware
washing?
Requirement
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
Draft
June
2003
12
13
Purification
and
Slide
Preparation
Technician:

13.1
What
IMS
kit/
manufacturer
is
used?

13.2
Is
the
supernatant
from
the
centrifuged
sample
aspirated
no
lower
than
5
mL
above
the
pellet?

[
Section
13.2.2]
Requirement
13.3
Is
the
pellet
vortexed
a
sufficient
time
for
resuspension?
[
Section
13.2.3]
Method
Procedure
13.4
Does
the
lab
have
an
appropriate
SOP
for
dividing
pellets
greater
than
0.5mL
into
subsamples
and
analyzing?
Requirement
13.5
Is
no
more
than
0.5
mL
of
pellet
used
per
IMS?
[
Section
13.2.4]
Method
Procedure
13.6
Is
the
resuspended
pellet
volume
quantitatively
transferred
to
the
Leighton
tube
(
2
rinses)?

[
Section
13.3.2.1]
Method
Procedure
13.7
Are
the
IMS
beads
thoroughly
resuspended
prior
to
addition
to
the
Leighton
tube?
[
Section
13.3.2.2]
Method
Procedure
13.8
Is
the
leighton
tube
rotated
at
18
rpm
for
1
hour
at
room
temperature?
Method
Procedure
13.9
Is
the
sample
quantitatively
transferred
from
the
Leighton
tube
to
the
microcentrifuge
tube
(
2
rinses)?
[
Section
13.3.2.13]
Method
Procedure
13.10
Is
standard
NaOH
(
5
µ
L,
1N)
and
standard
HCl
(
50
µ
L,
0.1N)
used?
[
See
note
on
pg
37]
Requirement
13.11
Is
sample
vortexed
vigorously
for
50
seconds
immediately
after
the
addition
of
acid
and
30
seconds
after
the
sample
has
set
for
10
minutes
at
room
temperature?
[
Section
13.3.3]
Method
Procedure
13.12
Is
a
second
dissociation
performed?
[
Section
13.3.3.10]
Method
Procedure
13.13
When
the
second
dissociation
is
performed,
does
the
laboratory:
(
A)
use
a
second
slide,
or
(
B)
add
the
additional
volume
to
the
original
slide?
Circle
one:
A
B
13.14
Are
the
slides
clearly
labeled
so
they
can
be
associated
with
the
correct
sample?
Requirement
13.15
What
type
of
slides
are
used?

13.16
Is
slide
dried
at
a)
room
temperature
or
b)
35
to
42
C?
[
Section
13.3.3.12]
Circle
one:
A
B
13.17
If
the
slide
is
warmed,
is
incubator
or
slide
tray
calibrated
and
labeled?
Requirement
13.18
Does
the
laboratory
have
an
acceptable
SOP
for
sample
purification?
Requirement
13.19
Other
than
the
issues
noted
in
items
13.1
through
13.18
(
if
any)
were
sample
purification
and
slide
preparation
performed
successfully?

14
Sample
staining
Technician:

14.1
What
staining
kit/
manufacturer
is
used?
[
Section
14.3]

14.2
Is
FITC
stain
applied
according
to
manufacturer's
directions?
[
Section
14.1]
Method
Procedure
14.3
Are
positive
and
negative
staining
controls
performed?
Requirement
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
Draft
June
2003
13
14.4
Are
the
slides
incubated
in
a
humid
chamber
in
the
dark
at
room
temperature
for
approximately
30
minutes
or
per
manufacturer's
directions?
[
Section
14.4]
Method
Procedure
14.5
Are
the
labeling
reagents
rinsed
away
properly
after
incubation,
without
disturbing
the
sample?

[
Section
14.5]
Method
Procedure
14.6
Was
the
working
DAPI
stain
prepared
the
day
it
was
used?
[
Section
7.7.2]
Method
Procedure
14.7
Is
stock
DAPI
stored
at
0
to
8oC
in
the
dark?
[
Section
7.7.2]
Method
Procedure
14.8
Is
the
DAPI
stain
applied
properly
and
allowed
to
stand
for
a
minimum
of
1
minute?
[
Section
14.6]
Method
Procedure
14.9
Is
the
DAPI
stain
rinsed
away
properly
without
disturbing
the
sample?
[
Section
14.7]
Method
Procedure
14.10
Is
the
mounting
media
applied
properly?
Method
Procedure
14.10.1
What
type
of
mounting
media
is
used?

14.10.2
Are
all
the
edges
of
the
cover
slip
sealed
well
with
clear
fingernail
polish,
unless
Elvenol
is
used?
[
Section
14.9]
Method
Procedure
14.11
Are
the
finished
slides
stored
in
a
humid
chamber
in
the
dark
at
0
to
8oC
(
humid
chamber
not
required
for
Evenol)?
[
Section
14.10]
Method
Procedure
14.12
Does
the
laboratory
have
an
acceptable
SOP
for
sample
staining?
Requirement
14.13
Other
than
the
issues
noted
in
items
14.2
through
14.13
(
if
any)
was
sample
staining
demonstrated
successfully?

15
Microscope
and
Examination
15.1
Is
microscope
equipped
with
appropriate
excitation
and
band
pass
filters
for
examining
FITC
labeled
specimens?
(
Exciter
filter
­
450­
490
nm,
dichroic
beam­
splitting
mirror
­
510
nm,
barrier
or
suppression
filter:
515­
520
nm)?
[
Section
6.9.2]
Requirement
15.2
Is
microscope
is
equipped
with
appropriate
excitation
and
band
pass
filters
for
examining
DAPI
labeled
specimens?
(
Exciter
filter
­
340­
380
nm,
dichroic
beam­
splitting
mirror
­
400
nm,
barrier
or
suppression
filter
­
420
nm)
[
Section
6.9.3]
Requirement
15.3
Does
the
microscope
have
HMO
or
DIC,
objectives?
[
Section
6.9.1]
Requirement
15.4
Is
microscope
operation
easily
changed
from
epifluorescence
to
DIC/
HMO?
Recommendation
15.5
Does
the
microscope
have
a
20
X
scanning
objective?
[
Section
6.9.1]
Requirement
15.6
Does
the
microscope
have
a
100
X
oil
immersion
objective?
[
Section
6.9.1]
Requirement
15.7
Is
the
microscope
equipped
with
an
ocular
micrometer?
[
Section
6.9.1]
Requirement
15.8
Is
a
stage
micrometer
available
to
laboratory?
[
Section
10.3.5]
Requirement
15.9
Is
a
calibration
table
for
each
objective
located
close
to
the
microscope(
s)?
[
Section
10.3.5]
Requirement
15.10
Has
the
mercury
bulb
been
used
less
than
the
maximum
hours
recommended
by
the
manufacturer?
[
Section
10.3.2.11]
Recommendation
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
Draft
June
2003
14
15.11
Does
the
positive
control
contain
Cryptosporidium
oocysts
at
the
appropriate
fluorescence
intensity
for
both
FITC
and
DAPI?
Requirement
15.12
Does
the
laboratory
have
an
acceptable
SOP
for
sample
examination?
Requirement
15.13
Other
than
the
issues
noted
for
items
15.1
through
15.13
(
if
any)
were
other
microscope
or
examination
issues
acceptable?

Note:
All
section
references
in
[
]
refer
to
Method
1623
April
2001
Draft
15
16
Evaluation
of
sample
processing
Method
Step
(
filtration,
elution,
concentration,
purification
or
staining)
Name
Position
Demonstrated
Technique
Successfully
yes/
no
Recorded
data
as
sample
was
analyzed
yes/
no
Draft
June
2003
16
17
Was
analyst
microscope
examination
acceptable?
(
yes/
no)

Classification
Requirement
Requirement
Requirement
Requirement
Requirement
Requirement
Requirement
Requirement
Recommendation
Name
Position
Adjust
Interpupillary
Distance
Focus
both
eyepieces
Establish
Kohler
Illumination
Examine
and
Record
Characteristics
of
Three
Oocysts
Crypto
Count
Measurement
with
100X
objective
Demonstrate
d
Internal
Structures
Examines
Slides
<
4
hrs
per
day
Comments: