Document ID: EPA-HQ-OW-2002-0039-0083
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-07-09T04:00Z

OMB
Control
Number:
2040­
0246
Expiration
Date:
07/
31/
02
United
States
Environmental
Protection
Agency
Office
of
Ground
Water
and
Drinking
Water
Technical
Support
Center
April
29,
2002
Dear
Laboratory
Manager:

Thank
you
for
your
interest
in
the
U.
S.
EPA 
s
Laboratory
Quality
Assurance
Evaluation
Program
for
Analysis
of
Cryptosporidium
under
the
Safe
Drinking
Water
Act
(
Lab
QA
Program).
This
is
a
voluntary
program
open
to
laboratories
analyzing
Cryptosporidium
in
water
using
EPA
Method
1622
and
EPA
Method
1623.
To
increase
the
likelihood
that
laboratories
analyzing
water
samples
for
Cryptosporidium
generate
reliable
data,
EPA
has
established
the
following
process
for
evaluating
laboratory
performance
and
quality
assurance
practices:

Step
1.
Application.
Laboratories
must
first
submit
the
Laboratory
QA
Program
application.
The
application
forms
are
enclosed
with
this
letter,
and
the
application
requirements
are
described
in
detail
below.
EPA
will
evaluate
laboratory
applications
to
confirm
the
following:
(
1)
the
laboratory
has
the
equipment
required
in
EPA
Method
1622
and/
or
EPA
Method
1623
(
April
2001),
(
2)
laboratory
personnel
have
the
recommended
experience
to
analyze
samples,
and
(
3)
the
laboratory
has
successfully
completed
the
initial
precision
and
recovery
(
IPR)
and
matrix
spike/
matrix
spike
duplicate
(
MS/
MSD)
tests
specified
in
the
method.
Laboratories
that
do
not
meet
these
requirements
will
be
requested
to
correct
any
deficiencies
before
proceeding
to
the
next
step
in
the
evaluation
process.

Step
2.
Performance
evaluation.
After
an
application
has
been
accepted,
the
laboratory
will
be
sent
a
set
of
eight
initial
proficiency
testing
(
IPT)
samples
consisting
of
a
suspension
of
oocysts
in
a
concentrated
matrix.
Laboratories
will
resuspend
these
spikes
in
reagent
water
to
produce
simulated
source
water
samples,
and
analyze
the
samples
using
the
version
of
Method
1622/
1623
(
April
2001)
that
the
laboratory
plans
to
use
for
routine
Cryptosporidium
analyses.
If
a
laboratory
wishes
to
be
evaluated
for
more
than
one
version
of
the
method,
the
laboratory
will
receive
a
set
of
eight
PT
samples
for
each
version.

Laboratory
IPT
data
will
be
evaluated
against
the
mean
recovery
and
precision
(
as
relative
standard
deviation)
criteria
for
the
IPT
samples.
Laboratories
will
receive
two
opportunities
to
pass
the
IPT
test.
If
a
laboratory
fails
two
times,
it
will
not
be
eligible
for
another
set
until
after
the
laboratory
staff
has
received
additional
training
in
performing
the
method.

Laboratories
already
participating
in
the
EPA
Protozoa
Performance
Evaluation
(
PE)
Program,
may
use
the
initial
round
of
samples
from
the
PE
program
to
meet
the
IPT
sample
requirement.

Step
3.
On­
site
evaluation.
After
a
laboratory
passes
the
IPT,
an
on­
site
evaluation
of
the
laboratory
will
be
scheduled.
The
on­
site
evaluation
will
include
two
separate,
but
concurrent
assessments:
(
1)
assessment
of
the
laboratory 
s
sample
processing
and
analysis
procedures,
including
microscopic
examination,
and
(
2)
evaluation
of
the
laboratory 
s
personnel
qualifications,
quality
control
program,
equipment,
and
record
keeping
procedures.
Each
laboratory
will
receive
an
audit
report,
which
will
document
deficiencies,
if
any,
that
should
be
corrected
by
the
laboratory.
After
a
laboratory
has
corrected
any
deficiencies
noted
in
the
audit
report,
EPA
will
confirm
that
the
laboratory
meets
the
performance
criteria
of
the
Laboratory
Quality
Assurance
Program
for
Analysis
of
Cryptosporidium
under
the
Safe
Drinking
Water
Act.

Laboratories
that
meet
the
program
performance
criteria
will
also
receive
a
set
of
three
ongoing
proficiency
testing
(
OPT)
samples
approximately
every
four
months
that
must
be
analyzed
in
the
same
manner
as
the
IPT
samples.
EPA
will
evaluate
the
precision
and
recovery
data
for
OPT
samples
to
determine
if
the
laboratory
continues
to
meet
the
performance
criteria
of
the
Laboratory
QA
Program.

Application
Requirements
The
first
step
in
the
laboratory
evaluation
process
is
submission
of
a
laboratory
application
package.
The
following
materials
should
be
submitted
for
each
laboratory
application
package:

1.
Signed,
completed
application
form
(
attached).

2.
Completed
self­
audit
checklist
(
attached).
This
checklist
is
similar
to
the
checklist
that
will
be
used
to
audit
your
laboratory
during
the
on­
site
evaluation.

3.
Resumes
detailing
qualifications
of
your
laboratory 
s
proposed
principal
analyst/
supervisor
and
each
analyst
and
technician
listed
on
the
application
form
and
documentation
of
the
training,
including
the
number
of
samples
analyzed
by
each
(
the
list
for
each
analyst
and
technician
should
include
at
a
minimum
the
number
of
samples
specified
below
for
personnel
prerequisites).

The
recommended
personnel
prerequisites
for
the
laboratory
evaluation
program
are
as
follows:

Principal
Analyst/
Supervisor
(
one
required
per
laboratory)
 
BS/
BA
in
microbiology
or
closely
related
field
 
A
minimum
of
1
year
of
continuous
bench
experience
with
Cryptosporidium
and
IFA
microscopy
 
A
minimum
of
6
months
experience
using
EPA
Method
1622
and/
or
EPA
Method
1623
 
A
minimum
of
100
samples
analyzed
using
EPA
Method
1622
and/
or
EPA
Method
1623
(
minimum
50
samples
if
the
person
was
an
approved
analyst
for
Cryptosporidium
under
the
Information
Collection
Rule(
ICR))

Other
Analysts
(
no
minimum
requirement
per
laboratory)
 
Two
years
of
college
in
microbiology
or
equivalent
or
closely
related
field
 
A
minimum
of
6
months
of
continuous
bench
experience
with
Cryptosporidium
and
IFA
microscopy
 
A
minimum
of
3
months
experience
using
EPA
Method
1622
and/
or
EPA
Method
1623
 
A
minimum
of
50
samples
analyzed
using
EPA
Method
1622
and/
or
EPA
Method
1623
(
minimum
25
samples
if
the
person
was
an
ICR­
approved
analyst)

Technician
(
no
minimum
requirement
per
laboratory)
 
Three
months
experience
with
the
specific
parts
of
the
procedure
he/
she
will
be
performing
 
A
minimum
of
50
samples
analyzed
using
EPA
Method
1622
and/
or
EPA
Method
1623
(
minimum
25
samples
if
the
person
was
an
ICR­
approved
technician)
for
the
specific
analytical
procedures
they
will
be
using.
4.
Detailed
laboratory
standard
operating
procedures
for
each
version
of
the
method
your
laboratory
plans
on
using
for
routine
Cryptosporidium
analyses.
SOP 
s
for
the
following
should
be
included:

 
Performance
of
each
method
step
including,
sample
spiking,
filtration,
elution,
concentration,
purification,
slide
preparation,
sample
staining
and
examination
 
Dividing
pellets
greater
than
0.5mL
 
Preparation
of
reagents
 
Dishwashing
 
Staff
training
 
Corrective
action
procedures
for
failing
to
meet
OPR,
method
blank,
staining
controls,
sample
acceptance,
and
performance
verification
criteria
 
Sampling
procedures
to
be
followed
by
field
or
utility
personnel
 
Procedures
for
data
recording,
checking
manual
calculations,
and
checking
accuracy
of
all
data
transcriptions
5.
EPA
Method
1622
or
EPA
Method
1623
initial
demonstration
of
capability
(
IDC)
data
which
include
initial
precision
and
recovery
(
IPR)
test
results
and
matrix
spike
and
matrix
spike
duplicate
(
MS/
MSD)
test
results
for
Cryptosporidium.
The
IPR
test
consists
of
four
reagent
water
samples
spiked
with
between
100
­
500
oocysts
and
one
method
blank.
The
MS/
MSD
test
consists
of
one
unspiked
and
two
spiked
source
water
samples.
These
tests
are
described
in
Section
9
of
EPA
Method
1622
and
EPA
Method
1623
and
the
results
should
meet
the
criteria
in
the
method
(
April
2001
version).
The
following
data
should
be
submitted:

 
Completed
EPA
Method
1622/
1623
bench
sheets
and
report
forms
for
each
of
the
eight
samples
(
attached)
 
Initial
demonstration
of
capability
summary
form
(
attached)
 
Spiking
suspension
preparation
data.
This
should
include
completed
spiking
suspension
sheets
(
attached)
if
spikes
were
prepared
by
your
laboratory
or
flow­
cytometer
calibration
forms
if
spikes
were
obtained
from
an
external
source.

Laboratories
wishing
to
be
evaluated
for
more
than
one
version
of
the
method
(
different
volumes,
filters,
elution
and
concentration
procedures,
and
immunomagnetic
separation
kits)
should
submit
a
complete
set
of
IPR
and
MS
data
for
each
version.

If
your
laboratory
currently
participates
in
the
EPA
PT
sample
program
and
the
required
IDC
data
have
already
been
submitted,
the
data
do
not
need
to
be
resubmitted.
Please
indicate
this
is
the
case
on
the
initial
demonstration
of
capability
summary
form.

6.
Table
of
contents
from
your
laboratory 
s
quality
assurance
plan.
The
quality
assurance
plan
should
specifically
address
the
requirements
of
protozoa
analysis
under
the
programs
for
which
the
laboratory
intends
to
analyze
samples.

7.
An
example
of
the
data
reporting
form
used
to
submit
Cryptosporidium
results
to
your
clients.

8.
A
statistical
summary
of
percent
recoveries
for
all
OPR
and
MS
samples
analyzed
at
your
laboratory
for
the
past
six
months.

Application
materials
should
be
submitted
to
the
following
address:
Jennifer
Scheller
Cryptosporidium
Laboratory
QA
Program
Coordinator
DynCorp
Biology
Studies
Group
6101
Stevenson
Avenue
Alexandria,
VA
22304
Send
comments
on
the
Agency's
need
for
this
information,
the
accuracy
of
the
provided
burden
estimates,
and
any
suggested
methods
for
minimizing
respondent
burden,
including
through
the
use
of
automated
collection
techniques
to
the
Director,
Collection
Strategies
Division,
U.
S.
Environmental
Protection
Agency
(
2822T),
1200
Pennsylvania
Ave.,
NW,
Washington,
D.
C.
20460.
Include
the
OMB
control
number
in
any
correspondence.
Do
not
send
the
completed
form
to
this
address.

When
your
application
package
has
been
received
and
reviewed,
you
will
be
notified
whether
it
is
complete
or
has
any
deficiencies.
After
your
application
has
been
accepted,
you
will
be
notified
of
when
you
should
expect
your
initial
set
of
PT
samples.
If
you
have
any
questions
about
the
laboratory
application
materials
or
evaluation
process,
please
feel
free
to
contact
either
me
at
513­
569­
7944
or
Jennifer
Scheller
at
703­
461­
2118.

Sincerely,

Mary
Ann
Feige
Cryptosporidium
Laboratory
Quality
Assurance
Evaluation
Program
Manager
26
West
Martin
Luther
King
Drive
Cincinnati,
OH
45268
Attachments
Burden
Statement:
The
public
reporting
and
recordkeeping
burden
for
this
collection
of
information
is
estimated
to
average
18
hours
per
response
or
72
hours
per
respondent
annually.
Burden
means
the
total
time,
effort,
or
financial
resources
expended
by
persons
to
generate,
maintain,
retain,
or
disclose
or
provide
information
to
or
for
a
Federal
agency.
This
includes
the
time
needed
to
review
instructions;
develop,
acquire,
install,
and
utilize
technology
and
systems
for
the
purposes
of
collecting,
validating,
and
verifying
information,
processing
and
maintaining
information,
and
disclosing
and
providing
information;
adjust
the
existing
ways
to
comply
with
any
previously
applicable
instructions
and
requirements;
train
personnel
to
be
able
to
respond
to
a
collection
of
information;
search
data
sources;
complete
and
review
the
collection
of
information;
and
transmit
or
otherwise
disclose
the
information.
An
agency
may
not
conduct
or
sponsor,
and
a
person
is
not
required
to
respond
to,
a
collection
of
information
unless
it
displays
a
currently
valid
OMB
control
number.
OMB
Control
Number:
2040­
0246
Expiration
Date:
07/
31/
02
Application
for
the
Laboratory
Quality
Assurance
Evaluation
Program
for
Analysis
of
Cryptosporidium
under
the
Safe
Drinking
Water
Act
Part
1.
Laboratory
Information
Laboratory
Name:

Address:

City:
State:
Zip:

Contact
Person:

Title:

Telephone:
Fax:

Email
address:

Type
of
laboratory
(
circle
one):
Commercial
Utility
State
Academic
Other
Was
your
laboratory
ICR­
approved
for
protozoa?
9
Yes
9
No
Is
your
laboratory
currently
participating
in
the
EPA
PE
Program?
9
Yes
9
No
Number
of
field
samples
analyzed
by
your
laboratory
using
Method
1622/
1623:

Number
of
spiked
samples
analyzed
by
your
laboratory
using
Method
1622/
23:

Number
of
fields
samples
your
laboratory
can
currently
analyze
per
month
using
Method
1622/
1623:
Number
of
field
samples
your
laboratory
could
potentially
analyze
per
month
using
Method
1622/
1623
during
LT2:

1
April
30,
2002
Part
2.
Method
Information:
Versions
of
Method
1622/
1623
for
which
the
lab
is
seeking
evaluation
Method
step
Method
1622
(
Cryptosporidium
only)
Method
1623
(
Cryptosporidium
&
Giardia)

Filtration
(
check
all
that
apply
and
indicate
the
volume
filtered
for
each)

Gelman
Envirochek
Gelman
HV
Envirochek
IDEXX
FiltaMax
Whatman
CrypTest
Other
(
describe)

Elution
(
check
all
that
apply)

Wrist
action
shaker
(
Envirochek)

Stomaching
of
FiltaMax
filter
FiltaMax
wash
station
plunger
Back
Wash/
Sonication
(
CrypTest)

Other
(
describe)

Concentration
(
check
all
that
apply)

Centrifugation
Filtration
through
membrane
Other
(
describe)

Purification
(
check
all
that
apply)

Dynal
anti­
Crypto,
Dynal
GC­
combo
Other
(
describe)

Staining
(
check
all
that
apply)

Waterborne
AquaGlo
Waterborne
Crypt­
a­
Glo
Waterborne
Giardi­
a­
Glo
Meridian
Merifluor
Other
(
describe)

Descriptions
of
 
other 
method
steps
and
other
comments:

2
April
30,
2002
Part
3.
Personnel
Information
(
attach
additional
sheets
if
necessary)
1.
Principal
Analyst/
Supervisor
:
one
required
per
approved
laboratory
Name
Current
position
Academic
training/
degree(
s)
Time
in
current
position
No.
of
samples
processed
for
protozoa
analyses
No.
of
samples
processed
using
Methods
1622/
1623
Was
this
person
approved
as
an
analyst
under
the
ICR?
9
Yes
9
No
Portions
of
method
currently
performed
(
circle
all
that
apply):
Filtration
Elution
Concentration
IMS
Staining
Examination
2.
Analyst
or
Technician
(
circle
one)

Name
Current
position
Academic
training/
degree(
s)
Time
in
current
position
No.
of
samples
processed
for
protozoa
analyses
No.
of
samples
processed
using
Methods
1622/
1623
Was
this
person
approved
under
the
ICR?
9
Yes
9
No
If
yes
then
check
one:
9
Analyst
9
Technician
Portions
of
method
currently
performed
(
circle
all
that
apply):
Filtration
Elution
Concentration
IMS
Staining
Examination
3.
Analyst
or
Technician
(
circle
one)

Name
Current
position
Academic
training/
degree(
s)
Time
in
current
position
No.
of
samples
processed
for
protozoa
analyses
No.
of
samples
processed
using
Methods
1622/
1623
Was
this
person
approved
under
the
ICR?
9
Yes
9
No
If
yes
then
check
one:
9
Analyst
9
Technician
Portions
of
method
currently
performed
(
circle
all
that
apply):
Filtration
Elution
Concentration
IMS
Staining
Examination
4.
Analyst
or
Technician
(
circle
one)

Name
Current
position
Academic
training/
degree(
s)
Time
in
current
position
No.
of
samples
processed
for
protozoa
analyses
No.
of
samples
processed
using
Methods
1622/
1623
Was
this
person
approved
under
the
ICR?
9
Yes
9
No
If
yes
then
check
one:
9
Analyst
9
Technician
Portions
of
method
currently
performed
(
circle
all
that
apply):
Filtration
Elution
Concentration
IMS
Staining
Examination
5.
Analyst
or
Technician
(
circle
one)

Name
Current
position
Academic
training/
degree(
s)
Time
in
current
position
No.
of
samples
processed
for
protozoa
analyses
No.
of
samples
processed
using
Methods
1622/
1623
Was
this
person
approved
under
the
ICR?
9
Yes
9
No
If
yes
then
check
one:
9
Analyst
9
Technician
Portions
of
method
currently
performed
(
circle
all
that
apply):
Filtration
Elution
Concentration
IMS
Staining
Examination
3
April
30,
2002
Part
4.
Laboratory
Equipment
Confirmation
Checklist
for
Methods
1622
and
1623
Key
Equipment
and
Reagents
Manufacturer/
Model
If
not
Present,
Proof
of
Purchase
Attached
(
Y/
N)

Filtration
and
elution
Flow
control
valve
­
0.5
gpm
Centrifugal
or
other
pump
Low­
flow
meter
or
graduated
container
Laboratory
shaker
for
agitating
capsule
filters
(
Envirochek
only)

Laboratory
shaker
side
arms
(
Envirochek
only)

Filter
housing
(
CrypTest
or
Filta­
Max)

Wash
station
(
Filta­
Max
only)

Stomacher
(
Filta­
Max
only)

Compressed
air
source
(
CrypTest
only)

Sonicator
(
CrypTest
only)

Concentration
Concentrator
apparatus
(
Filta­
Max
only)

1500
X
G,
swinging­
bucket
centrifuge
for
15
mL
­
250­
mL
tubes
Immunomagnetic
separation
Sample
mixer/
rotator
for
10­
mL
tubes
Magnetic
particle
concentrator
for
10­
mL
tubes
Magnetic
particle
concentrator
for
1.5­
mL
tubes
Flat­
sided
sample
tubes
Examination
Epifluorescence/
differential
interference
contrast
microscope
with
stage
and
ocular
micrometers
and
20X
to
100X
objectives
Excitation/
band
pass
microscope
filters
for
fluorescein
isothiocyanate
(
FITC)
assay
Excitation/
band­
pass
filters
for
4',
6­
diamidino­
2­
phenylindole
(
DAPI)
assay
The
above
application
information
is
complete
and
accurate
to
the
best
of
my
knowledge.

Name
and
Signature
Laboratory
Manager
or
Designee
Date
Submit
application
package
to:
Jennifer
Scheller,
Cryptosporidium
Laboratory
Quality
Assurance
Evaluation
Program
Coordinator,
DynCorp
I&
ET,
6101
Stevenson
Avenue,
Suite
500,
Alexandria,
VA
22304
4
April
30,
2002
Audit
Checklist
(
To
Be
Used
for
Self­
Audit)

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
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:

1.7.1
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
5
April
29,
2002
Item
to
be
Evaluated
Classification
Yes,
No,
Unknown*,
or
NA
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
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]

6
April
29,
2002
Item
to
be
Evaluated
Classification
Yes,
No,
Unknown*,
or
NA
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
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
7
April
29,
2002
Item
to
be
Evaluated
Classification
Yes,
No,
Unknown*,
or
NA
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
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
8
April
29,
2002
Part
B:
Sample
Processing
and
Examination
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)
well­
slide­
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
meter
or
calibrated
carboy?
[
Section
12.2.4.2]
Requirement
8.1.6
Is
the
system
well
maintained
and
cleaned
appropriately
following
use?
Requirement
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
April
29,
2002
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
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
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
April
29,
2002
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
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
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
Filta­
max
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?

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
April
29,
2002
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
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
Is
the
centrifugation
nomograph
for
determining
relative
centrifugal
force
located
close
to
the
centrifuge(
s)?
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
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
leighton
tube
rotated
at
18
rpm
for
1
hour
at
room
temperature?
Method
Procedure
13.7
Is
the
resuspended
pellet
volume
quantitatively
transferred
to
the
Leighton
tube
(
2
rinses)?
[
Section
13.3.2.1]
Method
Procedure
13.8
Are
the
IMS
beads
thoroughly
resuspended
prior
to
addition
to
the
Leighton
tube?
[
Section
13.3.2.2]
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
12
April
29,
2002
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
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?

14.2
Is
FITC
stain
applied
according
to
manufacturer 
s
directions?
Method
Procedure
14.3
Are
positive
and
negative
staining
controls
performed?
Requirement
14.4
Are
the
direct
labeling
reagents
applied
properly?
[
Section
15.2.1]
Method
Procedure
14.5
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
14.6
Are
the
labeling
reagents
rinsed
away
properly
after
incubation,
without
disturbing
the
sample?
[
Section
14.5]
Method
Procedure
14.7
Was
the
working
DAPI
stain
prepared
the
day
it
was
used?
[
Section
7.7.2]
Method
Procedure
14.8
Is
stock
DAPI
stored
at
0
to
8oC
in
the
dark?
[
Section
7.7.2]
Method
Procedure
14.9
Is
the
DAPI
stain
applied
properly
and
allowed
to
stand
for
a
minimum
of
1
minute?
[
Section
14.6]
Method
Procedure
14.10
Is
the
DAPI
stain
rinsed
away
properly
without
disturbing
the
sample?
[
Section
14.7]
Method
Procedure
14.11
Is
the
mounting
media
applied
properly?
Method
Procedure
14.11.1
What
type
of
mounting
media
is
used?

14.11.2
Are
all
the
edges
of
the
cover
slip
sealed
well
with
clear
fingernail
polish,
unless
Elvenol
is
used?
[
Section
14.9]
Method
Procedure
14.12
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
14.13
Does
the
laboratory
have
an
acceptable
SOP
for
sample
staining?
Requirement
14.14
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
13
April
29,
2002
Item
to
be
evaluated
Classification
Yes,
No,
NA
or
Unknown
15.10
Does
the
wattage
of
the
mercury
lamp
meet
the
microscope
specifications?
Requirement
15.11
Has
the
mercury
bulb
been
used
less
than
the
maximum
hours
recommended
by
the
manufacturer?
[
Section
10.3.2.11]
Recommendation
15.12
Does
the
positive
control
contain
Cryptosporidium
oocysts
at
the
appropriate
fluorescence
intensity
for
both
FITC
and
DIC?
Requirement
15.13
Does
the
laboratory
have
an
acceptable
SOP
for
sample
examination?
Requirement
15.14
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
14
April
29,
2002
Initial
Demonstration
of
Capability
Data
Summary
Form
Laboratory
Name
Laboratory
ID
Date
Method
Information
Which
method
was
used?
9
Method
1622
9
Method
1623
Filter
used:
Elution
method:
Concentration
method:

IMS
kit
used:
Staining
kit
used:

Volume
of
water
spiked
(
L):
Volume
of
water
filtered
(
L):

Initial
Demonstration
of
Capability
Summary
Data
Sample
Giardia
(
not
required)
Cryptosporidium
Equivalent
Sample
Volume
Analyzed
(
to
nearest
1/
4
L)
Turbidity
(
NTU)
Estimated
No.
of
Cysts
Spiked
No.
of
Cysts
Detected
Estimated
No.
of
Oocysts
Spiked
No.
of
Oocysts
Detected
Method
blank
Spiked
reagent
water
1
Spiked
reagent
water
2
Spiked
reagent
water
3
Spiked
reagent
water
4
Mean
recovery
Precision
(
RSD)

Matrix
unspiked
Matrix
spike
1
Matrix
spike
2
Mean
recovery
Precision
(
RPD)

15
April
29,
2002
Burden
Statement:
The
public
reporting
and
recordkeeping
burden
for
this
collection
of
information
is
estimated
to
average
18
hours
per
response
or
72
hours
per
respondent
annually.
Burden
means
the
total
time,
effort,
or
financial
resources
expended
by
persons
to
generate,
maintain,
retain,
or
disclose
or
provide
information
to
or
for
a
Federal
agency.
This
includes
the
time
needed
to
review
instructions;
develop,
acquire,
install,
and
utilize
technology
and
systems
for
the
purposes
of
collecting,
validating,
and
verifying
information,
processing
and
maintaining
information,
and
disclosing
and
providing
information;
adjust
the
existing
ways
to
comply
with
any
previously
applicable
instructions
and
requirements;
train
personnel
to
be
able
to
respond
to
a
collection
of
information;
search
data
sources;
complete
and
review
the
collection
of
information;
and
transmit
or
otherwise
disclose
the
information.
An
agency
may
not
conduct
or
sponsor,
and
a
person
is
not
required
to
respond
to,
a
collection
of
information
unless
it
displays
a
currently
valid
OMB
control
number.

16
April
29,
2002