Method
1623:
Cryptosporidium
and
Giardia
in
Water
by
Filtration/
IMS/
FA
June
2003
Draft
for
Comment
Office
of
Water
(
4607)
EPA
815­
R­
03­
XXX
http://
www.
epa.
gov/
microbes/
June
2003
Printed
on
Recycled
Paper
i
Acknowledgments
This
method
was
prepared
under
the
direction
of
William
A.
Telliard
of
the
Engineering
and
Analysis
Division
within
the
U.
S.
Environmental
Protection
Agency
(
U.
S.
EPA)
Office
of
Water.
This
document
was
prepared
by
DynCorp
under
a
U.
S.
EPA
contract,
with
assistance
from
its
subcontractor,
Interface,
Inc.

The
U.
S.
EPA
Office
of
Water
gratefully
acknowledges
the
contributions
of
the
following
persons
and
organizations
to
the
development
of
this
method:

Mike
Arrowood,
Centers
for
Disease
Control
and
Prevention,
Division
of
Parasitic
Diseases
(
MS­
F13),
4770
Buford
Highway,
N.
E.,
Atlanta,
GA
30341­
3724,
USA
Phil
Berger,
Office
of
Groundwater
and
Drinking
Water,
U.
S.
Environmental
Protection
Agency,
401
M
Street,
S.
W.,
Washington,
DC
20460,
USA
Jennifer
Clancy,
Clancy
Environmental
Consultants,
Inc.,
P.
O.
Box
314,
St.
Albans,
VT
05478,
USA
Kevin
Connell,
DynCorp,
6101
Stevenson
Avenue,
Alexandria,
VA
22314,
USA
Ricardo
DeLeon,
Metropolitan
Water
District
of
Southern
California,
700
Moreno
Avenue,
LaVerne,
CA
91760,
USA
Shirley
Dzogan,
EnviroTest
Laboratories,
745
Logan
Avenue,
Winnipeg,
Manitoba
R3E
3L5,
Canada
Mary
Ann
Feige,
Technical
Support
Center,
Office
of
Ground
Water
and
Drinking
Water,
U.
S.
Environmental
Protection
Agency,
26
W.
Martin
Luther
King
Drive,
Cincinnati,
OH
45268­
1320,
USA
Colin
Fricker,
Thames
Water
Utilities,
Manor
Farm
Road,
Reading,
Berkshire,
RG2
0JN,
England
Carrie
Hancock,
CH
Diagnostic
&
Consulting
Service,
Inc.,
214
S.
E.
Nineteenth
Street,
Loveland,
CO
80537,
USA
Stephanie
Harris,
Manchester
Laboratory,
U.
S.
Environmental
Protection
Agency,
Region
10,
7411
Beach
Drive
East,
Port
Orchard,
WA
98366,
USA
Dale
Rushneck,
Interface,
Inc.,
3194
Worthington
Avenue,
Fort
Collins,
CO
80526,
USA
Frank
Schaefer
III,
National
Exposure
Research
Laboratory,
U.
S.
Environmental
Protection
Agency,
26
W.
Martin
Luther
King
Drive,
Cincinnati,
OH
45268­
1320,
USA
Steve
Schaub,
Health
and
Ecological
Criteria
Division
(
4304),
Office
of
Science
and
Technology,
U.
S.
Environmental
Protection
Agency,
401
M
Street,
S.
W.,
Washington,
DC
20460,
USA
Ajaib
Singh,
City
of
Milwaukee
Health
Department,
841
North
Broadway,
Milwaukee,
WI
53202,
USA
Huw
Smith,
Department
of
Bacteriology,
Scottish
Parasite
Diagnostic
Laboratory,
Stobhill
NHS
Trust,
Springburn,
Glasgow,
G21
3UW,
Scotland
Timothy
Straub,
Lockheed
Martin,
7411
Beach
Drive
East,
Port
Orchard,
WA
98366,
USA
William
A.
Telliard,
Office
of
Science
and
Technology,
U.
S.
Environmental
Protection
Agency,
401
M
Street,
S.
W.,
Washington,
DC
20460,
USA
Cryptosporidium
cover
photo
courtesy
of
the
U.
S.
Centers
for
Disease
Control
Giardia
cover
photo
courtesy
of
CH
Diagnostic
&
Consulting
Service,
Inc.
ii
Disclaimer
This
method
has
been
reviewed
by
the
U.
S.
EPA
Office
of
Water
and
approved
for
publication.
Mention
of
trade
names
or
commercial
products
does
not
constitute
endorsement
or
recommendation
for
use.

Questions
regarding
this
method
or
its
application
should
be
addressed
to:

Laboratory
Quality
Assurance
Program
for
the
Analysis
of
Cryptosporidium
in
Water
Coordinator
Office
of
Ground
Water
and
Drinking
Water
Technical
Support
Center
U.
S.
EPA
Office
of
Water
26
West
Martin
Luther
King
Drive
Cincinnati,
OH
45268­
1320
William
A.
Telliard
U.
S.
EPA
Office
of
Water
Analytical
Methods
Staff
Mail
Code
4303
Washington,
DC
20460
Email:
telliard.
william@
epa.
gov
iii
Introduction
To
support
future
regulation
of
protozoa
in
drinking
water,
the
Safe
Drinking
Water
Act
Amendments
of
1996
require
the
U.
S.
Environmental
Protection
Agency
(
EPA)
to
evaluate
the
risk
to
public
health
posed
by
drinking
water
contaminants,
including
waterborne
parasites,
such
as
Cryptosporidium
and
Giardia.
To
implement
these
requirements,
EPA
must
assess
Cryptosporidium
and
Giardia
occurrence
in
raw
surface
waters
used
as
source
waters
for
drinking
water
treatment
plants.
EPA
Method
1623
was
developed
to
support
this
assessment.

Method
Development
and
Validation
EPA
initiated
an
effort
in
1996
to
identify
new
and
innovative
technologies
for
protozoan
monitoring
and
analysis.
After
evaluating
potential
alternatives
to
the
then­
current
method
through
literature
searches,
discussions
with
research
and
commercial
laboratories,
and
meetings
with
experts
in
the
field,
the
Engineering
and
Analysis
Division
within
the
Office
of
Science
and
Technology
within
EPA's
Office
of
Water
developed
draft
Method
1622
for
Cryptosporidium
detection
in
December
1996.
This
Cryptosporidium­
only
method
was
validated
through
an
interlaboratory
study
in
August
1998,
and
was
revised
as
a
final,
valid
method
for
detecting
Cryptosporidium
in
water
in
January
1999.

Although
development
of
an
acceptable
immunomagnetic
separation
system
for
Giardia
lagged
behind
development
of
an
acceptable
system
for
Cryptosporidium,
an
acceptable
system
was
identified
in
October
1998,
and
EPA
validated
a
method
for
simultaneous
detection
of
Cryptosporidium
and
Giardia
in
February
1999
and
developed
quality
control
(
QC)
acceptance
criteria
for
the
method
based
on
this
validation
study.
To
avoid
confusion
with
Method
1622,
which
already
had
been
validated
and
was
in
use
both
domestically
and
internationally
as
a
stand­
alone
Cryptosporidium­
only
detection
method,
EPA
designated
the
new
combined
procedure
EPA
Method
1623.

The
interlaboratory
validated
versions
of
Method
1622
(
January
1999;
EPA­
821­
R­
99­
001)
and
Method
1623
(
April
1999;
EPA­
821­
R­
99­
006)
were
used
to
analyze
approximately
3,000
field
and
QC
samples
during
the
Information
Collection
Rule
Supplemental
Surveys
(
ICRSS)
between
March
1999
and
February
2000.
Method
1622
was
used
to
analyze
samples
from
March
1999
to
mid­
July
1999;
Method
1623
was
used
from
mid­
July
1999
to
February
2000.
The
April
2001
revision
of
both
methods
include
updated
QC
acceptance
criteria
based
on
analysis
of
the
QC
samples
analyzed
during
the
ICRSS.

Changes
in
the
April
2001
Versions
of
the
Methods
Both
methods
were
revised
in
April
2001,
after
completion
of
the
ICRSS
and
multiple
meetings
with
researchers
and
experienced
laboratory
staff
to
discuss
potential
method
updates.
Changes
incorporated
in
the
April
2001
revisions
of
the
methods
(
EPA­
821­
R­
01­
025
and
EPA­
821­
R­
01­
026)
included
the
following:

C
Nationwide
approval
of
modified
versions
of
the
methods
using
the
following
components:
(
a)
Whatman
Nuclepore
CrypTest
 
filter
(
b)
IDEXX
Filta­
Max
 
filter
(
c)
Waterborne
Aqua­
Glo
 
G/
C
Direct
FL
antibody
stain
(
d)
Waterborne
Crypt­
a­
Glo
 
and
Giardi­
a­
Glo
 
antibody
stains
C
Clarified
sample
acceptance
criteria
C
Modified
capsule
filter
elution
procedure
C
Modified
concentrate
aspiration
procedure
iv
C
Modified
IMS
acid
dissociation
procedure
C
Updated
QC
acceptance
criteria
for
IPR
and
OPR
tests
C
Addition
of
a
troubleshooting
section
for
QC
failures
C
Modified
holding
times
C
Inclusion
of
flow
cytometry
 
sorted
spiking
suspensions
Changes
in
the
June
2003
Versions
of
the
Methods
Both
methods
were
revised
again
in
June
2003
to
support
proposal
of
EPA's
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule.
Changes
incorporated
into
the
December
2002
versions
include:

°
Nationwide
approval
of
a
modified
version
of
the
methods
using
the
Pall
Gelman
Envirochek
 
HV
filter
°
Removal
of
Whatman
Nuclepore
CrypTest
 
filter
from
the
methods
as
a
result
of
discontinuation
of
the
product
by
the
manufacturer
°
Nationwide
approval
of
the
use
of
BTF
EasySeed
 
irradiated
oocysts
and
cysts
for
use
in
routine
quality
control
(
QC)
samples
°
Minor
clarifications
and
corrections
°
Rejection
criteria
for
sample
condition
upon
receipt
°
Guidance
on
measuring
sample
temperatures
°
Clarification
of
QC
sample
requirements
and
use
of
QC
sample
results
°
Guidance
on
minimizing
carry­
over
debris
onto
microscope
slides
after
IMS
Performance­
Based
Method
Concept
and
Modifications
Approved
for
Nationwide
Use
EPA
Method
1623
is
a
performance­
based
method
applicable
to
the
determination
of
Cryptosporidium
and
Giardia
in
aqueous
matrices.
EPA
Method
1623
requires
filtration,
immunomagnetic
separation
of
the
oocysts
and
cysts
from
the
material
captured,
and
enumeration
of
the
target
organisms
based
on
the
results
of
immunofluorescence
assay,
4',
6­
diamidino­
2­
phenylindole
(
DAPI)
staining
results,
and
differential
interference
contrast
microscopy.

The
interlaboratory
validation
of
EPA
Method
1623
conducted
by
EPA
used
the
Pall
Gelman
capsule
filtration
procedure,
Dynal
immunomagnetic
separation
(
IMS)
procedure,
and
Meridian
sample
staining
procedure
described
in
this
document.
Alternate
procedures
are
allowed,
provided
that
required
quality
control
tests
are
performed
and
all
quality
control
acceptance
criteria
in
this
method
are
met.

Since
the
interlaboratory
validation
of
EPA
Method
1623,
interlaboratory
validation
studies
have
been
performed
to
demonstrate
the
equivalency
of
modified
versions
of
the
method
using
the
following
components:
°
Whatman
Nuclepore
CryptTest
 
filter
(
no
longer
available)
°
IDEXX
Filta­
Max
 
filter
°
Pall
Gelman
Envirochek
 
HV
filter
°
Waterborne
Aqua­
Glo
 
G/
C
Direct
FL
antibody
stain
°
Waterborne
Crypt­
a­
Glo
 
and
Giardi­
a­
Glo
 
antibody
stains
°
BTF
EasySeed
 
irradiated
oocysts
and
cysts
for
use
in
routine
QC
samples
v
The
validation
studies
for
these
modified
versions
of
the
method
met
EPA's
performance­
based
measurement
system
Tier
2
validation
for
nationwide
use
(
see
Section
9.1.2
for
details),
and
have
been
accepted
by
EPA
as
equivalent
in
performance
to
the
original
version
of
the
method
validated
by
EPA.
The
equipment
and
reagents
used
in
these
modified
versions
of
the
method
are
noted
in
Sections
6
and
7
of
the
method;
the
procedures
for
using
these
equipment
and
reagent
options
are
available
from
the
manufacturers.

Because
this
is
a
performance­
based
method,
other
alternative
components
not
listed
in
the
method
may
be
available
for
evaluation
and
use
by
the
laboratory.
Confirming
the
acceptable
performance
of
a
modified
version
of
the
method
using
alternate
components
in
a
single
laboratory
does
not
require
that
an
interlaboratory
validation
study
be
conducted.
However,
method
modifications
validated
only
in
a
single
laboratory
have
not
undergone
sufficient
testing
to
merit
inclusion
in
the
method.
Only
those
modified
versions
of
the
method
that
have
been
demonstrated
as
equivalent
at
multiple
laboratories
on
multiple
water
sources
through
a
Tier
2
interlaboratory
study
will
be
cited
in
the
method.
vi
Table
of
Contents
1.0
Scope
and
Application
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1
2.0
Summary
of
Method
.
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1
3.0
Definitions
.
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2
4.0
Contamination,
Interferences,
and
Organism
Degradation
.
.
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2
5.0
Safety
.
.
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.
3
6.0
Equipment
and
Supplies
.
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3
7.0
Reagents
and
Standards
.
.
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.
.
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.
8
8.0
Sample
Collection
and
Storage
.
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10
9.0
Quality
Control
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13
10.0
Microscope
Calibration
and
Analyst
Verification
.
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.
20
11.0
Oocyst
and
Cyst
Suspension
Enumeration
and
Spiking
.
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26
12.0
Sample
Filtration
and
Elution
.
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35
13.0
Sample
Concentration
and
Separation
(
Purification)
.
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44
14.0
Sample
Staining
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49
15.0
Examination
.
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50
16.0
Analysis
of
Complex
Samples
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53
17.0
Method
Performance
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53
18.0
Pollution
Prevention
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53
19.0
Waste
Management
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53
20.0
References
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54
21.0
Tables
and
Figures
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55
22.0
Glossary
of
Definitions
and
Purposes
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63
June
2003
1
Method
1623:
Cryptosporidium
and
Giardia
in
Water
by
Filtration/
IMS/
FA
1.0
Scope
and
Application
1.1
This
method
is
for
determination
of
the
identity
and
concentration
of
Cryptosporidium
(
CAS
Registry
number
137259­
50­
8)
and
Giardia
(
CAS
Registry
number
137259­
49­
5)
in
water
by
filtration
concentration,
immunomagnetic
separation
(
IMS),
and
immunofluorescence
assay
(
FA)
microscopy.
Cryptosporidium
and
Giardia
may
be
confirmed
using
4',
6­
diamidino­
2­
phenylindole
(
DAPI)
staining
and
differential
interference
contrast
(
DIC)
microscopy.
The
method
has
been
validated
in
surface
water,
but
may
be
used
in
other
waters,
provided
the
laboratory
demonstrates
that
the
method's
performance
acceptance
criteria
are
met.

1.2
This
method
is
designed
to
meet
the
survey
and
monitoring
requirements
of
the
U.
S.
Environmental
Protection
Agency
(
EPA).
It
is
based
on
laboratory
testing
of
recommendations
by
a
panel
of
experts
convened
by
EPA.
The
panel
was
charged
with
recommending
an
improved
protocol
for
recovery
and
detection
of
protozoa
that
could
be
tested
and
implemented
with
minimal
additional
research.

1.3
This
method
will
not
identify
the
species
of
Cryptosporidium
or
Giardia
or
the
host
species
of
origin,
nor
can
it
determine
the
viability
or
infectivity
of
detected
oocysts
and
cysts.

1.4
This
method
is
for
use
only
by
persons
experienced
in
the
determination
of
Cryptosporidium
and
Giardia
by
filtration,
IMS,
and
FA.
Experienced
persons
are
defined
in
Section
22.2
as
analysts
(
and
principal
analysts,
in
applicable
programs).
Laboratories
unfamiliar
with
analyses
of
environmental
samples
by
the
techniques
in
this
method
should
gain
experience
using
water
filtration
techniques,
IMS,
fluorescent
antibody
staining
with
monoclonal
antibodies,
and
microscopic
examination
of
biological
particulates
using
bright­
field
and
DIC
microscopy.

1.5
Any
modification
of
the
method
beyond
those
expressly
permitted
is
subject
to
the
application
and
approval
of
alternative
test
procedures
under
40
CFR
Part
141.27.

2.0
Summary
of
Method
2.1
A
water
sample
is
filtered
and
the
oocysts,
cysts,
and
extraneous
materials
are
retained
on
the
filter.
Although
EPA
has
only
validated
the
method
using
laboratory
filtration
of
bulk
water
samples
shipped
from
the
field,
field­
filtration
also
can
may
be
used.

2.2
Elution
and
separation
2.2.1
Materials
on
the
filter
are
eluted
and
the
eluate
is
centrifuged
to
pellet
the
oocysts
and
cysts,
and
the
supernatant
fluid
is
aspirated.

2.2.2
The
oocysts
and
cysts
are
magnetized
by
attachment
of
magnetic
beads
conjugated
to
anti­
Cryptosporidium
and
anti­
Giardia
antibodies.
The
magnetized
oocysts
and
cysts
are
separated
from
the
extraneous
materials
using
a
magnet,
and
the
extraneous
materials
are
discarded.
The
magnetic
bead
complex
is
then
detached
from
the
oocysts
and
cysts.

2.3
Enumeration
2.3.1
The
oocysts
and
cysts
are
stained
on
well
slides
with
fluorescently
labeled
monoclonal
antibodies
and
4',
6­
diamidino­
2­
phenylindole
(
DAPI).
The
stained
sample
is
examined
using
fluorescence
and
differential
interference
contrast
(
DIC)
microscopy.

2.3.2
Qualitative
analysis
is
performed
by
scanning
each
slide
well
for
objects
that
meet
the
size,
shape,
and
fluorescence
characteristics
of
Cryptosporidium
oocysts
or
Giardia
cysts.
Potential
oocysts
or
cysts
are
confirmed
through
DAPI
staining
characteristics
and
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
2
DIC
microscopy.
Oocysts
and
cysts
are
identified
when
the
size,
shape,
color,
and
morphology
agree
with
specified
criteria
and
examples
in
a
photographic
library.

2.3.3
Quantitative
analysis
is
performed
by
counting
the
total
number
of
objects
on
the
slide
confirmed
as
oocysts
or
cysts.

2.4
Quality
is
assured
through
reproducible
calibration
and
testing
of
the
filtration,
immunomagnetic
separation
(
IMS),
staining,
and
microscopy
systems.
Detailed
information
on
these
tests
is
provided
in
Section
9.0.

3.0
Definitions
3.1
Cryptosporidium
is
defined
as
a
protozoan
parasite
potentially
found
in
water
and
other
media.
The
six
species
of
Cryptosporidium
and
their
potential
hosts
are
C.
parvum
(
mammals,
including
humans);
C.
baileyi
and
C.
meleagridis
(
birds);
C.
muris
(
rodents);
C.
serpentis
(
reptiles);
and
C.
nasorum
(
fish).
Cryptosporidium
is
defined
in
this
method
as
those
determined
by
brilliant
apple
green
fluorescence
under
UV
light,
size
(
4
to
6
µ
m),
and
shape
(
round
to
oval),
excluding
atypical
organisms
specifically
identified
as
other
microbial
organisms
by
FITC
and
DIC
(
for
example,
those
possessing
spikes,
stalks,
appendages,
pores,
one
or
two
large
nuclei
filling
the
cell,
red
fluorescing
chloroplasts,
crystals,
spores,
etc.).

3.2
Giardia
is
a
protozoan
parasite
potentially
found
in
water
and
other
media.
The
two
species
of
Giardia
and
their
potential
hosts
are
G.
intestinalis
(
humans)
and
G.
muris
(
mice).
Giardia
is
defined
in
this
method
as
those
determined
by
brilliant
apple
green
fluorescence
under
UV
light,
size
(
8
to
18
µ
m
long
by
5
to
15
µ
m
wide),
and
shape
(
oval),
excluding
atypical
organisms
specifically
identified
as
other
microbial
organisms
by
FITC
and
DIC
(
for
example,
those
possessing
spikes,
stalks,
appendages,
pores,
one
or
two
large
nuclei
filling
the
cell,
red
fluorescing
chloroplasts,
crystals,
spores,
etc.).

3.3
Definitions
for
other
terms
used
in
this
method
are
given
in
the
glossary
(
Section
22.0).

4.0
Contamination,
Interferences,
and
Organism
Degradation
4.1
Turbidity
caused
by
inorganic
and
organic
debris
can
interfere
with
the
concentration,
separation,
and
examination
of
the
sample
for
Cryptosporidium
oocysts
and
Giardia
cysts.
In
addition
to
naturally­
occurring
debris,
such
as
clays
and
algae,
chemicals,
such
as
iron
and
alum
coagulants
and
polymers,
may
be
added
to
finished
waters
during
the
treatment
process,
which
may
result
in
additional
interference.

4.2
Organisms
and
debris
that
autofluoresce
or
demonstrate
non­
specific
fluorescence,
such
as
algal
and
yeast
cells,
when
examined
by
epifluorescent
microscopy,
may
interfere
with
the
detection
of
oocysts
and
cysts
and
contribute
to
false
positives
by
immunofluorescence
assay
(
FA)
(
Reference
20.1).

4.3
Solvents,
reagents,
labware,
and
other
sample­
processing
hardware
may
yield
artifacts
that
may
cause
misinterpretation
of
microscopic
examinations
for
oocysts
and
cysts.
All
materials
used
shall
be
demonstrated
to
be
free
from
interferences
under
the
conditions
of
analysis
by
running
a
method
blank
(
negative
control
sample)
initially
and
a
minimum
of
every
week
or
after
changes
in
source
of
reagent
water.
Specific
selection
of
reagents
and
purification
of
solvents
and
other
materials
may
be
required.

4.4
Interferences
co­
extracted
from
samples
will
vary
considerably
from
source
to
source,
depending
on
the
water
being
sampled.
Experience
suggests
that
high
levels
of
algae,
bacteria,
and
other
protozoa
can
interfere
in
the
identification
of
oocysts
and
cysts
(
Reference
20.1).
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
3
4.4
Freezing
samples,
filters,
eluates,
concentrates,
or
slides
may
interfere
with
the
detection
and/
or
identification
of
oocysts
and
cysts.

4.5
All
equipment
should
be
cleaned
according
to
manufacturers'
instructions.
Disposable
supplies
should
be
used
wherever
possible.

5.0
Safety
5.1
The
biohazard
associated
with,
and
the
risk
of
infection
from,
oocysts
and
cysts
is
high
in
this
method
because
live
organisms
are
handled.
This
method
does
not
purport
to
address
all
of
the
safety
problems
associated
with
its
use.
It
is
the
responsibility
of
the
laboratory
to
establish
appropriate
safety
and
health
practices
prior
to
use
of
this
method.
In
particular,
laboratory
staff
must
know
and
observe
the
safety
procedures
required
in
a
microbiology
laboratory
that
handles
pathogenic
organisms
while
preparing,
using,
and
disposing
of
sample
concentrates,
reagents
and
materials,
and
while
operating
sterilization
equipment.

5.2
The
toxicity
or
carcinogenicity
of
each
compound
or
reagent
used
in
this
method
has
not
been
precisely
determined;
however,
each
chemical
compound
should
be
treated
as
a
potential
health
hazard.
Exposure
to
these
compounds
should
be
reduced
to
the
lowest
possible
level.
The
laboratory
is
responsible
for
maintaining
a
current
awareness
file
of
Occupational
Safety
and
Health
Administration
regulations
regarding
the
safe
handling
of
the
chemicals
specified
in
this
method.
A
reference
file
of
material
safety
data
sheets
should
be
made
available
to
all
personnel
involved
in
these
analyses.
Additional
information
on
laboratory
safety
can
be
found
in
References
20.2
through
20.5.

5.3
Samples
may
contain
high
concentrations
of
biohazards
and
toxic
compounds,
and
must
be
handled
with
gloves
and
opened
in
a
biological
safety
cabinet
to
prevent
exposure.
Reference
materials
and
standards
containing
oocysts
and
cysts
must
also
be
handled
with
gloves
and
laboratory
staff
must
never
place
gloves
in
or
near
the
face
after
exposure
to
solutions
known
or
suspected
to
contain
oocysts
and
cysts.
Do
not
mouth­
pipette.

5.4
Laboratory
personnel
must
change
gloves
after
handling
filters
and
other
contaminant­
prone
equipment
and
reagents.
Gloves
must
be
removed
or
changed
before
touching
any
other
laboratory
surfaces
or
equipment.

5.5
Centers
for
Disease
Control
(
CDC)
regulations
(
42
CFR
72)
prohibit
interstate
shipment
of
more
than
4
L
of
solution
known
to
contain
infectious
materials
(
see
http://
www.
cdc.
gov/
od/
ohs/
biosfty/
shipregs.
htm
for
details).
State
regulations
may
contain
similar
regulations
for
intrastate
commerce.
Unless
the
sample
is
known
or
suspected
to
contain
Cryptosporidium,
Giardia,
or
other
infectious
agents
(
e.
g.,
during
an
outbreak),
samples
should
be
shipped
as
noninfectious
and
should
not
be
marked
as
infectious.
If
a
sample
is
known
or
suspected
to
be
infectious,
and
the
sample
must
be
shipped
to
a
laboratory
by
a
transportation
means
affected
by
CDC
or
state
regulations,
the
sample
should
be
shipped
in
accordance
with
these
regulations.

6.0
Equipment
and
Supplies
NOTE:
Brand
names,
suppliers,
and
part
numbers
are
for
illustrative
purposes
only.
No
endorsement
is
implied.
Equivalent
performance
may
be
achieved
using
apparatus
and
materials
other
than
those
specified
here,
but
demonstration
of
equivalent
performance
that
meets
the
requirements
of
this
method
is
the
responsibility
of
the
laboratory.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
4
6.1
Sample
collection
equipment
for
shipment
of
bulk
water
samples
for
laboratory
filtration.
Collapsible
LDPE
cubitainer
for
collection
of
10­
L
bulk
sample(
s)
 
Cole
Parmer
cat.
no.
U­
06100­
30
or
equivalent.
Fill
completely
to
ensure
collection
of
a
full
10­
L
sample.
Discard
after
one
use.

6.2
Equipment
for
sample
filtration.
Three
options
have
been
demonstrated
to
be
acceptable
for
use
with
Method
1623.
Other
options
may
be
used
if
their
acceptability
is
demonstrated
according
to
the
procedures
outlined
in
Section
9.1.2.

6.2.1
Cubitainer
spigot
to
facilitate
laboratory
filtration
of
sample
(
for
use
with
any
filtration
option)
 
Cole
Parmer
cat.
no.
U­
06061­
01,
or
equivalent.

6.2.2
Original
Envirochek
 
sampling
capsule
or
Envirochek
 
HV
sampling
capsule
equipment
requirements
(
for
use
with
the
procedure
described
in
Section
12.2).
The
versions
of
the
method
using
this
filter
was
these
filters
were
validated
using
10­
L
and
50­
L
sample
volumes,
respectively.
Alternate
sample
volumes
may
be
used,
provided
the
laboratory
demonstrates
acceptable
performance
on
initial
and
ongoing
spiked
reagent
water
and
source
water
samples
(
Section
9.1.2).

6.2.2.1
Sampling
capsule
6.2.2.1.1
Envirochek
 
,
Pall
Gelman
Laboratory,
Ann
Arbor,
MI,
product
no.
12110
(
individual
filter)
and
or
product
no.
12107
(
box
of
25
filters)
(
www.
pall.
com/
gelman
or
(
800)
521­
1520
ext.
2)

6.2.2.1.2
Envirochek
 
HV,
Pall
Gelman
Laboratory,
Ann
Arbor,
MI,
product
no.
12099
(
individual
filter)
or
product
no.
12098
(
box
of
25
filters)
(
www.
pall.
com/
gelman
or
(
800)
521­
1520
ext.
2)

6.2.2.2
Laboratory
shaker
with
arms
for
agitation
of
sampling
capsules
6.2.2.2.1
Laboratory
shaker
 
Lab­
Line
model
3589,
VWR
Scientific
cat.
no.
57039­
055,
Fisher
cat.
no.
14260­
11,
or
equivalent
6.2.2.2.2
Side
arms
for
laboratory
shaker
 
Lab­
Line
Model
3587­
4,
VWR
Scientific
cat.
no.
57039­
045,
Fisher
cat.
no.
14260­
13,
or
equivalent
6.2.3
CrypTest
 
capsule
filter
equipment
requirements.
Follow
the
manufacturer's
instructions
when
using
this
filtration
option.
The
version
of
the
method
using
this
filter
was
validated
using
10­
L
sample
volumes;
alternate
sample
volumes
may
be
used,
provided
the
laboratory
demonstrates
acceptable
performance
on
initial
and
ongoing
spiked
reagent
water
and
matrix
samples
(
Section
9.1.2).

6.2.3.1
Capsule
filter
 
CrypTest
 
,
Whatman
Inc,
Clifton,
NJ,
product
no.
610064
6.2.3.2
Cartridge
housing
 
Ametek
5­
in.
clear
polycarbonate,
Whatman
cat.
no.
71503,
or
equivalent
6.2.3.3
Ultrasonic
bath
 
VWR
Model
75T#
21811­
808,
or
equivalent
6.2.3.4
Laboratory
tubing
 
Tygon
formula
R­
3603,
or
equivalent
6.2.3
Filta­
Max
 
foam
filter
equipment
requirements
(
for
use
with
the
procedure
described
in
Section
12.3).
The
version
of
the
method
using
this
filter
was
validated
using
50­
L
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
5
sample
volumes;
alternate
sample
volumes
may
be
used,
provided
the
laboratory
demonstrates
acceptable
performance
on
initial
and
ongoing
spiked
reagent
water
and
matrix
samples
(
Section
9.1.2).

6.2.3.1
Foam
filter
 
Filta­
Max
 
,
IDEXX,
Westbrook,
ME.
Filter
module
and
membrane:
product
code
FMC
10601;
filter
membranes
(
100
pack),
product
code
FMC
10800
NOTE:
Check
at
least
one
filter
per
batch
to
ensure
that
the
filters
have
not
been
affected
by
improper
storage
or
other
factors
that
could
result
in
brittleness
or
other
problems.
At
a
minimum
confirm
that
the
test
filter
expands
properly
in
water
before
using
the
batch
or
shipping
filters
to
the
field.

6.2.3.2
Filter
processing
equipment
 
Filta­
Max
starter
kit,
IDEXX,
Westbrook,
ME,
cat.
no.
FMC
11002.
Includes
all
equipment
required
to
run
and
process
Filta­
Max
filter
modules
(
manual
wash
station
(
FMC
10102)
including
plunger
head
(
FMC
12001),
elution
tubing
set
(
FMC
10301),
vacuum
set
(
FMC
10401),
filter
housing
(
FMC
10501),
and
magnetic
stirrer
(
FMC
10901).
6.3
Ancillary
sampling
equipment
6.3.1
Tubing
 
Glass,
polytetrafluoroethylene
(
PTFE),
high­
density
polyethylene
(
HDPE),
or
other
tubing
to
which
oocysts
and
cysts
will
not
easily
adhere
 
Tygon
formula
R­
3603,
or
equivalent.
If
rigid
tubing
(
glass,
PTFE,
HDPE)
is
used
and
the
sampling
system
uses
a
peristaltic
pump,
a
minimum
length
of
compressible
tubing
may
be
used
in
the
pump.
Before
use,
the
tubing
must
be
autoclaved,
thoroughly
rinsed
with
detergent
solution,
followed
by
repeated
rinsing
with
reagent
water
to
minimize
sample
contamination.
Alternately,
decontaminate
using
hypochlorite
solution,
sodium
thiosulfate,
and
multiple
reagent
water
rinses;
dispose
of
tubing
when
wear
is
evident.
Dispose
of
tubing
after
one
use
whenever
possible.
6.3.2
Flow
control
valve
 
0.5
gpm
(
0.03
L/
s),
Bertram
Controls,
Plast­
O­
Matic
cat.
no.
FC050B
½
­
PV,
or
equivalent;
or
0.4­
to
4­
Lpm
flow
meter
with
valve
 
Alamo
Water
Treatment,
San
Antonio,
TX,
cat.
no.
R5310,
or
equivalent
6.3.3
Centrifugal
pump
 
Grainger,
Springfield,
VA,
cat.
no.
2P613
Simer,
model
number,
M40,
or
equivalent.
6.3.4
Flow
meter
 
Sameco
cold
water
totalizer,
E.
Clark
and
Associates,
Northboro,
MA,
product
no.
WFU
10.110,
or
equivalent
6.4
Equipment
for
spiking
samples
in
the
laboratory
6.4.1
10­
L
carboy
with
bottom
delivery
port
(
½
"
)
 
Cole­
Palmer
cat.
no.
06080­
42,
or
equivalent;
calibrate
to
10.0
L
and
mark
level
with
waterproof
marker
6.4.2
Stir
bar
 
Fisher
cat.
no.
14­
511­
93,
or
equivalent
6.4.3
Stir
plate
 
Fisher
cat.
no.
14­
493­
120S,
or
equivalent
6.4.4
Hemacytometer
 
Neubauer
type,
Hauser
Scientific,
Horsham,
PA,
cat.
no.
3200
or
1475,
or
equivalent
6.4.5
Hemacytometer
coverslip
 
Hauser
Scientific,
cat.
no.
5000
(
for
hemacytometer
cat.
no.
3200)
or
1461
(
for
hemacytometer
cat.
no
1475),
or
equivalent
6.4.6
Lens
paper
without
silicone
 
Fisher
cat.
no.
11­
995,
or
equivalent
6.4.7
Polystyrene
or
polypropylene
conical
tubes
with
screw
caps
 
15­
and
50­
mL
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
6
6.4.8
Equipment
required
for
enumeration
of
spiking
suspensions
using
membrane
filters
6.4.8.1
Glass
microanalysis
filter
holder
 
25­
mm­
diameter,
with
fritted
glass
support,
Fisher
cat.
no.
09­
753E,
or
equivalent.
Replace
stopper
with
size
8,
one­
hole
rubber
stopper,
Fisher
Cat.
No.
14­
135M,
or
equivalent.
6.4.8.2
Three­
port
vacuum
filtration
manifold
and
vacuum
source
 
Fisher
Cat.
No.
09­
753­
39A,
or
equivalent
6.4.8.3
Cellulose
acetate
support
membrane
 
1.2­
µ
m­
pore­
size,
25­
mmdiameter
Fisher
cat.
no.
A12SP02500,
or
equivalent
6.4.8.4
Polycarbonate
track­
etch
hydrophilic
membrane
filter
 
1­
µ
m­
pore­
size,
25­
mm­
diameter,
Fisher
cat.
no.
K10CP02500,
or
equivalent
6.4.8.5
100
×
15
mm
polystyrene
petri
dishes
(
bottoms
only)
6.4.8.6
60
×
15
mm
polystyrene
petri
dishes
6.4.8.7
Glass
microscope
slides
 
1
in.
×
3
in
or
2
in.
×
3
in.
6.4.8.8
Coverslips
 
25
mm2
6.5
Immunomagnetic
separation
(
IMS)
apparatus
6.5.1
Sample
mixer
 
Dynal
Inc.,
Lake
Success,
NY,
cat.
no.
947.01,
or
equivalent
6.5.2
Magnetic
particle
concentrator
for
10­
mL
test
tubes
 
Dynal
MPC­
1
®
,
cat.
no.
120.01,
or
equivalent
6.5.3
Magnetic
particle
concentrator
for
microcentrifuge
tubes
 
Dynal
MPC­
M
®
,
cat.
no.
120.09,
or
equivalent
6.5.4
Flat­
sided
sample
tubes
 
16
×
125
mm
Leighton­
type
tubes
with
60
×
10
mm
flat­
sided
magnetic
capture
area,
Dynal
L10,
cat.
no.
740.03,
or
equivalent
6.6
Powder­
free
latex
gloves
 
Fisher
cat
no.
113945B,
or
equivalent
6.7
Graduated
cylinders,
autoclavable
 
10­,
100­,
and
1000­
mL
6.8
Centrifuges
6.8.1
Centrifuge
capable
of
accepting
15­
to
250­
mL
conical
centrifuge
tubes
and
achieving
1500
×
G
 
International
Equipment
Company,
Needham
Heights,
MA,
Centrifuge
Size
2,
Model
K
with
swinging
bucket,
or
equivalent
6.8.2
Centrifuge
tubes
 
Conical,
graduated,
1.5­,
50­,
and
250­
mL
6.9
Microscope
6.9.1
Epifluorescence/
differential
interference
contrast
(
DIC)
with
stage
and
ocular
micrometers
and
20X
(
N.
A.=
0.4)
to
100X
(
N.
A.=
1.3)
objectives
 
Zeiss
 
Axioskop,
Olympus
 
BH,
or
equivalent
6.9.2
Excitation/
band­
pass
filters
for
immunofluorescence
assay
(
FA)
 
Zeiss
 
487909
or
equivalent,
including,
450­
to
490­
nm
exciter
filter,
510­
nm
dicroic
beam­
splitting
mirror,
and
515­
to
520­
nm
barrier
or
suppression
filter
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
7
6.9.3
Excitation/
band­
pass
filters
for
DAPI
 
Filters
cited
below
(
Chroma
Technology,
Brattleboro,
VT),
or
equivalent
Microscope
model
Fluoro­
chrome
Excitation
filter
(
nm)
Dichroic
beamsplitting
mirror
(
nm)
Barrier
or
suppression
filter
(
nm)
Chroma
catalog
number
Zeiss
 
­
Axioskop
DAPI
(
UV)
340­
380
400
420
CZ902
Zeiss
 
­
IM35
DAPI
(
UV)
340­
380
400
420
CZ702
Olympus
 
BH
DAPI
(
UV)
340­
380
400
420
11000
Filter
holder
91002
Olympus
 
BX
DAPI
(
UV)
340­
380
400
420
11000
Filter
holder
91008
Olympus
 
IMT2
DAPI
(
UV)
340­
380
400
420
11000
Filter
holder
91003
6.10
Ancillary
equipment
for
microscopy
6.10.1
Well
slides
 
Spot­
On
well
slides,
Dynal
cat.
no.
740.04;
treated,
12­
mm
diameter
well
slides,
Meridian
Diagnostics
Inc.,
Cincinnati,
OH,
cat.
no.
R2206;
or
equivalent
6.10.2
Glass
coverslips
 
22
×
50
mm
6.10.3
Nonfluorescing
immersion
oil
 
Type
FF,
Cargille
cat.
no.
16212,
or
equivalent
6.10.4
Micropipette,
adjustable:
0­
to
10­
µ
L
with
0­
to
10­
µ
L
tips
10­
to
100­
µ
L,
with
10­
to
200­
µ
L
tips
100­
to
1000­
µ
L
with
100­
to
1000­
µ
L
tips
6.10.5
Forceps
 
Splinter,
fine
tip
6.10.6
Forceps
 
Blunt­
end
6.10.7
Desiccant
 
Drierite
 
Absorbent,
Fisher
cat.
no.
07­
577­
1A,
or
equivalent
6.10.8
Humid
chamber
 
A
tightly
sealed
plastic
container
containing
damp
paper
towels
on
top
of
which
the
slides
are
placed
6.11
Pipettes
 
Glass
or
plastic
6.11.1
5­,
10­,
and
25­
mL
6.11.2
Pasteur,
disposable
6.12
Balances
6.12.1
Analytical
 
Capable
of
weighing
0.1
mg
6.12.2
Top
loading
 
Capable
of
weighing
10
mg
6.13
pH
meter
6.14
Incubator
 
Fisher
Scientific
Isotemp
 
,
or
equivalent
6.15
Vortex
mixer
 
Fisons
Whirlmixer,
or
equivalent
6.16
Vacuum
source
 
Capable
of
maintaining
25
in.
Hg,
equipped
with
shutoff
valve
and
vacuum
gauge
6.17
Miscellaneous
labware
and
supplies
6.17.1
Test
tubes
and
rack
6.17.2
Flasks
 
Suction,
Erlenmeyer,
and
volumetric,
various
sizes
6.17.3
Beakers
 
Glass
or
plastic,
5­,
10­,
50­,
100­,
500­,
1000­,
and
2000­
mL
6.17.4
Lint­
free
tissues
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
8
6.18
10­
to
15­
L
graduated
container
 
Fisher
cat.
no.
02­
961­
50B,
or
equivalent;
calibrate
to
9.0,
9.5,
10.0,
10.5,
and
11.0
L
and
mark
levels
with
waterproof
marker
6.19
Filters
for
filter­
sterilizing
reagents
 
Sterile
Acrodisc,
0.45
µ
m,
Gelman
Sciences
cat
no.
4184,
or
equivalent
7.0
Reagents
and
Standards
7.1
Reagents
for
adjusting
pH
7.1.1
Sodium
hydroxide
(
NaOH)
 
ACS
reagent
grade,
6.0
N
and
1.0
N
in
reagent
water
7.1.2
Hydrochloric
acid
(
HCl)
 
ACS
reagent
grade,
6.0
N,
1.0
N,
and
0.1
N
in
reagent
water
NOTE:
Due
to
the
low
volumes
of
pH­
adjusting
reagents
used
in
this
method,
and
the
impact
that
changes
in
pH
have
on
the
immunofluorescence
assay,
the
laboratory
should
purchase
standards
at
the
required
normality
directly
from
a
vendor.
Normality
should
not
be
adjusted
by
the
laboratory.

7.2
Solvents
 
Acetone,
glycerol,
ethanol,
and
methanol,
ACS
reagent
grade
7.3
Reagent
water
 
Water
in
which
oocysts
and
cysts
and
interfering
materials
and
substances,
including
magnetic
minerals,
are
not
detected
by
this
method
7.4
Reagents
for
eluting
filters
NOTE:
Laboratories
should
store
prepared
eluting
solution
for
no
more
than
1
week
or
when
noticeably
turbid,
whichever
comes
sooner.

7.4.1
Reagents
for
eluting
Envirochek
 
sampling
capsules
(
Section
6.2.2)
7.4.1.1
Laureth­
12
 
PPG
Industries,
Gurnee,
IL,
cat.
no.
06194,
or
equivalent.
Store
Laureth­
12
as
a
10%
solution
in
reagent
water.
Weigh
10
g
of
Laureth­
12
and
dissolve
using
a
microwave
or
hot
plate
in
90
mL
of
reagent
water.
Dispense
10­
mL
aliquots
into
sterile
vials
and
store
at
room
temperature
for
up
to
2
months,
or
in
the
freezer
for
up
to
a
year.
7.4.1.2
1
M
Tris,
pH
7.4
 
Dissolve
121.1
g
Tris
(
Fisher
cat.
no.
BP152)
in
700
mL
of
reagent
water
and
adjust
pH
to
7.4
with
1
N
HCl
or
NaOH.
Dilute
to
a
final
1000
mL
with
reagent
water
and
adjust
the
final
pH.
Filtersterilize
through
a
0.2­
µ
m
membrane
into
a
sterile
plastic
container
and
store
at
room
temperature.
7.4.1.3
0.5
M
EDTA,
2
Na,
pH
8.0
 
Dissolve
186.1
g
ethylenediamine
tetraacetic
acid,
disodium
salt
dihydrate
(
Fisher
cat.
no.
S311)
in
800
mL
of
reagent
water
and
adjust
pH
to
8.0
with
6.0
N
HCl
or
NaOH.
Dilute
to
a
final
volume
of
1000
mL
with
reagent
water
and
adjust
to
pH
8.0
with
1.0
N
HCl
or
NaOH.
7.4.1.4
Antifoam
A
 
Sigma
Chemical
Co.
cat.
no.
A5758,
or
equivalent
7.4.1.5
Preparation
of
elution
buffer
solution
 
Add
the
contents
of
a
preprepared
Laureth­
12
vial
(
Section
7.4.1.1)
to
a
1000­
mL
graduated
cylinder.
Rinse
the
vial
several
times
to
ensure
the
transfer
of
the
detergent
to
the
cylinder.
Add
10
mL
of
Tris
solution
(
Section
7.4.1.2),
2
mL
of
EDTA
solution
(
Section
7.4.1.3),
and
150
µ
L
Antifoam
A
(
Section
7.4.1.4).
Dilute
to
1000
mL
with
reagent
water.
7.4.2
Reagents
for
eluting
CrypTest
 
capsule
filters
(
Section
6.2.3).
To
900
mL
of
reagent
water
add
8.0
g
NaCl,
0.2
g
KH2PO4,
2.9
g
Na2HPO4
(
12H2O)
0.2
g
KCl,
0.2
g
sodium
lauryl
sulfate
(
SDS),
0.2
mL
Tween
80,
and
0.02
mL
Antifoam
A
(
Sigma
Chemical
Co.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
9
cat.
no.
A5758,
or
equivalent).
Adjust
volume
to
1
L
with
reagent
water
and
adjust
pH
to
7.4
with
1
N
NaOH
or
HCl.
7.4.2
Reagents
for
eluting
Filta­
Max
 
foam
filters
(
Section
6.2.3)
7.4.2.1
Phosphate
buffered
saline
(
PBS),
pH
7.4
 
Sigma
Chemical
Co.
cat.
no.
P­
3813,
or
equivalent.
Alternately,
prepare
PBS
by
adding
the
following
to
1
L
of
reagent
water:
8
g
NaCl;
0.2
g
KCl;
1.15
g
Na2HPO4,
anhydrous;
and
0.2
g
KH2PO4.
7.4.2.2
Tween
20
 
Sigma
Chemical
Co.
cat.
no.
P­
7949,
or
equivalent
7.4.2.3
High­
vacuum
grease
 
BDH/
Merck.
cat.
no.
636082B,
or
equivalent
7.4.2.4
Preparation
of
PBST
elution
buffer.
Add
100
µ
L
of
Tween
20
to
prepared
PBS
(
Section
7.4.3.1
7.4.2.1).
Alternatively,
add
the
contents
of
one
sachet
packet
of
PBS
to
1.0
L
of
reagent
water.
Dissolve
by
stirring
for
30
minutes.
Add
100
µ
L
of
Tween
20.
Mix
by
stirring
for
5
minutes.
7.5
Reagents
for
immunomagnetic
separation
(
IMS)
 
Dynabeads
®
GC­
Combo,
Dynal
cat.
nos.
730.02,
730.12,
or
equivalent
7.6
Direct
antibody
labeling
reagents
for
detection
of
oocysts
and
cysts.
Store
reagents
at
0
°
C
to
8
°
C
and
return
promptly
to
this
temperature
after
each
use.
Do
not
allow
any
of
the
reagents
to
freeze.
The
reagents
should
be
protected
from
exposure
to
light.
Diluted,
unused
working
reagents
should
be
discarded
after
48
hours.
Discard
reagents
after
the
expiration
date
is
reached.
The
labeling
reagents
in
Sections
7.6.1­
7.6.3
have
been
approved
for
use
with
this
method.
7.6.1
Merifluor
Cryptosporidium/
Giardia,
Meridian
Diagnostics
cat.
no.
250050,
Cincinnati,
OH,
or
equivalent
7.6.2
Aqua­
Glo
 
G/
C
Direct
FL,
Waterborne
cat.
no.
A100FLR,
New
Orleans,
LA,
or
equivalent
7.6.3
Crypt­
a­
Glo
 
and
Giardi­
a­
Glo
 
,
Waterborne
cat.
nos.
A400FLR
and
A300FLR,
respectively,
New
Orleans,
LA,
or
equivalent
NOTE:
If
a
laboratory
will
use
multiple
types
of
labeling
reagents,
the
laboratory
must
demonstrate
acceptable
performance
through
an
initial
precision
and
recovery
test
(
Section
9.4)
for
each
type,
and
must
perform
positive
and
negative
staining
controls
for
each
batch
of
slides
stained
using
each
product.
However,
the
laboratory
is
not
required
to
analyze
additional
ongoing
precision
and
recovery
samples
or
method
blank
samples
for
each
type.
The
performance
of
each
labeling
reagent
used
also
should
be
monitored
in
each
source
water
type.

7.6.4
Diluent
for
labeling
reagents
 
Phosphate
buffered
saline
(
PBS)
(
Section
7.4.1).
,
pH
7.4
 
Sigma
Chemical
Co.
cat.
no.
P­
3813,
or
equivalent.
Alternately,
prepare
PBS
by
adding
the
following
to
1
L
of
reagent
water:
8
g
NaCl;
0.2
g
KCl;
1.15
g
Na2HPO4,
anhydrous;
and
0.2
g
KH2PO4.
Filter­
sterilize
(
Section
6.19)
or
autoclave.
Discard
if
growth
is
detected
or
after
6
months,
whichever
comes
first.
7.7
4',
6­
diamidino­
2­
phenylindole
(
DAPI)
stain
 
Sigma
Chemical
Co.
cat.
no.
D9542,
or
equivalent
7.7.1
Stock
solution
 
Dissolve
2
mg/
mL
DAPI
in
absolute
methanol.
Prepare
volume
consistent
with
minimum
use.
Store
at
0
°
C
to
8
°
C
in
the
dark.
Do
not
allow
to
freeze.
Discard
unused
solution
when
positive
staining
control
fails.
7.7.2
Staining
solution
(
1/
5000
dilution
in
PBS
[
Section
7.6.4])
 
Add
10
µ
L
of
2
mg/
mL
DAPI
stock
solution
to
50
mL
of
PBS.
Prepare
daily.
Store
at
0
°
C
to
8
°
C
in
the
dark
except
when
staining.
Do
not
allow
to
freeze.
The
solution
concentration
may
be
increased
up
to
1
µ
g/
mL
if
fading/
diffusion
of
DAPI
staining
is
encountered,
but
the
staining
solution
must
be
tested
first
on
expendable
environmental
samples
to
confirm
that
staining
intensity
is
appropriate.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
10
7.8
Mounting
medium
7.8.1
DABCO/
glycerol
mounting
medium
(
2%)
 
Dissolve
2
g
of
DABCO
(
Sigma
Chemical
Co.
cat
no.
D­
2522,
or
equivalent)
in
95
mL
of
warm
glycerol/
PBS
(
60%
glycerol,
40%
PBS
[
Section
7.6.4]).
After
the
DABCO
has
dissolved
completely,
adjust
the
solution
volume
to
100
mL
by
adding
an
appropriate
volume
of
glycerol/
PBS
solution.
Alternately,
dissolve
the
DABCO
in
40
mL
of
PBS,
then
add
azide
(
1
mL
of
100X,
or
10%
solution),
then
60
mL
of
glycerol.
7.8.2
Mounting
medium
supplied
with
Merifluor
direct
labeling
kit
(
Section
7.6.1)
7.9
Clear
fingernail
polish
or
clear
fixative,
PGC
Scientifics,
Gaithersburg,
MD,
cat.
no.
60­
4890,
or
equivalent
7.10
Oocyst
and
cyst
suspensions
for
spiking
7.10.1
Enumerated
spiking
suspensions
prepared
by
flow
cytometer
 
not
heat­
fixed
or
formalin
fixed:
Wisconsin
State
Laboratory
of
Hygiene
Flow
Cytometry
Unit
or
equivalent.
7.10.1.1
Live,
flow
cytometer
 
sorted
oocysts
and
cysts
 
Wisconsin
State
Laboratory
of
Hygiene
Flow
Cytometry
Unit
([
608]
224­
6260),
or
equivalent
7.10.1.2
Irradiated,
flow
cytometer
 
sorted
oocysts
and
cysts
 
flow
cytometer
 
sorted
oocysts
and
cysts
 
BTF
EasySeed
 
(
www.
biotechnologyfrontiers.
com),
or
equivalent
7.10.2
Materials
for
manual
enumeration
of
spiking
suspensions
7.10.2.1
Purified
Cryptosporidium
oocyst
stock
suspension
for
manual
enumeration
 
not
heat­
fixed
or
formalin­
fixed:
Sterling
Parasitology
Laboratory,
University
of
Arizona,
Tucson,
or
equivalent
7.10.2.2
Purified
Giardia
cyst
stock
suspension
for
manual
enumeration
 
not
heat­
fixed
or
formalin­
fixed:
Waterborne,
Inc.,
New
Orleans,
LA;
Hyperion
Research,
Medicine
Hat,
Alberta,
Canada;
or
equivalent
7.10.2.3
Tween­
20,
0.01%
 
Dissolve
1.0
mL
of
a
10%
solution
of
Tween­
20
in
1
L
of
reagent
water
7.10.3
Storage
procedure
 
Store
oocyst
and
cyst
suspensions
at
0
°
C
to
810
°
C,
until
ready
to
use;
do
not
allow
to
freeze
7.11
Additional
reagents
for
enumeration
of
spiking
suspensions
using
membrane
filtration
(
Section
11.3.6)
 
Sigmacote
®
Sigma
Company
Product
No.
SL­
2,
or
equivalent
8.0
Sample
Collection
and
Storage
8.1
Sample
collection,
shipment,
and
receipt
8.1.1
Sample
collection.
Samples
are
collected
as
bulk
samples
and
shipped
to
the
laboratory
for
processing
through
the
entire
method,
or
are
filtered
in
the
field
and
shipped
to
the
laboratory
for
processing
from
elution
(
Section
12.2.6)
onward.
8.1.2
Sample
shipment.
Ambient
water
samples
are
dynamic
environments
and,
depending
on
sample
constituents
and
environmental
conditions,
Cryptosporidium
oocysts
or
Giardia
cysts
present
in
a
sample
can
degrade,
potentially
biasing
analytical
results.
Samples
that
are
not
analyzed
the
same
day
they
are
collected
should
be
chilled
to
reduce
biological
activity,
and
preserve
the
state
of
source
water
samples
between
collection
and
analysis.
Samples
must
analyzed
by
an
off­
site
laboratory
should
be
shipped
via
overnight
service
on
the
day
they
are
collected.
Overnight
service
may
not
be
necessary
if
the
samples
are
maintained
below
10
°
C
(
but
not
frozen)
and
holding
times
are
met.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
11
NOTE:
See
transportation
precautions
in
Section
5.5.

8.1.2.1
If
samples
are
collected
early
in
the
day,
chill
samples
as
much
as
possible
between
collection
and
shipment
by
storing
in
a
refrigerator
or
pre­
icing
the
sample
in
a
cooler.
If
the
sample
is
pre­
iced
before
shipping,
replace
with
fresh
ice
immediately
before
shipment.
8.1.2.2
If
samples
are
collected
later
in
the
day,
these
samples
may
be
held
overnight
in
a
refrigerator.
This
should
be
considered
for
bulk
water
samples
that
will
be
shipped
off­
site,
as
this
minimizes
the
potential
for
water
samples
collected
during
the
summer
to
melt
the
ice
in
which
they
are
packed
and
arrive
at
the
laboratory
at
>
10
°
C.
8.1.2.3
Samples
should
be
shipped
at
0
°
C
to
<
10
°
C
8
°
C
and
not
frozen,
unless
the
time
required
to
chill
the
sample
to
8
°
C
10
°
C
would
prevent
the
sample
from
being
shipped
overnight
for
receipt
at
the
laboratory
the
day
after
collection.
Samples
must
not
be
allowed
to
freeze.
8.1.2.4
Public
water
systems
shipping
samples
to
off­
site
laboratories
for
analysis
should
include
in
the
shipping
container
a
means
for
monitoring
the
temperature
of
the
sample
during
shipping
to
verify
that
the
sample
did
not
freeze
or
exceed
10
°
C.
Suggested
approaches
for
monitoring
sample
temperature
during
shipping
are
discussed
in
Section
8.1.4.
8.1.3
Sample
receipt.
Upon
receipt,
the
laboratory
must
record
the
temperature.
Samples
that
were
not
collected
the
same
day
they
were
received,
and
that
are
received
at
>
10
°
C
or
frozen,
or
samples
that
the
laboratory
has
determined
exceeded
>
10
°
C
or
froze
during
shipment,
must
be
rejected.
After
receipt,
samples
must
be
stored
at
the
laboratory
at
<
10
°
C,
and
not
frozen,
until
processed.
Results
from
samples
shipped
overnight
to
the
laboratory
and
received
at
>
8
°
C
should
be
qualified
by
the
laboratory..
8.1.4
Suggestions
on
measuring
sample
temperature.
Given
the
importance
of
maintaining
sample
temperatures
for
Cryptosporidium
and
Giardia
determination,
laboratories
performing
analyses
using
this
method
must
establish
acceptance
criteria
for
receipt
of
samples
transported
to
their
laboratory.
Several
options
are
available
to
measure
sample
temperature
upon
receipt
at
the
laboratory
and,
in
some
cases,
during
shipment:
8.1.4.1
Temperature
sample.
One
option,
for
filtered
samples
only
(
not
for
10­
L
bulk
samples),
is
for
the
sampler
to
fill
a
small,
inexpensive
sample
bottle
with
water
and
pack
this
"
temperature
sample"
next
to
the
filtered
sample.
The
temperature
of
this
extra
sample
volume
is
measured
upon
receipt
to
estimate
the
temperature
of
the
filter.
Temperature
sample
bottles
are
not
appropriate
for
use
with
bulk
samples
because
of
the
potential
effect
that
the
difference
in
sample
volume
may
have
in
temperature
equilibration
in
the
sample
cooler.
Example
product:
Cole
Parmer
cat.
no.
U­
06252­
20.
8.1.4.2
Thermometer
vial.
A
similar
option
is
to
use
a
thermometer
that
is
securely
housed
in
a
liquid­
filled
vial.
Unlike
temperature
samples,
the
laboratory
does
not
need
to
perform
an
additional
step
to
monitor
the
temperature
of
the
vial
upon
receipt,
but
instead
just
needs
to
read
the
thermometer.
Example
product:
Eagle­
Picher
Sentry
Temperature
Vial
3TR­
40CS­
F
or
3TR­
40CS.
8.1.4.3
iButton.
Another
option
for
measuring
the
sample
temperature
during
shipment
and
upon
receipt
is
a
Thermocron
®
iButton.
An
iButton
is
a
small,
waterproof
device
that
contains
a
computer
chip
that
can
be
programmed
to
record
temperature
at
different
time
intervals.
The
information
is
then
downloaded
from
the
iButton
onto
a
computer.
The
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
12
iButton
should
be
placed
in
a
temperature
sample
(
Section
8.1.4.1),
rather
than
placed
loose
in
the
cooler.
Information
on
Thermocron
®
iButtons
is
available
from
http://
www.
ibutton.
com/.
Distributors
include
http://
www.
pointsix.
com/,
http://
www.
rdsdistributing.
com,
and
http://
www.
scigiene.
com/.
8.1.4.4
Stick­
on
temperature
strips.
Another
option
is
for
the
laboratory
to
apply
a
stick­
on
temperature
strip
to
the
outside
of
the
sample
container
upon
receipt
at
the
laboratory.
This
option
does
not
measure
temperature
as
precisely
as
the
other
options,
but
still
mitigates
the
risk
of
sample
contamination
while
providing
an
indication
of
sample
temperature
to
verify
that
the
sample
temperature
is
acceptable.
Example
product:
Cole
Parmer
cat.
no.
U­
90316­
00.
8.1.4.5
Infrared
thermometers.
A
final
option
is
to
measure
the
temperature
of
the
surface
of
the
sample
container
or
filter
using
an
infrared
thermometer.
The
thermometer
is
pointed
at
the
sample,
and
measures
the
temperature
without
coming
in
contact
with
the
sample
volume.
Example
product:
Cole
Parmer
cat.
no.
EW­
39641­
00.
As
with
other
laboratory
equipment,
all
temperature
measurement
devices
must
be
calibrated
routinely
to
ensure
accurate
measurements.
See
the
EPA
Manual
for
the
Certification
of
Laboratories
Analyzing
Drinking
Water
(
Reference
20.6)
for
more
information.
8.2
Sample
holding
times.
Samples
must
be
processed
or
examined
within
each
of
the
holding
times
specified
in
Sections
8.2.1
­
8.2.4.
Sample
processing
should
be
completed
as
soon
as
possible
by
the
laboratory.
The
laboratory
should
complete
sample
filtration,
elution,
concentration,
purification,
and
staining
the
day
the
sample
is
received
wherever
possible.
However,
the
laboratory
is
permitted
to
split
up
the
sample
processing
steps
if
processing
a
sample
completely
in
one
day
is
not
possible.
If
this
is
necessary,
sample
processing
can
be
halted
after
filtration,
application
of
the
purified
sample
onto
the
slide,
or
staining.
Table
1,
in
Section
21.0
provides
a
breakdown
of
the
holding
times
for
each
set
of
steps.
Sections
8.2.1
through
8.2.4
provide
descriptions
of
these
holding
times.
8.2.1
Sample
collection
and
filtration.
Sample
elution
must
be
initiated
within
96
hours
of
sample
collection
(
if
shipped
to
the
laboratory
as
a
bulk
sample)
or
filtration
(
if
filtered
in
the
field).
8.2.2
Sample
elution,
concentration,
and
purification.
The
laboratory
must
complete
the
elution,
concentration,
and
purification
(
Sections
12.2.6
through
13.3.3.11)
in
one
work
day.
It
is
critical
that
these
steps
be
completed
in
one
work
day
to
minimize
the
time
that
any
target
organisms
present
in
the
sample
sit
in
eluate
or
concentrated
matrix.
This
process
ends
with
the
application
of
the
purified
sample
on
the
slide
for
drying.
8.2.3
Staining.
The
sample
must
be
stained
within
72
hours
of
application
of
the
purified
sample
to
the
slide.
8.2.4
Examination.
Although
immunofluorescence
assay
(
FA)
and
4',
6­
diamidino­
2­
phenylindole
(
DAPI)
and
differential
interference
contrast
(
DIC)
microscopy
examination
and
confirmation
should
be
performed
immediately
after
staining
is
complete,
laboratories
have
up
to
7
days
from
completion
of
sample
staining
to
complete
the
examination
and
confirmation
of
samples.
However,
if
fading/
diffusion
of
FITC
or
DAPI
staining
is
noticed,
the
laboratory
must
reduce
this
holding
time.
In
addition
the
laboratory
may
adjust
the
concentration
of
the
DAPI
staining
solution
(
Sections
7.7.2)
so
that
fading/
diffusion
does
not
occur.
8.5
Spiking
suspension
enumeration
holding
times.
Flow­
cytometer­
sorted
spiking
suspensions
(
Sections
7.10.1
and
11.2)
used
for
spiked
quality
control
(
QC)
samples
(
Section
9)
must
be
used
within
the
expiration
date
noted
on
the
suspension.
Laboratories
should
use
flow­
cytometer­
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
13
sorted
spiking
suspensions
containing
live
organisms
within
two
weeks
of
preparation
at
the
flow
cytometry
laboratory.
Manually
enumerated
spiking
suspensions
must
be
used
within
24
hours
of
enumeration
of
the
spiking
suspension
if
the
hemacytometer
chamber
technique
is
used
(
Section
11.3.4);
or
within
24
hours
of
application
of
the
spiking
suspension
to
the
slides
if
the
well
slide
or
membrane
filter
enumeration
technique
is
used
(
Sections
11.3.5
and
11.3.6).
Oocyst
and
cyst
suspensions
must
be
stored
at
0
°
C
to
10
°
C,
until
ready
to
use;
do
not
allow
to
freeze
9.0
Quality
Control
9.1
Each
laboratory
that
uses
this
method
is
required
to
operate
a
formal
quality
assurance
(
QA)
program
(
Reference
20.7).
The
minimum
requirements
of
this
program
consist
of
an
initial
demonstration
of
laboratory
capability
through
performance
of
the
initial
precision
and
recovery
(
IPR)
test
(
Section
9.4),
analysis
of
spiked
samples
to
evaluate
and
document
data
quality,
and
analysis
of
standards
and
blanks
as
tests
of
continued
performance.
Laboratory
performance
is
compared
to
established
performance
criteria
to
determine
if
the
results
of
analyses
meet
the
performance
characteristics
of
the
method.
9.1.1
A
test
of
the
microscope
used
for
detection
of
oocysts
and
cysts
is
performed
prior
to
examination
of
slides.
This
test
is
described
in
Section
10.0.
9.1.2
In
recognition
of
advances
that
are
occurring
in
analytical
technology,
the
laboratory
is
permitted
to
modify
certain
method
procedures
to
improve
recovery
or
lower
the
costs
of
measurements,
provided
that
all
required
quality
control
(
QC)
tests
are
performed
and
all
QC
acceptance
criteria
are
met.
Method
procedures
that
can
be
modified
include
front­
end
techniques,
such
as
filtration
or
immunomagnetic
separation
(
IMS).
The
laboratory
is
not
permitted
to
use
an
alternate
determinative
technique
to
replace
immunofluorescence
assay
in
this
method
(
the
use
of
different
determinative
techniques
are
considered
to
be
different
methods,
rather
than
modified
version
of
this
method).
However,
the
laboratory
is
permitted
to
modify
the
immunofluorescence
assay
procedure,
provided
that
all
required
QC
tests
are
performed
(
Section
9.1.2.1)
and
all
QC
acceptance
criteria
are
met
(
see
guidance
on
the
use
of
multiple
labeling
reagents
in
Section
7.6).

NOTE:
Method
modifications
should
be
considered
only
to
improve
method
performance,
reduce
cost,
or
reduce
sample
processing
time.
Method
modifications
that
reduce
cost
or
sample
processing
time,
but
that
result
in
poorer
method
performance
should
not
be
used.

9.1.2.1
Method
modification
validation/
equivalency
demonstration
requirements
9.1.2.1.1
Method
modifications
at
a
single
laboratory.
Each
time
a
modification
is
made
to
this
method
for
use
in
single
laboratory,
the
laboratory
must,
at
a
minimum,
validate
the
modification
according
to
Tier
1
of
EPA's
performance­
based
measurement
system
(
PBMS)
(
Table
2
and
Reference
20.8)
to
demonstrate
that
the
modification
produces
results
equivalent
or
superior
to
results
produced
by
this
method
as
written.
Briefly,
each
time
a
modification
is
made
to
this
method,
the
laboratory
is
required
to
demonstrate
acceptable
modified
method
performance
through
the
IPR
test
(
Section
9.4).
IPR
results
must
meet
the
QC
acceptance
criteria
in
Tables
3
and
4
in
Section
21.0,
and
should
be
comparable
to
previous
results
using
the
unmodified
procedure.
Although
not
required,
the
laboratory
also
should
perform
a
matrix
spike/
matrix
spike
duplicate
(
MS/
MSD)
test
to
demonstrate
the
performance
of
the
modified
method
in
at
least
one
real­
world
matrix
before
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
14
analyzing
field
samples
using
the
modified
method.
The
laboratory
is
required
to
perform
MS
samples
using
the
modified
method
at
the
frequency
noted
in
Section
9.1.8.
If
the
modified
method
involves
changes
that
cannot
be
adequately
evaluated
through
these
tests,
additional
tests
may
be
required
to
demonstrate
acceptability.
9.1.2.1.2
Method
modifications
for
nationwide
approval.
If
the
laboratory
or
a
manufacturer
seeks
EPA
approval
of
a
method
modification
for
nationwide
use,
the
laboratory
or
manufacturer
must,
at
a
minimum,
validate
the
modification
according
to
Tier
2
of
EPA's
PBMS
(
Table
2
and
Reference
20.8).
Briefly,
at
least
three
laboratories
must
perform
IPR
tests
(
Section
9.4)
and
MS/
MSD
(
Section
9.5)
tests
using
the
modified
method,
and
all
tests
must
meet
the
QC
acceptance
criteria
specified
in
Tables
3
and
4
in
Section
21.0.
Upon
nationwide
approval,
laboratories
electing
to
use
the
modified
method
still
must
demonstrate
acceptable
performance
in
their
own
laboratory
according
to
the
requirements
in
Section
9.1.2.1.1.
If
the
modified
method
involves
changes
that
cannot
be
adequately
evaluated
through
these
tests,
additional
tests
may
be
required
to
demonstrate
acceptability.
9.1.2.2
The
laboratory
is
required
to
maintain
records
of
modifications
made
to
this
method.
These
records
include
the
following,
at
a
minimum:
9.1.2.2.1
The
names,
titles,
addresses,
and
telephone
numbers
of
the
analyst(
s)
who
performed
the
analyses
and
modification,
and
of
the
quality
control
officer
who
witnessed
and
will
verify
the
analyses
and
modification.
9.1.2.2.2
A
listing
of
the
analyte(
s)
measured
(
Cryptosporidium
and
Giardia).
9.1.2.2.3
A
narrative
stating
reason(
s)
for
the
modification.
9.1.2.2.4
Results
from
all
QC
tests
comparing
the
modified
method
to
this
method,
including:
(
a)
IPR
(
Section
9.4)
(
b)
MS/
MSD
(
Section
9.5)
(
c)
Analysis
of
method
blanks
(
Section
9.6)
9.1.2.2.5
Data
that
will
allow
an
independent
reviewer
to
validate
each
determination
by
tracing
the
following
processing
and
analysis
steps
leading
to
the
final
result:
(
a)
Sample
numbers
and
other
identifiers
(
b)
Source
of
spiking
suspensions,
as
well
as
lot
number
and
date
received
(
Section
7.10)
(
c)
Spike
enumeration
date
and
time
(
d)
All
spiking
suspension
enumeration
counts
and
calculations
(
Section
11.0)
(
e)
Sample
spiking
dates
and
times
(
f)
Volume
filtered
(
Section
12.2.5.2)
(
g)
Filtration
and
elution
dates
and
times
(
h)
Pellet
volume,
resuspended
concentrate
volume,
resuspended
concentrate
volume
transferred
to
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
15
IMS,
and
all
calculations
required
to
verify
the
percent
of
concentrate
examined
(
Section
13.2)
(
i)
Purification
completion
dates
and
times
(
Section
13.3.3.11)
(
j)
Staining
completion
dates
and
times
(
Section
14.10)
(
k)
Staining
control
results
(
Section
15.2.1)
(
l)
All
required
examination
information
(
Section
15.2.2)
(
m)
Examination
completion
dates
and
times
(
Section
15.2.4)
(
n)
Analysis
sequence/
run
chronology
(
o)
Lot
numbers
of
elution,
IMS,
and
staining
reagents
(
p)
Copies
of
bench
sheets,
logbooks,
and
other
recordings
of
raw
data
(
q)
Data
system
outputs,
and
other
data
to
link
the
raw
data
to
the
results
reported
9.1.3
The
laboratory
shall
spike
a
separate
sample
aliquot
from
the
same
source
to
monitor
method
performance.
This
MS
test
is
described
in
Section
9.5.1.
9.1.4
Analysis
of
method
blanks
is
required
to
demonstrate
freedom
from
contamination.
The
procedures
and
criteria
for
analysis
of
a
method
blank
are
described
in
Section
9.6.
9.1.5
The
laboratory
shall,
on
an
ongoing
basis,
demonstrate
through
analysis
of
the
ongoing
precision
and
recovery
(
OPR)
sample
that
the
analysis
system
is
in
control.
These
procedures
are
described
in
Section
9.7.
9.1.6
The
laboratory
shall
maintain
records
to
define
the
quality
of
data
that
are
generated.
Development
of
accuracy
statements
is
described
in
Sections
9.5.1.4
and
9.7.3.
9.1.7
The
laboratory
shall
analyze
one
method
blank
(
Section
9.6)
and
one
OPR
sample
(
Section
9.7)
each
week
during
which
samples
are
analyzed
if
20
or
fewer
field
samples
are
analyzed
during
this
period.
The
laboratory
shall
analyze
one
laboratory
blank
and
one
OPR
sample
for
every
20
samples
if
more
than
20
samples
are
analyzed
in
a
week.
9.1.8
The
laboratory
shall
analyze
MS
samples
(
Section
9.5.1)
at
a
minimum
frequency
of
1
MS
sample
per
20
field
samples
and
at
least
12
months
must
elapse
between
the
first
and
last
MS
sample.
The
laboratory
should
analyze
an
MS
sample
when
samples
are
first
received
from
a
PWS
for
which
the
laboratory
has
never
before
analyzed
samples
to
identify
potential
method
performance
issues
with
the
matrix
(
Section
9.5.1;
Tables
3
and
4).
If
an
MS
sample
cannot
be
analyzed
on
the
first
sampling
event,
the
first
MS
sample
should
be
analyzed
as
soon
as
possible
to
identify
potential
method
performance
issues
with
the
matrix.
9.2
Micropipette
calibration
9.2.1
Micropipettes
must
be
sent
to
the
manufacturer
for
calibration
annually.
Alternately,
a
qualified
independent
technician
specializing
in
micropipette
calibration
can
be
used,
or
the
calibration
can
be
performed
by
the
laboratory,
provided
the
laboratory
maintains
a
detailed
procedure
that
can
be
evaluated
by
an
independent
auditor.
Documentation
on
the
precision
of
the
recalibrated
micropipette
must
be
obtained
from
the
manufacturer
or
technician.
9.2.2
Internal
and
external
calibration
records
must
be
kept
on
file
in
the
laboratory's
QA
logbook.
9.2.3
If
a
micropipette
calibration
problem
is
suspected,
the
laboratory
shall
tare
an
empty
weighing
boat
on
the
analytical
balance
and
pipette
the
following
volumes
of
reagent
water
into
the
weigh
boat
using
the
pipette
in
question:
100%
of
the
maximum
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
16
dispensing
capacity
of
the
micropipette,
50%
of
the
capacity,
and
10%
of
the
capacity.
Ten
replicates
should
be
performed
at
each
weight.
Record
the
weight
of
the
water
(
assume
that
1.00
mL
of
reagent
water
weighs
1.00
g)
and
calculate
the
relative
standard
deviation
(
RSD)
for
each.
If
the
weight
of
the
reagent
water
is
within
1%
of
the
desired
weight
(
mL)
and
the
RSD
of
the
replicates
at
each
weight
is
within
1%,
then
the
pipette
remains
acceptable
for
use.
9.2.4
If
the
weight
of
the
reagent
water
is
outside
the
acceptable
limits,
consult
the
manufacturer's
instruction
manual
troubleshooting
section
and
repeat
steps
described
in
Section
9.2.3.
If
problems
with
the
pipette
persist,
the
laboratory
must
send
the
pipette
to
the
manufacturer
for
recalibration.
9.3
Microscope
adjustment
and
certification:
Adjust
the
microscope
as
specified
in
Section
10.0.
All
of
the
requirements
in
Section
10.0
must
be
met
prior
to
analysis
of
IPRs,
blanks,
OPRs,
field
samples,
and
MS/
MSDs.
9.4
Initial
precision
and
recovery
(
IPR)
 
To
establish
the
ability
to
demonstrate
control
over
the
analytical
system
and
to
generate
acceptable
precision
and
recovery,
the
laboratory
shall
perform
the
following
operations:
9.4.1
Using
the
spiking
procedure
in
Section
11.4
and
enumerated
spiking
suspensions
(
Section
7.10.1
or
Section
11.3),
spike,
filter,
elute,
concentrate,
separate
(
purify),
stain,
and
examine
four
reagent
water
samples
spiked
with
100
to
500
oocysts
and
100
to
500
cysts.
9.4.1.1
The
laboratory
is
permitted
to
analyze
the
four
spiked
reagent
samples
on
the
same
day
or
on
as
many
as
four
different
days
(
provided
that
the
spiked
reagent
samples
are
analyzed
consecutively),
and
also
may
use
different
analysts
and/
or
reagent
lots
for
each
sample
(
however,
the
procedures
used
for
all
analyses
must
be
identical).
Laboratories
should
note
that
the
variability
of
four
measurements
performed
on
multiple
days
or
using
multiple
analysts
or
reagent
lots
may
be
greater
than
the
variability
of
measurements
performed
on
the
same
day
with
the
same
analysts
and
reagent
lots.
As
a
result,
the
laboratory
is
at
a
greater
risk
of
generating
unacceptably
variable
IPR
results
if
the
test
is
performed
across
multiple
days,
analysts,
and
/
or
reagent
lots.
9.4.1.2
If
more
than
one
process
will
be
used
for
filtration
and/
or
separation
of
samples,
a
separate
set
of
IPR
samples
must
be
prepared
for
each
process.
9.4.1.3
The
set
of
four
IPR
tests
samples
must
be
accompanied
by
analysis
of
a
an
acceptable
method
blank
(
Section
9.6).
9.4.2
Using
results
of
the
four
analyses,
calculate
the
average
percent
recovery
and
the
relative
standard
deviation
(
RSD)
of
the
recoveries
for
Cryptosporidium
and
for
Giardia.
The
RSD
is
the
standard
deviation
divided
by
the
mean,
times
100.
9.4.3
Compare
the
RSD
and
the
mean
with
the
corresponding
limits
for
initial
precision
and
recovery
in
Tables
3
and
4
in
Section
21.0.
If
the
RSD
and
the
mean
meet
the
acceptance
criteria,
system
performance
is
acceptable
and
analysis
of
blanks
and
samples
may
begin.
If
the
RSD
or
the
mean
falls
outside
the
range
for
recovery,
system
performance
is
unacceptable.
In
this
event,
trouble­
shoot
the
problem
by
starting
at
the
end
of
the
method
(
see
guidance
in
Section
7.9.5
9.7.5),
correct
the
problem
and
repeat
the
test
(
Section
9.4.1).
9.5
Matrix
spike
(
MS)
and
matrix
spike
duplicate
(
MSD):
9.5.1
Matrix
spike
 
The
laboratory
shall
spike
and
analyze
a
separate
field
sample
aliquot
to
determine
the
effect
of
the
matrix
on
the
method's
oocyst
and
cyst
recovery.
The
MS
and
field
sample
must
be
collected
from
the
same
sampling
location
as
split
samples
or
as
samples
sequentially
collected
immediately
after
one
another.
The
MS
sample
volume
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
17
analyzed
must
be
within
10%
of
the
field
sample
volume.
The
MS
shall
be
analyzed
according
to
the
frequency
in
Section
9.1.8.
9.5.1.1
Analyze
an
unspiked
field
sample
according
to
the
procedures
in
Sections
12.0
to
15.0.
Using
the
spiking
procedure
in
Section
11.4
and
enumerated
spiking
suspensions
(
Section
7.10.1
or
Section
11.3),
spike,
filter,
elute,
concentrate,
separate
(
purify),
stain,
and
examine
a
second
field
sample
aliquot
with
a
similar
the
number
of
organisms
as
that
used
in
the
IPR
or
OPR
tests
(
Sections
9.4
and
9.7).
9.5.1.2
For
each
organism,
calculate
the
percent
recovery
(
R)
using
the
following
equation.

R
=
100
x
Nsp
­
Ns
T
where
R
is
the
percent
recovery
Nsp
is
the
number
of
oocysts
or
cysts
detected
in
the
spiked
sample
Ns
is
the
number
of
oocysts
or
cysts
detected
in
the
unspiked
sample
T
is
the
true
value
of
the
oocysts
or
cysts
spiked
9.5.1.3
Compare
the
recovery
for
each
organism
with
the
corresponding
limits
in
Tables
3
and
4
in
Section
21.0.

NOTE:
Some
sample
matrices
may
prevent
the
acceptance
criteria
in
Tables
3
and
4
from
being
met.
An
assessment
of
the
distribution
of
MS
recoveries
across
430
MS
samples
from
87
sites
during
the
ICR
Supplemental
Surveys
is
provided
in
Table
5.

9.5.1.4
As
part
of
the
QA
program
for
the
laboratory,
method
precision
for
samples
should
be
assessed
and
records
maintained.
After
the
analysis
of
five
samples
for
which
the
spike
recovery
for
each
organism
passes
the
tests
in
Section
9.5.1.3,
the
laboratory
should
calculate
the
average
percent
recovery
(
P)
and
the
standard
deviation
of
the
percent
recovery
(
sr).
Express
the
precision
assessment
as
a
percent
recovery
interval
from
P
!
2
sr
to
P
+
2
sr
for
each
matrix.
For
example,
if
P
=
80%
and
sr
=
30%,
the
accuracy
interval
is
expressed
as
20%
to
140%.
The
precision
assessment
should
be
updated
regularly
across
all
MS
samples
and
stratified
by
MS
samples
for
each
source.
9.5.2
Matrix
spike
duplicate
 
MSD
analysis
is
required
as
part
of
nationwide
approval
of
a
modified
version
of
this
method
to
demonstrate
that
the
modified
version
of
this
method
produces
results
equal
or
superior
to
results
produced
by
the
method
as
written
(
Section
9.1.2.1.2).
At
the
same
time
the
laboratory
spikes
and
analyzes
the
second
field
sample
aliquot
in
Section
9.5.1.1,
the
laboratory
shall
spike
and
analyze
a
third,
identical
field
sample
aliquot.

NOTE:
Matrix
spike
duplicate
samples
are
only
required
for
Tier
2
validation
studies.
They
are
recommended
for
Tier
1
validation,
but
not
required.

9.5.2.1
For
each
organism,
calculate
the
percent
recovery
(
R)
using
the
equation
in
Section
9.5.1.2.
9.5.2.2
Calculate
the
mean
of
the
number
of
oocysts
or
cysts
in
the
MS
and
MSD
(
Xmean)
(=
[
MS+
MSD]/
2).
Method
1623
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Cryptosporidium
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June
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18
9.5.2.3
Calculate
the
relative
percent
difference
(
RPD)
of
the
recoveries
using
the
following
equation:

RPD
=
100
x
|
NMS
­
NMSD
|

XMEAN
where
RPD
is
the
relative
percent
difference
NMS
is
the
number
of
oocysts
or
cysts
detected
in
the
MS
NMSD
is
the
number
of
oocysts
or
cysts
detected
in
the
MSD
Xmean
is
the
mean
number
of
oocysts
or
cysts
detected
in
the
MS
and
MSD
9.5.2.4
Compare
the
mean
MS/
MSD
recovery
and
RPD
with
the
corresponding
limits
in
Tables
3
and
4
in
Section
21.0
for
each
organism.
9.6
Method
blank
(
negative
control
sample,
laboratory
blank):
Reagent
water
blanks
are
analyzed
to
demonstrate
freedom
from
contamination.
Analyze
the
blank
immediately
prior
to
after
analysis
of
the
IPR
test
(
Section
9.4)
and
OPR
test
(
Section
9.7)
and
prior
to
analysis
of
samples
for
the
week
to
demonstrate
freedom
from
contamination.
9.6.1
Filter,
elute,
concentrate,
separate
(
purify),
stain,
and
examine
at
least
one
reagent
water
blank
per
week
(
Section
9.1.7)
according
to
the
procedures
in
Sections
12.0
to
15.0.
If
more
than
20
samples
are
analyzed
in
a
week,
process
and
analyze
one
reagent
water
blank
for
every
20
samples.
9.6.2
Actions
9.6.2.1
If
Cryptosporidium
oocysts,
Giardia
cysts,
or
potentially
interfering
organisms
or
materials
that
may
be
misidentified
as
oocysts
or
cysts
are
not
found
in
the
method
blank,
the
method
blank
test
is
acceptable
and
analysis
of
samples
may
proceed
Any
method
blank
in
which
oocysts
or
cysts
are
not
detected
is
assumed
to
be
uncontaminated
and
may
be
reported.
9.6.2.2
If
Cryptosporidium
oocysts,
or
Giardia
cysts
(
as
defined
in
Section
3)
or
any
potentially
interfering
organism
or
materials
that
may
be
misidentified
as
oocysts
or
cysts
are
found
in
the
method
blank,
the
method
blank
test
is
unacceptable.
Any
field
sample
in
a
batch
associated
with
an
unacceptable
method
blank
a
contaminated
blank
that
shows
the
presence
of
one
or
more
oocysts
or
cysts
is
assumed
to
be
contaminated
and
should
be
recollected,
if
possible.
Analysis
of
additional
samples
is
halted
until
the
source
of
contamination
is
eliminated,
the
method
blank
test
is
performed
again,
and
no
evidence
of
contamination
is
detected.
9.7
Ongoing
precision
and
recovery
(
OPR;
positive
control
sample;
laboratory
control
sample):
Using
the
spiking
procedure
in
Section
11.4
and
enumerated
spiking
suspensions
(
Section
7.10.1
or
Section
11.3),
filter,
elute,
concentrate,
separate
(
purify),
stain,
and
examine
at
least
one
reagent
water
sample
spiked
with
100
to
500
oocysts
and
100
to
500
cysts
each
week
to
verify
all
performance
criteria.
The
laboratory
must
analyze
one
OPR
sample
for
every
20
samples
if
more
than
20
samples
are
analyzed
in
a
week.
If
multiple
method
variations
are
used,
separate
OPR
samples
must
be
prepared
for
each
method
variation.
Adjustment
and/
or
recalibration
of
the
analytical
system
shall
be
performed
until
all
performance
criteria
are
met.
Only
after
all
performance
criteria
are
met
may
should
samples
be
analyzed.
9.7.1
Examine
the
slide
from
the
OPR
prior
to
analysis
of
samples
from
the
same
batch.
9.7.1.1
Using
200X
to
400X
magnification,
more
than
50%
of
the
oocysts
or
cysts
must
appear
undamaged
and
morphologically
intact;
otherwise,
the
Method
1623
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Cryptosporidium
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June
2003
19
organisms
in
the
spiking
suspension
may
be
of
unacceptable
quality
or
the
analytical
process
is
may
be
damaging
the
organisms.
Examine
the
spiking
suspension
organisms
directly
(
by
centrifuging,
if
possible,
to
concentrate
the
organisms
in
a
volume
that
can
be
applied
directly
to
a
slide).
If
the
organisms
appear
undamaged
and
morphologically
intact
under
DIC,
determine
the
step
or
reagent
that
is
causing
damage
to
the
organisms.
Correct
the
problem
and
repeat
the
OPR
test.
9.7.1.2
Identify
and
enumerate
each
organism
using
epifluorescence
microscopy.
The
first
three
presumptive
Cryptosporidium
oocysts
and
three
Giardia
cysts
identified
in
the
OPR
sample
must
be
examined
using
FITC,
DAPI,
and
DIC,
as
per
Section
15.2,
and
the
detailed
characteristics
(
size,
shape,
DAPI
category,
and
DIC
category)
reported
on
the
Cryptosporidium
and
Giardia
report
form,
as
well
as
any
additional
comments
on
organism
appearance,
if
notable.
9.7.2
For
each
organism,
calculate
the
percent
recovery
(
R)
using
the
following
equation:

R
=
100
x
N
T
where:
R
=
the
percent
recovery
N
=
the
number
of
oocysts
or
cysts
detected
T
=
the
number
of
oocysts
or
cysts
spiked
9.7.3
Compare
the
recovery
with
the
limits
for
ongoing
precision
and
recovery
in
Tables
3
and
4
in
Section
21.0.
9.7.4
Actions
9.7.4.1
If
the
recoveries
for
Cryptosporidium
and
Giardia
meet
the
acceptance
criteria,
system
performance
is
acceptable
and
analysis
of
blanks
and
samples
may
proceed.
9.7.4.2
If,
however,
the
recovery
for
Cryptosporidium
or
Giardia
falls
outside
of
the
criteria
range
given,
system
performance
is
unacceptable.
Any
sample
in
a
batch
associated
with
an
unacceptable
OPR
sample
is
unacceptable.
Analysis
of
additional
samples
is
halted
until
the
analytical
system
is
brought
under
control.
In
this
event,
there
may
be
a
problem
with
the
microscope
or
with
the
filtration
or
separation
systems.
Troubleshoot
the
problem
using
the
procedures
at
Section
9.7.5
as
a
guide.
After
assessing
the
issue,
perform
another
OPR
test
and
verify
that
Cryptosporidium
and
Giardia
recoveries
meet
the
acceptance
criteria.
All
samples
must
be
associated
with
an
OPR
that
passes
the
criteria
in
Section
21.0.
Samples
that
are
not
associated
with
an
acceptable
OPR
must
be
flagged
accordingly.
9.7.5
Troubleshooting.
If
an
OPR
sample
has
failed,
and
the
cause
of
the
failure
is
not
known,
the
laboratory
generally
should
identify
the
problem
working
backward
in
the
analytical
process
from
the
microscopic
examination
to
filtration.
9.7.5.1
Quality
of
spiked
organisms.
Examine
the
spiking
suspension
organisms
directly
(
by
centrifuging,
if
possible,
to
concentrate
the
organisms
in
a
volume
that
can
be
applied
directly
to
a
slide).
If
the
organisms
appear
damaged
under
DIC,
obtain
fresh
spiking
materials.
If
the
organisms
appear
undamaged
and
morphologically
intact,
determined
whether
the
problem
is
associated
with
the
microscope
system
or
antibody
stain
(
Section
9.7.5.2).
Method
1623
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Cryptosporidium
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Giardia
June
2003
20
9.7.5.2
Microscope
system
and
antibody
stain:
To
determine
if
the
failure
of
the
OPR
test
is
due
to
changes
in
the
microscope
or
problems
with
the
antibody
stain,
re­
examine
the
positive
staining
control
(
Section
15.2.1),
check
Köhler
illumination,
and
check
the
fluorescence
of
the
fluoresceinlabeled
monoclonal
antibodies
(
Mabs)
and
4',
6­
diamidino­
2­
phenylindole
(
DAPI).
If
results
are
unacceptable,
re­
examine
the
previously­
prepared
positive
staining
control
to
determine
whether
the
problem
is
associated
with
the
microscope
or
the
antibody
stain.
9.7.5.3
Separation
(
purification)
system:
To
determine
if
the
failure
of
the
OPR
test
is
attributable
to
the
separation
system,
check
system
performance
by
spiking
a
10­
mL
volume
of
reagent
water
with
100
­
500
oocysts
and
cysts
and
processing
the
sample
through
the
IMS,
staining,
and
examination
procedures
in
Sections
13.3
through
15.0.
Recoveries
should
be
greater
than
70%.
9.7.5.4
Filtration/
elution/
concentration
system:
If
the
failure
of
the
OPR
test
is
attributable
to
the
filtration/
elution/
concentration
system,
check
system
performance
by
processing
spiked
reagent
water
according
to
the
procedures
in
Section
12.2
through
13.2.2.1
13.2.2,
and
filter,
stain,
and
examine
the
sample
concentrate
according
to
Section
11.3.6.
9.7.6
The
laboratory
should
add
results
that
pass
the
specifications
in
Section
9.7.3
to
initial
and
previous
ongoing
data
and
update
the
QC
chart
to
form
a
graphic
representation
of
continued
laboratory
performance.
The
laboratory
should
develop
a
statement
of
laboratory
accuracy
(
reagent
water,
raw
surface
water)
by
calculating
the
average
percent
recovery
(
R)
and
the
standard
deviation
of
percent
recovery
(
sr).
Express
the
accuracy
as
a
recovery
interval
from
R
!
2
sr
to
R
+
2
sr.
For
example,
if
R
=
95%
and
sr
=
25%,
the
accuracy
is
45%
to
145%.
9.8
The
laboratory
should
periodically
analyze
an
external
QC
sample,
such
as
a
performance
evaluation
or
standard
reference
material,
when
available.
The
laboratory
also
should
periodically
participate
in
interlaboratory
comparison
studies
using
the
method.
9.9
The
specifications
contained
in
this
method
can
be
met
if
the
analytical
system
is
under
control.
The
standards
used
for
initial
(
Section
9.4)
and
ongoing
(
Section
9.7)
precision
and
recovery
should
be
identical,
so
that
the
most
precise
results
will
be
obtained.
The
microscope
in
particular
will
provide
the
most
reproducible
results
if
dedicated
to
the
settings
and
conditions
required
for
the
determination
of
Cryptosporidium
and
Giardia
by
this
method.
9.10
Depending
on
specific
program
requirements,
field
replicates
may
be
collected
to
determine
the
precision
of
the
sampling
technique,
and
duplicate
spiked
samples
may
be
required
to
determine
the
precision
of
the
analysis.

10.0
Microscope
Calibration
and
Analyst
Verification
10.1
In
a
room
capable
of
being
darkened
to
near­
complete
darkness,
assemble
the
microscope,
all
filters,
and
attachments.
The
microscope
should
be
placed
on
a
solid
surface
free
from
vibration.
Adequate
workspace
should
be
provided
on
either
side
of
the
microscope
for
taking
notes
and
placement
of
slides
and
ancillary
materials.
10.2
Using
the
manuals
provided
with
the
microscope,
all
analysts
must
familiarize
themselves
with
operation
of
the
microscope.
10.3
Microscope
adjustment
and
calibration
(
adapted
from
Reference
20.7)
10.3.1
Preparations
for
adjustment
10.3.1.1
The
microscopy
portion
of
this
procedure
depends
upon
proper
alignment
and
adjustment
of
very
sophisticated
optics.
Without
proper
alignment
and
adjustment,
the
microscope
will
not
function
at
maximal
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
21
efficiency,
and
reliable
identification
and
enumeration
of
oocysts
and
cysts
will
not
be
possible.
Consequently,
it
is
imperative
that
all
portions
of
the
microscope
from
the
light
sources
to
the
oculars
are
properly
adjusted.
10.3.1.2
While
microscopes
from
various
vendors
are
configured
somewhat
differently,
they
all
operate
on
the
same
general
physical
principles.
Therefore,
slight
deviations
or
adjustments
may
be
required
to
make
the
procedures
below
work
for
a
particular
instrument.
10.3.1.3
The
sections
below
assume
that
the
mercury
bulb
has
not
exceeded
time
limits
of
operation,
that
the
lamp
socket
is
connected
to
the
lamp
house,
and
that
the
condenser
is
adjusted
to
produce
Köhler
illumination.
10.3.1.4
Persons
with
astigmatism
should
always
wear
contact
lenses
or
glasses
when
using
the
microscope.

CAUTION:
In
the
procedures
below,
do
not
touch
the
quartz
portion
of
the
mercury
bulb
with
your
bare
fingers.
Finger
oils
can
cause
rapid
degradation
of
the
quartz
and
premature
failure
of
the
bulb.

WARNING:
Never
look
at
the
ultraviolet
(
UV)
light
from
the
mercury
lamp,
lamp
house,
or
the
UV
image
without
a
barrier
filter
in
place.
UV
radiation
can
cause
serious
eye
damage.

10.3.2
Epifluorescent
mercury
bulb
adjustment:
The
purpose
of
this
procedure
is
to
ensure
even
field
illumination.
This
procedure
must
be
followed
when
the
microscope
is
first
used,
when
replacing
bulbs,
and
if
problems
such
as
diminished
fluorescence
or
uneven
field
illumination
are
experienced.
10.3.2.1
Remove
the
diffuser
lens
between
the
lamp
and
microscope
or
swing
it
out
of
the
transmitted
light
path.
10.3.2.2
Using
a
prepared
microscope
slide,
adjust
the
focus
so
the
image
in
the
oculars
is
sharply
defined.
10.3.2.3
Replace
the
slide
with
a
business
card
or
a
piece
of
lens
paper.
10.3.2.4
Close
the
field
diaphragm
(
iris
diaphragm
in
the
microscope
base)
so
only
a
small
point
of
light
is
visible
on
the
card.
This
dot
of
light
indicates
the
location
of
the
center
of
the
field
of
view.
10.3.2.5
Mount
the
mercury
lamp
house
on
the
microscope
without
the
UV
diffuser
lens
in
place
and
turn
on
the
mercury
bulb.
10.3.2.6
Remove
the
objective
in
the
light
path
from
the
nosepiece.
A
primary
(
brighter)
and
secondary
image
(
dimmer)
of
the
mercury
bulb
arc
should
appear
on
the
card
after
focusing
the
image
with
the
appropriate
adjustment.
10.3.2.7
Using
the
lamp
house
adjustments,
adjust
the
primary
and
secondary
mercury
bulb
images
so
they
are
side
by
side
(
parallel
to
each
other)
with
the
transmitted
light
dot
in
between
them.
10.3.2.8
Reattach
the
objective
to
the
nosepiece.
10.3.2.9
Insert
the
diffuser
lens
into
the
light
path
between
the
mercury
lamp
house
and
the
microscope.
10.3.2.10
Turn
off
the
transmitted
light
and
replace
the
card
with
a
slide
of
fluorescent
material.
Check
the
field
for
even
fluorescent
illumination.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
22
Adjustment
of
the
diffuser
lens
probably
will
be
required.
Additional
slight
adjustments
as
in
Section
10.3.2.7
above
may
be
required.
10.3.2.11
Maintain
a
log
of
the
number
of
hours
the
UV
bulb
has
been
used.
Never
use
the
bulb
for
longer
than
it
has
been
rated.
Fifty­
watt
bulbs
should
not
be
used
longer
than
100
hours;
100­
watt
bulbs
should
not
be
used
longer
than
200
hours.
10.3.3
Transmitted
bulb
adjustment:
The
purpose
of
this
procedure
is
to
center
the
filament
and
ensure
even
field
illumination.
This
procedure
must
be
followed
when
the
bulb
is
changed.
10.3.3.1
Remove
the
diffuser
lens
between
the
lamp
and
microscope
or
swing
it
out
of
the
transmitted
light
path.
10.3.3.2
Using
a
prepared
microscope
slide
and
a
40X
(
or
similar)
objective,
adjust
the
focus
so
the
image
in
the
oculars
is
sharply
defined.
10.3.3.3
Without
the
ocular
or
Bertrand
optics
in
place,
view
the
pupil
and
filament
image
at
the
bottom
of
the
tube.
10.3.3.4
Focus
the
lamp
filament
image
with
the
appropriate
adjustment
on
the
lamp
house.
10.3.3.5
Similarly,
center
the
lamp
filament
image
within
the
pupil
with
the
appropriate
adjustment(
s)
on
the
lamp
house.
10.3.3.6
Insert
the
diffuser
lens
into
the
light
path
between
the
transmitted
lamp
house
and
the
microscope.
10.3.4
Adjustment
of
the
interpupillary
distance
and
oculars
for
each
eye:
These
adjustments
are
necessary
so
that
eye
strain
is
reduced
to
a
minimum,
and
must
be
made
for
each
individual
using
the
microscope.
Section
10.3.4.2
assumes
use
of
a
microscope
with
both
oculars
adjustable;
Section
10.3.4.3
assumes
use
of
a
microscope
with
a
single
adjustable
ocular.
The
procedure
must
be
followed
each
time
an
analyst
uses
the
microscope.
10.3.4.1
Interpupillary
distance
10.3.4.1.1
Place
a
prepared
slide
on
the
microscope
stage,
turn
on
the
transmitted
light,
and
focus
the
specimen
image
using
the
coarse
and
fine
adjustment
knobs.
10.3.4.1.2
Using
both
hands,
move
the
oculars
closer
together
or
farther
apart
until
a
single
circle
of
light
is
observed
while
looking
through
the
oculars
with
both
eyes.
Note
interpupillary
distance.
10.3.4.2
Ocular
adjustment
for
microscopes
capable
of
viewing
a
photographic
frame
through
the
viewing
binoculars:
This
procedure
assumes
both
oculars
are
adjustable.
10.3.4.2.1
Place
a
card
between
the
right
ocular
and
eye
keeping
both
eyes
open.
Adjust
the
correction
(
focusing)
collar
on
the
left
ocular
by
focusing
the
left
ocular
until
it
reads
the
same
as
the
interpupillary
distance.
Bring
an
image
located
in
the
center
of
the
field
of
view
into
as
sharp
a
focus
as
possible.
10.3.4.2.2
Transfer
the
card
to
between
the
left
eye
and
ocular.
Again
keeping
both
eyes
open,
bring
the
same
image
into
as
sharp
a
focus
for
the
right
eye
as
possible
by
adjusting
the
ocular
correction
(
focusing)
collar
at
the
top
of
the
right
ocular.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
23
10.3.4.3
Ocular
adjustment
for
microscopes
without
binocular
capability:
This
procedure
assumes
a
single
focusing
ocular.
The
following
procedure
assumes
that
only
the
right
ocular
is
capable
of
adjustment.
10.3.4.3.1
Place
a
card
between
the
right
ocular
and
eye
keeping
both
eyes
open.
Using
the
fine
adjustment,
focus
the
image
for
the
left
eye
to
its
sharpest
point.
10.3.4.3.2
Transfer
the
card
to
between
the
left
eye
and
ocular.
Keeping
both
eyes
open,
bring
the
image
for
the
right
eye
into
sharp
focus
by
adjusting
the
ocular
collar
at
the
top
of
the
ocular
without
touching
the
coarse
or
fine
adjustment.
10.3.5
Calibration
of
an
ocular
micrometer:
This
section
assumes
that
a
reticle
has
been
installed
in
one
of
the
oculars
by
a
microscopy
specialist
and
that
a
stage
micrometer
is
available
for
calibrating
the
ocular
micrometer
(
reticle).
Once
installed,
the
ocular
reticle
should
be
left
in
place.
The
more
an
ocular
is
manipulated
the
greater
the
probability
is
for
it
to
become
contaminated
with
dust
particles.
This
calibration
should
be
done
for
each
objective
in
use
on
the
microscope.
If
there
is
a
top
lens
on
the
microscope,
the
calibration
procedure
must
be
done
for
the
respective
objective
at
each
top
lens
setting.
The
procedure
must
be
followed
when
the
microscope
is
first
used
and
each
time
the
objective
is
changed.
10.3.5.1
Place
the
stage
micrometer
on
the
microscope
stage,
turn
on
the
transmitted
light,
and
focus
the
micrometer
image
using
the
coarse
and
fine
adjustment
knobs
for
the
objective
to
be
calibrated.
Continue
adjusting
the
focus
on
the
stage
micrometer
so
you
can
distinguish
between
the
large
(
0.1
mm)
and
the
small
(
0.01
mm)
divisions.
10.3.5.2
Adjust
the
stage
and
ocular
with
the
micrometer
so
the
"
0"
line
on
the
ocular
micrometer
is
exactly
superimposed
on
the
"
0"
line
on
the
stage
micrometer.
10.3.5.3
Without
changing
the
stage
adjustment,
find
a
point
as
distant
as
possible
from
the
two
0
lines
where
two
other
lines
are
exactly
superimposed.
10.3.5.4
Determine
the
number
of
ocular
micrometer
spaces
as
well
as
the
number
of
millimeters
on
the
stage
micrometer
between
the
two
points
of
superimposition.
For
example:
Suppose
48
ocular
micrometer
spaces
equal
0.6
mm.
10.3.5.5
Calculate
the
number
of
mm/
ocular
micrometer
space.
For
example:

0.6
mm
=
0.0125
mm
48
ocular
micrometer
spaces
ocular
micrometer
space
10.3.5.6
Because
most
measurements
of
microorganisms
are
given
in
µ
m
rather
than
mm,
the
value
calculated
above
must
be
converted
to
µ
m
by
multiplying
it
by
1000
µ
m/
mm.
For
example:

0.0125
mm
x
1,000
µ
m
=
12.5
µ
m
ocular
micrometer
space
mm
ocular
micrometer
space
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
24
10.3.5.7
Follow
the
procedure
below
for
each
objective.
Record
the
information
as
shown
in
the
example
below
and
keep
the
information
available
at
the
microscope.

Item
no.
Objective
power
Description
No.
of
ocular
micrometer
spaces
No.
of
stage
micrometer
mm1
µ
m/
ocular
micrometer
space2
1
10X
N.
A.
3=

2
20X
N.
A.=

3
40X
N.
A.=

4
100X
N.
A.=

11000
µ
m/
mm
2(
Stage
micrometer
length
in
mm
×
(
1000
µ
m/
mm))
÷
no.
ocular
micrometer
spaces
3N.
A.
refers
to
numerical
aperature.
The
numerical
aperature
value
is
engraved
on
the
barrel
of
the
objective.

10.3.6
Köhler
illumination:
This
section
assumes
that
Köhler
illumination
will
be
established
for
only
the
100X
oil
DIC
objective
that
will
be
used
to
identify
internal
morphological
characteristics
in
Cryptosporidium
oocysts
and
Giardia
cysts.
If
more
than
one
objective
is
to
be
used
for
DIC,
then
each
time
the
objective
is
changed,
Köhler
illumination
must
be
reestablished
for
the
new
objective
lens.
Previous
sections
have
adjusted
oculars
and
light
sources.
This
section
aligns
and
focuses
the
light
going
through
the
condenser
underneath
the
stage
at
the
specimen
to
be
observed.
If
Köhler
illumination
is
not
properly
established,
then
DIC
will
not
work
to
its
maximal
potential.
These
steps
need
to
become
second
nature
and
must
be
practiced
regularly
until
they
are
a
matter
of
reflex
rather
than
a
chore.
The
procedure
must
be
followed
each
time
an
analyst
uses
the
microscope
and
each
time
the
objective
is
changed.
10.3.6.1
Place
a
prepared
slide
on
the
microscope
stage,
place
oil
on
the
slide,
move
the
100X
oil
objective
into
place,
turn
on
the
transmitted
light,
and
focus
the
specimen
image
using
the
coarse
and
fine
adjustment
knobs.
10.3.6.2
At
this
point
both
the
radiant
field
diaphragm
in
the
microscope
base
and
the
aperture
diaphragm
in
the
condenser
should
be
wide
open.
Now
close
down
the
radiant
field
diaphragm
in
the
microscope
base
until
the
lighted
field
is
reduced
to
a
small
opening.
10.3.6.3
Using
the
condenser
centering
screws
on
the
front
right
and
left
of
the
condenser,
move
the
small
lighted
portion
of
the
field
to
the
center
of
the
visual
field.
10.3.6.4
Now
look
to
see
whether
the
leaves
of
the
iris
field
diaphragm
are
sharply
defined
(
focused)
or
not.
If
they
are
not
sharply
defined,
then
they
can
be
focused
distinctly
by
changing
the
height
of
the
condenser
up
and
down
with
the
condenser
focusing
knob
while
you
are
looking
through
the
binoculars.
Once
you
have
accomplished
the
precise
focusing
of
the
radiant
field
diaphragm
leaves,
open
the
radiant
field
diaphragm
until
the
leaves
just
disappear
from
view.
10.3.6.5
The
aperture
diaphragm
of
the
condenser
should
is
now
be
adjusted
to
make
it
compatible
with
the
total
numerical
aperture
of
the
optical
system.
This
is
done
by
removing
an
ocular,
looking
into
the
tube
at
the
rear
focal
plane
of
the
objective,
and
stopping
down
the
aperture
diaphragm
iris
leaves
until
they
are
visible
just
inside
the
rear
plane
of
the
objective.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
25
10.3.6.6
After
completing
the
adjustment
of
the
aperture
diaphragm
in
the
condenser,
return
the
ocular
to
its
tube
and
proceed
with
the
adjustments
required
to
establish
DIC.
10.4
Microscope
cleaning
procedure
10.4.1
Use
canned
air
to
remove
dust
from
the
lenses,
filters,
and
microscope
body.
10.4.2
Use
a
Kimwipe­
dampened
with
a
microscope
cleaning
solution
(
MCS)
(
consisting
of
2
parts
90%
isoproponal
and
1
part
acetone)
to
wipe
down
all
surfaces
of
the
microscope
body.
Dry
off
with
a
clean,
dry
Kimwipe.
10.4.3
Protocol
for
cleaning
oculars
and
condenser
10.4.3.1
Use
a
new,
clean
Q­
tip
dampened
with
MCS
to
clean
each
lense.
Start
at
the
center
of
the
lens
and
spiral
the
Q­
tip
outward
using
little
to
no
pressure.
Rotate
the
Q­
tip
head
while
spiraling
to
ensure
a
clean
surface
is
always
contacting
the
lens.
10.4.3.2
Repeat
the
procedure
using
a
new,
dry
Q­
tip.
10.4.3.3
Repeat
Sections
10.4.3.1
and
10.4.3.2.
10.4.3.4
Remove
the
ocular
and
repeat
the
cleaning
procedure
on
the
bottom
lens
of
the
ocular.
10.4.4
Protocol
for
cleaning
objective
lenses
10.4.4.1
Wipe
100X
oil
objective
with
lense
paper
to
remove
the
bulk
of
the
oil
from
the
objective.
10.4.4.2
Hold
a
new
Q­
tip
dampened
with
MCS
at
a
45
°
angle
on
the
objective
and
twirl.
10.4.4.3
Repeat
Sections
10.4.4.2
with
a
new,
dry
Q­
tip.
10.4.4.4
Repeat
Sections
10.4.4.2
and
10.4.4.3.
10.4.4.5
Clean
all
objectives
whether
they
are
used
or
not.
10.4.5
Protocol
for
cleaning
light
source
lens
and
filters
10.4.5.1
Using
a
Kimwipe
dampened
with
microscope
cleaning
solution,
wipe
off
the
surface
of
each
lens
and
filter.
10.4.5.2
Repeat
the
procedure
using
a
dry
Kimwipe.
10.4.5.3
Repeat
Sections
10.4.5.1
and
10.4.5.2.
10.4.6
Protocol
for
cleaning
microscope
stage
10.4.6.1
Using
a
Kimwipe
dampened
with
microscope
cleaning
solution,
wipe
off
the
stage
and
stage
clip.
Be
sure
to
clean
off
any
residual
immersion
oil
or
fingernail
polish.
Remove
the
stage
clip
if
necessary
to
ensure
that
it
is
thoroughly
cleaned.
10.4.7
Use
409
and
a
paper
towel
to
clean
the
bench
top
surrounding
the
microscope.
10.4.8
Frequency
10.4.8.1
Perform
Sections
10.4.2,
10.4.3,
10.4.4,
10.4.5
and
10.4.7
after
each
microscope
session.
10.4.8.2
Perform
complete
cleaning
each
week.
10.5
Protozoa
libraries:
Each
laboratory
is
encouraged
to
develop
libraries
of
photographs
and
drawings
for
identification
of
protozoa.
10.5.1
Take
color
photographs
of
Cryptosporidium
oocysts
and
Giardia
cysts
by
FA,
and
4',
6­
diamidino­
2­
phenylindole
(
DAPI),
and
DIC
that
the
analysts
(
Section
22.2)
determine
are
accurate
(
Section
15.2).
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
26
10.5.2
Similarly,
take
color
photographs
of
interfering
organisms
and
materials
by
FA,
and
DAPI
,
and
DIC
that
the
analysts
believe
are
not
Cryptosporidium
oocysts
or
Giardia
cysts.
Quantify
the
size,
shape,
microscope
settings,
and
other
characteristics
that
can
be
used
to
differentiate
oocysts
and
cysts
from
interfering
debris
and
that
will
result
in
accurate
positive
identification
of
DAPI
positive
or
negative
organisms.
10.6
Verification
of
analyst
performance:
Until
standard
reference
materials,
such
as
National
Institute
of
Standards
and
Technology
standard
reference
materials,
are
available
that
contain
a
reliable
number
of
DAPI
positive
or
negative
oocysts
and
cysts,
this
method
shall
rely
upon
the
ability
of
the
analyst
for
identification
and
enumeration
of
oocysts
and
cysts.
10.6.1
At
least
monthly
when
microscopic
examinations
are
being
performed,
the
laboratory
shall
prepare
a
slide
containing
40
to
100
oocysts
and
40
to
100
cysts.
More
than
50%
of
the
oocysts
and
cysts
must
be
DAPI
positive
and
undamaged
under
DIC.
10.6.2
Each
analyst
shall
determine
the
total
number
of
oocysts
and
cysts
and
the
number
that
are
DAPI
positive
and
DAPI
negative
for
the
entire
slide.
For
10
oocysts
and
10
cysts,
each
analyst
shall
determine
using
the
slide
prepared
in
Section
10.5.1.
the
number
of
nuclei
by
DAPI
and
the
DIC
category
(
empty,
containing
amorphous
structures,
or
containing
identifiable
internal
structures)
of
each.
10.6.3
Requirements
for
laboratories
with
multiple
analysts
10.6.3.1
The
total
number
and
the
number
of
DAPI
positive
or
and
DAPI
negative
oocysts
and
cysts
determined
by
each
analyst
(
Section
10.5.2.)
must
be
within
±
10%
of
each
other.
If
the
number
is
not
within
this
range,
the
analysts
must
identify
the
source
of
any
variability
between
analysts'
examination
criteria,
prepare
a
new
slide,
and
repeat
the
performance
verification
(
Sections
10.5.1
to
10.5.2).
Differences
in
the
number
of
nuclei
by
DAPI
and
in
DIC
categorizations
among
analysts
must
be
discussed
and
resolved,
and
these
resolutions
must
be
documented.
10.6.3.2
Document
the
date,
name(
s)
of
analyst(
s),
number
of
total,
DAPI
positive
or
negative
oocysts
and
cysts
determined
by
the
analyst(
s),
whether
the
test
was
passed/
failed
and
the
results
of
attempts
before
the
test
was
passed.
10.6.3.3
Only
after
an
analyst
has
passed
the
criteria
in
Section
10.6.3,
may
oocysts
and
cysts
in
QC
samples
and
field
samples
be
identified
and
enumerated.
10.6.4
Laboratories
with
only
one
analyst
should
maintain
a
protozoa
library
(
Section
10.5)
and
compare
the
results
of
the
examinations
performed
in
Sections
10.6.1
and
10.6.2
to
photographs
of
oocysts
and
cysts
and
interfering
organisms
to
verify
that
examination
results
are
consistent
with
these
references.
These
laboratories
also
should
perform
repetitive
counts
of
a
single
verification
slide
for
FITC
and
DAPI.
These
laboratories
should
also
coordinate
with
other
laboratories
to
share
slides
and
compare
counts.

11.0
Oocyst
and
Cyst
Suspension
Enumeration
and
Spiking
11.1
This
method
requires
routine
analysis
of
spiked
QC
samples
to
demonstrate
acceptable
initial
and
ongoing
laboratory
and
method
performance
(
initial
precision
and
recovery
samples
[
Section
9.4],
matrix
spike
and
matrix
spike
duplicate
samples
[
Section
9.5],
and
ongoing
precision
and
recovery
samples
[
Section
9.7]).
The
organisms
used
for
these
samples
must
be
enumerated
to
calculate
recoveries
and
precision.
EPA
recommends
that
flow
cytometry
be
used
for
this
enumeration,
rather
than
manual
techniques.
Flow
cytometer
 
sorted
spikes
generally
are
characterized
by
a
relative
standard
deviation
of
#
2.5%,
versus
greater
variability
for
manual
enumeration
techniques
(
Reference
20.8).
Guidance
on
preparing
spiking
suspensions
using
a
flow
cytometer
is
provided
in
Section
11.2.
Manual
enumeration
procedures
are
provided
in
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
27
Section
11.3.
The
procedure
for
spiking
bulk
samples
in
the
laboratory
is
provided
in
Section
11.4.
11.2
Flow
cytometry
enumeration
guidelines.
Although
it
is
unlikely
that
many
laboratories
performing
Method
1623
will
have
direct
access
to
a
flow
cytometer
for
preparing
spiking
suspensions,
flow­
sorted
suspensions
are
available
from
commercial
vendors
and
other
sources
(
Section
7.10.1).
The
information
provided
in
Sections
11.2.1
through
11.2.4
is
simply
meant
as
a
guideline
for
preparing
spiking
suspensions
using
a
flow
cytometer.
Laboratories
performing
flow
cytometry
must
develop
and
implement
detailed
standardized
protocols
for
calibration
and
operation
of
the
flow
cytometer.
11.2.1
Spiking
suspensions
should
be
prepared
using
unstained
organisms
that
have
not
been
heat­
fixed
or
formalin­
fixed.
11.2.2
Spiking
suspensions
should
be
prepared
using
Cryptosporidium
parvum
oocysts
<
3
months
old,
and
Giardia
intestinalis
cysts
<
2
weeks
old.
11.2.3
Initial
calibration.
Immediately
before
sorting
spiking
suspensions,
an
initial
calibration
of
the
flow
cytometer
should
be
performed
by
conducting
10
sequential
sorts
directly
onto
membranes
or
well
slides.
The
oocyst
and
cyst
levels
used
for
the
initial
calibration
should
be
the
same
as
the
levels
used
for
the
spiking
suspensions.
Each
initial
calibration
sample
should
be
stained
and
manually
counted
microscopically
and
the
manual
counts
used
to
verify
the
accuracy
of
the
system.
The
relative
standard
deviation
(
RSD)
of
the
10
counts
should
be
#
2.5%.
If
the
RSD
is
>
2.5%,
the
laboratory
should
perform
the
initial
calibration
again,
until
the
RSD
of
the
10
counts
is
#
2.5%.
In
addition
to
counting
the
organisms,
the
laboratory
also
should
evaluate
the
quality
of
the
organisms
using
DAPI
and
DIC
to
confirm
that
the
organisms
are
in
good
condition.
11.2.4
Ongoing
calibration.
When
sorting
the
spiking
suspensions
for
use
in
QC
samples,
the
laboratory
should
perform
ongoing
calibration
samples
at
a
10%
frequency,
at
a
minimum.
The
laboratory
should
sort
the
first
run
and
every
eleventh
sample
directly
onto
a
membrane
or
well
slide.
Each
ongoing
calibration
sample
should
be
stained
and
manually
counted
microscopically
and
the
manual
counts
used
to
verify
the
accuracy
of
the
system.
The
mean
of
the
ongoing
calibration
counts
also
should
be
used
as
the
estimated
spike
dose,
if
the
relative
standard
deviation
(
RSD)
of
the
ongoing
calibration
counts
is
#
2.5%.
If
the
RSD
is
>
2.5%,
the
laboratory
should
discard
the
batch.
11.2.5
Method
blanks.
Depending
on
the
operation
of
the
flow
cytometer,
method
blanks
should
be
prepared
and
examined
at
the
same
frequency
as
the
ongoing
calibration
samples
(
Section
11.2.4).
11.2.6
Holding
time
criteria.
Flow­
cytometer­
sorted
spiking
suspensions
(
Sections
7.10.1
and
11.2)
used
for
spiked
quality
control
(
QC)
samples
(
Section
9)
must
be
used
within
the
expiration
date
noted
on
the
suspension.
The
holding
time
specified
by
the
flow
cytometry
laboratory
should
be
determined
based
on
a
holding
time
study.
Laboratories
should
use
flow­
cytometer­
sorted
spiking
suspensions
containing
live
organisms
within
two
weeks
of
preparation
at
the
flow
cytometry
laboratory.
11.3
Manual
enumeration
procedures.
Two
sets
of
manual
enumerations
are
required
per
organism
before
purified
Cryptosporidium
oocyst
and
Giardia
cyst
stock
suspensions
(
Sections
7.9.2.1
and
7.9.2.2
Section
7.10.2)
received
from
suppliers
can
be
used
to
spike
samples
in
the
laboratory.
First,
the
stock
suspension
must
be
diluted
and
enumerated
(
Section
11.3.3)
to
yield
a
suspension
at
the
appropriate
oocyst
or
cyst
concentration
for
spiking
(
spiking
suspension).
Then,
10
aliquots
of
spiking
suspension
must
be
enumerated
to
calculate
a
mean
spike
dose.
Spiking
suspensions
can
be
enumerated
using
hemacytometer
chamber
counting
(
Section
11.3.4),
well
slide
counting
(
Section
11.3.5),
or
membrane
filter
counting
(
Section
11.3.6).
11.3.1
Precision
criteria.
The
relative
standard
deviation
(
RSD)
of
the
calculated
mean
spike
dose
for
manually
enumerated
spiking
suspensions
must
be
#
16%
for
Cryptosporidium
and
#
19%
for
Giardia
before
proceeding
(
these
criteria
are
based
on
the
pooled
RSDs
of
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
28
105
manual
Cryptosporidium
enumerations
and
104
manual
Giardia
enumerations
submitted
by
20
different
laboratories
under
the
EPA
Protozoa
Performance
Evaluation
Program).
11.3.2
Holding
time
criteria.
Manually
enumerated
spiking
suspensions
must
be
used
within
24
hours
of
enumeration
of
the
spiking
suspension
if
the
hemacytometer
chamber
technique
is
used
(
Section
11.3.4);
or
within
24
hours
of
application
of
the
spiking
suspension
or
membrane
filter
to
the
slides
if
the
well
slide
or
membrane
filter
enumeration
technique
is
used
(
Sections
11.3.5
and
11.3.6).
11.3.3
Enumerating
and
diluting
stock
suspensions
11.3.3.1
Purified,
concentrated
stock
suspensions
(
Sections
7.10.2.1
and
7.10.2.2)
must
be
diluted
and
enumerated
before
the
diluted
suspensions
are
used
to
spike
samples
in
the
laboratory.
Stock
suspensions
should
be
diluted
with
reagent
water/
Tween­
20,
0.01%
(
Section
7.10.2.3),
to
a
concentration
of
20
to
50
organisms
per
large
hemacytometer
square
before
proceeding
to
Section
11.3.3.2.
11.3.3.2
Apply
a
clean
hemacytometer
coverslip
(
Section
6.4.5)
to
the
hemacytometer
and
load
the
hemacytometer
chamber
with
10
µ
L
of
vortexed
suspension
per
chamber.
If
this
operation
has
been
properly
executed,
the
liquid
should
amply
fill
the
entire
chamber
without
bubbles
or
overflowing
into
the
surrounding
moats.
Repeat
this
step
with
a
clean,
dry
hemacytometer
and
coverslip
if
loading
has
been
incorrectly
performed.
See
Section
11.3.3.13,
below,
for
the
hemacytometer
cleaning
procedure.
11.3.3.3
Place
the
hemacytometer
on
the
microscope
stage
and
allow
the
oocysts
or
cysts
to
settle
for
2
minutes.
Do
not
attempt
to
adjust
the
coverslip,
apply
clips,
or
in
any
way
disturb
the
chamber
after
it
has
been
filled.
11.3.3.4
Use
200X
magnification.
11.3.3.5
Move
the
chamber
so
the
ruled
area
is
centered
underneath
it
the
objective.
11.3.3.6
Move
the
objective
close
to
the
coverslip
while
watching
it
from
the
side
of
the
microscope,
rather
than
through
the
microscope.
11.3.3.7
Focus
up
from
the
coverslip
until
the
hemacytometer
ruling
appears.
11.3.3.8
At
each
of
the
four
corners
of
the
chamber
is
a
1­
square­
mm
area
divided
into
16
squares
in
which
organisms
are
to
be
counted
(
Figure
1).
Beginning
with
the
top
row
of
four
squares,
count
with
a
hand­
tally
counter
in
the
directions
indicated
in
Figure
2.
Avoid
counting
organisms
twice
by
counting
only
those
touching
the
top
and
left
boundary
lines.
Count
each
square
millimeter
in
this
fashion.
11.3.3.9
Use
the
following
formula
to
determine
the
number
of
organisms
per
mL
of
suspension:

number
of
organisms
counted
×
10
×
dilution
factor
×
1000
mm2
=
number
of
organisms
number
of
mm2
counted
1
mm
1
1
µ
LmL
µ
LmL
11.3.3.10
Record
the
result
on
a
hemacytometer
data
sheet.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
29
11.3.3.11
A
total
of
six
different
hemacytometer
chambers
must
be
loaded,
counted,
and
averaged
for
each
suspension
to
achieve
optimal
counting
accuracy.
11.3.3.12
Based
on
the
hemacytometer
counts,
the
stock
suspension
should
be
diluted
to
a
final
concentration
of
between
8000
and
12,000
organisms
per
mL
(
80
to
120
organisms
per
10
µ
L);
however,
ranges
as
great
as
5000
to
15,000
organisms
per
mL
(
50
to
150
organisms
per
10
µ
L)
can
be
used.

NOTE:
If
the
diluted
stock
suspensions
(
the
spiking
suspensions)
will
be
enumerated
using
hemacytometer
chamber
counts
(
Section
11.3.4)
or
membrane
filter
counts
(
Section
11.3.6),
then
the
stock
suspensions
should
be
diluted
with
0.01%
Tween­
20.
If
the
spiking
suspensions
will
be
enumerated
using
well
slide
counts
(
Section
11.3.3
11.3.5),
then
the
stock
suspensions
should
be
diluted
in
reagent
water.

To
calculate
the
volume
(
in
µ
L)
of
stock
suspension
required
per
mL
of
reagent
water
(
or
reagent
water/
Tween­
20,
0.01%),
use
the
following
formula:

volume
of
stock
suspension
(
µ
L)
required
=
required
number
of
organisms
x
1000
:
L
number
of
organisms/
µ
mL
of
stock
suspension
If
the
volume
is
less
than
10
µ
L,
an
additional
dilution
of
the
stock
suspension
is
recommended
before
proceeding.
To
calculate
the
dilution
factor
needed
to
achieve
the
required
number
of
organisms
per
10
µ
L,
use
the
following
formula:

total
volume
(:
L)
=
number
of
organisms
required
x
10:
L
predicted
number
of
organisms
per
10:
L
(
80
to
120)

To
calculate
the
volume
of
reagent
water
(
or
reagent
water/
Tween­
20,
0.01%)
needed,
use
the
following
formula:

reagent
water
volume
(:
L)
=
total
volume
(:
L)
­
stock
suspension
volume
required
(:
L)

11.3.3.13
After
each
use,
the
hemacytometer
and
coverslip
must
be
cleaned
immediately
to
prevent
the
organisms
and
debris
from
drying
on
it.
Since
this
apparatus
is
precisely
machined,
abrasives
cannot
be
used
to
clean
it,
as
they
will
disturb
the
flooding
and
volume
relationships.
11.3.3.13.1
Rinse
the
hemacytometer
and
cover
glass
first
with
tap
water,
then
70%
ethanol,
and
finally
with
acetone.
11.3.3.13.2
Dry
and
polish
the
hemacytometer
chamber
and
cover
glass
with
lens
paper.
Store
it
in
a
secure
place.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
30
11.3.3.14
Several
factors
are
known
to
introduce
errors
into
hemacytometer
counts,
including:

C
Inadequate
mixing
of
suspension
before
flooding
the
chamber
C
Irregular
filling
of
the
chamber,
trapped
air
bubbles,
dust,
or
oil
on
the
chamber
or
coverslip
C
Total
number
of
organisms
counted
is
too
low
to
provide
statistical
confidence
in
the
result
C
Error
in
recording
tally
C
Calculation
error;
failure
to
consider
dilution
factor,
or
area
counted
C
Inadequate
cleaning
and
removal
of
organisms
from
the
previous
count
C
Allowing
filled
chamber
to
sit
too
long,
so
that
the
chamber
suspension
dries
and
concentrates.
11.3.4
Enumerating
spiking
suspensions
using
a
hemacytometer
chamber
NOTE:
Spiking
suspensions
enumerated
using
a
hemacytometer
chamber
must
be
used
within
24
hours
of
enumeration.

11.3.4.1
Vortex
the
tube
containing
the
spiking
suspension
(
diluted
stock
suspension;
Section
11.3.3)
for
a
minimum
of
2
minutes.
Gently
invert
the
tube
three
times.
11.3.4.2
To
an
appropriate­
size
beaker
containing
a
stir
bar,
add
enough
spiking
suspension
to
perform
all
spike
testing
and
the
enumeration
as
described.
The
liquid
volume
and
beaker
relationship
should
be
such
that
a
spinning
stir
bar
does
not
splash
the
sides
of
the
beaker,
the
stir
bar
has
unimpeded
rotation,
and
there
is
enough
room
to
draw
sample
from
the
beaker
with
a
10­
µ
L
micropipette
without
touching
the
stir
bar.
Cover
the
beaker
with
a
watch
glass
or
petri
dish
to
prevent
evaporation
between
sample
withdrawals.
11.3.4.3
Allow
the
beaker
contents
to
stir
for
a
minimum
of
30
minutes
before
beginning
enumeration.
11.3.4.4
While
the
stir
bar
is
still
spinning,
remove
a
10­
µ
L
aliquot
and
carefully
load
one
side
of
the
hemacytometer.
Count
all
organisms
on
the
platform,
at
200X
magnification
using
phase­
contrast
or
darkfield
microscopy.
The
count
must
include
the
entire
area
under
the
hemacytometer,
not
just
the
four
outer
1­
mm2
squares.
Repeat
this
procedure
nine
times.
This
step
allows
confirmation
of
the
number
of
organisms
per
10
µ
L
(
Section
11.3.3.12).
Based
on
the
10
counts,
calculate
the
mean,
standard
deviation,
and
RSD
of
the
counts.
Record
the
counts
and
the
calculations
on
a
spiking
suspension
enumeration
form.
The
relative
standard
deviation
(
RSD)
of
the
calculated
mean
spike
dose
must
be
#
16%
for
Cryptosporidium
and
#
19%
for
Giardia
before
proceeding.
If
the
RSD
is
unacceptable,
or
the
mean
number
is
outside
the
expected
range,
add
additional
oocysts
from
stock
suspension
or
dilute
the
contents
of
the
beaker
appropriately
with
reagent
water.
Repeat
the
process
to
confirm
counts.
Refer
to
Section
11.3.3.14
for
factors
that
may
introduce
errors.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
31
11.3.5
Enumerating
spiking
suspensions
using
well
slides
NOTE:
Spiking
suspensions
enumerated
using
well
slides
must
be
used
within
24
hours
of
application
of
the
spiking
suspension
to
the
slides.

11.3.5.1
Prepare
well
slides
for
sample
screening
and
label
the
slides.
Remove
well
slides
from
cold
storage
and
lay
the
slides
on
a
flat
surface
for
15
minutes
to
allow
them
to
warm
to
room
temperature.
11.3.5.2
Vortex
the
tube
containing
the
spiking
suspension
(
diluted
stock
suspension;
Section
11.3.3)
for
a
minimum
of
2
minutes.
Gently
invert
the
tube
three
times.
11.3.5.3
Remove
a
10­
µ
L
aliquot
from
the
spiking
suspension
and
apply
it
to
the
center
of
a
well.
11.3.5.4
Before
removing
subsequent
aliquots,
cap
the
tube
and
gently
invert
it
three
times
to
ensure
that
the
oocysts
or
cysts
are
in
suspension.
11.3.5.5
Ten
wells
must
be
prepared
and
counted,
and
the
counts
averaged,
to
sufficiently
enumerate
the
spike
dose.
Air­
dry
the
well
slides.
Because
temperature
and
humidity
varies
from
laboratory
to
laboratory,
no
minimum
time
is
specified.
However,
the
laboratory
must
take
care
to
ensure
that
the
sample
has
dried
completely
before
staining
to
prevent
losses
during
the
rinse
steps.
A
slide
warmer
set
at
35
°
C
to
42
°
C
also
can
be
used.
11.3.5.6
Positive
and
negative
controls
must
be
prepared.
11.3.5.6.1
For
the
positive
control,
pipette
10
µ
L
of
positive
antigen
or
200
to
400
intact
oocysts
or
cysts
to
the
center
of
a
well
and
distribute
evenly
over
the
well
area.
11.3.5.6.2
For
the
negative
control,
pipette
50
µ
L
of
PBS
onto
the
center
of
a
well
and
spread
it
over
the
well
area
with
a
pipette
tip.
11.3.5.6.3
Air­
dry
the
control
slides.
11.3.5.7
Apply
50­
µ
L
of
absolute
methanol
to
each
well
containing
the
dried
sample
and
allow
to
air­
dry
for
3
to
5
minutes.
11.3.5.7
Follow
the
manufacturer's
instructions
(
Section
7.6)
in
applying
the
stain
to
the
slide.
11.3.5.8
Place
the
slides
in
a
humid
chamber
in
the
dark
and
incubate
according
to
manufacturer's
directions.
at
room
temperature
for
approximately
30
minutes.
The
humid
chamber
consists
of
a
tightly
sealed
plastic
container
containing
damp
paper
towels
on
top
of
which
the
slides
are
placed.
11.3.5.9
Apply
one
drop
of
wash
buffer
(
prepared
according
to
the
manufacturer's
instructions
[
Section
7.6])
to
each
well.
Tilt
each
slide
on
a
clean
paper
towel,
long
edge
down.
Gently
aspirate
the
excess
detection
reagent
from
below
the
well
using
a
clean
Pasteur
pipette
or
absorb
with
a
paper
towel
or
other
absorbent
material.
Avoid
disturbing
the
sample.

NOTE:
If
using
the
Merifluor
stain
(
Section
7.6.1),
do
not
allow
slides
to
dry
completely.

11.3.5.10
Add
mounting
medium
(
Section
7.8)
to
each
well.
11.3.5.11
Apply
a
cover
slip.
Use
a
tissue
to
remove
excess
mounting
fluid
from
the
edges
of
the
coverslip.
Seal
the
edges
of
the
coverslip
onto
the
slide
using
clear
nail
polish.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
32
11.3.5.12
Record
the
date
and
time
that
staining
was
completed.
If
slides
will
not
be
read
immediately,
store
in
a
humid
chamber
in
the
dark
at
0
°
C
to
8
°
C
<
10
°
C
and
not
frozen
until
ready
for
examination.
11.3.5.13
After
examination
of
the
10
wells,
calculate
the
mean,
standard
deviation,
and
RSD
of
the
10
replicates.
Record
the
counts
and
the
calculations
on
a
spiking
suspension
enumeration
form.
The
relative
standard
deviation
(
RSD)
of
the
calculated
mean
spike
dose
must
be
#
16%
for
Cryptosporidium
and
#
19%
for
Giardia
before
proceeding.
If
the
RSD
is
unacceptable,
or
the
mean
number
is
outside
the
expected
range,
add
additional
oocysts
from
stock
suspension
or
dilute
the
contents
of
the
beaker
appropriately
with
reagent
water.
Repeat
the
process
to
confirm
counts.
11.3.6
Enumeration
of
spiking
suspensions
using
membrane
filters
NOTE:
Spiking
suspensions
enumerated
using
membrane
filters
must
be
used
within
24
hours
of
application
of
the
filters
to
the
slides.

11.3.6.1
Precoat
the
glass
funnels
with
Sigmacote
®
by
placing
the
funnel
in
a
large
petri
dish
and
applying
5­
mL
of
Sigmacoat
®
to
the
funnel
opening
using
a
pipette
and
allowing
it
to
run
down
the
inside
of
the
funnel.
Repeat
for
all
funnels
to
be
used.
The
pooled
Sigmacoat
®
may
be
returned
to
the
bottle
for
re­
use.
Place
the
funnels
at
35
°
C
or
41
°
C
for
approximately
5
minutes
to
dry.
11.3.6.2
Place
foil
around
the
bottoms
of
the
100
×
15
mm
petri
dishes.
11.3.6.3
Filter­
sterilize
(
Section
6.19)
approximately
10
mL
of
PBS
pH
7.2
(
Section
7.
9.
4
7.6.4).
Dilute
detection
reagent
(
Section
7.7)
as
per
manufacturer's
instructions
using
sterile
PBS.
Multiply
the
anticipated
number
of
filters
to
be
stained
by
100
mL
to
calculate
total
volume
of
stain
required.
Divide
the
total
volume
required
by
5
to
obtain
the
microliters
of
antibody
necessary.
Subtract
the
volume
of
antibody
from
the
total
stain
volume
to
obtain
the
required
microliters
of
sterile
PBS
to
add
to
the
antibody.
11.3.6.4
Label
the
tops
of
foil­
covered,
60
×
15
mm
petri
dishes
for
10
spiking
suspensions
plus
positive
and
negative
staining
controls
and
multiple
filter
blanks
controls
(
one
negative
control,
plus
a
blank
after
every
five
sample
filters
to
control
for
carry­
over).
Create
a
humid
chamber
by
laying
damp
paper
towels
on
the
bottom
of
a
stain
tray
(
the
inverted
foillined
petri
dishes
will
protect
filters
from
light
and
prevent
evaporation
during
incubation).
11.3.6.5
Place
a
decontaminated
and
cleaned
filter
holder
base
(
Section
6.4.8.1)
into
each
of
the
three
ports
of
the
vacuum
manifold
(
Section
6.4.8.2).
11.3.6.6
Pour
approximately
10
mL
of
0.01%
Tween
20
into
a
60
×
15
mm
petri
dish.
11.3.6.7
Using
forceps,
moisten
a
1.2­
µ
m
cellulose­
acetate
support
membrane
(
Section
6.4.8.3)
in
the
0.01%
Tween
20
and
place
it
on
the
fritted
glass
support
of
one
of
the
filter
bases.
Moisten
a
polycarbonate
filter
(
Section
6.4.8.4)
the
same
way
and
position
it
on
top
of
the
cellulose­
acetate
support
membrane.
Carefully
clamp
the
glass
funnel
to
the
loaded
filter
support.
Repeat
for
the
other
two
filters.
11.3.6.8
Add
5
mL
of
0.01%
Tween
20
to
each
of
the
three
filtration
units
and
allow
to
stand.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
33
11.3.6.9
Vortex
the
tube
containing
the
spiking
suspension
(
diluted
stock
suspension;
Section
11.3.3)
for
a
minimum
of
2
minutes.
Gently
invert
the
tube
three
times.
11.3.6.10
Using
a
micropipettor,
sequentially
remove
two,
10­
µ
L
aliquots
from
the
spiking
suspension
and
pipet
into
the
5
mL
of
0.01%
Tween
20
standing
in
the
unit.
Rinse
the
pipet
tip
twice
after
each
addition.
Apply
10
µ
L
of
0.01%
Tween
20
to
the
third
unit
to
serve
as
the
negative
control.
Apply
vacuum
at
2"
Hg
and
allow
liquid
to
drain
to
miniscus,
then
close
off
vacuum.
Pipet
10
mL
of
reagent
water
into
each
funnel
and
drain
to
miniscus,
closing
off
the
vacuum.
Repeat
the
rinse
and
drain
all
fluid,
close
off
the
vacuum.
11.3.6.11
Pipet
100
mL
of
diluted
antibody
to
the
center
of
the
bottom
of
a
60
×
15
mm
petri
dish
for
each
sample.
11.3.6.12
Unclamp
the
top
funnel
and
transfer
each
cellulose
acetate
support
membrane/
polycarbonate
filter
combination
onto
the
drop
of
stain
using
forceps
(
apply
each
membrane/
filter
combination
to
a
different
petri
dish
containing
stain).
Roll
the
filter
into
the
drop
to
exclude
air.
Place
the
small
petri
dish
containing
the
filter
onto
the
damp
towel
and
cover
with
the
corresponding
labeled
foil­
covered
top.
Incubate
for
approximately
45
minutes
at
room
temperature.
11.3.6.13
Reclamp
the
top
funnels,
apply
vacuum
and
rinse
each
three
times,
each
time
with
20
mL
of
reagent
water.
11.3.6.14
Repeat
Sections
11.3.6.4
through
11.3.6.10
for
the
next
three
samples
(
if
that
the
diluted
spiking
suspension
has
sat
less
than
15
minutes,
reduce
the
suspension
vortex
time
to
60
seconds).
Ten,
10­
µ
L
spiking
suspension
aliquots
must
be
prepared
and
counted,
and
the
counts
averaged,
to
sufficiently
enumerate
the
spike
dose.
Include
a
filter
blank
sample
at
a
frequency
of
every
five
samples;
rotate
the
position
of
filter
blank
to
eventually
include
all
three
filter
placements.
11.3.6.15
Repeat
Sections
11.3.6.4
through
11.3.6.10
until
the
10­
µ
L
spiking
suspensions
have
been
filtered.
The
last
batch
should
include
a
10­
µ
L
0.01
Tween
20
blank
control
and
20
µ
L
of
positive
control
antigen
as
a
positive
staining
control.
11.3.6.16
Label
slides.
After
incubation
is
complete,
for
each
sample,
transfer
the
cellulose
acetate
filter
support
and
polycarbonate
filter
from
drop
of
stain
and
place
on
fritted
glass
support.
Cycle
vacuum
on
and
off
briefly
to
remove
excess
fluid.
Peel
the
top
polycarbonate
filter
off
the
supporting
filter
and
place
on
labeled
slide.
Discard
cellulose
acetate
filter
support.
Mount
and
apply
coverslips
to
the
filters
immediately
to
avoid
drying.
11.3.6.17
To
each
slide,
add
20
µ
L
of
mounting
medium
(
Section
7.8).
11.3.6.18
Apply
a
coverslip.
Seal
the
edges
of
the
coverslip
onto
the
slide
using
clear
nail
polish.
(
Sealing
may
be
delayed
until
cover
slips
are
applied
to
all
slides.)
11.3.6.19
Record
the
date
and
time
that
staining
was
completed.
If
slides
will
not
be
read
immediately,
store
sealed
slides
in
a
closed
container
in
the
dark
at
0
°
C
to
<
8
°
C
(
but
not
frozen)
until
ready
for
examination.
11.3.6.20
After
examination
of
the
10
slides,
calculate
the
mean,
standard
deviation,
and
RSD
of
the
10
replicates.
Record
the
counts
and
the
calculations
on
a
spiking
suspension
enumeration
form.
The
relative
standard
deviation
(
RSD)
of
the
calculated
mean
spike
dose
must
be
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
34
#
16%
for
Cryptosporidium
and
#
19%
for
Giardia
before
proceeding.
If
the
RSD
is
unacceptable,
or
the
mean
number
is
outside
the
expected
range,
add
additional
oocysts
from
stock
suspension
or
dilute
the
contents
of
the
beaker
appropriately
with
reagent
water.
Repeat
the
process
to
confirm
counts.
11.3.6.21
If
oocysts
or
cysts
are
detected
on
the
filter
blanks,
modify
the
rinse
procedure
to
ensure
that
no
carryover
occurs
and
repeat
enumeration.
11.4
Procedure
for
spiking
samples
in
the
laboratory
with
enumerated
spiking
suspensions.
11.4.1
Arrange
a
bottom­
dispensing
container
to
feed
the
filter
or
insert
the
influent
end
of
the
tube
connected
to
the
filter
through
the
top
of
a
carboy
to
allow
siphoning
of
the
sample.
11.4.2
For
initial
precision
and
recovery
(
Section
9.4)
and
ongoing
precision
and
recovery
(
Section
9.7)
samples,
fill
the
container
with
10
L
of
reagent
water
or
a
volume
of
reagent
water
equal
to
the
volume
of
the
field
samples
analyzed
in
the
analytical
batch.
For
matrix
spike
samples
(
Section
9.5),
fill
the
container
with
the
field
sample
to
be
spiked.
Continuously
mix
the
sample
(
using
a
stir
bar
and
stir
plate
for
smaller­
volume
samples
and
alternate
means
for
larger­
volume
samples).
11.4.3
Follow
the
procedures
in
Section
11.4.3.1
for
flow
cytometer
 
enumerated
suspensions
and
the
procedures
in
Section
11.4.3.2
for
manually
enumerated
suspensions.
Vortex
the
spiking
suspension(
s)
(
Section
11.2
or
Section
11.3)
for
a
minimum
of
2
minutes.
11.4.3.1
For
flow
cytometer
 
enumerated
suspensions
(
where
the
entire
volume
of
a
spiking
suspension
tube
will
be
used):
11.4.3.1.1
Add
400
µ
L
of
Antifoam
A
to
100
mL
of
reagent
water,
and
mix
well
to
emulsify.
11.4.3.1.2
Add
500
µ
L
of
the
diluted
antifoam
to
the
tube
containing
the
spiking
suspension
and
vortex
for
30
seconds
2
minutes.
11.4.3.1.3
Pour
the
suspension
into
the
sample
container.
11.4.3.1.4
Add
20
mL
of
reagent
water
to
the
empty
tube,
cap,
vortex
10
seconds
to
rinse,
and
add
the
rinsate
to
the
carboy.
11.4.3.1.5
Repeat
this
rinse
using
another
20
mL
of
reagent
water.
11.4.3.1.6
Record
the
estimated
number
of
organisms
spiked,
the
date
and
time
the
sample
was
spiked,
and
the
sample
volume
spiked
on
a
bench
sheet.
Proceed
to
Section
11.4.4.
11.4.3.2
For
manually
enumerated
spiking
suspensions:
11.4.3.2.1
Vortex
the
spiking
suspension(
s)
(
Section
11.2
or
Section
11.3)
for
a
minimum
of
30
seconds.
11.4.3.2.2
Rinse
a
pipette
tip
with
0.01%
Tween­
20
once,
then
rinse
with
the
well­
mixed
spiking
suspension
a
minimum
of
five
times
before
pulling
an
aliquot
to
be
used
to
spike
the
container.
11.4.3.2.3
Add
the
spiking
suspension(
s)
to
the
carboy,
delivering
the
aliquot
below
the
surface
of
the
water.
11.4.3.2.4
Record
the
estimated
number
of
organisms
spiked,
the
date
and
time
the
sample
was
spiked,
and
the
sample
volume
spiked
on
a
bench
sheet.
Proceed
to
Section
11.4.4
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
35
11.4.4
Allow
the
spiking
suspensions
to
mix
for
approximately
1
minute
in
the
container.
11.4.5
Turn
on
the
pump
and
allow
the
flow
rate
to
stabilize.
Set
flow
at
the
rate
designated
for
the
filter
being
used.
As
the
carboy
is
depleted,
check
the
flow
rate
and
adjust
if
necessary.
11.4.6
When
the
water
level
approaches
the
discharge
port
of
the
carboy,
tilt
the
container
so
that
it
is
completely
emptied.
At
that
time,
turn
off
the
pump
and
add
sufficient
reagent
water
to
the
container
to
rinse.
Swirl
the
contents
to
rinse
down
the
sides.
Additional
rinses
may
be
performed.
11.4.7
Turn
on
the
pump.
Allow
all
of
the
water
to
flow
through
the
filter
and
turn
off
the
pump.

12.0
Sample
Filtration
and
Elution
12.1
A
water
sample
is
filtered
according
to
the
procedures
in
Section
12.2
or
Section
12.3.
Alternate
procedures
may
be
used
if
the
laboratory
first
demonstrates
that
the
alternate
procedure
provides
equivalent
or
superior
performance
per
Section
9.1.2.

NOTE:
Sample
elution
must
be
initiated
within
96
hours
of
sample
collection
(
if
shipped
to
the
laboratory
as
a
bulk
sample)
or
filtration
(
if
filtered
in
the
field).

12.2
Capsule
filtration
(
adapted
from
Reference
20.9).
This
procedure
was
validated
using
10­
L
sample
volumes
(
for
the
original
Envirochek
 
filter)
and
50­
L
sample
volumes
(
for
the
Envirochek
 
HV
filter).
Alternate
sample
volumes
may
be
used,
provided
the
laboratory
demonstrates
acceptable
performance
on
initial
and
ongoing
spiked
reagent
water
and
source
water
samples
(
Section
9.1.2).

NOTE:
The
filtration
procedures
specified
in
Section
12.2.1
­
12.2.5.3
are
specific
to
laboratory
filtration
of
a
bulk
sample,
and
reflect
the
procedures
used
during
the
interlaboratory
validation
of
this
method
(
Reference
20.10).
These
procedures
may
require
modification
if
samples
will
be
filtered
in
the
field.

12.2.1
Flow
rate
adjustment
12.2.1.1
Connect
the
sampling
system,
minus
the
capsule,
to
a
carboy
filled
with
reagent
water
(
Figure
3).
12.2.1.2
Turn
on
the
pump
and
adjust
the
flow
rate
to
2.0
L/
min.
12.2.1.3
Allow
2
to
10
L
of
reagent
water
to
flush
the
system.
Adjust
the
pump
speed
as
required
during
this
period.
Turn
off
the
pump
when
the
flow
rate
has
been
adjusted.
12.2.2
Install
the
capsule
filter
in
the
line,
securing
the
inlet
and
outlet
ends
with
the
appropriate
clamps/
fittings.
12.2.3
Record
the
sample
number,
sample
turbidity
(
if
not
provided
with
the
field
sample),
sample
type,
and
sample
filtration
start
date
and
time
on
a
bench
sheet.
12.2.4
Filtration
12.2.4.1
Mix
the
sample
well
by
shaking
and
connect
the
sampling
system
to
the
field
carboy
of
sample
water,
or
transfer
the
sample
water
to
the
laboratory
carboy
used
in
Section
12.2.1.1.
If
the
sample
will
be
filtered
from
a
field
carboy,
a
spigot
(
Section
6.2.1)
can
be
used
with
the
carboy
to
facilitate
sample
filtration.

NOTE:
If
the
bulk
field
sample
is
transferred
to
a
laboratory
carboy,
the
laboratory
carboy
must
be
cleaned
and
disinfected
before
it
is
used
with
another
field
sample.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
36
12.2.4.2
Place
the
drain
end
of
the
sampling
system
tubing
into
an
empty
graduated
container
with
a
capacity
of
10
to
15
L,
calibrated
at
9.0,
9.5,
10.0,
10.5,
and
11.0
L
(
Section
6.18).
This
container
will
be
used
to
determine
the
sample
volume
filtered.
Alternately,
connect
a
flow
meter
(
Section
6.3.4)
downstream
of
the
filter,
and
record
the
initial
meter
reading.
12.2.4.3
Allow
the
carboy
discharge
tube
and
capsule
to
fill
with
sample
water
by
gravity.
Vent
residual
air
using
the
bleed
valve/
vent
port,
gently
shaking
or
tapping
the
capsule,
if
necessary.
Turn
on
the
pump
to
start
water
flowing
through
the
filter.
Verify
that
the
flow
rate
is
2
L/
min.
12.2.4.4
After
all
of
the
sample
has
passed
through
the
filter,
turn
off
the
pump.
Allow
the
pressure
to
decrease
until
flow
stops.
(
If
the
sample
was
filtered
in
the
field,
and
excess
sample
remains
in
the
filter
capsule
upon
receipt
in
the
laboratory,
pull
the
remaining
sample
volume
through
the
filter
before
eluting
the
filter
[
Section
12.2.6].)
12.2.5
Disassembly
12.2.5.1
Disconnect
the
inlet
end
of
the
capsule
filter
assembly
while
maintaining
the
level
of
the
inlet
fitting
above
the
level
of
the
outlet
fitting
to
prevent
backwashing
and
the
loss
of
oocysts
and
cysts
from
the
filter.
Restart
the
pump
and
allow
as
much
water
to
drain
as
possible.
Turn
off
the
pump.
12.2.5.2
Based
on
the
water
level
in
the
graduated
container
and
½
­
L
hash
marks
or
meter
reading,
record
the
volume
filtered
on
the
bench
sheet
to
the
nearest
quarter
liter.
Discard
the
contents
of
the
graduated
container.
12.2.5.3
Loosen
the
outlet
fitting,
then
cap
the
inlet
and
outlet
fittings.
12.2.6
Elution
NOTE:
The
laboratory
must
complete
the
elution,
concentration,
and
purification
(
Sections
12.2.6
through
13.3.3.11)
in
one
work
day.
It
is
critical
that
these
steps
be
completed
in
one
work
day
to
minimize
the
time
that
any
target
organisms
present
in
the
sample
sit
in
eluate
or
concentrated
matrix.
This
process
ends
with
the
application
of
the
purified
sample
on
the
slide
for
drying.

12.2.6.1
Setup
12.2.6.1.1
Assemble
the
laboratory
shaker
with
the
clamps
aligned
vertically
so
that
the
filters
will
be
aligned
horizontally.
Extend
the
clamp
arms
to
their
maximum
distance
from
the
horizontal
shaker
rods
to
maximize
the
shaking
action.
12.2.6.1.2
Prepare
sufficient
elution
buffer
so
that
all
samples
to
be
eluted
that
day
can
be
eluted
with
the
same
batch
of
buffer.
Elution
may
require
up
to
275
mL
of
buffer
per
sample.
12.2.6.1.3
Designate
at
least
one
250­
mL
conical
centrifuge
tube
for
each
sample
and
label
with
the
sample
number.
12.2.6.2
Elution
12.2.6.2.1
Record
the
elution
date
and
time
on
the
bench
sheet.
Using
a
ring
stand
or
other
means,
clamp
each
capsule
in
a
vertical
position
with
the
inlet
end
up.
Remove
the
inlet
cap
and
allow
the
liquid
level
to
stabilize.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
37
12.2.6.2.2
Pour
elution
buffer
through
the
inlet
fitting.
Remove
the
inlet
cap
and
allow
the
liquid
level
to
stabilize.
Sufficient
elution
buffer
must
be
added
to
cover
the
pleated
white
membrane
with
buffer
solution.
Replace
the
inlet
cap
and
clamp
the
cap
in
place.
12.2.6.2.3
Securely
clamp
the
capsule
in
one
of
the
clamps
on
the
laboratory
shaker
with
the
bleed
valve
positioned
at
the
top
on
a
vertical
axis
(
in
the
12
o'clock
position).
Turn
on
the
shaker
and
set
the
speed
to
maximum
(
approximately
900
rpm).
Agitate
the
capsule
for
approximately
5
minutes.
Time
the
agitation
using
a
lab
timer,
rather
than
the
timer
on
the
shaker
to
ensure
accurate
time
measurement.
12.2.6.2.4
Remove
the
filter
from
the
shaker,
remove
the
inlet
cap,
and
pour
the
contents
of
the
capsule
into
the
250­
mL
conical
centrifuge
tube.
12.2.6.2.5
Clamp
the
capsule
vertically
with
the
inlet
end
up
and
add
sufficient
volume
of
elution
buffer
through
the
inlet
fitting
to
cover
the
pleated
membrane.
Replace
the
inlet
cap.
12.2.6.2.6
Return
the
capsule
to
the
shaker
with
the
bleed
valve
positioned
at
the
4
o'clock
position.
Turn
on
the
shaker
and
agitate
the
capsule
for
approximately
5
minutes.
12.2.6.2.7
Remove
the
filter
from
the
shaker,
but
leave
the
elution
buffer
in
the
capsule.
Re­
clamp
the
capsule
to
the
shaker
at
the
8
o'clock
position.
Turn
on
the
shaker
and
agitate
the
capsule
for
a
final
5
minutes.
12.2.6.2.8
Remove
the
filter
from
the
shaker
and
pour
the
contents
into
the
250­
mL
centrifuge
tube.
Rinse
down
the
inside
of
the
capsule
filter
walls
with
reagent
water
or
elution
buffer
using
a
squirt
bottle
inserted
in
the
inlet
end
of
the
capsule.
Invert
the
capsule
filter
over
the
centrifuge
tube
and
ensure
that
as
much
of
the
eluate
as
possible
has
been
transferred.
12.2.7
Proceed
to
Section
13.0
for
concentration
and
separation
(
purification).
12.3
Sample
filtration
using
the
Filta­
Max
 
foam
filter.
This
procedure
was
validated
using
50­
L
sample
volumes.
Alternate
sample
volumes
may
be
used,
provided
the
laboratory
demonstrates
acceptable
performance
on
initial
and
ongoing
spiked
reagent
water
and
source
water
samples
(
Section
9.1.2).

NOTE:
The
filtration
procedures
specified
in
Sections
12.3.1.2
­
12.3.1.6.3
are
specific
to
laboratory
filtration
of
a
bulk
sample.
These
procedures
may
require
modification
if
samples
will
be
filtered
in
the
field.

12.3.1
Filtration
12.3.1.1
Flow
rate
adjustment
12.3.1.1.1
Connect
the
sampling
system,
minus
the
filter
housing,
to
a
carboy
filled
with
reagent
water.
12.3.1.1.2
Place
the
peristaltic
pump
upstream
of
the
filter
housing.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
38
12.3.1.1.3
Turn
on
the
pump
and
adjust
the
flow
rate
to
1
to
4
L
per
minute.

NOTE:
A
head
pressure
of
0.5
bar
(
7.5
psi)
is
required
to
create
flow
through
the
filter,
and
the
recommended
pressure
of
5
bar
(
75
psi)
should
produce
the
flow
rate
of
3
to
4
L
per
minute.
The
maximum
operating
pressure
of
8
bar
(
120
psi)
should
not
be
exceeded.

12.3.1.1.4
Allow
2
to
10
L
of
reagent
water
to
flush
the
system.
Adjust
the
pump
speed
as
necessary
during
this
period.
Turn
off
the
pump
when
the
flow
rate
has
been
adjusted.
12.3.1.2
Place
filter
module
into
the
filter
housing
and
secure
lid,
hand
tighten
housings,
apply
gentle
pressure
to
create
the
seal
between
the
module
and
the
`
O'
rings
in
the
base
and
the
lid
of
the
housing.
Excessive
tightening
is
not
necessary,
and
may
shorten
the
life
of
the
`
O'
rings.
Tools
may
be
used
to
tighten
housing
to
the
alignment
marks
(
refer
to
manufacturer's
instructions).
`
O'
rings
should
be
lightly
greased
before
use
(
refer
to
manufacturer's
instructions).
12.3.1.3
Install
the
filter
housing
in
the
line,
securing
the
inlet
and
outlet
ends
with
the
appropriate
clamps/
fittings.
Verify
that
the
filter
housing
is
installed
so
that
the
end
closest
to
the
screw
top
cap
is
the
inlet
and
the
opposite
end
is
the
outlet.
12.3.1.4
Record
the
sample
number,
sample
turbidity
(
if
not
provided
with
the
field
sample),
and
the
name
of
the
analyst
filtering
the
sample
on
a
bench
sheet.
12.3.1.5
Filtration
12.3.1.5.1
Connect
the
sampling
system
to
the
field
carboy
of
sample
water,
or
transfer
the
sample
water
to
the
laboratory
carboy
used
in
Section
12.3.1.1.1.
If
the
sample
will
be
filtered
from
a
field
carboy,
a
spigot
can
be
used
with
the
carboy
to
facilitate
sample
filtration.

NOTE:
If
the
bulk
field
sample
is
transferred
to
a
laboratory
carboy,
the
laboratory
carboy
must
be
cleaned
and
disinfected
before
it
is
used
with
another
field
sample.

12.3.1.5.2
Place
the
drain
end
of
the
sampling
system
tubing
into
an
empty
graduated
container
with
a
capacity
greater
than
or
equal
to
the
volume
to
be
filtered.
This
container
will
be
used
to
determine
the
sample
volume
filtered.
Alternately,
connect
a
flow
meter
downstream
of
the
filter,
and
record
the
initial
meter
reading.
12.3.1.5.3
Allow
the
carboy
discharge
tube
and
filter
housing
to
fill
with
sample
water.
Turn
on
the
pump
to
start
water
flowing
through
the
filter.
Verify
that
the
flow
rate
is
between
1
and
4
L
per
min.
12.3.1.5.4
After
all
of
the
sample
has
passed
through
the
filter,
turn
off
the
pump.
Allow
the
pressure
to
decrease
until
flow
stops.
12.3.1.6
Disassembly
12.3.1.6.1
Disconnect
the
inlet
end
of
the
filter
housing
assembly
while
maintaining
the
level
of
the
inlet
fitting
above
the
level
of
the
outlet
fitting
to
prevent
backwashing
and
the
loss
of
oocysts
and
cysts
from
the
filter.
Restart
the
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
39
pump
and
allow
as
much
water
to
drain
as
possible.
Turn
off
the
pump.
12.3.1.6.2
Based
on
the
water
level
in
the
graduated
container
or
the
meter
reading,
record
the
volume
filtered
on
a
bench
sheet
to
the
nearest
quarter
liter.
12.3.1.6.3
Loosen
the
outlet
fitting,
the
filter
housing
should
be
sealed
with
rubber
plugs.

NOTE:
Filters
should
be
prevented
from
drying
out,
as
this
can
impair
their
ability
to
expand
when
decompressed.

12.3.2
Elution
12.3.2.1
The
filter
is
eluted
to
wash
the
oocysts
from
the
filter.
This
can
be
accomplished
using
the
Filta­
Max
 
wash
station,
which
moves
a
plunger
up
and
down
a
tube
containing
the
filter
and
eluting
solution
(
Section
12.3.2.2),
or
a
stomacher,
which
uses
paddles
to
agitate
the
stomacher
bag
containing
the
foam
filter
in
the
eluting
solution
(
Section
12.3.2.3).
If
the
Filta­
MaxTM
automatic
wash
station
is
used
please
see
the
manufacturer's
operator's
guide
for
instructions
on
its
use.
12.3.2.2
Filta­
Max
 
wash
station
elution
procedure
12.3.2.2.1
First
wash
(
a)
Detach
the
removable
plunger
head
using
the
tool
provided,
and
remove
the
splash
guard.
(
b)
Place
the
filter
membrane
flat
in
the
concentrator
base
with
the
rough
side
up.
Locate
the
concentrator
base
in
the
jaws
of
the
wash
station
and
screw
on
the
concentrator
tube
(
the
longer
of
the
two
tubes),
creating
a
tight
seal
at
the
membrane.
Take
the
assembled
concentrator
out
of
the
jaws
and
place
on
the
bench.
(
c)
Replace
the
splash
guard
and
temporarily
secure
it
at
least
15
cm
above
the
end
of
the
rack.
Secure
the
plunger
head
with
the
tool
provided
ensuring
that
the
lever
is
fully
locked
down.
(
d)
Remove
the
filter
module
from
the
filter
housing
or
transportation
container.
Pour
excess
liquid
into
the
assembled
concentrator,
then
rinse
the
housing
or
container
with
PBST
and
add
the
rinse
to
the
concentrator
tube.
Screw
the
filter
module
onto
the
base
of
the
plunger.
Locate
the
elution
tube
base
in
the
jaws
of
the
wash
station
and
screw
the
elution
tube
(
the
shorter
of
the
two
tubes)
firmly
in
place.
(
e)
Pull
the
plunger
down
until
the
filter
module
sits
at
the
bottom
of
the
elution
tube;
the
locking
pin
(
at
the
top
left
of
the
wash
station)
should
"
click"
to
lock
the
plunger
in
position.
(
f)
Remove
the
filter
module
bolt
by
turning
the
adapted
allen
key
(
provided)
in
a
clockwise
direction
(
as
seen
from
above).
Attach
the
steel
tube
to
the
elution
tube
base.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
40
(
g)
Add
600
mL
of
PBST
to
the
assembled
concentrator.
If
more
than
50
mL
of
liquid
has
been
recovered
from
the
shipped
filter
module,
reduce
the
volume
of
PBST
accordingly.
Screw
the
concentrator
tube
onto
the
base
beneath
the
elution
tube.
Release
the
locking
pin.

NOTE:
Gentle
pressure
on
the
lever,
coupled
with
a
pulling
action
on
the
locking
pin
should
enable
the
pin
to
be
easily
released.

(
h)
Wash
the
foam
disks
by
moving
the
plunger
up
and
down
20
times.
Gentle
movements
of
the
plunger
are
recommended
to
avoid
generating
excess
foam.

NOTE:
The
plunger
has
an
upper
movement
limit
during
the
wash
process
to
prevent
it
popping
out
of
the
top
of
the
chamber.

(
i)
Detach
the
concentrator
and
hold
it
such
that
the
stainless
steel
tube
is
just
above
the
level
of
the
liquid.
Purge
the
remaining
liquid
from
the
elution
tube
by
moving
the
plunger
up
and
down
5
times,
then
lock
the
plunger
in
place.
To
prevent
drips,
place
the
plug
provided
in
the
end
of
the
steel
tube.
(
j)
Prior
to
the
second
wash
the
eluate
from
the
first
wash
can
be
concentrated
using
the
Filta­
Max
 
apparatus
according
to
Section
12.3.3.2.1
or
the
eluate
can
be
decanted
into
a
2­
L
pooling
beaker
and
set
aside.
13.3.2.2.2
Second
wash
(
a)
Add
an
additional
600
mL
of
PBST
to
the
concentrator
module,
remove
the
plug
from
the
end
of
the
steel
tube
and
screw
the
concentrator
tube
back
onto
the
elution
module
base.
Release
the
locking
pin.
(
b)
Wash
the
foam
disks
by
moving
the
plunger
up
and
down
10
times.
Gentle
movements
of
the
plunger
are
recommended
to
avoid
generating
excess
foam.
(
c)
The
eluate
can
be
concentrated
using
the
Filta­
Max
 
apparatus
according
to
Section
12.3.3.2.2
or
the
eluate
can
be
decanted
into
the
2­
L
pooling
beaker
containing
the
eluate
from
the
first
wash
and
concentrated
using
centrifugation,
as
described
in
Section
12.3.3.3.
12.3.2.3
Stomacher
elution
procedure
12.3.2.3.1
First
wash
(
a)
Place
the
filter
module
in
the
stomacher
bag
then
use
the
allen
key
to
remove
the
bolt
from
the
filter
module,
allowing
the
rings
to
expand.
Remove
the
end
caps
from
the
stomacher
bag
and
rinse
with
PBST
into
the
stomacher
bag.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
41
(
b)
Add
600
mL
of
PBST
to
stomacher
bag
containing
the
filter
pads.
Place
bag
in
stomacher
and
wash
for
5
minutes
on
a
normal
setting.
(
c)
Remove
the
bag
from
the
stomacher
and
decant
the
eluate
into
a
2­
L
pooling
beaker.
12.3.2.3.2
Second
wash
(
a)
Add
a
second
600­
mL
aliquot
of
PBST
to
the
stomacher
bag.
Place
bag
in
stomacher
and
wash
for
5
minutes
on
a
normal
setting.
Remove
the
bag
from
the
stomacher
and
decant
the
eluate
from
the
stomacher
bag
into
the
2­
L
pooling
beaker.
Wring
the
stomacher
bag
by
hand
to
remove
eluate
from
the
foam
filter
and
add
to
the
pooling
beaker.
Remove
the
foam
filter
from
the
bag
and
using
a
squirt
bottle,
rinse
the
stomacher
bag
with
reagent
water
and
add
the
rinse
to
the
pooling
beaker.
(
b)
Proceed
to
concentration
(
Section
12.3.3).
12.3.3
Concentration
12.3.3.1
The
eluate
can
be
concentrated
using
the
Filta­
Max
 
concentrator
apparatus,
which
pulls
most
of
the
eluate
through
a
membrane
filter
leaving
the
oocysts
concentrated
in
a
small
volume
of
the
remaining
eluting
solution
(
Section
12.3..
2),
or
by
directly
centrifuging
all
of
the
eluting
solution
used
to
wash
the
filter
(
Section
12.3.2.3).
12.3.3.2
The
Filta­
Max
 
concentrator
procedure
12.3.3.2.1
Concentration
of
first
wash
(
a)
If
the
stomacher
was
used
to
elute
the
sample
(
Section
12.3.2.3),
transfer
600
mL
of
eluate
from
the
pooling
beaker
to
the
concentrator
tube.
Otherwise
proceed
to
Step
(
b).
(
b)
Stand
the
concentrator
tube
on
a
magnetic
stirring
plate
and
attach
the
lid
(
with
magnetic
stirrer
bar).
Connect
the
waste
bottle
trap
and
hand
or
electric
vacuum
pump
to
the
valve
on
the
concentrator
base.
Begin
stirring
and
open
the
tap.
Increase
the
vacuum
using
the
hand
pump.

NOTE:
The
force
of
the
vacuum
should
not
exceed
30
cmHg.

(
c)
Allow
the
liquid
to
drain
until
it
is
approximately
level
with
the
middle
of
the
stirrer
bar
then
close
the
valve.
Remove
the
magnetic
stirrer,
and
rinse
it
with
PBST
or
distilled
water
to
recover
all
oocysts.
Decant
the
concentrate
into
a
50­
mL
tube,
then
rinse
the
sides
of
the
concentration
tube
and
add
the
rinsate
to
the
50­
mL
tube.
12.3.3.2.2
Concentration
of
second
wash
(
a)
If
the
stomacher
was
used
to
elute
the
sample
(
Section
12.3.2.3),
transfer
the
remaining
600
mL
of
eluate
from
the
pooling
beaker
to
the
concentrator
tube.
Otherwise
proceed
to
Step
(
b).
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
42
(
b)
Add
the
concentrate,
in
the
50­
mL
tube,
retained
from
the
first
concentration
(
Section
12.3.3.2.1
(
c))
to
the
600
mL
of
eluate
from
the
second
wash,
then
repeat
concentration
steps
from
Sections
12.3.3.2.1
(
b)
and
12.3.3.2.1
(
c).
The
final
sample
can
be
poured
into
the
same
50­
mL
tube
used
to
retain
the
first
concentrate.
Rinse
the
sides
of
the
concentrator
tube
with
PBST
and
add
the
rinse
to
the
50­
mL
tube.
(
c)
Remove
the
magnetic
stirrer.
Insert
the
empty
concentrator
module
into
the
jaws
of
the
wash
station
and
twist
off
the
concentrator
tube.
(
d)
Transfer
the
membrane
from
the
concentrator
base
to
the
bag
provided
using
membrane
forceps.
12.3.3.2.3
Membrane
elution.
The
membrane
can
be
washed
manually
or
using
a
stomacher:
°
Manual
wash.
Add
5
mL
of
PBST
to
the
bag
containing
the
membrane.
Rub
the
surface
of
the
membrane
through
the
bag
until
the
membrane
appears
clean.
Using
a
pipette,
transfer
the
eluate
to
a
50­
mL
tube.
Repeat
the
membrane
wash
with
another
5
mL
of
PBST
and
transfer
the
eluate
to
the
50­
mL
tube.
(
Optional:
Perform
a
third
wash
using
another
5
mL
of
PBST,
by
hand­
kneading
an
additional
minute
or
placing
the
bag
on
a
flat­
headed
vortexer
and
vortexing
for
one
minute.
Transfer
the
eluate
to
the
50­
mL
tube.)

NOTE:
Mark
the
bag
with
an
"
X"
to
note
which
side
of
the
membrane
has
the
oocysts
to
encourage
the
hand­
kneading
to
focus
on
the
appropriate
side
of
the
membrane.

°
Stomacher
wash.
Add
5
mL
of
PBST
to
the
bag
containing
the
membrane.
Place
the
bag
containing
the
membrane
into
a
small
stomacher
and
stomach
for
3
minutes.
Using
a
pipette
transfer
the
eluate
to
a
50­
mL
tube.
Repeat
the
wash
two
times
using
the
stomacher
and
5­
mL
aliquots
of
PBST.
(
Optional:
Perform
a
fourth
wash
using
another
5
mL
of
PBST,
by
hand­
kneading
an
additional
minute
or
placing
the
bag
on
a
flat­
headed
vortexer
and
vortexing
for
one
minute.
Transfer
the
eluate
to
the
50­
mL
tube.)
12.3.3.2.4
If
the
membrane
filter
clogs
before
concentration
is
complete,
there
are
two
possible
options
for
completion
of
concentration.
One
option
is
replacing
the
membrane
as
often
as
necessary.
Filter
membranes
may
be
placed
smooth
side
up
during
the
second
concentration
step.
Another
option
is
concentrating
the
remaining
eluate
using
centrifugation.
Both
options
are
provided
below.
°
Using
multiple
membranes.
Disassemble
the
concentrator
tube
and
pour
any
remaining
eluate
back
into
the
pooling
beaker.
Remove
the
membrane
using
membrane
forceps,
placing
it
in
the
bag
provided.
Place
a
new
membrane
in
the
concentrator
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
43
tube
smooth
side
up,
reassemble,
return
the
eluate
to
the
concentrator
tube,
rinse
the
pooling
beaker
and
add
rinse
to
the
eluate,
and
continue
the
concentration.
Replace
the
membrane
as
often
as
necessary.
°
Centrifuging
remaining
volume.
Decant
the
remaining
eluate
into
a
2­
L
pooling
beaker.
Rinse
the
sides
of
the
concentrator
tube
and
add
to
the
pooling
beaker.
Remove
the
filter
membrane
and
place
it
in
the
bag
provided.
Wash
the
membrane
as
described
in
Section
12.3.3.2.3,
then
concentrate
the
sample
as
described
in
Section
12.3.3.3.1.
12.3.3.3
If
the
Filta­
Max
 
concentrator
is
not
used
for
sample
concentration,
or
if
the
membrane
filter
clogs
before
sample
concentration
is
complete,
then
the
procedures
described
in
Section
12.3.3.3.1
should
be
used
to
concentrate
the
sample.
If
less
than
50
mL
of
concentrate
has
been
generated,
the
sample
can
be
further
concentrated,
as
described
in
Section
12.3.3.3.2,
to
reduce
the
volume
of
sample
to
be
processed
through
IMS.

NOTE:
The
volume
must
not
be
reduced
to
less
than
5
mL
above
the
packed
pellet.
The
maximum
amount
of
pellet
that
should
be
processed
through
IMS
is
0.5
mL.
If
the
packed
pellet
is
greater
than
0.5
mL
then
the
pellet
may
be
subsampled
as
described
in
Section
13.2.4.

12.3.3.3.1
Centrifugation
of
greater
than
50
mL
of
eluate
(
a)
Decant
the
eluate
from
the
2­
L
pooling
beaker
into
250­
mL
conical
centrifuge
tubes.
Make
sure
that
the
centrifuge
tubes
are
balanced.

(
b)
Centrifuge
the
250­
mL
centrifuge
tubes
containing
the
eluate
at
1500
×
G
for
15
minutes.
Allow
the
centrifuge
to
coast
to
a
stop.

(
c)
Using
a
Pasteur
pipette,
carefully
aspirate
off
the
supernatant
to
5
mL
above
the
pellet.
If
the
sample
is
reagent
water
(
e.
g.
initial
or
ongoing
precision
and
recovery
sample)
extra
care
must
be
taken
to
avoid
aspirating
oocysts
and
cysts
during
this
step.

(
d)
Vortex
each
250­
mL
tube
vigorously
until
pellet
is
completely
resuspended.
Swirl
the
centrifuge
tube
gently
to
reduce
any
foaming
after
vortexing.
Combine
the
contents
of
each
250­
mL
centrifuge
tube
into
a
50­
mL
centrifuge
tube.
Rinse
each
of
the
250­
mL
centrifuge
tubes
with
PBST
and
add
the
rinse
to
the
50­
mL
tube.

(
e)
Proceed
to
Section
12.3.3.3.2.
12.3.3.3.2
Centrifugation
of
less
than
50
mL
of
eluate
(
a)
Centrifuge
the
50­
mL
centrifuge
tube
containing
the
combined
concentrate
at
1500
x
G
for
15
minutes.
Allow
the
centrifuge
to
coast
to
a
stop.
Record
the
initial
pellet
volume
(
volume
of
solids)
and
the
date
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
44
and
time
that
concentration
was
completed
on
a
bench
sheet.
(
b)
Proceed
to
Section
13.0
for
concentration
and
separation
(
purification).
12.3.4
Maintenance
and
cleaning
12.3.4.1
Maintenance
of
O­
rings
12.3.4.1.1
Check
all
rubber
O­
rings
for
wear
or
deterioration
prior
to
each
use
and
replace
as
necessary.
12.3.4.1.2
Lubricate
the
plunger
head
O­
ring
inside
and
out
with
silicon
before
each
use.
12.3.4.1.3
Lubricate
all
other
O­
rings
(
concentrator
tube
set,
filter
housing)
regularly
in
order
to
preserve
their
condition.
12.3.4.2
Cleaning
12.3.4.2.1
All
components
of
the
Filta­
MaxTM
system
can
be
cleaned
using
warm
water
and
laboratory
detergent.
After
washing,
rinse
all
components
with
oocyst
and
cyst
free
reagent
water
and
dry
them.
All
O­
rings
should
be
re­
lubricated.
Alternatively
a
mild
(
40
°
C)
dishwasher
cycle
without
bleach
or
rinse
aid
can
be
used.
12.3.4.2.2
To
wash
the
detachable
plunger
head
slide
the
locking
pin
out
and
wash
the
plunger
head
and
locking
pin
in
warm
water
and
laboratory
detergent.
Rinse
the
plunger
head
and
locking
pin
with
oocyst
and
cyst
free
reagent
water
and
dry.
Lightly
lubricate
the
locking
pin
and
reassemble
the
plunger
head.

13.0
Sample
Concentration
and
Separation
(
Purification)
13.1
During
concentration
and
separation,
the
filter
eluate
is
concentrated
through
centrifugation,
and
the
oocysts
and
cysts
in
the
sample
are
separated
from
other
particulates
through
immunomagnetic
separation
(
IMS).
Alternate
procedures
and
products
may
be
used
if
the
laboratory
first
demonstrates
equivalent
or
superior
performance
as
per
Section
9.1.2.
13.2
Adjustment
of
pellet
volume
13.2.1
Centrifuge
the
250­
mL
centrifuge
tube
containing
the
capsule
filter
eluate
at
1500
×
G
for
15
minutes.
Allow
the
centrifuge
to
coast
to
a
stop
 
do
not
use
the
brake.
Record
the
pellet
volume
(
volume
of
solids)
on
the
bench
sheet.

NOTE:
Recoveries
may
be
improved
if
centrifugation
force
is
increased
to
2000
×
G.
However,
do
not
use
this
higher
force
if
the
sample
contains
sand
or
other
gritty
material
that
may
degrade
the
condition
of
any
oocysts
and/
or
cysts
in
the
sample.

13.2.2
Using
a
Pasteur
pipette,
carefully
aspirate
the
supernatant
to
5
mL
above
the
pellet.
Extra
care
must
be
taken
to
avoid
aspirating
oocysts
and
cysts
during
this
step,
particularly
if
the
sample
is
reagent
water
(
e.
g.
initial
or
ongoing
precision
and
recovery
sample).
13.2.3
If
the
packed
pellet
volume
is
#
0.5
mL,
vortex
the
tube
vigorously
until
pellet
is
completely
resuspended.
Swirl
the
centrifuge
tube
gently
to
reduce
any
foaming
after
vortexing.
Record
the
resuspended
pellet
volume
on
the
bench
sheet.
Proceed
to
Section
13.3.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
45
NOTE:
Extra
care
must
be
taken
with
samples
containing
sand
or
other
gritty
material
when
vortexing
to
ensure
that
the
condition
of
any
oocysts
and/
or
cysts
in
the
sample
is
not
compromised.

13.2.4
If
the
packed
pellet
volume
is
>
0.5
mL,
the
concentrate
needs
to
must
be
separated
into
multiple
subsamples
(
a
subsample
is
equivalent
to
no
greater
than
0.5
mL
of
packed
pellet
material,
the
recommended
maximum
amount
of
particulate
material
to
process
through
the
subsequent
purification
and
examination
steps
in
the
method).
Use
the
following
formula
to
determine
the
total
volume
required
in
the
centrifuge
tube
before
separating
the
concentrate
into
two
or
more
subsamples:

total
volume
(
mL)
required
=
pellet
volume
x
5
mL
0.5
mL
(
For
example,
if
the
packed
pellet
volume
is
1.2
mL,
the
total
volume
required
is
12
mL.)
Add
reagent
water
to
the
centrifuge
tube
to
bring
the
total
volume
to
the
level
calculated
above.
Vortex
the
tube
vigorously
for
10
to
15
seconds
to
completely
resuspend
the
pellet.
Record
the
resuspended
pellet
volume
on
the
bench
sheet.

NOTE:
Extra
care
must
be
taken
with
samples
containing
sand
or
other
gritty
material
when
vortexing
to
ensure
that
the
condition
of
any
oocysts
in
the
sample
is
not
compromised.

13.2.4.1
Analysis
of
entire
sample.
If
analysis
of
the
entire
sample
is
required,
determine
the
number
of
subsamples
to
be
processed
independently
through
the
remainder
of
the
method:
13.2.4.1.1
Calculate
number
of
subsamples:
Divide
the
total
volume
in
the
centrifuge
tube
by
5
mL
and
round
up
to
the
nearest
integer
(
for
example,
if
the
resuspended
volume
in
Section
13.2.4
is
12
mL,
then
the
number
of
subsamples
would
be
12
mL
/
5
mL
=
2.4,
rounded
=
3
subsamples).
13.2.4.1.2
Determine
volume
of
resuspended
concentrate
per
subsample.
Divide
the
total
volume
in
the
centrifuge
tube
by
the
calculated
number
of
subsamples
(
for
example,
if
the
resuspended
volume
in
Section
13.2.4
is
12
mL,
then
the
volume
to
use
for
each
subsample
=
12
mL
/
3
subsamples
=
4
mL).
13.2.4.1.3
Process
subsamples
through
IMS.
Vortex
the
tube
vigorously
for
10
to
15
seconds
to
completely
resuspend
the
pellet.
Record
the
resuspended
pellet
volume
on
the
bench
sheet.
Proceed
immediately
to
Section
13.3,
and
transfer
aliquots
of
the
resuspended
concentrate
equivalent
to
the
volume
in
the
previous
step
to
multiple,
flat­
sided
sample
tubes
in
Section
13.3.2.1.
Process
the
sample
as
multiple,
independent
subsamples
from
Section
13.3
onward,
including
the
preparation
and
examination
of
separate
slides
for
each
aliquot.
Record
the
volume
of
resuspended
concentrate
transferred
to
IMS
on
the
bench
sheet
(
this
will
be
equal
to
the
volume
recorded
in
Section
13.2.4).
Also
record
the
number
of
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
46
subsamples
processed
independently
through
the
method
on
the
bench
sheet.
13.2.4.2
Analysis
of
partial
sample.
If
not
all
of
the
concentrate
will
be
examined,
vortex
the
tube
vigorously
for
10
to
15
seconds
to
completely
resuspend
the
pellet.
Record
the
resuspended
pellet
volume
on
the
bench
sheet.
Proceed
immediately
to
Section
13.3,
and
transfer
one
or
more
5­
mL
aliquots
of
the
resuspended
concentrate
to
one
or
more
flat­
sided
sample
tubes
in
Section
13.3.2.1.
Record
the
volume
of
resuspended
concentrate
transferred
to
IMS
on
the
bench
sheet.
To
determine
the
volume
analyzed,
calculate
the
percent
of
the
concentrate
examined
using
the
following
formula:

percent
examined
=
total
volume
of
resuspended
concentrate
transferred
to
IMS
x
100%
total
volume
of
resuspended
concentrate
in
Section
13.2.4
Then
multiply
the
volume
filtered
(
Section
12.2.5.2)
by
this
percentage
to
determine
the
volume
analyzed.
13.3
IMS
procedure
(
adapted
from
Reference
20.11)

NOTE:
The
IMS
procedure
should
be
performed
on
a
bench
top
with
all
materials
at
room
temperature,
ranging
from
15
°
C
to
25
°
C.

13.3.1
Preparation
and
addition
of
reagents
13.3.1.1
Prepare
a
1X
dilution
of
SL­
buffer­
A
from
the
10X
SL­
buffer­
A
(
clear,
colorless
solution)
supplied.
Use
reagent
water
(
demineralized;
Section
7.3)
as
the
diluent.
For
every
1
mL
of
1X
SL­
buffer­
A
required,
take
100
µ
L
of
10X
SL­
buffer­
A
and
make
up
to
1
mL
with
the
diluent
water.
A
volume
of
1.5
mL
of
1X
SL­
buffer­
A
will
be
required
per
sample
or
subsample
on
which
the
Dynal
IMS
procedure
is
performed.
13.3.1.2
For
each
sample
or
subsample
(
Section
13.2)
to
be
processed
through
IMS,
add
1
mL
of
the
10X
SL­
buffer­
A
(
supplied
 
not
the
diluted
1X
SL­
buffer­
A)
to
a
flat­
sided
tube
(
Section
6.5.4).
13.3.1.3
For
each
subsample,
add
1
mL
of
the
10X
SL­
buffer­
B
(
supplied
 
magenta
solution)
to
the
flat­
sided
tube
containing
the
10X
SL­
buffer­
A.
13.3.2
Oocyst
and
cyst
capture
13.3.2.1
Use
a
graduated,
10­
mL
pipette
that
has
been
pre­
rinsed
with
elution
buffer
to
transfer
the
water
sample
concentrate
from
Section
13.2
to
the
flat­
sided
tube(
s)
containing
the
SL­
buffers.
If
all
of
the
concentrate
is
used,
rinse
the
centrifuge
tube
twice
with
reagent
water
and
add
the
rinsate
to
the
flat­
sided
tube
containing
the
concentrate
(
or
to
the
tube
containing
the
first
subsample,
if
multiple
subsamples
will
be
processed).
Each
of
the
two
rinses
should
be
half
the
volume
needed
to
bring
the
volume
in
the
flat­
sided
sample
tube
to
10
mL
(
including
the
buffers
added
in
Sections
13.3.1.2
and
13.3.1.3).
(
For
example,
if
the
tube
contained
1
mL
of
SL­
buffer­
A
and
1
mL
of
SL­
buffer­
B,
and
5
mL
of
sample
was
transferred
after
resuspension
of
the
pellet,
for
a
total
of
7
mL,
the
centrifuge
tube
would
be
rinsed
twice
with
1.5
mL
of
reagent
water
to
bring
the
total
volume
in
the
flat­
sided
tube
to
10
mL.)
Visually
inspect
the
centrifuge
tube
after
completing
the
transfer
to
ensure
that
no
concentrate
remains.
If
multiple
subsamples
will
be
processed,
bring
the
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
47
volume
in
the
remaining
flat­
sided
tubes
to
10
mL
with
reagent
water.
Label
the
flat­
sided
tube(
s)
with
the
sample
number
(
and
subsample
letters).
13.3.2.2
Vortex
the
Dynabeads
®
Crypto­
Combo
vial
from
the
IMS
kit
for
approximately
10
seconds
to
suspend
the
beads.
Ensure
that
the
beads
are
fully
resuspended
by
inverting
the
sample
tube
and
making
sure
that
there
is
no
residual
pellet
at
the
bottom.
13.3.2.3
Add
100
µ
L
of
the
resuspended
Dynabeads
®
Crypto­
Combo
(
Section
13.3.2.2)
to
the
sample
tube(
s)
containing
the
water
sample
concentrate
and
SL­
buffer.
13.3.2.4
Vortex
the
Dynabeads
®
Giardia­
Combo
vial
from
the
IMS
kit
for
approximately
10
seconds
to
suspend
the
beads.
Ensure
that
the
beads
are
fully
resuspended
by
inverting
the
tube
and
making
sure
that
there
is
no
residual
pellet
at
the
bottom.
13.3.2.5
Add
100
µ
L
of
the
resuspended
Dynabeads
®
Giardia­
Combo
(
Section
13.3.2.4)
to
the
sample
tube(
s)
containing
the
water
sample
concentrate,
Dynabeads
®
Crypto­
Combo,
and
SL­
buffer.
13.3.2.6
Affix
the
sample
tube(
s)
to
a
rotating
mixer
and
rotate
at
approximately
18
rpm
for
1
hour
at
room
temperature.
13.3.2.7
After
rotating
for
1
hour,
remove
each
sample
tube
from
the
mixer
and
place
the
tube
in
the
magnetic
particle
concentrator
(
MPC­
1)
with
flat
side
of
the
tube
toward
the
magnet.
13.3.2.8
Without
removing
the
sample
tube
from
the
MPC­
1,
place
the
magnet
side
of
the
MPC­
1
downwards,
so
the
tube
is
horizontal
and
the
flat
side
of
the
tube
is
facing
down.
13.3.2.9
Gently
rock
the
sample
tube
by
hand
end­
to­
end
through
approximately
90
°
,
tilting
the
cap­
end
and
base­
end
of
the
tube
up
and
down
in
turn.
Continue
the
tilting
action
for
2
minutes
with
approximately
one
tilt
per
second.
13.3.2.10
Ensure
that
the
tilting
action
is
continued
throughout
this
period
to
prevent
binding
of
low­
mass,
magnetic
or
magnetizable
material.
If
the
sample
in
the
MPC­
1
is
allowed
to
stand
motionless
for
more
than
10
seconds,
remove
the
flat­
sided
tube
from
the
MPC­
1,
shake
the
tube
to
resuspend
all
material,
replace
the
sample
tube
in
the
MPC­
1
and
repeat
Section
13.3.2.9
before
continuing
to
Section
13.3.2.11.
13.3.2.11
Return
the
MPC­
1
to
the
upright
position,
sample
tube
vertical,
with
cap
at
top.
Immediately
remove
the
cap
and,
keeping
the
flat
side
of
the
tube
on
top,
pour
off
all
of
the
supernatant
from
the
tube
held
in
the
MPC­
1
into
a
suitable
container.
Do
not
shake
the
tube
and
do
not
remove
the
tube
from
MPC­
1
during
this
step.
Allow
more
supernatant
to
settle;
aspirate
additional
supernatant
with
pipette.
13.3.2.12
Remove
the
sample
tube
from
the
MPC­
1
and
resuspend
the
sample
in
1­
mL
0.5
mL
1X
SL­
buffer­
A
(
prepared
from
10X
SL­
buffer­
A
stock
 
supplied).
Mix
very
gently
to
resuspend
all
material
in
the
tube.
Do
not
vortex.
13.3.2.13
Quantitatively
transfer
(
transfer
followed
by
two
rinses)
all
the
liquid
from
the
sample
tube
to
a
labeled,
1.5­
mL
microcentrifuge
tube.
Use
1
mL
0.5
mL
of
1X
SL­
buffer­
A
to
perform
the
first
rinse
and
0.5
mL
of
reagent
water
for
the
second
rinse.
Liberally
rinse
down
the
sides
of
the
Leighton
tube
before
transferring
and
transfer
the
rinsate
to
the
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
48
microcentrifuge
tube.
Allow
the
flat­
sided
sample
tube
to
sit
for
a
minimum
of
1
minute
after
transfer
of
the
second
rinse
volume,
then
use
a
pipette
to
collect
any
residual
volume
that
drips
down
to
the
bottom
of
the
tube
to
ensure
that
as
much
sample
volume
is
recovered
as
possible.
Ensure
that
all
of
the
liquid
and
beads
are
transferred.
13.3.2.14
Place
the
microcentrifuge
tube
into
the
second
magnetic
particle
concentrator
(
MPC­
M),
with
its
magnetic
strip
in
place.
13.3.2.15
Without
removing
the
microcentrifuge
tube
from
MPC­
M,
gently
rock/
roll
the
tube
through
180
°
by
hand.
Continue
for
approximately
1
minute
with
approximately
one
180
°
roll/
rock
per
second.
At
the
end
of
this
step,
the
beads
should
produce
a
distinct
brown
dot
at
the
back
of
the
tube.
13.3.2.16
Immediately
aspirate
the
supernatant
from
the
tube
and
cap
held
in
the
MPC­
M.
If
more
than
one
sample
is
being
processed,
conduct
three
90
°
rock/
roll
actions
before
removing
the
supernatant
from
each
tube.
Take
care
not
to
disturb
the
material
attached
to
the
wall
of
the
tube
adjacent
to
the
magnet.
Do
not
shake
the
tube.
Do
not
remove
the
tube
from
MPC­
M
while
conducting
these
steps.
13.3.3
Dissociation
of
beads/
oocyst/
cyst
complex
NOTE:
Two
acid
dissociations
are
required.

13.3.3.1
Remove
the
magnetic
strip
from
the
MPC­
M.
13.3.3.2
Add
50
µ
L
of
0.1
N
HCl,
then
vortex
at
the
highest
setting
for
approximately
50
seconds.

NOTE:
The
laboratory
should
must
use
0.1­
N
standards
purchased
directly
from
a
vendor,
rather
than
adjusting
the
normality
in­
house.

13.3.3.3
Place
the
tube
in
the
MPC­
M
without
the
magnetic
strip
in
place
and
allow
to
stand
in
a
vertical
position
for
at
least
10
minutes
at
room
temperature.
13.3.3.4
Vortex
vigorously
for
approximately
30
seconds.
13.3.3.5
Ensure
that
all
of
the
sample
is
at
the
base
of
the
tube.
Place
the
microcentrifuge
tube
in
the
MPC­
M.
13.3.3.6
Replace
magnetic
strip
in
MPC­
M
and
allow
the
tube
to
stand
undisturbed
for
a
minimum
of
10
seconds.
13.3.3.7
Prepare
a
well
slide
for
sample
screening
and
label
the
slide.
13.3.3.8
Add
5
µ
L
of
1.0
N
NaOH
to
the
sample
wells
of
two
well
slides
(
add
10
µ
L
to
the
sample
well
of
one
well
slide
if
the
volume
from
the
two
required
dissociations
will
be
added
to
the
same
slide).

NOTE:
The
laboratory
should
must
use
1.0­
N
standards
purchased
directly
from
a
vendor
rather
than
adjusting
the
normality
in­
house.

13.3.3.9
Without
removing
the
microcentrifuge
tube
from
the
MPC­
M,
transfer
all
of
the
sample
from
the
microcentrifuge
tube
in
the
MPC­
M
to
the
sample
well
with
the
NaOH.
Do
not
disturb
the
beads
at
the
back
wall
of
the
tube.
Ensure
that
all
of
the
fluid
is
transferred.
13.3.3.10
Do
not
discard
the
beads
or
microcentrifuge
tube
after
transferring
the
volume
from
the
first
acid
dissociation
to
the
well
slide.
Perform
the
steps
in
Sections
13.3.3.1
through
13.3.3.9
a
second
time.
The
volume
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
49
from
the
second
dissociation
can
be
added
to
the
slide
containing
the
volume
from
the
first
dissociation,
or
can
be
applied
to
a
second
slide.

NOTE:
If
one
slide
is
used,
exert
extra
care
when
using
The
wells
on
Dynal
Spot­
On
slides
are
likely
to
be
too
small
to
accommodate
the
volumes
from
both
dissociations.

13.3.3.11
Record
the
date
and
time
the
purified
sample
was
applied
to
the
slide(
s).
13.3.3.12
Air­
dry
the
sample
on
the
well
slide(
s).
Because
temperature
and
humidity
varyies
from
laboratory
to
laboratory,
no
minimum
time
is
specified.
However,
the
laboratory
must
take
care
to
ensure
that
the
sample
has
dried
completely
before
staining
to
prevent
losses
during
the
rinse
steps.
A
slide
warmer
set
at
35
°
C
to
42
°
C
also
can
be
used.
13.3.4
Tips
for
minimizing
carry­
over
of
debris
onto
microscope
slides
after
IMS
°
Make
sure
the
resuspended
pellet
is
fully
homogenized
before
placing
the
tube
in
the
MPC­
1
or
MPC­
M
to
avoid
trapping
"
clumps"
or
a
dirty
layer
between
the
beads
and
the
side
of
the
tube.

°
When
using
the
MPC­
1
magnet,
make
sure
that
the
tube
is
snugged
flat
against
the
magnet.
Push
the
tube
flat
if
necessary.
Sometimes
the
magnet
is
not
flush
with
the
outside
of
the
holder
and,
therefore,
the
attraction
between
the
beads
and
the
magnet
is
not
as
strong
as
it
should
be.
However,
it
can
be
difficult
to
determine
this
if
you
do
not
have
more
than
one
MPC­
1
to
make
comparisons.

°
After
the
supernatant
has
been
poured
off
at
Section
13.3.2.11,
leave
the
tube
in
the
MPC­
1
and
allow
time
for
any
supernatant
remaining
in
the
tube
to
settle
down
to
the
bottom.
Then
aspirate
the
settled
supernatant
and
associated
particles
from
the
bottom
of
the
tube.
The
same
can
be
done
at
Section
13.3.2.16
with
the
microcentrifuge
tube.

°
An
additional
rinse
can
also
be
performed
at
Section
13.3.2.11.
After
the
supernatant
has
been
poured
off
and
any
settled
material
is
aspirated
off
the
bottom,
leave
the
tube
in
the
MPC­
1
and
add
an
additional
10
mL
of
reagent
water
or
PBS
to
the
tube
and
repeat
Sections
13.3.2.9
and
13.3.2.11.
Although
labs
have
reported
successfully
using
this
technique
to
reduce
carryover,
because
the
attraction
between
the
MPC­
1
and
the
beads
is
not
as
great
as
the
attraction
between
the
MPC­
M
and
the
beads,
the
chances
would
be
greater
for
loss
of
cysts
and
oocysts
doing
the
rinse
at
this
step
instead
of
at
Section
13.3.2.16.

°
After
the
supernatant
has
been
aspirated
from
the
tube
at
Section
13.3.2.16,
add
0.1
mL
of
PBS,
remove
the
tube
from
the
MPC­
M,
and
resuspend.
Repeat
Sections
13.3.2.15
and
13.3.2.16.

°
Use
a
slide
with
the
largest
diameter
well
available
to
spread
out
the
sample
as
much
as
possible.

14.0
Sample
Staining
NOTE:
The
sample
must
be
stained
within
72
hours
of
application
of
the
purified
sample
to
the
slide.

14.1
Prepare
positive
and
negative
controls.
14.1.1
For
the
positive
control,
pipette
10
µ
L
of
positive
antigen
or
200
to
400
intact
oocysts
and
200
to
400
cysts
to
the
center
of
a
well.
14.1.2
For
the
negative
control,
pipette
50
µ
L
of
150
mM
PBS
(
Section
7.6.4)
into
the
center
of
a
well
and
spread
it
over
the
well
area
with
a
pipette
tip.
14.1.3
Air­
dry
the
control
slides
(
see
Section
13.3.3.12
for
guidance).
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
50
NOTE:
If
the
laboratory
has
a
large
batch
of
slides
that
will
be
examined
over
several
days,
and
is
concerned
that
a
single
positive
control
may
fade,
due
to
multiple
examinations,
the
laboratory
should
prepare
multiple
control
slides
with
the
batch
of
field
slides
and
alternate
between
the
positive
controls
when
performing
the
positive
control
check.

14.2
Apply
50­
µ
L
of
absolute
methanol
to
each
well
containing
the
dried
sample
and
allow
to
air­
dry
for
3
to
5
minutes.
14.2
Follow
manufacturer's
instructions
in
applying
stain
to
slides.
14.3
Place
the
slides
in
a
humid
chamber
in
the
dark
and
incubate
at
room
temperature
for
approximately
30
minutes.
The
humid
chamber
consists
of
a
tightly
sealed
plastic
container
containing
damp
paper
towels
on
top
of
which
the
slides
are
placed.
14.4
Remove
slides
from
humid
chamber
and
allow
condensation
to
evaporate,
if
present.
14.5
Apply
one
drop
of
wash
buffer
(
prepared
according
to
the
manufacturer's
instructions
[
Section
7.6])
to
each
well.
Tilt
each
slide
on
a
clean
paper
towel,
long
edge
down.
Gently
aspirate
the
excess
detection
reagent
from
below
the
well
using
a
clean
Pasteur
pipette
or
absorb
with
paper
towel
or
other
absorbent
material
placed
at
edge
of
slide.
Avoid
disturbing
the
sample.

NOTE:
If
using
the
Merifluor
stain
(
Section
7.6.1),
do
not
allow
slides
to
dry
completely.

14.6
Apply
50
µ
L
of
4',
6­
diamidino­
2­
phenylindole
(
DAPI)
staining
solution
(
Section
7.7.2)
to
each
well.
Allow
to
stand
at
room
temperature
for
a
minimum
of
1
minute.
(
The
solution
concentration
may
be
increased
up
to
1
µ
g/
mL
if
fading/
diffusion
of
DAPI
staining
is
encountered,
but
the
staining
solution
must
be
tested
first
on
expendable
environmental
samples
to
confirm
that
staining
intensity
is
appropriate.)
14.7
Apply
one
drop
of
wash
buffer
(
prepared
according
to
the
manufacturer's
instructions
[
Section
7.6])
to
each
well.
Tilt
each
slide
on
a
clean
paper
towel,
long
edge
down.
Gently
aspirate
the
excess
DAPI
staining
solution
from
below
the
well
using
a
clean
Pasteur
pipette
or
absorb
with
paper
towel
or
other
absorbent
material
placed
at
edge
of
slide.
Avoid
disturbing
the
sample.

NOTE:
If
using
the
Merifluor
stain
(
Section
7.6.1),
do
not
allow
slides
to
dry
completely.

14.8
Add
mounting
medium
(
Section
7.8)
to
each
well.
14.9
Apply
a
cover
slip.
Use
a
tissue
to
remove
excess
mounting
fluid
from
the
edges
of
the
coverslip.
Seal
the
edges
of
the
coverslip
onto
the
slide
using
clear
nail
polish.
14.10
Record
the
date
and
time
that
staining
was
completed
on
the
bench
sheet.
If
slides
will
not
be
read
immediately,
store
in
a
humid
chamber
in
the
dark
at
0
°
C
to
<
8
°
C
(
but
not
frozen)
until
ready
for
examination.

15.0
Examination
NOTE:
Although
immunofluorescence
assay
(
FA)
and
4',
6­
diamidino­
2­
phenylindole
(
DAPI)
and
differential
interference
contrast
(
DIC)
microscopy
examination
and
confirmation
should
be
performed
immediately
after
staining
is
complete,
laboratories
have
up
to
7
days
from
completion
of
sample
staining
to
complete
the
examination
and
confirmation
of
samples.
However,
if
fading/
diffusion
of
FITC
or
DAPI
staining
is
noticed,
the
laboratory
must
reduce
this
holding
time.
In
addition
the
laboratory
may
adjust
the
concentration
of
the
DAPI
staining
solution
(
Sections
7.7.2)
so
that
fading/
diffusion
does
not
occur.

15.1
Scanning
technique:
Scan
each
well
in
a
systematic
fashion.
An
up­
and­
down
or
a
side­
to­
side
scanning
pattern
may
be
used
(
Figure
4).
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
51
15.2
Examination
using
immunofluorescence
assay
(
FA),
4',
6­
diamidino­
2­
phenylindole
(
DAPI)
staining
characteristics,
and
differential
interference
contrast
(
DIC)
microscopy.
The
minimum
magnification
requirements
for
each
type
of
examination
are
noted
below.

NOTE:
All
shape
characterization
and
size
measurements
must
be
determined
using
1000X
magnification
and
reported
to
the
nearest
0.5
µ
m.

Record
examination
results
for
Cryptosporidium
oocysts
on
a
Cryptosporidium
report
examination
results
form;
record
examination
results
for
Giardia
cysts
on
a
Giardia
report
examination
results
form.
All
oocysts
and
cysts
that
meet
the
criteria
specified
in
Sections
15.2.2
and
15.2.3,
less
atypical
organisms
specifically
identified
as
non­
target
organisms
by
DIC
or
DAPI
(
e.
g.
possessing
spikes,
stalks,
appendages,
pores,
one
or
two
large
nuclei
filling
the
cell,
red
fluorescing
chloroplasts,
crystals,
spores,
etc),
must
be
reported.
15.2.1
Positive
and
negative
staining
control.
Positive
and
negative
staining
controls
must
be
acceptable.
15.2.1.1
Each
analyst
must
characterize
a
minimum
of
three
Cryptosporidium
oocysts
and
three
Giardia
cysts
on
the
positive
staining
control
slide
before
examining
field
sample
slides.
This
characterization
must
be
performed
by
each
analyst
during
each
microscope
examination
session.
FITC
examination
must
be
conducted
at
a
minimum
of
200X
total
magnification,
DAPI
examination
must
be
conducted
at
a
minimum
of
400X,
and
DIC
examination
and
size
measurements
must
be
conducted
at
a
minimum
of
1000X.
Size,
shape,
and
DIC
and
DAPI
characteristics
of
the
three
Cryptosporidium
oocysts
and
Giardia
cysts
must
be
recorded
by
the
analyst
on
a
microscope
log.
The
analyst
also
must
indicate
on
each
sample
report
examination
results
form
whether
the
positive
staining
control
was
acceptable.
15.2.1.2
Examine
the
negative
staining
control
to
confirm
that
it
does
not
contain
any
oocysts
or
cysts
(
Section
14.1).
Indicate
on
each
sample
report
examination
results
form
whether
the
negative
staining
control
was
acceptable.
15.2.1.3
If
the
positive
staining
control
contains
oocysts
and
cysts
within
the
expected
range
and
at
the
appropriate
fluorescence
for
both
FA
and
DAPI,
and
the
negative
staining
control
does
not
contain
any
oocysts
or
cysts
(
Section
14.1),
proceed
to
Sections
15.2.2
and
15.2.3.
15.2.2
Sample
examination
 
Cryptosporidium
15.2.2.1
FITC
examination
(
the
analyst
must
use
a
minimum
of
200X
total
magnification).
Use
epifluorescence
to
scan
the
entire
well
for
applegreen
fluorescence
of
oocyst
and
cyst
shapes.
When
brilliant
apple­
green
fluorescing
ovoid
or
spherical
objects
4
to
6
µ
m
in
diameter
are
observed
with
brightly
highlighted
edges,
increase
magnification
to
400X
and
switch
the
microscope
to
the
UV
filter
block
for
DAPI
(
Section
15.2.2.2),
then
to
DIC
(
Section
15.2.2.3)
at
1000X.
15.2.2.2
DAPI
examination
(
the
analyst
must
use
a
minimum
of
400X
total
magnification).
Using
the
UV
filter
block
for
DAPI,
the
object
will
exhibit
one
of
the
following
characteristics:
(
a)
Light
blue
internal
staining
(
no
distinct
nuclei)
with
a
green
rim
(
b)
Intense
blue
internal
staining
(
c)
Up
to
four
distinct,
sky­
blue
nuclei
Record
oocysts
in
category
(
a)
as
DAPI
negative;
record
oocysts
in
categories
(
b)
and
(
c)
as
DAPI
positive.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
52
15.2.2.3
DIC
examination
(
the
analyst
must
use
a
minimum
of
1000X
total
magnification
[
oil
immersion
lens]).
Using
DIC,
look
for
external
or
internal
morphological
characteristics
atypical
of
Cryptosporidium
oocysts
(
e.
g.,
spikes,
stalks,
appendages,
pores,
one
or
two
large
nuclei
filling
the
cell,
red
fluorescing
chloroplasts,
crystals,
spores,
etc.)
(
adapted
from
Reference
20.7).
If
atypical
structures
are
not
observed,
then
categorize
each
apple­
green
fluorescing
object
as:
(
a)
An
empty
Cryptosporidium
oocyst
(
b)
A
Cryptosporidium
oocyst
with
amorphous
structure
(
c)
A
Cryptosporidium
oocyst
with
internal
structure
(
one
to
four
sporozoites/
oocyst)
Using
1000X
total
magnification,
record
the
shape,
measurements
(
to
the
nearest
0.5
µ
m),
and
number
of
sporozoites
(
if
applicable)
for
each
apple­
green
fluorescing
object
meeting
the
size
and
shape
characteristics.
Although
not
a
defining
characteristic,
surface
oocyst
folds
may
be
observed
in
some
specimens.

NOTE:
All
measurements
must
be
made
at
1000X
magnification.

15.2.3
Sample
examination
 
Giardia
15.2.3.1
FITC
examination
(
the
analyst
must
use
a
minimum
of
200X
total
magnification).
When
brilliant
apple­
green
fluorescing
round
to
oval
ovoid
objects
(
8
­
18
µ
m
long
by
5
­
15
µ
m
wide)
are
observed,
increase
magnification
to
400X
and
switch
the
microscope
to
the
UV
filter
block
for
DAPI
(
Section
15.2.3.2)
then
to
DIC
(
Section
15.2.3.3)
at
1000X.
15.2.3.2
DAPI
examination
(
the
analyst
must
use
a
minimum
of
400X
total
magnification).
Using
the
UV
filter
block
for
DAPI,
the
object
will
exhibit
one
or
more
of
the
following
characteristics:
(
a)
Light
blue
internal
staining
(
no
distinct
nuclei)
and
a
green
rim
(
b)
Intense
blue
internal
staining
(
c)
Two
to
four
sky­
blue
nuclei
Record
cysts
in
category
(
a)
as
DAPI
negative;
record
cysts
in
categories
(
b)
and
(
c)
as
DAPI
positive.
15.2.3.3
DIC
examination
(
the
analyst
must
use
a
minimum
of
1000X
total
magnification
[
oil
immersion
lens]).
Using
DIC,
look
for
external
or
internal
morphological
characteristics
atypical
of
Giardia
cysts
(
e.
g.,
spikes,
stalks,
appendages,
pores,
one
or
two
large
nuclei
filling
the
cell,
red
fluorescing
chloroplasts,
crystals,
spores,
etc.)
(
adapted
from
Reference
20.7).
If
atypical
structures
are
not
observed,
then
categorize
each
object
meeting
the
criteria
specified
in
Sections
15.2.3.1
­
15.2.3.3
as
one
of
the
following,
based
on
DIC
examination:
(
a)
An
empty
Giardia
cyst
(
b)
A
Giardia
cyst
with
amorphous
structure
(
c)
A
Giardia
cyst
with
one
type
of
internal
structure
(
nuclei,
median
body,
or
axonemes),
or
(
d)
A
Giardia
cyst
with
more
than
one
type
of
internal
structure
Using
1000X
total
magnification,
record
the
shape,
measurements
(
to
the
nearest
0.5
µ
m),
and
number
of
nuclei
and
presence
of
median
body
or
axonemes
(
if
applicable)
for
each
apple­
green
fluorescing
object
meeting
the
size
and
shape
characteristics.

NOTE:
All
measurements
must
be
made
at
1000X
magnification.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
53
15.2.4
Record
the
date
and
time
that
sample
examination
was
completed
on
the
report
examination
results
form.
15.2.5
Report
Cryptosporidium
and
Giardia
concentrations
as
oocysts/
L
and
cysts/
L.
15.2.6
Record
analyst
name
16.0
Analysis
of
Complex
Samples
16.1
Some
samples
may
contain
high
levels
(>
1000/
L)
of
oocysts
and
cysts
and/
or
interfering
organisms,
substances,
or
materials.
Some
samples
may
clog
the
filter
(
Section
12.0);
others
will
not
allow
separation
of
the
oocysts
and
cysts
from
the
retentate
or
eluate;
and
others
may
contain
materials
that
preclude
or
confuse
microscopic
examination.
16.2
If
the
sample
holding
time
has
not
been
exceeded
and
a
full­
volume
sample
cannot
be
filtered,
dilute
an
aliquot
of
sample
with
reagent
water
and
filter
this
smaller
aliquot
(
Section
12.0).
This
dilution
must
be
recorded
and
reported
with
the
results.
16.3
If
the
holding
times
for
the
sample
and
for
microscopic
examination
of
the
cleaned
up
retentate/
eluate
have
been
exceeded,
the
site
should
be
re­
sampled.
If
this
is
not
possible,
the
results
should
be
qualified
accordingly.

17.0
Method
Performance
17.1
Method
acceptance
criteria
are
shown
in
Tables
3
and
4
in
Section
21.0.
The
initial
and
ongoing
precision
and
recovery
criteria
are
based
on
the
results
of
spiked
reagent
water
samples
analyzed
during
the
Information
Collection
Rule
Supplemental
Surveys
(
Reference
20.8).
The
matrix
spike
and
matrix
spike
duplicate
criteria
are
based
on
spiked
source
water
data
generated
during
the
interlaboratory
validation
study
of
Method
1623
involving
11
laboratories
and
11
raw
surface
water
matrices
across
the
U.
S.
(
Reference
20.10).

NOTE:
Some
sample
matrices
may
prevent
the
MS
acceptance
criteria
in
Tables
3
and
4
to
be
met.
An
assessment
of
the
distribution
of
MS
recoveries
across
430
MS
samples
from
87
sites
during
the
ICR
Supplemental
Surveys
is
provided
in
Table
5.

18.0
Pollution
Prevention
18.1
The
solutions
and
reagents
used
in
this
method
pose
little
threat
to
the
environment
when
recycled
and
managed
properly.
18.2
Solutions
and
reagents
should
be
prepared
in
volumes
consistent
with
laboratory
use
to
minimize
the
volume
of
expired
materials
that
need
to
be
discarded
to
be
disposed.

19.0
Waste
Management
19.1
It
is
the
laboratory's
responsibility
to
comply
with
all
federal,
state,
and
local
regulations
governing
waste
management,
particularly
the
biohazard
and
hazardous
waste
identification
rules
and
land
disposal
restrictions,
and
to
protect
the
air,
water,
and
land
by
minimizing
and
controlling
all
releases
from
fume
hoods
and
bench
operations.
Compliance
with
all
sewage
discharge
permits
and
regulations
is
also
required.
An
overview
of
these
requirements
can
be
found
in
the
Environmental
Management
Guide
for
Small
Laboratories
(
EPA
233­
B­
98­
001).
19.2
Samples,
reference
materials,
and
equipment
known
or
suspected
to
have
viable
oocysts
or
cysts
attached
or
contained
must
be
sterilized
prior
to
disposal.
19.3
For
further
information
on
waste
management,
consult
The
Waste
Management
Manual
for
Laboratory
Personnel
and
Less
is
Better:
Laboratory
Chemical
Management
for
Waste
Reduction,
both
available
from
the
American
Chemical
Society's
Department
of
Government
Relations
and
Science
Policy,
1155
16th
Street
N.
W.,
Washington,
D.
C.
20036.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
54
20.0
References
20.1
Rodgers,
Mark
R.,
Flanigan,
Debbie
J.,
and
Jakubowski,
Walter,
1995.
Applied
and
Environmental
Microbiology
61
(
10),
3759­
3763.
20.2
Fleming,
Diane
O.,
et
al.(
eds.),
Laboratory
Safety:
Principles
and
Practices,
2nd
edition.
1995.
ASM
Press,
Washington,
DC
20.3
"
Working
with
Carcinogens,"
DHEW,
PHS,
CDC,
NIOSH,
Publication
77­
206,
(
1977).
20.4
"
OSHA
Safety
and
Health
Standards,
General
Industry,"
OSHA
2206,
29
CFR
1910
(
1976).
20.5
"
Safety
in
Academic
Chemistry
Laboratories,"
ACS
Committee
on
Chemical
Safety
(
1979).
20.6
USEPA.
Manual
for
the
Certification
of
Laboratories
Analyzing
Drinking
Water:
Criteria
and
Procedures;
Quality
Assurance;
Fourth
Edition,
EPA
815­
B­
97­
001,
Office
of
Ground
Water
and
Drinking
Water,
U.
S.
Environmental
Protection
Agency,
26
West
Martin
Luther
King
Drive,
Cincinnati,
OH
45268
(
1997).
20.7
ICR
Microbial
Laboratory
Manual,
EPA/
600/
R­
95/
178,
National
Exposure
Research
Laboratory,
Office
of
Research
and
Development,
U.
S.
Environmental
Protection
Agency,
26
Martin
Luther
King
Drive,
Cincinnati,
OH
45268
(
1996).
20.8
USEPA.
EPA
Guide
to
Method
Flexibility
and
Approval
of
EPA
Water
Methods,
EPA
821­
D­
96­
004.
Office
of
Water,
Engineering
and
Analysis
Division,
Washington,
DC
20460
(
1996).
20.8
Connell,
K.,
C.
C.
Rodgers,
H.
L.
Shank­
Givens,
J
Scheller,
M.
L
Pope,
and
K.
Miller,
2000.
Building
a
Better
Protozoa
Data
Set.
Journal
AWWA,
92:
10:
30.
20.9
"
Envirochek
 
Sampling
Capsule,"
PN
32915,
Gelman
Sciences,
600
South
Wagner
Road,
Ann
Arbor,
MI
48103­
9019
(
1996).
20.10
USEPA.
Results
of
the
Interlaboratory
Method
Validation
Study
for
Determination
of
Cryptosporidium
and
Giardia
Using
USEPA
Method
1623,
EPA­
821­
R­
01­
028.
Office
of
Water,
Office
of
Science
and
Technology,
Engineering
and
Analysis
Division,
Washington,
DC
(
2001).
20.11
"
Dynabeads
®
GC­
Combo,"
Dynal
Microbiology
R&
D,
P.
O.
Box
8146
Dep.,
0212
Oslo,
Norway
(
September
1998,
Revision
no.
01).
20.12
USEPA.
Implementation
and
Results
of
the
Information
Collection
Rule
Supplemental
Surveys.
EPA­
815­
R­
01­
003.
Office
of
Water,
Office
of
Ground
Water
and
Drinking
Water,
Standards
and
Risk
Management
Division,
Washington,
DC
(
2001).
20.13
Connell,
K.,
J.
Scheller,
K.
Miller,
and
C.
C.
Rodgers,
2000.
Performance
of
Methods
1622
and
1623
in
the
ICR
Supplemental
Surveys.
Proceedings,
American
Water
Works
Association
Water
Quality
Technology
Conference,
November
5
­
9,
2000,
Salt
Lake
City,
UT.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
55
21.0
Tables
and
Figures
Table
1.
Method
Holding
Times
(
See
Section
8.2
for
details)

Sample
Processing
Step
Maximum
Allowable
Time
between
Breaks
(
Samples
should
be
processed
as
soon
as
possible)

Collection
Filtration
'
Up
to
96
hours
are
permitted
between
sample
collection
(
if
shipped
to
the
laboratory
as
a
bulk
sample)
or
filtration
(
if
filtered
in
the
field)
and
initiation
of
elution
Elution
These
steps
must
be
completed
in
1
working
day
Concentration
Purification
Application
of
purified
sample
to
slide
Drying
of
sample
'
Up
to
72
hours
are
permitted
from
application
of
the
purified
sample
to
the
slide
to
staining
Staining
'
Up
to
7
days
are
permitted
between
sample
staining
and
examination
Examination
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
56
Table
2.
Tier
1
and
Tier
2
Validation/
Equivalency
Demonstration
Requirements
Test
Description
Tier
1
modification(
1)
Tier
2
modification(
2)

IPR
(
Section
9.4)
4
replicates
of
spiked
reagent
water
Required.
Must
be
accompanied
by
a
method
blank.
Required
per
laboratory
Method
blank
(
Section
9.6)
Unspiked
reagent
water
Required
Required
per
laboratory
MS
(
Section
9.5.1)
Spiked
matrix
water
Required
on
each
water
to
which
the
modification
will
be
applied
and
on
every
20th
sample
of
that
water
thereafter.
Must
be
accompanied
by
an
unspiked
field
sample
collected
at
the
same
time
as
the
MS
sample
Not
required
MS/
MSD
(
Section
9.5)
2
replicates
of
spiked
matrix
water
Recommended,
but
not
required.
Must
be
accompanied
by
an
unspiked
field
sample
collected
at
the
same
time
as
the
MS
sample
Required
per
laboratory.
Each
laboratory
must
analyze
a
different
water.
(
1)
If
a
modification
will
be
used
only
in
one
laboratory,
these
tests
must
be
performed
and
the
results
must
meet
all
of
the
QC
acceptance
criteria
in
the
method
(
these
tests
also
are
required
the
first
time
a
laboratory
uses
the
validated
version
of
the
method)
(
2)
If
nationwide
approval
of
a
modification
is
sought
for
one
type
of
water
matrix
(
such
as
surface
water),
a
minimum
of
3
laboratories
must
perform
the
tests
and
the
results
from
each
lab
individually
must
meet
all
QC
acceptance
criteria
in
the
method.
If
more
than
3
laboratories
are
used
in
a
study,
a
minimum
of
75%
of
the
laboratories
must
meet
all
QC
acceptance
criteria.

NOTE:
The
initial
precision
and
recovery
and
ongoing
precision
and
recovery
(
OPR)
acceptance
criteria
listed
in
Tables
3
and
4
are
based
on
results
from
293
Cryptosporidium
OPR
samples
and
186
Giardia
OPR
samples
analyzed
by
six
laboratories
during
the
Information
Collection
Rule
Supplemental
Surveys
(
Reference
20.13).
The
matrix
spike
acceptance
criteria
are
based
on
data
generated
through
interlaboratory
validation
of
Method
1623
(
Reference
20.11).
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
57
Table
3.
Quality
Control
Acceptance
Criteria
for
Cryptosporidium
Performance
test
Section
Acceptance
criteria
Initial
precision
and
recovery
Mean
recovery
(
percent)

Precision
(
as
maximum
relative
standard
deviation)
9.4
9.4.2
24
­
100
9.4.2
55
Ongoing
precision
and
recovery
(
percent)
9.7
11
­
100
Matrix
spike/
matrix
spike
duplicate
(
for
method
modifications)
9.5
Mean
recovery1,
2
(
as
percent)
9.5.2
13
­
111
Precision
(
as
maximum
relative
percent
difference)
9.5.2
61
(
1)
The
acceptance
criteria
for
mean
MS/
MSD
recovery
serves
as
the
acceptance
criteria
for
MS
recovery
during
routine
use
of
the
method
(
Section
9.5.1).
(
2)
Some
sample
matrices
may
prevent
the
acceptance
criteria
from
being
met.
An
assessment
of
the
distribution
of
MS
recoveries
from
multiple
MS
samples
from
87
sites
during
the
ICR
Supplemental
Surveys
is
provided
in
Table
5.

Table
4.
Quality
Control
Acceptance
Criteria
for
Giardia
Performance
test
Section
Acceptance
criteria
Initial
precision
and
recovery
Mean
recovery
(
percent)

Precision
(
as
maximum
relative
standard
deviation)
9.4
9.4.2
24
­
100
9.4.2
49
Ongoing
precision
and
recovery
(
percent)
9.7
14
­
100
Matrix
spike/
matrix
spike
duplicate
(
for
method
modifications)
9.5
Mean
recovery*
(
as
percent)
9.5.2
15
­
118
Precision
(
as
maximum
relative
percent
difference)
9.5.2
30
(
1)
The
acceptance
criteria
for
mean
MS/
MSD
recovery
serves
as
the
acceptance
criteria
for
MS
recovery
during
routine
use
of
the
method
(
Section
9.5.1).
(
2)
Some
sample
matrices
may
prevent
the
acceptance
criteria
from
being
met.
An
assessment
of
the
distribution
of
MS
recoveries
across
multiple
MS
samples
from
87
sites
during
the
ICR
Supplemental
Surveys
is
provided
in
Table
5.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
58
Table
5.
Distribution
of
Matrix
Spike
Recoveries
from
Multiple
Samples
Collected
from
87
Source
Waters
During
the
ICR
Supplemental
Surveys
(
Adapted
from
Reference
20.14)

MS
Recovery
Range
Percent
of
430
Cryptosporidium
MS
Samples
in
Recovery
Range
Percent
of
270
Giardia
MS
Samples
in
Recovery
Range
<
10%
6.7%
5.2%

>
10%
­
20%
6.3%
4.8%

>
20%
­
30%
14.9%
7.0%

>
30%
­
40%
14.2%
8.5%

>
40%
­
50%
18.4%
17.4%

>
50%
­
60%
17.4%
16.3%

>
60%
­
70%
11.2%
16.7%

>
70%
­
80%
8.4%
14.1%

>
80%
­
90%
2.3%
6.3%

>
90%
0.2%
3.7%
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
59
A
B
E
D
C
1.

3.
4.
2.
1
mm
1/
5
mm
Figure
1.
Hemacytometer
Platform
Ruling.
Squares
1,
2,
3,
and
4
are
used
to
count
stock
suspensions
of
Cryptosporidium
oocysts
and
Giardia
cysts
(
after
Miale,
1967)
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
60
Figure
2.
Manner
of
Counting
Oocysts
and
Cysts
in
1
Square
mm.
Dark
organisms
are
counted
and
light
organisms
are
omitted
(
after
Miale,
1967).
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
61
Figure
3.
Laboratory
Filtration
System
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
62
Figure
4.
Methods
for
Scanning
a
Well
Slide
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
63
22.0
Glossary
of
Definitions
and
Purposes
These
definitions
and
purposes
are
specific
to
this
method
but
have
been
conformed
to
common
usage
as
much
as
possible.

22.1
Units
of
weight
and
measure
and
their
abbreviations
22.1.1
Symbols
°
C
degrees
Celsius
µ
L
microliter
<
less
than
>
greater
than
%
percent
22.1.2
Alphabetical
characters
cm
centimeter
g
gram
G
acceleration
due
to
gravity
hr
hour
ID
inside
diameter
in.
inch
L
liter
m
meter
MCS
microscope
cleaning
solution
mg
milligram
mL
milliliter
mm
millimeter
mM
millimolar
N
normal;
gram
molecular
weight
of
solute
divided
by
hydrogen
equivalent
of
solute,
per
liter
of
solution
RSD
relative
standard
deviation
sr
standard
deviation
of
recovery
X
average
percent
recovery
22.2
Definitions,
acronyms,
and
abbreviations
(
in
alphabetical
order)

Analyst
 
The
analyst
should
have
at
least
2
years
of
college
in
microbiology
or
equivalent
or
closely
related
field.
The
analyst
also
should
have
a
minimum
of
6
months
of
continuous
bench
experience
with
Cryptosporidium
and
IFA
microscopy.
The
analyst
shouldhave
a
minimum
of
3
months
experience
using
EPA
Method
1622
and/
or
EPA
Method
1623
and
should
have
successfully
analyzed
a
minimum
of
50
samples
using
EPA
Method
1622
and/
or
EPA
Method
1623.
The
analyst
must
have
at
least
2
years
of
college
lecture
and
laboratory
course
work
in
microbiology
or
a
closely
related
field.
The
analyst
also
must
have
at
least
6
months
of
continuous
bench
experience
with
environmental
protozoa
detection
techniques
and
IFA
microscopy,
and
must
have
successfully
analyzed
at
least
50
water
and/
or
wastewater
samples
for
Cryptosporidium
and
Giardia.
Six
months
of
additional
experience
in
the
above
areas
may
be
substituted
for
two
years
of
college.

Analyte
 
A
protozoan
parasite
tested
for
by
this
method.
The
analytes
in
this
method
are
Cryptosporidium
and
Giardia.

Axoneme
 
An
internal
flagellar
structure
that
occurs
in
some
protozoa,
such
as
Giardia,
Spironucleous,
and
Trichonmonas.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
64
Cyst
 
A
phase
or
a
form
of
an
organism
produced
either
in
response
to
environmental
conditions
or
as
a
normal
part
of
the
life
cycle
of
the
organism.
It
is
characterized
by
a
thick
and
environmentally
resistant
cell
wall.

Flow
cytometer
 
A
particle­
sorting
instrument
capable
of
counting
protozoa.

Immunomagnetic
separation
(
IMS)
 
A
purification
procedure
that
uses
microscopic,
magnetically
responsive
particles
coated
with
an
antibodies
targeted
to
react
with
a
specific
pathogen
in
a
fluid
stream.
Pathogens
are
selectively
removed
from
other
debris
using
a
magnetic
field.

Initial
precision
and
recovery
(
IPR)
 
Four
aliquots
of
spiking
suspension
analyzed
to
establish
the
ability
to
generate
acceptable
precision
and
accuracy.
An
IPR
is
performed
prior
to
the
first
time
this
method
is
used
and
any
time
the
method
or
instrumentation
is
modified.

Laboratory
blank
 
See
Method
blank
Laboratory
control
sample
(
LCS)
 
See
Ongoing
precision
and
recovery
(
OPR)
standard
Matrix
spike
(
MS)
 
A
sample
prepared
by
adding
a
known
quantity
of
organisms
to
a
specified
amount
of
sample
matrix
for
which
an
independent
estimate
of
target
analyte
concentration
is
available.
A
matrix
spike
is
used
to
determine
the
effect
of
the
matrix
on
a
method's
recovery
efficiency.

May
 
This
action,
activity,
or
procedural
step
is
neither
required
nor
prohibited.

May
not
 
This
action,
activity,
or
procedural
step
is
prohibited.

Median
bodies
 
Prominent,
dark­
staining,
paired
organelles
consisting
of
microtubules
and
found
in
the
posterior
half
of
Giardia.
In
G.
intestinalis
(
from
humans),
these
structures
often
have
a
claw­
hammer
shape,
while
in
G.
muris
(
from
mice),
the
median
bodies
are
round.

Method
blank
 
An
aliquot
of
reagent
water
that
is
treated
exactly
as
a
sample,
including
exposure
to
all
glassware,
equipment,
solvents,
and
procedures
that
are
used
with
samples.
The
method
blank
is
used
to
determine
if
analytes
or
interferences
are
present
in
the
laboratory
environment,
the
reagents,
or
the
apparatus.

Must
 
This
action,
activity,
or
procedural
step
is
required.

Negative
control
 
See
Method
blank
Nucleus
 
A
membrane­
bound
organelle
containing
genetic
material.
Nuclei
are
a
prominent
internal
structure
seen
both
in
Cryptosporidium
oocysts
and
Giardia
cysts.
In
Cryptosporidium
oocysts,
there
is
one
nucleus
per
sporozoite.
One
to
four
nuclei
can
be
seen
in
Giardia
cysts.

Oocyst
 
The
encysted
zygote
of
some
sporozoa;
e.
g.,
Cryptosporidium.
The
oocyst
is
a
phase
or
form
of
the
organism
produced
as
a
normal
part
of
the
life
cycle
of
the
organism.
It
is
characterized
by
a
thick
and
environmentally
resistant
outer
wall.

Ongoing
precision
and
recovery
(
OPR)
standard
 
A
method
blank
spiked
with
known
quantities
of
analytes.
The
OPR
is
analyzed
exactly
like
a
sample.
Its
purpose
is
to
assure
that
the
results
produced
by
the
laboratory
remain
within
the
limits
specified
in
this
method
for
precision
and
recovery.
Method
1623
­
Cryptosporidium
and
Giardia
June
2003
65
Oocyst
and
cyst
spiking
suspension
 
See
Spiking
suspension
Oocyst
and
cyst
stock
suspension
 
See
Stock
suspension
Positive
control
 
See
Ongoing
precision
and
recovery
standard
Principle
analyst
 
The
principle
analyst
(
may
not
be
applicable
to
all
monitoring
programs)
should
have
a
BS/
BA
in
microbiology
or
closely
related
field
and
a
minimum
of
1
year
of
continuous
bench
experience
with
Cryptosporidium
and
IFA
microscopy.
The
principle
analyst
also
should
have
a
minimum
of
6
months
experience
using
EPA
Method
1622
and/
or
EPA
Method
1623
and
should
have
analyzed
a
minimum
of
100
samples
using
EPA
Method
1622
and/
or
EPA
Method
1623.

PTFE
 
Polytetrafluoroethylene
Quantitative
transfer
 
The
process
of
transferring
a
solution
from
one
container
to
another
using
a
pipette
in
which
as
much
solution
as
possible
is
transferred,
followed
by
rinsing
of
the
walls
of
the
source
container
with
a
small
volume
of
rinsing
solution
(
e.
g.,
reagent
water,
buffer,
etc.),
followed
by
transfer
of
the
rinsing
solution,
followed
by
a
second
rinse
and
transfer.

Reagent
water
 
Water
demonstrated
to
be
free
from
the
analytes
of
interest
and
potentially
interfering
substances
at
the
method
detection
limit
for
the
analyte.

Reagent
water
blank
 
see
Method
blank
Relative
standard
deviation
(
RSD)
 
The
standard
deviation
divided
by
the
mean
times
100.

RSD
 
See
Relative
standard
deviation
Should
 
This
action,
activity,
or
procedural
step
is
suggested
but
not
required.

Spiking
suspension
 
Diluted
stock
suspension
containing
the
organism(
s)
of
interest
at
a
concentration
appropriate
for
spiking
samples.

Sporozoite
 
A
motile,
infective
stage
of
certain
protozoans;
e.
g.,
Cryptosporidium.
There
are
four
sporozoites
in
each
Cryptosporidium
oocyst,
and
they
are
generally
banana­
shaped.

Stock
suspension
 
A
concentrated
suspension
containing
the
organism(
s)
of
interest
that
is
obtained
from
a
source
that
will
attest
to
the
host
source,
purity,
authenticity,
and
viability
of
the
organism(
s).
