United
States
Office
of
Water
EPA­
821­
B­
03­
004
Environmental
Protection
4303T
Agency
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
for
Drinking
Water,
Ambient
Water,
and
Wastewater
Monitoring
Methods
Guidance
July
2003
ACKNOWLEDGMENTS
This
protocol
was
prepared
under
the
direction
of
the
U.
S.
Environmental
Protection
Agency's
(
EPA's)
Office
of
Water.
This
document
was
prepared
by
the
DynCorp
Biology
Studies
Group.

DISCLAIMER
The
Engineering
and
Analysis
Division,
of
the
Office
of
Science
and
Technology,
has
reviewed
and
approved
this
report
for
publication.
The
Office
of
Science
and
Technology
directed,
managed,
and
reviewed
the
work
of
DynCorp
in
preparing
this
report.
Neither
the
United
States
Government
nor
any
of
its
employees,
contractors,
or
their
employees
make
any
warranty,
expressed
or
implied,
or
assumes
any
legal
liability
or
responsibility
for
any
third
party's
use
of
or
the
results
of
such
use
of
any
information,
apparatus,
product,
or
process
discussed
in
this
report,
or
represents
that
its
use
by
such
party
would
not
infringe
on
privately
owned
rights.
Mention
of
company
names,
trade
names,
or
commercial
products
in
this
protocol
does
not
constitute
endorsement
or
recommendation
for
use.

Questions
concerning
this
report
should
be
addressed
to:

Robin
K.
Oshiro
Engineering
and
Analysis
Division
(
4303T)
U.
S.
EPA
Office
of
Water,
Office
of
Science
and
Technology
1200
Pennsylvania
Avenue,
NW
Washington,
DC
20460
oshiro.
robin@
epa.
gov
202­
566­
1075
202­
566­
1053
(
facsimile)
FOREWORD
This
document
describes
a
process
for
seeking
EPA
approval
of
microbiological
alternate
test
procedures
(
ATPs)
or
new
methods
for
use
in
monitoring
drinking
water,
ambient
water,
and
wastewater.
This
document
serves
as
a
supplement
to
the
ATP
guidelines
at
40
CFR
136.4,
136.5,
and
141.27.

With
this
guidance
document,
EPA
has
revised
and
combined
the
three
existing
microbiological
ATP
protocols
to
make
the
ATP
process
more
transparent
to
applicants
while
maintaining
the
same
level
of
data
quality.
The
guidelines
in
Quantitative
Membrane
Filter
Methods
(
Reference
10.16),
Presence/
Absence
Liquid
Culture
Methods
for
Finished
Water
(
Reference
10.17),
and
Presence/
Absence
Membrane
Filter
Methods
for
Finished
Waters
(
Reference
10.15)
have
been
incorporated
into
this
single
protocol,
which
is
applicable
to
a
wider
range
of
analytes
and
techniques.

This
revised
ATP
protocol
describes
a
process
for
conducting
side­
by­
side
method
comparisons
and
for
conducting
quality
control
(
QC)
acceptance
criteria­
based
method
studies
for
EPA­
approved
reference
methods
with
QC
acceptance
criteria.
Additionally,
in
some
cases
the
revised
protocol
provides
applicants
an
opportunity
to
demonstrate
comparability
by
meeting
QC
acceptance
criteria
associated
with
the
EPA­
approved
reference
methods
for
different
combinations
of
analyte
and
determinative
technique.

Under
EPA's
ATP
program,
any
person
may
apply
for
approval
of
the
use
of
an
ATP
or
new
method
to
test
for
a
regulated
analyte.
EPA
anticipates
that
the
standardized
procedures
described
herein
should
generally
expedite
the
approval
of
ATPs,
encourage
the
development
of
innovative
technologies,
and
enhance
the
overall
utility
of
the
EPA­
approved
methods
for
compliance
monitoring
under
the
National
Pollution
Discharge
Elimination
System
(
NPDES)
permit
program
and
national
primary
drinking
water
regulations
(
NPDWRs).

This
document
is
not
a
legal
instrument
and
does
not
establish
or
affect
legal
obligations
under
Federal
regulations.
EPA
reserves
the
right
to
change
this
protocol
without
prior
notice.

Questions
or
comments
regarding
this
document
should
be
directed
to:

Robin
K.
Oshiro
Engineering
and
Analysis
Division
(
4303T)
U.
S.
EPA
Office
of
Water,
Office
of
Science
and
Technology
1200
Pennsylvania
Avenue,
NW
Washington,
DC
20460
oshiro.
robin@
epa.
gov
202­
566­
1075
202­
566­
1053
(
facsimile)
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
i
TABLE
OF
CONTENTS
Section
1.0
Introduction
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1­
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1.1
Background
and
Objectives
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1­
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1.2
Types
of
Applications
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1­
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1.2.1
Limited
Use
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1.2.2
Nationwide
Use
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1­
3
1.3
Types
of
Studies
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1­
4
1.4
Scope
of
Alternate
Test
Procedures
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1­
4
1.4.1
EPA­
Approved
Reference
Methods
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1­
4
1.4.2
Modifications
to
Sample
Preparation
Techniques
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1­
5
Section
2.0
Application
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2­
1
2.1
Submission
Addresses
and
Approval
Authority
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2­
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2.2
Application
Information
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2­
1
2.3
Reason
for
ATP
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2­
2
2.4
Standard
EPA
Method
Format
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2­
3
2.5
Method
Comparison
Table
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2­
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2.6
Method
Development
Information
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2­
3
2.7
Study
Plan
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2­
3
2.8
Study
Report
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2­
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2.9
Proprietary
Information
in
Applications
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2­
4
Section
3.0
Method
Format
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3­
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3.1
Scope
and
Application
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3­
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3.2
Summary
of
Method
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3­
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3.3
Method
Definitions
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3­
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3.4
Interferences
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3­
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3.5
Safety
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3­
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3.6
Equipment
and
Supplies
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3­
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3.7
Reagents
and
Standards
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3­
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3.8
Sample
Collection,
Preservation,
and
Storage
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3­
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3.9
Quality
Control
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3­
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3.10
Calibration
and
Standardization
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3­
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3.11
Procedure
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3­
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3.12
Data
Analysis
and
Calculations
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3­
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3.13
Method
Performance
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3­
3
3.14
Pollution
Prevention
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3­
3
3.15
Waste
Management
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3­
3
3.16
References
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3­
3
3.17
Tables,
Diagrams,
Flowcharts,
and
Validation
Data
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3­
3
Section
4.0
Study
Plan
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4­
1
4.1
Background
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4­
1
4.2
Objectives
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4­
1
4.3
Study
Design
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4­
1
4.4
Coordination
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4­
2
4.4.1
Study
Management
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4­
2
4.4.2
Technical
Approach
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4­
2
4.5
Data
Reporting
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4­
2
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
ii
Section
5.0
Quality
Assurance/
Quality
Control
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5­
1
5.1
Quality
Assurance
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5­
1
5.2
Quality
Control
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5­
1
5.2.1
Analyst
Counting
Variability
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5­
3
5.2.2
Autoclave
Sterilization
Verification
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5­
3
5.2.3
Dilution/
Rinse
Water
Blanks
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5­
3
5.2.4
Incubator/
Waterbath
Temperatures
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5­
3
5.2.5
Initial
Demonstration
of
Capability
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5­
3
5.2.6
Initial
Precision
and
Recovery
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5­
4
5.2.7
Matrix
Spike
and
Matrix
Spike
Duplicate
Samples
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5­
4
5.2.8
Media
Sterility
Checks
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5­
4
5.2.9
Method
Blank
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5­
4
5.2.10
Ongoing
Demonstration
of
Capability
(
ODC)
Samples
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5­
5
5.2.11
Ongoing
Precision
and
Recovery
(
OPR)
Samples
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5­
5
5.2.12
Positive/
Negative
Controls
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5­
5
5.2.13
Preparation
Blanks
(
PB)
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5­
6
5.2.14
Refrigerator/
Freezer
Temperatures
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5­
6
5.2.15
Sample
Processing
Equipment
Sterility
Checks
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5­
6
Section
6.0
Study
Design
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6­
1
6.1
Side­
by­
Side
Comparison
Studies
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6­
1
6.1.1
Number
of
Laboratories
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6­
1
6.1.2
Number
of
Samples
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6­
1
6.1.3
Verification
of
Results
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6­
3
6.2
QC
Acceptance
Criteria­
Based
Comparison
Studies
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6­
4
6.2.1
Number
of
laboratories
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6­
4
6.2.2
Number
of
Matrices
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6­
4
6.2.3
Number
of
Replicates
per
Matrix
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6­
4
Section
7.0
Sample
Preparation
and
Analysis
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7­
1
7.1
Collection
of
Samples
for
Analysis
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7­
1
7.1.1
Source
Water
Characterization
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7­
1
7.2
Sample
Spiking
and
"
Stressing"
Procedures
for
Bacteriological
Methods
.
.
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.
7­
1
7.2.1
Drinking
Water:
Spiking
and
Chlorine­
Stressing
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7­
2
7.2.2
Preparation
of
Enumerated
Spiking
Suspension
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7­
4
7.2.3
Log
Phase
Growth
Curve
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7­
5
7.2.4
Commercially
Available
Enumerated
Spikes
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.
7­
5
7.3
Spiking
Procedures
for
Virus
Methods
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7­
6
7.3.1
Cell
Monolayer
Propagation
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7­
6
7.3.2
Propagation
of
Virus
Stock
Suspension
.
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.
7­
6
7.3.3
Titering
of
the
Virus
Stock
Suspension
.
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.
7­
6
7.4
Spiking
Procedures
for
Cryptosporidium
and
Giardia
.
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.
7­
6
7.5
Analysis
of
Samples
.
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.
7­
7
7.5.1
Side­
by­
Side
Comparison
Studies
.
.
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.
7­
7
7.5.2
QC
Acceptance
Criteria­
Based
Comparison
Studies
.
.
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.
7­
7
7.6
Verification
of
Results
.
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.
7­
7
7.6.1
Verification
of
Results
from
Bacteriological
Methods
.
.
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.
.
7­
8
7.6.2
Verification
of
Results
from
Virus
Methods
.
.
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.
.
7­
8
7.6.3
Verification
of
Results
from
Cryptosporidium
and
Giardia
Methods
.
.
.
.
.
.
.
7­
8
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
iii
Section
8.0
Review
of
Study
Results
.
.
.
.
.
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.
.
8­
1
8.1
Assessment
of
Compliance
with
Approved
Study
Plan
.
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8­
1
8.2
Data
Review
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8­
1
8.3
Data
Validation
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8­
1
8.4
Development
of
Descriptive
Statistics
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8­
1
8.4.1
Mean
Recovery
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8­
1
8.4.2
Precision
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8­
2
8.4.3
False
Positive
Rates,
False
Negative
Rates,
Sensitivity,
and
Specificity
.
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8­
2
8.5
Statistical
Assessment
of
Method
Comparability
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8­
3
8.5.1
Presence
/
Absence
Methods
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8­
3
8.5.2
Quantitative
Methods
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8­
5
8.5.3
QC
Acceptance
Criteria­
Based
Comparison
Studies
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8­
10
8.6
Method
Recommendation
and
Approval
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8­
12
Section
9.0
Study
Report
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9­
1
9.1
Background
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9­
1
9.2
Study
Objectives
and
Design
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9­
2
9.3
Study
Implementation
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9­
2
9.4
Data
Reporting
and
Validation
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9­
2
9.5
Results
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9­
2
9.6
Data
Analysis
and
Discussion
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9­
3
9.7
Conclusions
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9­
3
9.8
Appendix
A
­
Method
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9­
3
9.9
Appendix
B
­
Study
Plan
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9­
3
9.10
Appendix
C
­
Supporting
Data
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9­
3
9.10.1
Raw
Data
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9­
3
9.10.2
Electronic
Data
Reporting
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9­
4
9.10.3
Example
Calculations
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9­
4
9.11
Appendix
D
­
Supporting
References
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9­
4
Section
10.0
References
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10­
1
LIST
OF
APPENDICES
Appendix
A
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.
Glossary
Appendix
B
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.
ATP
Application
Form
Appendix
C
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.
Application
Inventory
Form
Appendix
D
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.
Data
Elements
and
Example
Bench
Sheets
Appendix
E
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.
EPA
Headquarters
and
Regional
ATP
Contacts
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
1­
1
LU
WW
&
AW
ATP
application
Apply
through
State
Director
RA
provides
letter
of
approval
or
disapproval
NW
DW,
WW,
&
AW
Regional
review
Forward
Forward
to
OW
for
review
OW
review
OW
recommends
approval
or
disapproval
Acronyms:
AW
=
Ambient
Water
DW
=
Drinking
Water
LU
=
Limited­
use
NW
=
Nationwide­
use
OW
=
Office
of
W
ater
RA
=
Regional
Administrator
WW
=
Wastewater
OW
recommendation
Optional
EPA
Administrator
approves
through
rulemaking
SECTION
1.0
INTRODUCTION
1.1
Background
and
Objectives
In
accordance
with
the
Clean
Water
Act
(
CWA)
and
Safe
Drinking
Water
Act
(
SDWA),
the
U.
S.
Environmental
Protection
Agency
(
EPA)
promulgates
guidelines
establishing
test
procedures
(
analytical
methods)
for
data
gathering
and
compliance
monitoring
under
National
Pollution
Discharge
Elimination
System
(
NPDES)
permits
and
national
primary
drinking
water
regulations
(
NPDWRs).
The
approved
test
procedures
can
be
found
in
the
Code
of
Federal
Regulations
(
CFR)
at
40
CFR
Part
136
for
wastewater
and
ambient
water
and
40
CFR
Part
141
for
drinking
water.
In
addition,
EPA's
regulations
at
40
CFR
136.4,
136.5,
and
40
CFR
141.27,
allow
entities
to
apply
for
Agency
permission
to
use
an
alternate
test
procedure
(
ATP)
in
place
of
an
EPA­
approved
reference
method.
Figure
1.1
below
summarizes
the
ATP
or
new
method
review
process
within
EPA.
These
regulations
are
the
basis
for
the
Agency's
alternate
test
procedure
(
ATP)
program
for
water
methods
that
is
administered
by
the
Office
of
Water,
Office
of
Science
and
Technology,
Director
of
Analytical
Methods.

An
ATP
is
a
modification
of
an
EPA­
approved
reference
method
or
a
procedure
that
uses
the
same
determinative
technique
(
i.
e.,
the
physical
and/
or
chemical
process
used
to
determine
the
identity
and
concentration
of
an
analyte)
and
measures
the
same
analyte(
s)
of
interest
as
the
EPA­
approved
reference
method.
The
use
of
a
different
determinative
technique
to
measure
the
same
analyte(
s)
of
interest
as
an
EPA­
approved
reference
method
is
considered
a
new
method.

Under
the
ATP
program,
an
organization
or
individual
may
apply
for
approval
of
an
ATP
or
new
method
to
be
used
as
an
alternate
to
an
EPA­
approved
reference
method.
The
applicant
is
generally
responsible
for
characterizing
method
performance
of
its
proposed
alternate
test
procedure
prior
to
submission
to
the
ATP
program.
EPA
can
provide
assistance
to
applicants
in
the
development
of
a
study
plan
to
demonstrate
comparability
with
the
EPA­
approved
reference
method.
Figure
1.1
summarizes
the
ATP
or
new
method
review
process
within
EPA.
The
Agency
reviews
the
ATP
package,
approves
or
disapproves
the
application,
and,
for
nationwide
applications,
will
generally
propose
to
include
successful
ATPs
in
the
CFR
(
unless
the
ATP
is
for
limited
use
or
constitutes
a
minor
modification­­
See
Appendix
A).

Figure
1­
1.
Summary
of
the
ATP
or
New
Method
Review
Process
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
1­
2
To
make
the
ATP
program
more
accessible
to
applicants
while
maintaining
data
quality,
EPA
has
revised
and
combined
the
three
microbiological
ATP
protocols
into
this
comprehensive
protocol.
The
guidelines
in
Quantitative
Membrane
Filter
Methods
(
Reference
10.16),
Presence/
Absence
Liquid
Culture
Methods
for
Finished
Water
(
Reference
10.17),
and
Presence/
Absence
Membrane
Filter
Methods
for
Finished
Waters
(
Reference
10.15)
have
been
incorporated
into
a
single
protocol
applicable
to
a
wider
range
of
analytes
and
techniques.
Additionally,
the
revised
protocol
illustrates
how
applicants
can
demonstrate
comparability
by
meeting
quality
control
(
QC)
acceptance
criteria
associated
with
EPA­
approved
reference
methods
for
which
those
criteria
have
been
developed.

The
ATP
program
provides
laboratories
and
regulated
facilities
with
an
opportunity
to
enhance
compliance
monitoring
and
encourages
the
use
of
innovative
technologies.
Approval
for
an
ATP
or
new
method
may
be
sought
when,
for
example,
the
alternate
procedure
reduces
analytical
costs,
overcomes
matrix
interferences
problems,
improves
laboratory
productivity,
or
reduces
the
amount
of
hazardous
materials
used
and/
or
produced
in
the
laboratory.

Any
person
or
organization
may
apply
to
gain
approval
for
the
use
of
an
ATP
or
new
method
for
determination
of
a
specific
constituent
that
is
regulated
under
the
NPDES
permit
program
or
the
NPDWRs.
The
ATP
applicants
generally
may
demonstrate
comparability
of
its
proposed
ATP
or
new
method
with
the
EPA­
approved
reference
method
using
the
procedures
described
in
this
protocol.
Other
possible
method
comparison
procedures
include
those
provided
by
organizations
such
as
ASTM
(
Reference
10.5),
AOAC­
International
(
Reference
10.1),
and
ISO
(
Reference
10.8).

1.2
Types
of
Applications
The
types
of
applications
submitted
may
depend
on
the
intended
use
of
the
ATP.
Methods
intended
for
use
in
demonstrating
compliance
with
the
NPDES
permit
program
(
wastewater
or
ambient
water
ATPs)
may
be
submitted
for
approval
for
limited­
use
(
single
laboratory)
or
for
nationwide­
use
(
all
laboratories).
Because
only
the
Administrator
has
the
authority
to
approve
an
alternate
analytical
technique
for
SWDA
purposes,
EPA
will
generally
consider
proposed
methods
intended
for
use
in
demonstrating
compliance
with
NPDWRs
(
drinking
water
ATPs)
that
are
submitted
for
approval
for
nationwide­
use
only.

1.2.1
Limited
Use
The
primary
intent
of
the
limited­
use
ATP
is
to
allow
use
of
an
ATP
or
new
method
by
a
single
laboratory.
Limited­
use
ATPs
can
be
applied
to
one
or
more
matrix
types,
excluding
drinking
water
matrices;
limited­
use
applications
generally
will
not
apply
to
Office
of
Ground
Water
and
Drinking
Water
(
OGWDW)
ATP
applications
(
Reference
10.18).
If
a
method
developer
intends
to
apply
the
method
to
more
than
one
matrix
type,
method
studies
should
be
conducted
on
each
matrix
type.
Generally,
nine
different
wastewater
types
should
be
analyzed
to
demonstrate
the
ATP
or
new
method
will
be
applicable
to
most
other
matrix
types.
If
method
modifications
are
within
the
specified
flexibility
of
the
EPAapproved
reference
method
and
all
QC
acceptance
criteria
are
met,
it
generally
will
not
be
necessary
to
submit
the
modification
to
the
ATP
program.

1.2.2
Nationwide
Use
The
primary
intent
of
a
nationwide­
use
ATP
is
to
allow
use
of
an
ATP
or
new
method
by
all
regulated
entities
and
laboratories
for
one
or
more
matrix
types
including
drinking
water.
Nationwide­
use
approval
allows
vendors
to
establish
that
new
devices
and
reagents
produce
results
that
are
acceptable
for
compliance
monitoring
purposes,
and
allows
environmental
laboratories
across
the
United
States
to
apply
new
technologies
or
modified
techniques
throughout
their
chain
of
laboratories
to
one
or
more
matrix
types.
If
a
method
developer
intends
to
apply
the
method
to
more
than
one
matrix
type,
method
studies
should
be
conducted
on
each
matrix
type.
Generally,
nine
different
wastewater
types
should
be
analyzed
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
1­
3
to
demonstrate
the
ATP
or
new
method
will
be
applicable
to
most
other
wastewaters.
If
method
modifications
are
within
the
specified
flexibility
of
the
EPA­
approved
reference
method
and
all
QC
acceptance
criteria
are
met,
it
generally
will
not
be
necessary
to
submit
the
modification
to
the
ATP
program.

1.3
Types
of
Studies
The
type
of
study
most
useful
in
seeking
approval
of
an
ATP
or
new
method
generally
depends
on
whether
or
not
the
EPA­
approved
reference
methods
contain
QC
acceptance
criteria.
There
are
two
basic
types
of
studies
described
in
this
protocol:
°
Side­
by­
Side
Method
Comparison
Study.
A
side­
by­
side
method
comparison
study
generally
consists
of
parallel
testing
of
an
ATP
or
new
method
along
side
an
EPA­
approved
reference
method
to
determine
whether
the
performance
of
the
new
or
modified
method
is
acceptable
compared
to
the
reference
method.
°
QC
Acceptance
Criteria­
Based
Method
Comparison
Study.
For
EPA­
approved
reference
methods
that
contain
(
or
are
supplemented
with)
QC
acceptance
criteria
for
most
combinations
of
analyte(
s)
and
determinative
technique(
s),
the
goal
of
the
study
is
for
the
applicant
to
demonstrate
that
its
ATP
or
new
method
is
able
to
meet
the
QC
acceptance
criteria
of
the
EPA­
approved
reference
method
(
or
other
EPA­
specified
document)
for
the
applicable
combination
of
analyte
and
determinative
technique
through
a
QC
acceptance
criteria­
based
comparison
study.

Specific
guidelines
for
the
studies
can
be
found
in
Section
5.0:
Quality
Control,
Section
6.0:
Study
Design,
Section
7.0:
Sample
Preparation
and
Analysis,
and
Section
9.0:
Review
of
Study
Results.

1.4
Scope
of
Alternate
Test
Procedures
This
protocol
for
demonstration
of
comparability,
submission,
and
approval
of
an
ATP
or
new
method
offers
flexibility
to
modify
EPA­
approved
reference
methods.
Generally,
an
applicant
should
demonstrate
and
document
that
the
modified
method
produces
results
better
than
or
equal
to
those
produced
by
an
appropriate
EPA­
approved
reference
method
for
the
applicable
combination
of
analyte
and
determinative
technique.

1.4.1
EPA­
Approved
Reference
Methods
The
ATP
process
is
based
on
comparing
the
performance
of
an
ATP
or
new
method
to
an
EPA­
approved
reference
method
through
a
side­
by­
side
comparison
study
or
a
QC
acceptance
criteria­
based
comparison
study.
Method
comparability
is
demonstrated
when
results
produced
by
an
ATP
meet
or
exceed
the
performance
criteria
associated
with
the
EPA­
approved
reference
method.
Table
1­
1
below
lists
the
EPAapproved
reference
methods
for
the
analytes
covered
by
this
protocol.
This
table
will
be
updated
as
necessary
as
additional
pathogens
are
added
to
the
list
or
advances
in
technology
merit
a
change
in
the
EPA­
approved
reference
method.
When
performing
a
study,
the
applicant
should
use
the
reference
method
that
uses
the
same
determinative
technique
(
e.
g.,
MF,
MPN)
as
the
ATP
for
the
analyte(
s)
of
interest.
If
the
applicant
is
validating
a
new
method,
which
generally
will
use
a
determinative
technique
that
is
not
currently
approved
for
use
with
the
analyte(
s)
of
interest,
then
the
applicant
should
consult
EPA
prior
to
commencing
the
study
to
determine
which
is
the
most
appropriate
reference
method.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
1­
4
Table
1­
1.
EPA­
Approved
Reference
Methods
Analyte
Method
Format1
EPA­
Approved
Reference
Method2
40
CFR
Citation
Total
coliforms
MPN
SM
9221B
136.3,
141.21,
141.74
MF
SM
9222B
Presence/
Absence
SM
9221D
141.21
Fecal
coliforms
MPN
SM
9221E
136.3,
141.21,
141.74,
503.8(
b)
MF
SM
9222D
E.
coli
MPN
SM
9221F
136.3,
141.21,
141.74
MF
SM
9222G,
SM
9213D
HPC
Pour
Plate
SM
9215B
141.74
Fecal
streptococcus
MPN
SM
9230B
136.3
MF
SM
9230C
Enterococcus
MPN
SM
9230B
136.3
MF
SM
9230C
Salmonella
MPN
Kenner
and
Clark
136.3,
503.8(
b)

Enteric
virus
Plaque
Assay
EPA
Document
3
503.8(
b)

Helminth
ova
Microscopy
EPA
Document
4
503.8(
b)

Aeromonas
MF
USEPA
Method
1605
141.40
Coliphage
Plaque
Assay
USEPA
Method
1601
141.403
Two­
Step
Enrichment
USEPA
Method
1602
Cryptosporidium
Filtration/
IMS/
FA
USEPA
Method
1622/
1623
136.3,
141.74
Giardia
Filtration/
IMS/
FA
USEPA
Method
1623
1MPN
=
most
probable
number,
MF
=
membrane
filtration,
IMS/
FA
=
immunomagnetic
separation/
fluorescent
antibody
2"
SM"
refers
to
Standard
Methods
for
the
Examination
of
Water
and
Wastewater.
For
the
edition(
s)
approved
for
a
specific
method,
consult
the
CFR
sections
referenced
in
the
column
headed
"
40
CFR
Citation."
(
References
10.2,
10.3,
and
10.4)
3
See
References
10.12
and
10.13
4
See
References
10.12
and
10.19
1.4.2
Modifications
to
Sample
Preparation
Techniques
A
sample
preparation
technique
is
any
technique
in
the
analytical
process
conducted
at
the
laboratory
that
precedes
the
determinative
technique
(
i.
e.,
the
physical
and/
or
chemical
process
by
which
measurement
of
the
identity
and
concentration
of
an
analyte
is
made).
Sample
preparation
techniques
include
the
procedures,
equipment,
reagents,
etc.,
that
are
used
in
the
preparation
and
cleanup
of
a
sample
for
analysis.
Laboratories
generally
may
modify
sample
preparation
techniques,
provided
the
modification
is
not
explicitly
prohibited
in
the
EPA­
approved
reference
method
that
is
being
modified
and
provided
the
modification
can
be
demonstrated
to
produce
results
equal
or
superior
to
results
produced
by
an
EPAapproved
reference
method
for
each
combination
of
analyte
and
determinative
technique.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
2­
1
SECTION
2.0
APPLICATION
ATP
applications
should
be
submitted
in
triplicate
to
EPA
to
facilitate
the
review
process.
The
application
consists
of
a
completed
ATP
application
form
(
a
sample
application
form
is
provided
in
Appendix
D)
with
any
attachments.
Electronic
submissions
are
also
generally
acceptable
and
often
may
accelerate
the
review
process.

2.1
Submission
Addresses
and
Approval
Authority
A
summary
of
ATP
submission
information
and
approval
authorities
is
provided
in
Table
2­
1.

Table
2­
1.
Submission
of
Alternate
Test
Procedure
Applications
Level
of
Use
Applicant
Submit
Application
To1
Approval
Authority
Limited
Use
for
Wastewater
or
Ambient
Water
EPA
Regional
laboratories
EPA
Regional
Administrator
(
Regional
ATP
Coordinator)
2
EPA
Regional
Administrator
States,
commercial
laboratories,
individual
dischargers,
or
permittees
in
States
that
do
not
have
the
authority
to
administer
Clean
Water
Act
and
Safe
Drinking
Water
Act
monitoring
programs
EPA
Regional
Administrator
(
Regional
ATP
Coordinator)
2
States,
commercial
laboratories,
individual
dischargers,
or
permittees
in
States
that
have
the
authority
to
administer
Clean
Water
Act
and
Safe
Drinking
Water
Act
monitoring
programs
Director
of
State
Agency
issuing
the
NPDES
permit2
Nationwide
Use
for
Drinking
Water,
Wastewater,
Ambient
Water
All
applicants
Director,
Analytical
Methods,
Attn:
ATP
Program
Coordinator,
EPA
Headquarters
EPA
Administrator
1
See
Appendix
E
for
EPA
addresses.
2
The
Regional
Administrator
or
the
Director
of
State
Agency
issuing
the
NPDES
permit
may
choose
to
forward
limited­
use
applications
to
the
Director
of
Analytical
Methods,
Attn:
ATP
Program
Coordinator
for
an
approval
recommendation.
Generally,
the
Regional
Administrator
or
the
Director
of
State
Agency
issuing
the
NPDES
permit
will
forward
a
copy
of
the
approval
to
the
Director
of
Analytical
Methods,
Attn:
ATP
Program
Coordinator.

Generally,
upon
receipt,
the
application
will
be
assigned
an
identification
number,
and
a
confirmation
letter
referencing
this
identification
number
will
be
sent
to
the
applicant.
The
applicant
should
use
the
identification
number
in
all
future
communications
concerning
the
application.

2.2
Application
Information
The
following
information
should
be
provided
on
the
ATP
application
form
(
Appendix
B):
°
Name,
mailing
address,
phone
number,
and
email
address
of
the
applicant
°
Date
of
submission
of
the
application
°
Method
number,
title,
and
revision
date
of
the
ATP
or
new
method
submitted
for
review
°
The
analyte(
s)
included
in
the
ATP
or
new
method
submitted
for
review
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
2­
2
°
The
matrix
or
matrices
to
which
the
ATP
applies
°
EPA­
approved
reference
method
used
for
demonstration
of
comparability
°
Type
of
application
(
i.
e.,
wastewater,
drinking
water,
ambient
water,
point
source
categories
regulated
at
40
CFR
Parts
400­
499)
°
The
level
of
use
desired
(
i.
e.,
limited
use
or
nationwide
use)
°
Type
of
study
(
side­
by­
side
comparison
or
QC
acceptance
criteria­
based
comparison
study)
°
Applicant's
NPDES
permit
number,
the
issuing
agency,
the
type
of
permit
and
the
discharge
serial
number
(
if
applicable)

The
following
items
should
be
submitted
as
attachments
to
the
initial
application:
°
Reason
for
proposing
the
ATP
or
new
method
°
The
proposed
ATP
or
new
method
prepared
in
standardized
format
(
Section
3.0)
°
A
method
comparison
table
that
gives
a
side­
by­
side
comparison
of
the
steps
of
the
proposed
ATP
or
new
method
and
the
EPA­
approved
reference
method
(
Section
2.5)
°
Method
development
information
°
Study
plan
for
EPA
review
and
comment
(
Section
4.0)

A
study
plan
is
generally
not
needed
with
the
application
if
an
applicant
is
unsure
whether
or
not
a
modification
is
allowed
within
the
method­
specified
flexibility.
In
such
cases,
the
applicant
may
request
that
EPA
determine
the
usefulness
of
a
full
ATP
comparability
assessment
based
on
the
other
information
submitted
with
the
application.
From
this
information,
EPA
can
determine
whether
a
full
ATP
assessment
will
be
helpful,
whether
the
proposed
modification
is
considered
to
be
a
minor
modification
(
i.
e.,
employs
the
same
chemistry
and/
or
biological
principles
as
the
EPA­
approved
reference
method
to
determine
the
presence/
absence
or
to
quantify
the
amount
of
the
target
organism
in
a
sample),
or
whether
the
proposed
modification
is
considered
to
be
within
the
specified
flexibility
of
the
EPA­
approved
reference
method.

The
elements
of
a
complete
application
are
presented
in
Table
2­
2.
A
list
of
the
information
discussed
in
detail
in
Sections
2.3
to
2.8
is
provided
in
Appendix
C.
EPA
will
generally
seek
all
application
information
and
attachments
before
the
application
is
considered
complete.

Table
2­
2.
Application
Information
Application
Information
°
Completed
application
form
°
Reason
for
ATP
°
Method
in
EPA
format
°
Method
comparison
table
°
Method
development
information
°
Study
plan
(
to
be
approved
by
EPA
before
proceeding
with
study)
°
Study
report
(
final
report
generally
considered
part
of
a
complete
application)

Note:
Although
the
application
process
generally
begins
with
the
initial
submission
of
ATP
materials,
the
application
is
not
usually
considered
to
be
complete
until
the
final
study
report
has
been
submitted,
and
all
EPA
questions
on
the
report
have
been
resolved.

2.3
Reason
for
ATP
The
entity
that
proposes
an
ATP
should
indicate
why
the
ATP
is
being
proposed.
Examples
include,
but
are
not
limited
to,
the
following:
°
The
ATP
improves
method
performance
°
The
ATP
provides
equivalent
method
components
for
a
lower
cost
°
The
ATP
enables
laboratories
to
perform
analyses
more
efficiently
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
2­
3
°
The
ATP
successfully
overcomes
some
or
all
of
the
interferences
associated
with
the
EPA­
approved
reference
method
°
The
ATP
significantly
reduces
the
amount
of
hazardous
wastes
generated
by
the
laboratory
°
The
ATP
provides
another
means
for
measuring
a
contaminant
(
i.
e.,
provides
more
choices)

2.4
Standard
EPA
Method
Format
In
accordance
with
the
standard
EPA
format
advocated
by
EPA's
Environmental
Monitoring
Management
Council
(
EMMC),
methods
should
contain
17
specific
topical
sections
in
a
designated
order.
The
17
sections
are
listed
in
Section
3.0
of
this
document.
Additional
numbered
sections
may
be
inserted
starting
with
Section
11.0,
Procedure,
as
appropriate
for
a
particular
method.
For
detailed
information
on
the
EPA
format
for
proposed
methods,
see
the
Guidelines
and
Format
document
(
Reference
10.14).

2.5
Method
Comparison
Table
As
part
of
the
application,
the
applicant
should
provide
a
two­
column
table
comparing
the
proposed
ATP
or
new
method
to
the
EPA­
approved
reference
method.
The
two­
column
method
comparison
table
should
include
the
number
and
title
of
each
method,
the
latest
revision
date
of
the
proposed
ATP,
and
a
detailed
discussion
of
each
of
the
17
topics
specified
by
the
standard
EPA
method
format
(
as
applicable).
Each
topic
should
be
discussed
on
a
separate
row
in
the
method
comparison
table.
The
applicant
should
highlight
any
differences
between
the
proposed
ATP
and
the
EPA­
approved
reference
method.

2.6
Method
Development
Information
Before
EPA
reviews
the
study
plan
and
works
with
applicants
on
ATP
studies,
the
applicant
should
provide
data
on
performance
of
the
modified
or
new
method
in
the
water
matrix
for
which
the
ATP
or
new
method
is
being
applied.
These
data
may
have
been
generated
during
method
development
by
the
vendor,
or
through
independent
tests
by
third­
party
laboratories.
Examples
include,
but
are
not
limited
to,
replicate
spiked
reagent
water
or
replicate
spiked
matrix
water
tests.
It
is
the
responsibility
of
the
applicant
to
provide
sufficient
data
to
demonstrate
that
the
ATP
or
new
method
performs
sufficiently
at
a
preliminary
level
in
the
matrix
of
interest
to
merit
evaluation
of
an
ATP.
If
sufficient
data
is
not
available,
EPA
may
request
additional
studies
be
conducted
prior
to
the
review
of
the
ATP
study
plan.

In
addition
to
data,
the
following
descriptive
method
information
will
facilitate
EPA's
evaluation
of
the
ATP
application:
°
The
purpose
and
intended
use
of
the
method
°
The
analytical
basis
for
the
method,
noting
any
relationship
of
the
method
to
other
existing
analytical
methods
and
indicating
whether
the
method
is
associated
with
a
sampling
method
°
Method
limitations
and
an
indication
of
any
means
of
recognizing
cases
where
the
method
may
not
be
applicable
to
specific
matrix
types
(
e.
g.,
turbidity
greater
than
50
NTU)
°
The
basic
steps
involved
in
performing
the
test
and
data
analysis
°
Options
to
the
method,
if
applicable
This
information
also
will
aid
EPA
in
preparing
the
docket
and
the
preamble
for
the
proposed
rule
that
will
be
published
in
the
Federal
Register
if
EPA
proposes
to
approve
the
ATP
for
nationwide
use.

2.7
Study
Plan
Prior
to
conducting
all
studies,
the
applicant
should
submit
a
study
design
for
EPA
review
and
comment.
A
detailed
procedure
(
Section
3.0)
for
the
new
method
or
the
modification
should
be
included
as
an
attachment
to
the
study
plan.
Generally,
EPA
will
evaluate
the
study
plan
to
ensure
that
the
appropriate
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
2­
4
data
quality
objectives
identified
in
this
protocol
are
defined
and
addressed.
EPA
comments
will
be
incorporated
into
the
study
design
and
this
process
will
be
repeated
until
EPA
has
approved
the
study
design.
Generally,
the
study
plan
should
contain
the
elements
listed
below:
°
Background
°
Objectives
°
Study
Design
°
Coordination
°
Data
Reporting
These
elements
are
further
described
in
Section
4.0.

2.8
Study
Report
The
applicant
should
conduct
a
study
and
provide
a
comprehensive
study
report
with
the
ATP
or
new
method
application.
The
study
report
should
include
the
following
elements:
°
Background
°
Study
Objectives
and
Design
°
Study
Implementation
°
Data
Reporting
and
Validation
°
Results
°
Data
Analysis
and
Discussion
°
Conclusions
°
Appendix
A
­
Method
°
Appendix
B
­
Study
Plan
°
Appendix
C
­
Supporting
Data
°
Appendix
D
­
Supporting
References
These
elements
are
further
described
in
Section
9.0.

2.9
Proprietary
Information
in
Applications
All
information
provided
to
the
Federal
government
is
subject
to
the
requirements
of
the
Freedom
of
Information
Act.
Therefore,
any
proprietary
information
submitted
with
the
proposed
ATP
application
should
be
marked
as
confidential.
However,
EPA
prefers
that
supporting
documentation
labeled
as
confidential
business
information
not
be
submitted
as
part
of
the
ATP
application.
If
proprietary
information
is
determined
to
be
essential
to
the
application,
EPA
staff
will
request
the
information
and
will
handle
such
information
according
to
the
regulations
in
subparts
A
and
B
of
40
CFR
Part
2.

Specifically,
in
accordance
with
40
CFR
§
2.203,
a
business
that
submits
information
to
EPA
may
assert
a
business
confidentiality
claim
covering
the
information
by
placing
on
(
or
attaching
to)
the
information
at
the
time
it
is
submitted
to
EPA,
a
cover
sheet,
stamped
or
typed
legend,
or
other
suitable
form
of
notice
employing
language
such
as
trade
secret,
proprietary,
or
company
confidential.
Allegedly
confidential
portions
of
otherwise
non­
confidential
documents
should
be
clearly
identified
by
the
business,
and
may
be
submitted
separately
to
facilitate
identification
and
handling
by
EPA.
If
the
business
desires
confidential
treatment
only
until
a
certain
date
or
until
the
occurrence
of
a
certain
event,
the
notice
should
so
state.
Please
be
advised,
however,
that
any
methods
proposed
in
the
Federal
Register
cannot
be
claimed
as
confidential
business
information.

If
a
claim
of
business
confidentiality
is
not
made
at
the
time
of
submission,
EPA
will
make
such
efforts
as
are
administratively
practicable
to
associate
a
late
claim
with
copies
of
previously
submitted
information
in
EPA
files.
However,
EPA
cannot
ensure
that
such
efforts
will
be
effective
due
to
the
nature
of
application
review
that
may
already
be
in
progress.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
3­
1
SECTION
3.0
METHOD
FORMAT
Because
alternate
test
procedures
may
be
approved
by
EPA
as
comparable
to
the
reference
methods,
and
may
be
implemented
by
multiple
laboratories,
it
is
important
that
the
written
procedures
include
all
of
the
information
necessary
to
use
the
technique
in
the
laboratory,
including
but
not
limited
to:
reagents
and
equipment,
sample
collection
and
preservation
procedures,
quality
control,
and
a
detailed
description
of
the
procedure.
The
information
described
below
should
be
provided
in
the
method.
In
addition,
EPA
recommends
the
Environmental
Monitoring
Methods
Council
(
EMMC)
format
described
below
be
used.

Sections
3.1
through
3.17
provide
a
list
of
EMMC
method
sections
and
a
general
description
of
the
type
of
information
that
should
be
included
in
each
section.
The
date
and
revision
number
of
the
method
should
be
included
on
the
cover
page.
In
addition,
the
date
should
be
included
as
a
footer
on
each
page
of
the
method.
A
detailed
description
of
method
format
guidelines,
as
well
as
an
example
of
a
formatted
method,
is
provided
in
Reference
10.14.
The
detailed
information
in
Reference
10.14
is
provided
as
guidance
for
the
method
write­
up
and
as
such,
specific
suggestions
for
font
size,
margins,
etc.
are
optional.

3.1
Scope
and
Application
Include
a
list
of
target
organisms
(
by
common
name),
taxonomic
group
and
their
CAS
registry
numbers
or
other
accepted
numbering
systems
(
if
available),
the
matrices
to
which
the
method
applies,
a
generic
description
of
method
sensitivity
(
the
minimum
number
of
organisms
the
method
can
detect
per
unit
volume
or
mass,
if
known),
and
the
data
quality
objectives
that
the
method
is
designed
to
meet
or
monitoring
programs
for
which
the
method
was
designed
to
support.

3.2
Summary
of
Method
Summarize
the
method
in
a
few
paragraphs.
The
purpose
of
the
summary
is
to
provide
a
succinct
overview
of
the
method
procedure
to
aid
the
reviewer
or
data
user
in
understanding
the
method
and
how
the
results
are
generated.
Include
a
general
description
of
the
method
procedure,
sample
volume,
type
of
media
used,
preparation
steps,
incubation
time
and
temperatures,
and
the
techniques
used
for
qualitative
or
quantitative
determinations.

3.3
Method
Definitions
Provide
definitions
of
terms
that
are
necessary
to
understand
how
the
method
is
used
or
what
the
results
represent.
This
should
include
a
definition
of
the
target
organism
or
group
of
organisms,
relative
to
the
determinative
step
of
the
method.
For
extensive
lists
of
definitions,
this
section
may
simply
refer
to
a
glossary
attached
at
the
end
of
the
method
document.

3.4
Interferences
This
section
should
discuss
any
known
method
interferences
such
as
toxic
materials,
particulates,
nontarget
organisms,
etc.
If
known
interferences
in
the
reference
method
are
not
interferences
in
the
alternate
method,
this
also
should
be
clearly
stated.

3.5
Safety
This
section
should
discuss
only
those
safety
issues
specific
to
the
method
and
beyond
the
scope
of
routine
laboratory
practices.
Target
analytes
or
reagents
that
pose
specific
health,
toxicity,
or
safety
issues
should
be
addressed
in
this
section.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
3­
2
3.6
Equipment
and
Supplies
For
critical
equipment
that
may
affect
the
performance
of
the
method,
cite
the
manufacturer,
model
name,
and
catalog
or
product
number
of
the
equipment
that
was
used
to
develop
or
validate
the
method;
note
that
equivalent
equipment
can
be
used,
if
applicable.
Use
generic
language
for
standard
laboratory
glassware
and
disposables.

3.7
Reagents
and
Standards
Provide
sufficient
details
on
the
concentration
and
preparation
of
reagents
and
standards
to
allow
the
work
to
be
duplicated,
but
avoid
lengthy
discussions
of
common
procedures.
If
only
pre­
prepared
proprietary
reagents
can
be
used,
specify
this.
Include
catalog
and/
or
product
numbers
where
appropriate.
Indicate
shelf
life
of
packaged
materials
and
special
storage
specifications.

3.8
Sample
Collection,
Preservation,
and
Storage
Provide
information
on
sample
collection,
preservation,
shipment,
storage
conditions,
and
holding
times.
If
effects
of
holding
time
were
specifically
evaluated,
provide
reference
to
relevant
data.

3.9
Quality
Control
Describe
specific
quality
control
(
QC)
measures
that
enable
one
to
establish
the
sensitivity,
specificity,
false
positive
rates,
false
negative
rates,
bias,
and
precision
of
measurements
using
the
method,
and
that
the
measurements
are
free
from
contamination.
Specific
QC
measures
may
include
positive
and
negative
controls,
duplicate
samples,
method
blanks,
and
media
sterility
checks.
Indicate
which
QC
measures
are
appropriate
initially,
before
a
laboratory
uses
the
method,
and
which
are
appropriate
on
an
ongoing
basis.
Indicate
frequencies
for
each
QC
measure
and
list
minimum
specifications
or
acceptance
ranges
(
see
Section
5.0).
Indicate
corrective
actions
that
should
be
taken
when
QC
measures
are
not
met.
Define
all
terms
in
method
definitions
section.

3.10
Calibration
and
Standardization
Discuss
initial
calibration
specifications
for
instruments
used
in
the
method
(
e.
g.,
water
baths,
refrigerators,
thermometers,
balances,
pH
meters,
microscopes,
etc.).
Indicate
frequency
of
such
calibrations;
refer
to
performance
specifications;
and
indicate
corrective
actions
that
should
be
taken
when
performance
specifications
are
not
met.
This
section
may
also
include
procedures
for
calibration,
verification,
or
continuing
calibration,
or
these
steps
may
be
included
in
the
procedure
section.

3.11
Procedure
Provide
a
detailed
description
of
the
sample
processing
and
analysis
steps.
Avoid
unnecessarily
restrictive
instructions,
but
provide
sufficient
detail
for
manual
procedures
so
that
analysts
in
other
laboratories
perform
the
method
consistently.
Ranges
should
be
provided
for
temperature
requirements,
time
requirements,
etc.

3.12
Data
Analysis
and
Calculations
Identify
qualitative
and
quantitative
aspects
of
the
method.
List
criteria
for
the
identification
of
target
organism(
s)
and
interpretation
of
results
for
all
steps
of
the
method,
including
criteria
for
presumptive
and
confirmed
results.
Provide
equations
used
to
derive
final
sample
results.
Provide
discussion
of
estimating
detection
limits,
recoveries,
specificity,
false
positive/
false
negative
rates,
etc.,
if
appropriate.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
3­
3
3.13
Method
Performance
Provide
detailed
information
on
method
performance,
including
data
on
precision,
bias
(
for
quantitative
methods),
specificity,
detection
limits
(
including
the
method
by
which
they
were
determined
and
matrices
to
which
they
apply),
statistical
procedures
used
to
develop
performance
specifications
(
i.
e.,
recovery,
precision,
specificity,
false
positive/
false
negative
rates,
etc.).
Where
performance
is
tested
relative
to
the
reference
method,
provide
a
summary
of
the
side­
by­
side
comparison
of
performance
versus
reference
method
specifications.

3.14
Pollution
Prevention
Describe
aspects
of
this
method
that
minimize
or
prevent
pollution
that
may
be
attributable
to
the
reference
method.

3.15
Waste
Management
Cite
how
waste
is
minimized
and
the
proper
disposal
of
samples
and
waste.

3.16
References
Include
source
documents,
publications,
etc.

3.17
Tables,
Diagrams,
Flowcharts,
and
Validation
Data
Additional
information
may
be
presented
at
the
end
of
the
method.
Lengthy
tables
may
be
included
here
and
referred
to
elsewhere
in
the
text
by
number.
Diagrams
should
only
include
new
or
unusual
equipment
or
aspects
of
the
method.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
4­
1
SECTION
4.0
STUDY
PLAN
Applicants
should
submit
a
study
design
for
EPA
review,
comment,
and
approval
prior
to
conducting
the
side­
by­
side
comparison
study
or
the
QC
acceptance
criteria­
based
comparison
study.
This
process
protects
the
applicant
by
providing
written
approval
of
the
study
design
before
resources
are
spent
to
conduct
the
study.
The
ATP
program
is
intended
to
be
flexible,
and
thus
EPA
may
modify
the
study
design
for
a
particular
proposed
method.
Data
from
studies
conducted
without
EPA
review
and
approval
may
not
meet
EPA's
criteria,
and
may
not
adequately
address
the
applicant's
study
objectives.
A
detailed
procedure
(
Section
3.0)
for
the
ATP
or
new
method
should
be
included
as
an
attachment
to
the
study
plan.
EPA
will
generally
evaluate
the
study
plan
to
verify
that
the
appropriate
data
quality
objectives
identified
in
this
protocol
are
defined
and
addressed.
EPA
comments
are
incorporated
into
the
study
design.
This
review/
revision
process
is
repeated
until
EPA
has
approved
the
study
design.

Generally,
the
study
design
should
include
the
information
described
in
Sections
4.1
through
4.5.

4.1
Background
This
section
of
the
study
plan
should
include
the
following
information:
°
A
statement
identifying
the
ATP
as
a
new
method
or
a
modification
of
a
reference
method
°
The
EPA
program(
s)
to
which
the
ATP
or
new
method
applies
(
e.
g.,
drinking
water,
wastewater,
ambient
water,
point
source
categories
regulated
at
40
CFR
Parts
400­
499,
etc.)
°
A
short
(
one
paragraph)
summary
of
the
ATP
or
new
method
°
The
organization
and
method
number
of
the
reference
method
if
applicable
°
A
description
of
the
reasons
for
the
extent
of
the
modification,
the
logic
behind
the
technical
approach
to
the
modification,
and
the
result
of
the
modification
°
The
matrices
(
e.
g.,
finished
water,
wastewater,
ambient
water,
etc.),
matrix
types
(
e.
g.,
turbidity
greater
than
10
NTU,
etc.),
and/
or
media
to
which
the
ATP
or
new
method
is
believed
to
be
applicable
°
A
list
of
the
analytes
measured
by
the
ATP
or
new
method,
including
the
corresponding
CAS
registry
number
(
if
available)
or
other
identification
number
4.2
Objectives
Include
a
description
of
the
new
method
or
modification,
describe
the
goals
of
the
study,
and
define
data
quality
objectives.

4.3
Study
Design
The
following
information
should
be
included
in
the
study
design:
°
Laboratories
that
will
participate
in
the
study
(
Sections
5.0
and
6.0)
°
Number
and
type
of
samples
to
be
analyzed
(
Section
6.0)
°
Description
of
the
matrices
that
will
be
used
(
Sections
6.0
and
7.0)
°
Description
of
the
spikes
that
will
be
used
(
Section
7.0)
°
Description
of
the
spiking
procedure
(
Section
7.0)
°
Positive
and
negative
control
organisms
(
Section
5.0)
°
Quality
control
procedures
that
will
be
followed
(
Section
5.0)
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
4­
2
4.4
Coordination
Describe
how
the
study
will
be
coordinated,
how
the
spikes
will
be
shipped
to
the
laboratories,
and
who
will
compile
the
data
for
submission.
Data
compilation
should
not
be
performed
by
any
of
the
analysts
conducting
the
sample
analyses.

4.4.1
Study
Management
This
section
of
the
study
plan
should
include
the
following
information:
°
The
organization
responsible
for
managing
the
study
°
The
laboratories,
facilities,
and
other
organizations
that
will
participate
in
the
study
°
A
delineated
study
schedule
including,
but
not
limited
to,
sample
collection,
start
of
sample
analysis,
interpretation
of
sample
results,
completion
of
study,
etc.

4.4.2
Technical
Approach
This
section
of
the
study
plan
should
include
the
following:
°
A
description
of
how
sample
matrices
and
participating
laboratories
will
be
selected
°
A
description
of
how
samples
will
be
collected
and
distributed
°
The
numbers
and
types
of
analyses
to
be
performed
by
the
participating
laboratories
°
A
description
of
sample
spiking
procedures
°
A
description
of
how
analyses
are
to
be
performed
4.5
Data
Reporting
List
the
data
elements
that
will
be
collected
and
provide
sample
bench
sheets
(
see
Appendix
D)
that
will
be
used
to
record
raw
data
during
the
study.
Raw
data
should
be
submitted
as
an
attachment
to
the
Study
Report
(
Section
9.0).
Address
the
statistical
analysis
of
the
study
results
that
will
be
performed,
if
the
statistical
analyses
will
differ
from
those
described
in
Section
8.0.
Please
note,
however,
that
EPA's
evaluation
of
method
performance
will
generally
be
based
on
the
statistical
analyses
described
in
Section
8.0.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
5­
1
SECTION
5.0
QUALITY
ASSURANCE/
QUALITY
CONTROL
For
side­
by­
side
comparison
studies
in
which
only
one
laboratory
is
performing
analyses,
the
comparability
study
should
be
conducted
at
an
independent
laboratory
and
the
laboratory
should
be
certified
to
perform
microbiological
analyses
under
EPA's
drinking
water
laboratory
certification
program.
A
laboratory
with
a
vested
interest
in
the
method,
instrumentation,
apparatus,
reagents,
media,
or
associated
kits
may
not
participate
in
the
side­
by­
side
comparison
study.

For
QC
acceptance
criteria­
based
comparison
studies,
at
least
three
independent
laboratories
should
participate.
A
laboratory
with
a
vested
interest
in
the
ATP
also
may
participate
in
the
study,
but
in
such
instances
there
should
be
at
least
three
independent
laboratories
participate
and
the
majority
of
the
laboratories
participating
are
independent
laboratories.
All
laboratories
should
be
certified
to
perform
microbiological
analyses
under
EPA's
drinking
water
certification
program,
or
a
comparable
certification
program,
if
a
laboratory
located
outside
of
the
U.
S.
is
included.
At
least
three
independent
laboratories
participating
in
the
study
should
to
be
certified
for
microbiological
analyses
under
EPA's
drinking
water
certification
program.
If
more
than
three
laboratories
participate,
the
majority
of
the
laboratories
should
be
certified
for
microbiological
analyses
under
EPA's
drinking
water
certification
program.

5.1
Quality
Assurance
The
laboratory
should
have
a
comprehensive
quality
assurance
(
QA)
program
in
place
and
operating
at
all
times
during
the
performance
of
the
comparability
study.
General
QA
program
guidance
is
provided
at
http://
www.
epa.
gov/
quality/
qs­
docs/
r2­
final.
pdf
(
Reference
10.11).
The
laboratory
should
adhere
closely
to
all
QA
and
quality
control
(
QC)
measures
in
this
protocol
as
well
as
the
QC
measures
in
the
method(
s).
Laboratory
QA/
QC
criteria
for
facilities,
personnel,
and
laboratory
equipment
are
included
in
Standard
Methods
9020­
Quality
Assurance
(
Reference
10.4)
and
the
U.
S.
EPA
Manual
for
the
Certification
of
Laboratories
Analyzing
Drinking
Water,
Fourth
Edition
(
March
1997)
(
Reference
10.13).

The
laboratory
should
adhere
to
standard
laboratory
practices
for
cleanliness
and
environment,
and
to
the
methods
for
glassware
and
apparatus,
reagents,
solvents,
and
safety.
Additional
guidelines
regarding
general
laboratory
procedures
should
generally
be
followed,
as
specified
in
Sections
4
and
5
of
the
Handbook
for
Analytical
Quality
Control
in
Water
and
Wastewater
Laboratories,
EPA­
600/
4­
79­
019
(
Reference
10.19).

5.2
Quality
Control
Laboratories
participating
in
a
comparability
study
should
perform
all
QC
procedures
specified
in
the
methods,
except
where
explicitly
stated
in
the
approved
study
plan.
The
QC
procedures
listed
in
Table
5­
1
and
described
below
should
be
performed,
as
appropriate,
based
on
the
technique
used
in
the
method.
Other
QC
procedures
may
be
necessary,
based
on
the
approved
study
plan.
Laboratories
participating
in
side­
by­
side
comparison
studies
or
QC
acceptance
criteria­
based
comparison
studies
should
perform
all
QC
procedures
specified
in
the
methods,
except
where
explicitly
stated
in
the
approved
study
plan.
The
laboratory
should
maintain
records
to
define
the
quality
of
data
that
are
generated.
The
laboratory
should
maintain
a
record
of
the
date
and
results
of
all
QC
sample
analyses.
Laboratories
should
maintain
reagent
and
material
lot
numbers
along
with
samples
analyzed
using
each
of
the
lots.
Laboratories
should
also
maintain
media
preparation
records.

Table
5­
1
lists
quality
control
measures
for
each
laboratory
participating
in
side­
by­
side
comparison
studies
and
QC
acceptance
criteria­
based
comparison
studies.
Detailed
descriptions
of
the
QC
measures
are
provided
in
Sections
5.2.1
to
5.2.15.
If
contamination
is
detected
in
any
of
the
blanks
or
sterility
checks
described
below,
the
source
of
contamination
should
be
identified
and
corrected.
The
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
5­
2
blank/
sterility
check
and
all
samples
associated
with
that
contaminated
blank/
sterility
check
should
be
reprepared
and
reanalyzed.
Measures
taken
to
eliminate
contamination
should
be
reported.

Table
5­
1.
Quality
control
measures
for
each
laboratory
involved
in
the
study
Quality
Control
Measure
Frequency
Suggested
for
Study
Side­
by­
Side
Comparison
Study
QC
acceptance
criteria­
based
Comparison
Study
Analyst
counting
variability
(
Section
5.2.1)
2
samples
per
study
T
T
Autoclave
sterilization
verification
(
Section
5.2.2)
Within
one
week
prior
to
the
start
of
the
study
T
T
Dilution/
rinse
water
blanks
(
Section
5.2.3)
1
per
every
20
samples
or
1
per
day
of
study,
whichever
is
greater
T
T
Incubator/
waterbath
temperatures
(
Section
5.2.4)
2
times
per
day
when
used
in
study
T
T
Initial
demonstration
of
capability
(
IDC)
(
Section
5.2.5)
or
Initial
precision
and
recovery
(
IPR)
(
Section
5.2.6)
Each
laboratory
participating
in
the
study
should
generate
acceptable
IDC/
IPR
data.
T
Matrix
spike/
matrix
spike
duplicate
(
Section
5.2.7)
Per
approved
study
plan
T
Media
sterility
checks
(
Section
5.2.8)
1
per
each
batch
of
media
used
in
the
study
or
per
each
test
run,
whichever
is
greater
T
T
Method
blank
(
Section
5.2.9)
Per
approved
study
plan
T
T
Ongoing
demonstration
of
capability
(
ODC)
(
Section
5.2.10)
or
Ongoing
precision
and
recovery
(
OPR)
(
Section
5.2.11)
Included
as
part
of
the
study;
not
needed
as
separate
QC.
Optional
Positive
and
negative
controls
(
Section
5.2.12)
1
positive
control
and
1
negative
control
for
each
media/
stain
used
in
the
study.
See
5.2.12
below
for
more
details.
T
T
Preparation
blanks
(
Section
5.2.13)
Frequency
depends
on
the
type
of
method.
See
5.2.13
below
for
more
details.
T
T
Refrigerator/
freezer
temperatures
(
Section
5.2.14)
Once
per
day
when
used
in
study
T
T
Sample
processing
equipment
sterility
checks
(
Section
5.2.15)
Prior
to
the
analysis
of
samples.
See
5.2.15
below
for
more
details.
T
T
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
5­
3
5.2.1
Analyst
Counting
Variability
If
the
laboratory
has
two
or
more
analysts,
each
should
count
colonies,
plaques,
or
positive
wells
on
the
same
plate/
tray
from
one
positive
field
sample
per
month.
Compare
each
analyst's
count
of
the
colonies,
plaques,
or
wells.
Counts
should
fall
within
10%
between
analysts.
If
counts
fail
to
fall
within
10%
of
each
other,
analysts
should
perform
additional
sets
of
counts,
until
the
number
of
target
colonies,
plaques,
or
positive
wells
counted
fall
within
10%
between
analysts
for
at
least
three
consecutive
samples.
If
there
is
only
one
analyst
replicate
counts
should
be
done
and
be
within
5%
of
original
counts.

5.2.2
Autoclave
Sterilization
Verification
Autoclave
sterilization
verification
should
be
performed
within
one
week
of
the
start
of
the
study
by
placing
Bacillus
stearothermophilus
spore
suspensions
or
strips
inside
glassware.
Autoclave
at
121
°
C
for
15
minutes.
Place
Bacillus
stearothermophilus
spore
suspensions
in
trypticase
soy
broth
tubes
and
incubate
at
55
°
C
for
48
hours.
Check
for
growth
to
verify
that
sterilization
was
adequate.
If
sterilization
was
inadequate,
determine
appropriate
time
for
autoclave
sterilization.
Repeat
spore
test.
The
laboratory
should
have
historical
data
verifying
that
at
a
minimum,
autoclave
sterilization
is
performed
on
a
monthly
basis.

5.2.3
Dilution/
Rinse
Water
Blanks
The
laboratory
should
analyze
dilution/
rinse
water
blanks
to
demonstrate
freedom
from
contamination.
An
aliquot
of
dilution/
rinse
water
which
is
analyzed
exactly
like
a
field
sample
should
be
analyzed
for
every
day
of
the
study
or
for
every
20
samples,
whichever
is
more
frequent,
and
observed
for
contamination
with
agent
of
interest.

5.2.4
Incubator/
Waterbath
Temperatures
Incubator
or
waterbath
temperatures
should
be
measured
and
recorded
two
times
per
day
when
in
use.
Temperatures
should
be
taken
at
least
4
hours
apart
and
should
be
within
the
range
of
the
desired
temperature
as
specified
in
each
method.
Thermometers
used
to
measure
"
in­
use"
temperatures
should
be
calibrated
yearly
against
an
NIST
traceable
thermometer.

5.2.5
Initial
Demonstration
of
Capability
Laboratories
participating
in
a
QC
acceptance
criteria­
based
comparison
study
should
have
successfully
performed
an
initial
demonstration
of
capability
(
IDC)
test
for
the
new
or
modified
method
under
evaluation
in
the
study.
An
IDC
test
is
performed
when
QC
acceptance
criteria
are
available
for
the
evaluation
of
precision
or
recovery,
but
not
both.
The
laboratory
should
perform
an
IDC
as
specified
in
the
method
to
demonstrate
acceptable
performance.
The
laboratory
should
complete
any
additional
analyses
as
specified
in
the
study
plan.

For
the
IDC
test,
the
laboratory
spikes
and
analyzes
reference
matrix
(
e.
g.,
reagent
water,
buffered
water,
etc.)
samples
to
demonstrate
acceptable
performance
with
the
method
prior
to
the
analysis
of
field
samples.
The
number
of
samples
involved
in
the
IDC
varies
by
method.
If
the
results
of
the
IDC
test
meets
all
IDC
acceptance
criteria
(
e.
g.,
RSD,
minimum
number
of
samples
positive,
etc.),
system
performance
will
generally
be
acceptable.
If
any
of
the
IDC
test
results
fail
to
meet
the
acceptance
criteria,
system
performance
will
generally
be
unacceptable.
In
this
event,
the
laboratory
should
identify
and
correct
the
problem
and
repeat
the
test.
IDC
tests
should
be
accompanied
by
a
method
blank
(
Section
5.2.9).
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
5­
4
5.2.6
Initial
Precision
and
Recovery
Laboratories
participating
in
a
QC
acceptance
criteria­
based
comparison
study
should
have
successfully
performed
an
initial
precision
and
recovery
(
IPR)
test
for
the
method
using
the
modified
version
of
the
method
under
evaluation
in
the
study.
An
IPR
test
is
performed
when
QC
acceptance
criteria
are
available
for
the
evaluation
of
both
precision
and
recovery.
The
laboratory
should
perform
an
IPR
as
specified
in
the
method
to
demonstrate
acceptable
performance.
The
laboratory
should
complete
any
additional
analyses
as
specified
in
the
study
plan.

For
the
IPR
test,
the
laboratory
spikes
and
analyzes
reference
matrix
(
e.
g.,
reagent
water,
buffered
water,
etc.)
samples
to
establish
the
laboratory's
ability
to
generate
acceptable
precision
and
recovery
prior
to
the
analysis
of
field
samples.
Using
results
of
the
analyses,
the
laboratory
calculates
mean
percent
recovery
and
relative
standard
deviation
(
RSD)
of
the
recoveries
for
the
analyte(
s)
and
compares
them
with
the
corresponding
limits
for
the
IPR
test
criteria
in
the
method.
If
the
RSD
and
the
mean
percent
recovery
meet
the
acceptance
criteria,
system
performance
is
acceptable
and
analysis
of
samples
may
begin.
If
the
RSD
or
the
mean
percent
recovery
are
unacceptable,
system
performance
will
generally
be
unacceptable.
In
this
event,
the
laboratory
should
identify
and
correct
the
problem
and
repeat
the
test.
IPR
tests
should
be
accompanied
by
method
blank
tests
(
Section
5.2.9).

5.2.7
Matrix
Spike
and
Matrix
Spike
Duplicate
Samples
Matrix
spike
(
MS)
and
matrix
spike
duplicate
(
MSD)
samples
are
spiked
matrix
water
samples
analyzed
by
the
laboratory
to
verify
acceptable
method
performance
in
the
matrix
being
monitored.
During
routine
performance
of
the
method,
MS/
MSD
samples
are
analyzed
by
the
laboratory
for
the
first
sample
of
any
new
matrix
that
will
be
monitored,
and
on
the
21st
sample
thereafter.
During
a
QC
acceptance
criteriabased
comparison
study
for
some
methods
(
e.
g.,
Cryptosporidium
and
Giardia
methods),
the
laboratory
should
analyze
MS
samples
as
part
of
routine
laboratory
QC,
however
these
analyses
may
not
be
necessary
during
this
study
because
MS/
MSD
samples
using
blinded
spiking
suspensions
distributed
by
the
study
coordinator
will
be
used
for
the
study.

5.2.8
Media
Sterility
Checks
Before
using
newly
prepared
media,
a
representative
portion
of
each
media
batch
needs
to
be
checked
for
sterility.
The
laboratory
should
test
media
sterility
by
incubating
one
unit
(
tube
or
plate)
from
each
batch
of
medium
specified
in
the
method
or
per
each
test
run,
whichever
is
more
frequent,
at
the
appropriate
temperature
for
the
length
of
the
method­
specified
incubation
time
and
observing
for
growth.

5.2.9
Method
Blank
Method
blanks
are
reagent
water
blanks
or
other
blanks
including
but
not
limited
to
buffered
water,
tap
water,
etc.,
depending
on
the
method
analyzed
to
demonstrate
freedom
from
contamination.
Method
blanks
should
be
analyzed
at
the
frequency
specified
in
the
approved
study
plan.
For
QC
acceptance
criteria­
based
comparison
studies,
the
laboratory
needs
to
analyze
a
method
blank
with
the
IPR
and
IDC
tests.
During
routine
performance
of
the
method,
the
laboratory
should
analyze
at
least
one
method
blank
per
every
20
test
samples
or
per
every
week.
During
a
QC
acceptance
criteria­
based
comparison
study
for
some
methods
(
e.
g.,
Cryptosporidium
and
Giardia
methods),
the
laboratory
should
analyze
method
blanks
as
part
of
routine
laboratory
QC,
but
method
blanks
also
should
be
shipped
to
the
laboratory
as
double­
blind
samples
by
the
study
coordinator.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
5­
5
5.2.10
Ongoing
Demonstration
of
Capability
(
ODC)
Samples
Ongoing
demonstration
of
capability
samples
are
spiked
reference
matrix
(
e.
g.,
reagent
water,
buffered
water,
etc.)
samples
that
are
analyzed
to
demonstrate
that
the
analytical
system
is
in
control
on
an
ongoing
basis.

5.2.11
Ongoing
Precision
and
Recovery
(
OPR)
Samples
Ongoing
precision
and
recovery
samples
are
spiked
reference
matrix
(
e.
g.,
reagent
water,
buffered
water,
etc.)
samples
that
are
analyzed
by
the
laboratory
to
verify
that
method
performance
criteria
are
being
met.
During
a
performance
based
comparison
study
for
some
methods
(
e.
g.,
Cryptosporidium
and
Giardia
methods),
the
laboratory
should
analyze
OPR
samples
as
part
of
routine
laboratory
QC
because
generally
OPR
samples
using
blinded
spiking
suspensions
distributed
by
the
spiking
coordinator
will
be
used
for
the
study.

5.2.12
Positive/
Negative
Controls
Positive
and
negative
controls
are
target
and
non­
target
organisms
processed
to
ensure
the
laboratories
are
familiar
with
the
identification
of
the
target
organism
and
to
ensure
that
confirmation
test
results
are
appropriate.

5.2.12.1
Positive/
Negative
Culture
Controls
(
Culture­
Based
Methods)

Positive
and
negative
culture
controls
refer
to
cultures
that,
when
analyzed
exactly
like
field
samples,
produce
a
known
positive
or
a
known
negative
result,
respectively,
for
a
given
type
of
media.
One
positive
culture
control
and
one
negative
culture
control
should
be
prepared
and
analyzed
for
every
media
(
including
confirmation
media)
used
in
the
method
whenever
a
new
batch
of
medium
or
reagents
is
used,
every
day
of
the
study,
or
every
20
samples,
or
as
specified
in
the
method,
whichever
is
more
frequent.
Each
control
should
be
carried
through
the
entire
procedure
and
should
exhibit
the
expected
positive
or
negative
result.

5.2.12.2
Positive/
Negative
Staining
Controls
(
Cryptosporidium
and
Giardia
Methods)

A
positive
staining
control
for
Cryptosporidium
and
Giardia
methods
is
a
slide
containing
positive
antigen
or
intact
Cryptosporidium
oocysts
and
Giardia
cysts,
and
that
is
stained
using
the
same
procedure
as
used
for
field
samples
or
test
samples.
A
negative
staining
control
is
a
slide
containing
only
phosphate
buffered
saline
(
PBS)
that
is
stained
using
the
same
procedure
as
used
for
field
samples
or
test
samples.
The
laboratory
should
prepare
and
examine
positive
and
negative
staining
controls
with
each
batch
of
slides
the
laboratory
prepares
during
the
study.
Positive
staining
controls
should
exhibit
acceptable
fluorescence
and
negative
staining
controls
should
not
exhibit
fluorescence.

5.2.12.3
Positive/
Negative
Staining
Controls
(
Other)

At
a
minimum,
the
laboratory
should
prepare
and
examine
a
positive
and
negative
control
using
the
same
procedure
as
used
for
field
or
test
samples
whenever
a
new
batch
of
media
or
reagents
is
used
every
day
of
the
study
or
every
20
samples,
whichever
is
more
frequent.
Each
control
should
be
carried
through
the
entire
procedure
and
should
exhibit
the
expected
positive
or
negative
result.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
5­
6
5.2.13
Preparation
Blanks
(
PB)

5.2.13.1
Membrane
Filter
Preparation
Blank
(
PB­
MF)

If
membrane
filtration
is
used,
at
the
beginning
and
the
end
of
each
filtration
series,
a
PB­
MF
is
performed
by
filtering
20­
30
mL
of
dilution
water
through
the
membrane
filter
and
testing
for
growth.
If
the
control
indicates
contamination
with
the
target
organism,
all
data
from
affected
samples
should
be
rejected.
A
filtration
series
ends
when
30
minutes
or
more
elapse
between
sample
filtrations.

5.2.13.2
Multiple­
Tube
Fermentation
Test
Preparation
Blank
(
PB­
MTF)

If
a
multiple­
tube
fermentation
test
is
used,
a
volume
of
sterile
buffered
water
that
is
analyzed
exactly
like
a
field
sample
should
be
analyzed
for
every
day
of
the
study
or
every
20
samples,
whichever
is
more
frequent.
The
preparation
blank
should
be
incubated
with
the
sample
batch
and
observed
for
growth
of
the
target
organism.
If
the
control
indicates
contamination
with
the
target
organism,
all
data
from
affected
samples
should
be
rejected.
If
buffered
water
is
not
used
for
dilutions,
only
the
multiple­
tube
fermentation
media
should
be
included.

5.2.13.3
Other
Preparation
Blank
(
PB­
Other)

A
volume
of
sterilized
water
(
e.
g.,
reagent
grade
as
defined
in
Specification
D
1193,
Annual
Book
of
ASTM
Standards)
that
is
analyzed
exactly
like
a
field
sample
should
be
analyzed
for
every
day
of
the
study
or
every
20
samples,
whichever
is
more
frequent.
The
preparation
blank
should
be
incubated
with
the
sample
batch
and
observed
for
growth
of
the
target
organism.
If
the
control
indicates
contamination
with
the
target
organism,
all
data
from
affected
samples
should
be
rejected.

5.2.14
Refrigerator/
Freezer
Temperatures
Refrigerator
and
freezer
temperatures
should
be
measured
and
recorded
once
per
day
when
in
use.
Refrigerator
temperature
should
be
maintained
at
1
°
C
to
4
°
C.
Freezer
temperatures
should
be
maintained
at
­
15
°
C
to
­
20
°
C.
Special
freezers
capable
of
long­
term
storage
of
cultures
or
virus
should
be
maintained
at
­
70
°
C
to
­
80
°
C.
Thermometers
used
to
measure
"
in­
use"
temperatures
should
be
calibrated
yearly
against
an
NIST
traceable
thermometer.

5.2.15
Sample
Processing
Equipment
Sterility
Checks
A
representative
portion
of
non­
disposable
items
such
as
sample
containers,
blender
jars,
etc.,
used
to
collect
or
process
samples
should
be
checked
for
sterility
prior
to
use
in
analyses.
To
test
for
sterility
add
approximately
500
mL
(
or
appropriate
volume
based
on
the
size
of
the
equipment
being
used)
of
a
sterile
non­
selective
broth
(
e.
g.,
tryptic
soy,
trypticase
soy,
or
tryptone
broth)
to
the
non­
disposable
item
and
incubate
at
35
°
C
±
0.5
°
C
for
24
hours
and
check
for
growth.
Depending
on
the
incubation
times
specified
in
the
method,
the
length
of
the
incubation
time
for
the
sample
processing
equipment
sterility
check
may
be
increased.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
6­
1
SECTION
6.0
STUDY
DESIGN
This
section
provides
a
description
of
the
study
design
that
is
important
for
assessing
comparability
using
either
side­
by­
side
method
comparison
studies
(
Section
6.1)
or
QC
acceptance
criteria­
based
studies
(
Section
6.2).
The
sections
below
address
the
number
of
laboratories,
number
of
matrices,
and
the
number
of
samples
that
should
be
analyzed
in
the
studies.

6.1
Side­
by­
Side
Comparison
Studies
Methods
are
compared
using
the
following
parameters:
°
Recovery.
Does
the
new
method
have
similar,
better
or
worse
recoveries
of
the
target
organism
as
the
reference
method?
°
Precision.
Are
the
recoveries
by
the
new
method
significantly
less
or
more
variable
than
the
reference
method?
°
False
positive
rate/
specificity.
Is
the
new
method
significantly
more
likely
or
less
likely
to
detect
non­
target
organisms
or
other
sample
constituents
that
would
be
reported
as
the
target
organism
by
the
analyst
when
compared
to
the
reference
method?
°
False
negative
rate/
sensitivity.
Is
the
new
method
significantly
more
likely
or
less
likely
to
exhibit
non­
detects
for
samples
with
the
target
organism
or
to
exhibit
results
that
are
biased
low
when
compared
to
the
reference
method?

To
generate
these
parameters,
samples
are
analyzed
by
a
single
laboratory
(
6.1.1).
The
number
of
samples
(
and
matrix
types)
used
in
the
study
are
determined
using
a
historical
EPA
standard
(
6.1.2.1)

6.1.1
Number
of
Laboratories
A
single
laboratory
should
be
used
for
a
side­
by­
side
comparability
study.
Since
the
study
should
be
conducted
in
a
single
independent
laboratory
with
no
conflict
of
interest;
the
laboratory
selected
cannot
be
the
method
developer's
laboratory
and
cannot
be
affiliated
with
the
method
developer.

6.1.2
Number
of
Samples
The
following
standards
generally
provide
the
minimum
number
of
samples
that
should
be
analyzed.
Additional
data
are
generally
acceptable
and
may
be
very
helpful
when
reviewing
an
ATP
or
new
method.

6.1.2.1
Method
Comparison
Study
Design
Summary
Table
6.1
provides
a
summary
of
the
method
comparability
requirements
for
nationwide
microbiological
ATPs
and
new
methods.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
6­
2
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
6­
3
Number
of
Matrices
Ten
different
water/
wastewater
matrices
from
geographically
diverse
locations
should
be
included
to
obtain,
as
much
as
is
practical,
a
good
presentation
of
the
wide
range
of
water
types
with
an
even
wider
range
of
target
organisms
to
which
the
method
should
appropriately
respond.
Each
water
or
wastewater
sample
should
be
collected
in
sufficient
volume
to
complete
all
replicate
analyses
of
sample
or
dilution
volumes
by
both
the
ATP
or
new
method
and
the
EPA­
approved
reference
method.
For
ambient
water
studies,
the
turbidity
of
at
least
one
matrix
should
be
greater
than
10
NTU.
Generally,
for
matrices
other
than
finished
drinking
water
or
ambient
water,
matrix
composition
will
be
addressed
on
a
study­
specific
basis.

Number
of
Samples
and
Replicates
Twenty
replicate
analyses
should
be
performed
by
each
method
for
each
of
the
10
matrices
for
a
total
of
200
replicate
analyses
per
method.
The
replicate
analyses
should
be
performed
on
the
same
day
for
both
the
proposed
and
reference
methods.

Verification
of
Results
For
quantitative
reference
methods,
10
typical
colonies
must
be
verified
from
4
randomly
chosen
replicates
(
of
the
20
replicates)
of
each
of
the
10
samples
for
a
total
of
400
colony
verifications.
For
presence/
absence
tests,
all
positive
samples
should
be
verified
for
up
to
200
samples.
See
Section
6.1.3
for
additional
information
pertaining
to
the
verification
of
results.
Calculations
for
false
positive
rates
and
false
negative
rates
are
described
in
Section
8.4.3.

6.1.3
Verification
of
Results
6.1.3.1
False
Positives
To
assess
whether
the
false
positive
rates
are
significantly
different
between
methods,
replicates
known
to
contain
non­
target
organisms
that
could
be
falsely
identified
as
the
target
organism
should
be
analyzed
by
both
the
ATP
or
new
method.
The
determination
that
the
samples
do
not
contain
the
target
organism
should
be
based
on
a
third
independent
standard
method
(
see
Section
7.6)
rather
than
by
the
EPAapproved
reference
method
being
used
in
the
comparison.
This
is
because
it
should
not
be
assumed
that
the
accepted
method
has
a
false
positive
or
negative
rate
of
zero.
In
side­
by­
side
comparison
studies
using
the
EPA
standard
for
determining
the
number
of
matrices
and
the
number
of
samples,
at
least
200
positive
results
should
be
verified
for
the
ATP
or
new
method
in
order
to
adequately
compare
false
positive
rates.

6.1.3.2
False
Negatives
To
assess
whether
the
false
negative
rates
are
significantly
different
between
methods,
replicates
known
to
contain
target
organisms
should
be
analyzed
by
both
the
ATP
or
new
method.
The
determination
that
the
samples
do
not
contain
the
target
organism
should
be
based
on
a
third
independent
standard
method
(
see
Section
7.6)
rather
than
by
the
EPA­
approved
reference
method
being
used
in
the
comparison.
This
is
because
it
should
not
be
assumed
that
the
accepted
method
has
a
false
positive
or
negative
rate
of
zero.
In
side­
by­
side
comparison
studies
using
the
EPA
standard
for
determining
the
number
of
matrices
and
the
number
of
samples,
at
least
200
negative
results
should
be
verified
for
the
ATP
or
new
method
in
order
to
adequately
compare
false
negative
rates.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
6­
4
6.2
QC
Acceptance
Criteria­
Based
Comparison
Studies
QC
acceptance
criteria­
based
comparison
studies
are
generally
conducted
to
demonstrate
that
the
ATP
or
new
method
is
able
to
meet
the
QC
acceptance
criteria
of
the
EPA­
approved
reference
method.
In
some
instances,
the
quality
control
(
QC)
acceptance
criteria
specified
in
a
method
may
not
be
sufficient
to
demonstrate
comparability
between
the
ATP
and
the
EPA­
approved
reference
method
and
a
side­
by­
side
comparison
study
should
be
conducted.
Generally,
EPA
will
make
this
decision
based
on
review
of
the
application
materials.

6.2.1
Number
of
laboratories
A
minimum
of
three
laboratories
should
be
used
for
a
QC
acceptance
criteria­
based
comparison
study.
All
three
laboratories
should
meet
all
QC
acceptance
criteria
in
the
EPA­
approved
reference
method.
If
more
than
three
laboratories
participate,
at
least
75%
of
the
participating
laboratories
should
meet
all
QC
acceptance
criteria
in
the
EPA­
approved
reference
method.

6.2.2
Number
of
Matrices
For
all
QC
acceptance
criteria­
based
comparison
studies,
there
should
be
one
matrix
per
laboratory.
For
ambient
water
studies,
the
turbidity
of
at
least
one
matrix
should
be
greater
than
10
NTU.
Generally,
for
matrices
other
than
finished
drinking
water
or
ambient
water,
matrix
composition
should
be
addressed
on
a
study­
specific
basis.

6.2.3
Number
of
Replicates
per
Matrix
The
number
of
replicates
per
matrix
and
laboratory
should
be
specific
to
the
QC
criteria
to
which
the
results
will
be
compared.
Generally,
the
number
of
reagent
results
should
be
4
and
the
number
of
source
water
results
per
matrix
should
be
2.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
7­
1
SECTION
7.0
SAMPLE
PREPARATION
AND
ANALYSIS
7.1
Collection
of
Samples
for
Analysis
Each
sample
should
be
collected
in
sufficient
volume
to
complete
all
replicate
analyses
by
both
the
ATP
or
new
method
and
the
EPA­
approved
reference
method.
Samples
should
be
spiked
(
if
necessary)
and
analyzed
as
soon
as
possible
after
collection.

7.1.1
Source
Water
Characterization
Source
water
characterization
information
should
be
collected
at
the
time
of
sample
collection.
This
information
will
be
useful
in
characterizing
the
matrix
to
identify
potential
interferences
and
generally
includes,
but
is
not
limited
to,
the
following:
°
Sample
collection
location
°
Source
of
water
(
e.
g.,
ground
water,
stream,
river,
lake,
etc.)
°
Plant
treatment
processes
(
if
sample
is
collected
from
a
water
treatment
facility)
°
Temperature
°
pH
°
Turbidity
°
Total
organic
carbon
(
TOC)
°
Free
and
total
disinfectant
residual
at
time
of
sample
collection
°
Heterotrophic
plate
count
(
HPC)
unless
heterotrophic
bacteria
are
the
target
analytes,
in
which
case
total
coliforms
should
be
measured
In
addition
to
this
information,
data
on
the
concentration
of
potential
interferences
(
e.
g.,
competing
bacteria,
interfering
chemicals,
etc.)
in
the
water
collected
for
analysis
may
be
necessary.
Known
interferences
should
be
discussed
in
the
method
and
addressed
in
the
study
plan.
The
final
results,
as
well
as
bench
sheets,
log
sheets,
instrument
printouts,
and
any
associated
quality
control
analyses
should
be
submitted
as
part
of
Appendix
C
of
the
study
report
(
Section
9.10).

7.2
Sample
Spiking
and
"
Stressing"
Procedures
for
Bacteriological
Methods
Depending
on
the
matrix
and
the
analyte
of
interest,
it
may
not
always
be
necessary
to
spike
samples
prior
to
analysis.
Rangefinding
analyses
should
be
performed
to
assess
the
ambient
concentration
of
target
organism(
s)
in
the
matrix
of
interest
to
determine
whether
sample
spiking
will
be
useful.
Samples
are
collected
and
dilutions
are
prepared
in
order
to
enumerate
the
number
of
organisms
in
the
sample
without
qualifiers
(
i.
e.
less
than,
greater
than,
or
too
numerous
to
count).
In
addition,
rangefinding
can
also
be
used
to
obtain
environmental
isolates
for
use
in
sample
spiking
or
to
determine
the
concentration
of
target
organism(
s)
in
a
spiking
suspension
or
spiked
sample.

If
samples
are
spiked,
environmental
isolates
should
be
used,
as
pure
strains
may
exhibit
different
recovery
and
precision
characteristics
than
natural
flora.
NELAC
(
http://
www.
epa.
gov/
ttn/
nelac/
standard/
5qs­
15­
0­
jhrev­
corr.
pdf,
Reference
10.9)
and
ATCC
(
http://
www.
atcc.
org/
SearchCatalogs/
faqBacteriology.
cfm#
Q15,
Reference
10.6)
recommend
that
bacterial
cultures
be
transferred
monthly
and
passed
no
more
than
five
times
before
returning
to
the
original
culture.

Sections
7.2.1
through
7.2.4
below,
detail
several
different
procedures
for
sample
spiking
and
stressing.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
7­
2
7.2.1
Drinking
Water:
Spiking
and
Chlorine­
Stressing
Drinking
water
samples
should
be
spiked
and
stressed
with
chlorine
as
described
in
Sections
7.2.1.1
and
7.2.1.2
below.

7.2.1.1
Drinking
Water
Spiking
Because
finished
drinking
water
does
not
typically
contain
the
target
analyte(
s)
of
interest,
finished
drinking
water
should
be
spiked.
For
the
evaluation
of
drinking
water
samples,
a
single
finished
drinking
water
matrix
is
spiked
with
target
organism(
s)
from
other
matrices,
such
as
non­
chlorinated
secondary
sewage
effluents
or
polluted
surface
waters.
Sewage
effluent
generally
has
the
advantage
of
providing
a
wide
range
of
strains,
whereas
surface
waters
typically
have
the
advantage
of
providing
organisms
more
variable
in
quality.
Depending
on
the
study
design
(
Section
6.0),
the
source
of
spiking
suspensions
(
i.
e.,
wastewaters)
should
come
from
geographically
dispersed
sites.

To
prepare
spiked
drinking
water
samples:

(
1)
Collect
at
least
five
liters
of
each
non­
chlorinated
secondary
sewage
effluents
or
polluted
surface
water
to
be
used
as
spiking
suspensions.

(
2)
Perform
rangefinding
analyses
as
described
above
on
each
spiking
suspension
to
determine
target
organism
density
as
soon
as
possible
after
receipt
of
the
sample
to
ensure
that
target
organism
density
and
diversity
are
not
reduced.

(
3)
For
each
spiking
suspension,
spike
a
sufficient
volume
of
drinking
water
with
a
sufficient
volume
of
spiking
suspension
(
based
on
rangefinding)
to
obtain
103­
105
target
organisms/
100
mL.
Please
note:
A
single
drinking
water,
with
negligible
concentrations
of
oxidants
and
reductants
(
e.
g.,
chlorine
and
sodium
thiosulfate,
respectively)
should
be
used
for
the
entire
study.
Refrigerate
all
spiked
drinking
waters
at
1
°
C
­
4
°
C
for
use
in
preliminary
chlorination
study
and
comparability
study.
High
oxidant
or
reductant
levels
in
the
drinking
water
could
interfere
with
organism
stressing.

7.2.1.2
Preliminary
Chlorination
Study
to
Determine
Appropriate
Exposure
Time
Microorganisms
in
the
spiked
drinking
water
samples
(
from
Section
7.2.1.1)
should
be
stressed
by
chlorination
at
ambient
temperatures
under
conditions
similar
to
those
in
drinking
water
treatment
facilities.
The
goal
of
chlorinating
the
spiked
samples
is
to
simulate
drinking
water
treatment
by
reducing
the
number
of
organisms
in
the
spiked
drinking
water
samples
from
103­
105
target
organisms/
100
mL
to
1­
10
chlorine­
stressed
target
organisms/
100
mL
for
most
probable
number
methods
or
to
20
­
100
chlorine­
stressed
target
organisms/
100
mL
for
membrane
filtration
methods
(
i.
e.,
a
2­
4
log
removal).

After
chlorination,
no
two
samples
are
expected
to
produce
the
same
levels
of
injured
(
stressed)
target
organisms
because
the
disinfection
process
is
impacted
by
physical
and
biological
factors.
These
include:
the
type
of
spiked
drinking
water
sample
to
be
disinfected
(
e.
g.,
spiked
with
sewage
effluent
or
polluted
source
water),
the
initial
concentration
of
the
target
organism(
s),
the
chlorine
demand
of
the
spiked
sample,
the
type
and
concentration
of
chlorinating
agent,
the
exposure
time,
the
sample
mixing
,
pH,
and
temperature.
As
a
result,
a
preliminary
chlorination
study
should
be
performed
to
establish
the
exposure
time
necessary
to
reduce
the
number
organisms
in
the
spiked
drinking
water
samples
from
103­
105
target
organisms/
100
mL
to
1­
10
chlorine­
stressed
target
organisms/
100mL
(
or
a
2­
4
log
removal).
During
this
preliminary
chlorination
exposure
time
study,
the
physical
and
biological
parameters
in
Section
7.1.1
should
be
carefully
monitored
and
recorded
for
each
sample.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
7­
3
The
exposure
time
is
directly
dependent
upon
the
initial
concentration
of
the
target
organism
present,
the
matrices'
chlorine
demand,
and
the
form
of
chlorine
present.
For
testing,
the
spiked
sample
is
generally
exposed
to
2.0­
2.5
mg
total
chlorine/
L
for
over
a
range
of
times
such
as
10,
20,
and
30
minutes
to
reduce
the
density
of
target
organisms
from
103­
105
CFU/
100mL
to
1­
10
CFU/
100
mL
sample.
However,
the
period
of
exposure
of
a
sample
with
a
low
chlorine
demand
may
be
significantly
shorter
than
20­
30
minutes.

Suggested
Preliminary
Chlorination
Study
(
1)
Determine
and
record
the
total
residual
and
free
residual
chlorine
concentrations
using
an
EPAapproved
N,
N
diethyl­
p­
phenylenediamine
(
DPD)
colorimetric
method
(
e.
g.,
Standard
Method
4500­
Cl­
G)
initially,
at
midpoint,
and
at
the
end
of
the
exposure
time
just
prior
to
dechlorination.

(
2)
For
each
exposure
time,
place
2
L
of
each
spiked
drinking
water
sample
(
from
Section
7.2.1.1)
in
a
glass
container.

(
3)
Add
an
appropriate
volume
of
a
diluted
solution
of
reagent
grade
sodium
hypochlorite
(
e.
g.,
a
1:
20
dilution
of
5%
(
w/
v)
stock
solution),
to
achieve
the
desired
level
of
chlorinating
agent
and
stir
the
sample
continuously
during
exposure
to
chlorination.
If
the
spiked
sample
has
an
appreciable
chlorine
demand
(
e.
g.,
spiked
with
a
primary
effluent
or
a
sewage
sample),
add
dilute
sodium
hypochlorite
solution
until
a
total
residual
chlorine
level
between
2.0
and
2.5
mg/
L
is
maintained
in
the
absence
of
free
chlorine.
If
a
sample
has
a
low
chlorine
demand,
avoid
overstressing
or
killing
the
organisms
by
prolonged
exposure
to
free
residual
chlorine.
The
free
residual
chlorine
concentration
should
not
exceed
0.5­
1.0
mg/
L.

(
4)
Stop
the
chlorine
oxidation
(
dechlorinate)
at
the
end
of
the
exposure
period
by
adding
0.8
mL
of
a
10%
(
w/
v)
sodium
thiosulfate
solution/
L
sample.

(
5)
Enumerate
the
target
organism
density
in
an
aliquot
of
the
spiked,
chlorine­
stressed,
dechlorinated
drinking
water
sample
using
the
appropriate
EPA­
approved
reference
method
from
Table
1­
1.

(
6)
For
each
exposure
time,
repeat
Steps
3
­
5,
above.

7.2.1.3
Suggested
Procedure
for
Chlorination
and
Dilution
of
Samples
for
Comparability
Study
(
1)
Determine
and
record
the
total
residual
and
free
residual
chlorine
concentrations
initially,
at
midpoint,
and
at
the
end
of
the
exposure
time
just
prior
to
dechlorination
using
an
EPAapproved
N,
N
diethyl­
p­
phenylenediamine
(
DPD)
colorimetric
method
(
e.
g.,
Standard
Method
4500­
Cl­
G).

(
2)
Place
a
sufficient
volume
of
each
spiked
drinking
water
in
a
glass
container
to
perform
sufficient
repeat
analyses
at
multiple
dilutions.

(
3)
Immediately
prior
to
chlorination,
perform
enumeration
to
determine
target
organism
density
as
described
in
7.2.1.1(
2).
This
value
should
be
used
to
determine
the
log
reduction
due
to
chlorination.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
7­
4
(
4)
Add
reagent­
grade
sodium
hypochlorite
to
achieve
the
same
concentration,
as
in
the
preliminary
chlorination
exposure
study.

(
5)
To
reduce
the
density
of
target
organisms
from
103­
105
CFU/
100mL
to
1­
10
CFU/
100
mL,
chlorinate
each
spiked
drinking
water
sample
for
the
appropriate
time,
based
upon
the
preliminary
chlorination
exposure
study.
Stir
the
sample
continuously
during
the
chlorination.

(
6)
Stop
the
chlorine
oxidation
(
dechlorinate)
at
the
end
of
the
exposure
period
by
adding
0.8
mL
of
a
10%
(
w/
v)
sodium
thiosulfate
solution/
L
sample.

(
7)
Enumerate
target
organism
density
in
an
aliquot
of
the
spiked,
chlorine­
stressed,
dechlorinated
drinking
water
sample
using
the
appropriate
EPA­
approved
reference
method
from
Table
1­
1.

(
8)
Refrigerate
the
spiked,
chlorine­
stressed,
dechlorinated
drinking
water
samples
at
1
°
C
­
4
°
C
for
use
in
comparability
testing.

(
9)
Read
the
plates
to
determine
the
approximate
density
of
the
target
organisms.
Use
these
results
to
estimate
the
appropriate
dilution
necessary
to
reach
the
target
organism
density
of
1­
10
CFU/
100
mL.

(
10)
Evaluate
three
dilutions
of
each
spiked
drinking
water
sample.
Samples
should
be
diluted
with
the
same,
original,
oxidant­
free
and
reductant­
free
drinking
water,
as
necessary
to
reach
a
target
organism
density
of
1­
10
target
organisms/
100
mL
for
most
probable
number
methods
or
20­
100
target
organisms/
100
mL
for
membrane
filtration
methods.
Make
the
dilution
and
at
least
two
others
that
bracket
the
target
density,
for
example,
half
and
double
that
dilution.
One
of
these
dilutions
should
contain
the
desired
1­
10
target
organisms
per
100
mL.
Immediately
conduct
the
comparability
analyses
with
each
sample
using
these
three
dilutions.

Please
note:
For
the
evaluation
of
presence/
absence
methods,
the
data
used
for
comparison
should
be
from
the
dilution(
s)
which
produces
results
closest
to
an
equal
number
of
positive
and
negative
results
for
the
reference
method.
A
25%
to
75%
split
in
responses
(
in
either
direction)
should
be
sought.
For
comparability,
the
evaluated
results
for
the
ATP
or
new
method
should
be
from
the
same
dilution
as
the
reference
method.
If
one
of
the
dilutions
does
not
produce
an
acceptable
split
in
positive
and
negative
results
for
the
reference
method,
the
applicant
should
return
to
the
original,
spiked
sample.

7.2.2
Preparation
of
Enumerated
Spiking
Suspension
This
dilution
scheme
is
adapted
from
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
19th
Edition,
Section
9020
B
(
Reference
10.3).
This
entire
process
should
be
performed
quickly
to
avoid
loss
of
viable
organisms.
There
should
be
approximately
1010
organisms
per
slant.
Therefore,
dilution
bottles
"
A"
through
"
E,"
below
contain
approximately
1010,
108,
106,
104,
and
103
organisms
per
dilution
bottle,
respectively.
Depending
on
the
growing
conditions,
these
numbers
may
vary.
As
a
result,
until
experience
has
been
gained,
more
dilutions
should
be
filtered
to
determine
the
appropriate
dilution.

Inoculate
bacterial
culture
onto
the
entire
surface
of
several
nutrient
agar
slants
with
a
slope
approximately
6.3
cm
long
in
a
125
×
16
mm
screw­
cap
tube.
Incubate
for
24
±
2
hours
at
35
°
C
±
0.5
°
C.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
7­
5
From
the
slant
that
has
the
best
growth,
prepare
serial
dilutions
using
four
dilution
bottles
with
99
mL
of
sterile
buffered
dilution
water
(
bottles
A,
B,
C,
and
D)
and
one
dilution
bottle
containing
90­
mL
of
sterile
buffered
dilution
water
(
bottle
E).

Pipette
1
mL
of
buffered
dilution
water
from
bottle
"
A"
to
one
of
the
slants.
Emulsify
the
growth
on
the
slant
by
gently
rubbing
the
bacterial
film
with
the
pipette,
being
careful
not
to
tear
the
agar.
Pipette
the
suspension
back
into
dilution
bottle
"
A."
Repeat
this
procedure
a
second
time
to
remove
any
remaining
growth
on
the
agar
slant,
without
disturbing
the
agar.

Make
serial
dilutions
as
follows:
(
1)
Shake
bottle
"
A"
vigorously
and
pipette
1
mL
to
bottle
"
B"
containing
99
mL
buffer
(
2)
Shake
bottle
"
B"
vigorously
and
pipette
1
mL
to
bottle
"
C"
containing
99
mL
buffer
(
3)
Shake
bottle
"
C"
vigorously
and
pipette
1
mL
to
bottle
"
D"
containing
99
mL
buffer
(
4)
Shake
bottle
"
D"
vigorously
and
pipette
10
mL
to
bottle
"
E"
containing
90
mL
buffer;
this
should
result
in
a
final
dilution
of
approximately
10
organisms
/
mL.
If
it
is
more
convenient
for
your
laboratory,
an
acceptable
alternative
to
the
dilution
scheme
presented
for
this
step,
is
to
pipette
11
mL
of
dilution
D
into
dilution
bottle
E,
which
contains
99
mL
of
dilution
water.

Filter
1­
to
5­
mL
portions
in
triplicate
from
bottles
"
D"
and
"
E"
according
to
standard
membrane
filtration
methods
to
determine
the
number
of
CFU
in
the
dilutions.
The
recommended
target
dilution
and
spike
volume
depends
on
the
method
and
the
target
analyte(
s).
Typically,
dilutions
should
be
stored
at
1
°
C
to
5
°
C
and
may
be
used
throughout
the
day
they
are
prepared,
however,
storage
conditions
should
be
adjusted
as
necessary
since
both
storage
conditions
and
viability
may
vary
from
organism
to
organism.

7.2.3
Log
Phase
Growth
Curve
Inoculate
100
mL
of
broth
media
with
a
single
isolated
colony
and
incubate
organisms
at
optimum
temperature.
If
possible,
shake
at
200
RPM
during
incubation.

Take
optical
density
(
OD)
measurements
at
550­
600
nm
at
30
minute
intervals
for
the
first
8
hours
and
record
readings.
In
addition
to
OD
readings
at
30
minute
intervals,
using
aseptic
technique,
remove
a
0.1­
mL
portion
of
the
culture
and
make
a
series
of
1:
10
dilutions
in
sterile
buffer.
Initially
plate
0.1
mL
of
the
1.0,
10­
1,
and
10­
2
dilutions
in
duplicate
and
incubate
overnight
at
optimum
temperature.
Count
colonies
and
record
data.
As
the
optical
density
increases,
evaluate
serial
dilutions
to
accommodate
the
increased
numbers
of
bacteria
(
i.
e.,
when
the
optical
density
exceeds
1.0
then
plate
0.1
mL
from
dilutions
10­
1,
10­
2,
and
10­
3).

Based
on
OD
and
the
CFU/
mL
results,
the
OD
of
fresh
cultures
(
in
log­
phase
growth)
can
be
used
to
determine
the
concentration
of
bacteria
in
the
tubes
by
a
simple
graphic
representation
of
the
combined
OD
and
CFU/
mL
results.

Commercially
available
McFarland
standards
may
be
used
to
determine
the
bacterial
density
instead
of
actually
doing
a
growth
curve
within
the
lab.
In
order
to
determine
bacterial
densities
using
McFarland
standards,
the
OD
of
the
standards
are
compared
to
the
OD
of
the
log
phase
culture.

7.2.4
Commercially
Available
Enumerated
Spikes
One
time
use,
commercially
available
spiking
suspensions
may
be
obtained
from
a
variety
of
vendors.
Prepare
spiking
suspensions
according
to
manufacturer's
instructions.
It
should
be
noted
that
a
different
spiking
volume
than
that
recommended
by
the
manufacturer
may
be
necessary
to
achieve
the
target
density.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
7­
6
7.3
Spiking
Procedures
for
Virus
Methods
7.3.1
Cell
Monolayer
Propagation
In
order
to
propagate
virus
stock
suspensions
cell
monolayers
should
be
propagated.
The
type
of
assay
cytopathic
effect
(
CPE)
or
plaque
assay
(
PA),
being
performed
determines
the
size
of
the
flask
and
the
number
of
days
the
monolayer
incubates
prior
to
use
for
the
assay.
Inoculate
T75
or
T25
flasks
with
1
x105
cells
containing
cell
specific
growth
medium.
Flasks
for
CPE
should
incubate
for
5­
7
days
at
37
°
C
±
0.5
°
C
with
95%
relative
humidity
(
and
5%
CO2
concentration,
if
necessary).
One
CPE
flask
should
be
stained
with
crystal
violet
and
microscopically
checked
to
ensure
a
95%
confluent
cell
monolayer
prior
to
use
in
the
assay.
Flasks
used
for
PA
incubate
for
7­
10
days
under
the
same
conditions
as
the
monolayers
used
for
CPE.
PA
flasks
should
also
be
checked
to
ensure
95%
confluency
before
use
for
assay.

7.3.2
Propagation
of
Virus
Stock
Suspension
For
(
CPE)
analysis,
utilizing
the
appropriate
cell
line
(
e.
g.,
Buffalo
green
monkey
kidney
cells;
BGMK),
inoculate
the
cell
monolayer
with
virus
and
incubate
culture
flasks
at
37
°
C
±
0.5
°
C
until
the
entire
monolayer
has
been
destroyed
by
virus
replication
(
approximately
72
­
96
hours).
Freeze
(
to
approximately
­
80
°
C)
and
thaw
the
flasks
three
times,
then
pool
contents
of
the
flasks
and
spin
at
10,000
x
g
for
30
minutes.
Filter
supernatant
using
a
0.2
µ
m
pore
nylon
filter
to
remove
any
remaining
cell
debris.
The
filtrate
is
the
virus
stock
suspension.

7.3.3
Titering
of
the
Virus
Stock
Suspension
Titer
the
virus
stock
suspension
(
from
Section
7.3.2)
by
performing
a
plaque
assay.
Inoculate
monolayers
with
0.2
mL
of
serial
diluted
viral
stock
suspension.
After
rocking
and
rinsing
the
monolayers,
add
the
agar
overlay
to
the
monolayer.
Incubate
the
culture
flasks
for
seven
days
while
reading
the
number
of
plaques
each
day
for
the
entire
seven
days.
After
the
viral
stock
suspension
has
been
titered,
appropriate
volumes
of
the
suspension
can
be
used
to
spike
test
matrices
to
obtain
plaque
forming
units
(
PFU)/
mL.

7.4
Spiking
Procedures
for
Cryptosporidium
and
Giardia
Enumerated
spiking
suspensions
are
needed
for
initial
and
ongoing
precision
and
recovery
(
IPR
and
OPR)
samples
(
often
referred
to
as
positive
controls)
and
matrix
spike
(
MS)
samples.
Flow
cytometer
 
sorted
organisms
are
necessary
for
these
spiking
suspensions,
rather
than
manual
techniques.
Flow
cytometer
 
sorted
spikes
generally
are
characterized
by
lower
variability
than
manually
enumerated
spikes.

Spiking
suspensions
should
be
prepared
using
unstained
organisms
that
have
not
been
irradiated,
heatfixed
or
formalin­
fixed.
Immediately
before
sorting
spiking
suspensions,
initial
calibration
of
the
flow
cytometer
should
be
performed
by
conducting
a
series
of
sequential
sorts
directly
onto
membranes
or
well
slides.
These
initial
sorts
should
be
stained
and
counted
microscopically
to
verify
the
accuracy
of
the
system.
When
sorting
the
spiking
suspensions,
ongoing
calibration
samples
should
also
be
prepared
and
counted
at
regular
intervals.
The
mean
of
the
ongoing
calibration
counts
should
be
used
as
the
estimated
spike
dose.
Flow­
cytometer­
sorted
spiking
suspensions
should
be
used
by
the
expiration
date
noted
on
the
suspension.
Flow­
cytometer­
sorted
spiking
suspensions
containing
live
organisms
should
be
used
within
two
weeks
of
the
preparation
date.
General
procedures
for
preparing
flow­
cytometer­
counted
spikes
for
Cryptosporidium
and
Giardia
can
be
found
in
EPA
Method
1622
and
1623
(
April
2001).
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
7­
7
A
potential
commercial
source
of
flow­
sorted
Cryptosporidium
and
Giardia
spiking
suspensions
is:

Wisconsin
State
Laboratory
of
Hygiene
Flow
Cytometry
Unit
2601
Agriculture
Drive
Madison,
WI
53718
Phone:
(
608)
224­
6260
Fax:
(
608)
224­
6213
The
Wisconsin
State
Laboratory
of
Hygiene
prepares
and
distributes
live
Cryptosporidium
parvum
oocysts
and
Giardia
intestinalis
cysts
that
have
not
been
treated
to
reduce
viability.

7.5
Analysis
of
Samples
Samples
should
be
analyzed
in
accordance
with
the
EPA­
approved
study
plan.
Any
deviations
from
the
study
plan
may
suggest
the
need
for
additional
analyses
and
potentially,
rejection
of
data
generated
from
the
study.
If
any
deviations
from
the
approved
study
plan
are
necessary
prior
to
or
during
the
study,
the
applicant
should
consult
EPA
and
receive
approval
for
the
modification
to
the
study
plan.
Deviations
from
the
approved
study
plan
should
be
documented
in
the
study
report
(
Section
9.0).

Example
analysis
schemes
are
provided
in
Sections
7.5.1
and
7.5.2
below.
Generally,
the
analysis
schemes
will
differ
depending
on
the
type
of
organism
and
the
type
of
method.
The
study
plan
(
Section
4.0)
should
detail
the
order
in
which
samples
will
be
analyzed.

7.5.1
Side­
by­
Side
Comparison
Studies
Method
Blank
1
Sample
1
up
to
Sample
20
Method
Blank
2
Positive
Control
Negative
Control
Method
Blank
3
Media
Sterility
Checks
7.5.2
QC
Acceptance
Criteria­
Based
Comparison
Studies
Replicate
1
for
IPR
Replicate
2
for
IPR
Replicate
3
for
IPR
Replicate
4
for
IPR
Method
Blank
Matrix
Spike
Matrix
Spike
Duplicate
Unspiked
Matrix
Sample
7.6
Verification
of
Results
The
number
of
positive
and
negative
results
to
be
verified
in
a
side­
by­
side
comparison
study
is
discussed
in
Section
6.1.3.
Sections
7.6.1
to
7.6.3
below
discuss
the
types
of
independent
standards
that
may
be
used
for
the
verification
of
results
from
bacteriological
methods,
virus
methods,
and
Cryptosporidium
and
Giardia
methods,
respectively.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
7­
8
7.6.1
Verification
of
Results
from
Bacteriological
Methods
7.6.1.1
Biochemical
Tests
that
May
be
Used
for
Verifications
Oxidase
Citrate
Catalase
Lysine
decarboxylase
ONPG
Methyl
Red
and
Voges
Proskauer
(
MRVP)
Indole
Triple
sugar
iron
(
TSI)
Coagulase
Lysine
iron
agar
(
LIA)
Esculin
Urease
Sugars
(
e.
g.,
Trehalose,
lactose,
mannitol,
and
sorbitol)

It
is
recommended
that
multiple
biochemical
tests
be
utilized
to
verify
colony
identification.
The
above
list
of
biochemical
tests
is
not
exhaustive.
The
choice
of
which
biochemical
tests
to
use
is
based
on
type
of
organism
(
i.
e.,
gram
stain
results)
being
identified/
verified.
Biochemical
tests
used
for
verification
should
be
discussed
in
the
study
plan
(
Section
4.0).
It
may
be
appropriate
to
perform
a
gram
stain
prior
to
biochemical
identification.
A
description
of
the
biochemical
tests
listed
above,
as
well
as
additional
tests
is
provided
in
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
Method
9225.

7.6.1.2
Commercially
Available
Biochemical
Testing
Products
Commercial
biochemical
test
systems
incorporate
multiple
biochemical
tests
to
allow
for
identification
to
the
genus
and/
or
species
level,
which
may
be
difficult
when
using
individual
biochemical
tests
prepared
in
house.

Commercial
biochemical
test
systems
are
available
in
two
formats:
systems
that
depend
on
the
analyst
to
manually
interpret
the
results
(
e.
g.,
API
strips,
BBL
crystal,
and
enterotube)
and
systems
that
automate
the
interpretation
of
results
(
e.
g.,
Vitek
and
Biolog).

7.6.2
Verification
of
Results
from
Virus
Methods
Viral
protocol
testing
confirmations
tend
to
be
method­
specific,
therefore
a
detailed
description
of
the
confirmation/
verification
procedure
should
be
included
in
the
study
plan
and
reviewed
on
a
study­
specific
basis.

7.6.3
Verification
of
Results
from
Cryptosporidium
and
Giardia
Methods
Verification
of
results
may
not
be
necessary
for
Cryptosporidium
and
Giardia
IFA
methods
beyond
the
analyst's
microscopic
examination
of
the
organism.
If
verification
is
necessary,
a
detailed
description
of
the
confirmation/
verification
procedure
should
be
included
in
the
study
plan
and
reviewed
on
a
studyspecific
basis.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
1
SECTION
8.0
REVIEW
OF
STUDY
RESULTS
Generally,
upon
receipt
of
the
applicant's
data,
EPA
will
perform
the
following
reviews
discussed
in
Sections
8.1
to
8.5.
1.
Assessment
of
compliance
with
the
approved
study
plan
2.
Data
review
3.
Data
validation
4.
Development
of
descriptive
statistics
5.
Statistical
assessment
of
method
comparability
Methods
that
are
deemed
acceptable
will
generally
be
recommended
for
approval
(
Section
8.6).

8.1
Assessment
of
Compliance
with
Approved
Study
Plan
Generally,
EPA
will
review
the
study
report
and
associated
data
to
ensure
the
study
was
conducted
according
to
the
approved
study
plan.
The
applicant
should
explain
and
justify
(
possibly
with
additional
studies)
any
deviations
from
the
study
plan.
Deviations
from
the
approved
study
plan
that
occur
without
prior
approval
from
EPA
may
result
in
the
rejection
of
some
or
all
study
data.

8.2
Data
Review
Upon
receipt
of
the
applicant's
data,
EPA
will
generally
verify
that
all
raw
data
described
in
Section
9.10
are
present
and
complete.
Generally,
all
calculations
used
in
the
method
will
be
verified.
This
may
include
calculations
used
for
spiking
enumeration,
preliminary
or
presumptive
stages
of
the
method,
and
the
determination
of
the
final
result.

8.3
Data
Validation
After
verifying
data
completeness
and
reviewing
of
all
calculations,
EPA
will
generally
verify
that
all
measurements
were
performed
in
accordance
with
the
method.
This
may
include,
but
is
not
limited
to,
the
following:
°
Temperature
logs
for
incubator/
waterbath/
refrigerator
°
Media
preparation
records
°
Sample
incubation
times
°
Associated
QC
samples
(
as
described
in
Section
5.0)
°
Method
blanks
°
Preparation
blanks
°
Sterility
checks
°
Positive
and
negative
controls
8.4
Development
of
Descriptive
Statistics
8.4.1
Mean
Recovery
To
determine
if
matrix
characteristics
effect
method
performance,
mean
recoveries
should
be
calculated
separately
for
each
matrix
and
method.
Mean
recoveries
should
also
be
calculated
for
each
method
over
all
matrices.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
2
8.4.2
Precision
Precision
can
be
expressed
both
on
an
absolute
scale
(
i.
e.,
standard
deviation)
and
on
a
relative
scale
(
i.
e.,
relative
standard
deviation).
The
RSD
(
sometimes
referred
to
as
coefficient
of
variation)
is
calculated
as
the
standard
deviation
divided
by
the
mean,
expressed
as
a
percent.
For
the
purpose
of
summarizing
the
data,
both
standard
deviations
and
RSDs
should
be
calculated,
and
the
one
which
is
most
appropriate
for
assessing
comparability
will
be
used
in
analyses.
Generally,
RSDs
are
most
appropriate
for
summarizing
precision
when
variability
increases
as
concentration
increases.

To
give
an
indication
of
the
effect
of
multiple
matrices
on
precision,
standard
deviations
should
be
calculated
separately
for
each
matrix
and
method.
Standard
deviations
also
should
be
calculated
for
each
method
over
all
matrices.

8.4.3
False
Positive
Rates,
False
Negative
Rates,
Sensitivity,
and
Specificity
False
positive
(
FP)
and
false
negative
(
FN)
rates
of
approved
and
reference
methods
should
be
evaluated
when
assessing
comparability.
An
independent
standard
(
described
in
Section
7.6)
may
sometimes
be
necessary
to
confirm
positives
and
negatives
of
both
the
ATP
or
new
method
and
the
EPA
reference
method.
The
confirmation
methods
used
should
be
discussed
in
the
validation
study
plan.

Generally,
performance
of
the
ATP
or
new
method
and
EPA­
approved
reference
methods
will
be
defined
in
terms
of
false
positive
rates
and
false
negative
rates.
For
the
purposes
of
the
ATP
protocol,
false
positive
rates
and
false
negative
rates
are
equivalent
to
(
1­
Specificity)
and
(
1­
Sensitivity),
respectively.
Specificity
is
defined
as
the
percent
of
negative
samples
correctly
identified
as
negative,
and
sensitivity
is
defined
as
the
percent
of
positive
samples
correctly
identified
as
positive
(
see
equations
on
the
next
page).
In
order
to
calculate
estimates
of
false
positives,
false
negatives,
sensitivity,
and
specificity
for
each
method,
2­
by­
2
tables
for
each
matrix,
and
over
all
matrices,
should
be
set
up
as
follows
in
Table
8­
1.

Table
8­
1.
Standard
Format
for
2­
by­
2
Tables
Independent
Standard
+
­
Total
ATP
or
New
Method
+
TP1
FP1
TP1
+
FP1
­
FN1
TN1
FN1
+
TN1
Total
TP1
+
FN1
FP1
+
TN1
TP1
+
FP1
+
TN1
+
FN1
Independent
Standard
+
­
Total
EPAApproved
Reference
Method
+
TP2
FP2
TP2
+
FP2
­
FN2
TN2
FN2
+
TN2
Total
TP2
+
FN2
FP2
+
TN2
TP2
+
FP2
+
TN2
+
FN2
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
3
Estimates
of
sensitivity,
specificity,
false
positive
rates,
and
false
negative
rates
as
percentages
for
the
two
methods
should
be
calculated
as
follows:

Sensitivityi
=
TPi
*
100
%
TPi
+
FNi
Specificityi
=
TNi
*
100
%
TNi
+
FPi
False
positive
ratei
=
FPi
*
100
%
=
(
1
­
TNi
)
*
100
%
=
1
­
Specificityi
TNi
+
FPi
TNi
+
FPi
False
negative
ratei
=
FNi
*
100
%
=
(
1
­
TPi
)
*
100
%
=
1
­
Sensitivityi
TPi
+
FNi
TPi
+
FNi
Where
F
=
False,
N
=
Negative,
T
=
True
and
"
i"
refers
to
the
specified
method
(
i=
1
for
new
method,
i=
2
for
reference
method)

8.5
Statistical
Assessment
of
Method
Comparability
8.5.1
Presence
/
Absence
Methods
For
presence/
absence
methods,
the
chi­
square
test
and
Breslow­
Day
test
will
generally
be
used
to
compare
the
percent
of
false
positives
and
false
negatives
in
the
proposed
method
analyses
with
the
percent
of
false
positives
and
false
negatives
in
the
reference
method
analyses.

Generally,
tests
for
significant
differences
in
false
positive
and
false
negative
rates
between
methods
will
be
run
using
the
results
summarized
in
Table
8­
1
in
Section
8.4.
To
perform
the
chi­
square
and
Breslow­
Day
tests,
it
may
help
to
first
rearrange
the
data
as
follows
in
Tables
8­
2
and
8­
3:
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
4
Table
8­
2.
False
Negative
Rate
Comparison
Method
New
Reference
Total
Result
True
+
TP1
TP2
TP1
+
TP2
False
­
FN1
FN2
FN1
+
FN2
Total
TP1+
FN1
TP2
+
FN2
TP1
+
TP2
+
FN1
+
FN2
Table
8­
3.
False
Positive
Rate
Comparison
Method
New
Reference
Total
Result
False
+
FP1
FP2
FP1
+
FP2
True
­
TN1
TN2
TN1
+
TN2
Total
FP1
+
TN1
FP2
+
TN2
FP1
+
FP2
+
TN1
+
TN2
Before
running
the
chi­
square
tests,
positives
and
negatives
should
be
confirmed
using
the
independent
standard
method.
The
chi­
square
test
will
be
used
to
determine
whether
the
false
positive
rate
and
false
negative
rate
have
a
statistically
significant
difference
between
the
ATP
or
new
method
and
the
EPAapproved
reference
method.

8.5.1.1
Assessing
Method
Differences
(
Chi­
Square
Test)

In
order
to
assess
whether
the
false
positive
and
false
negative
rates
differ
between
methods,
chi­
square
tests
should
be
run
over
all
matrices;
additional
tests
in
each
matrix
also
may
be
necessary
depending
on
the
presence
of
matrix
interactions
(
see
Section
8.5.1.2).
For
false
negative
rates,
the
chi­
square
test(
s)
indicate
whether
the
proportions
of
negative
samples
correctly
identified
as
negative
by
the
two
methods
are
significantly
different,
and
for
false
positive
rates,
the
chi­
square
test(
s)
indicate
whether
the
proportions
of
true
positive
results
correctly
identified
as
positive
for
the
two
methods
are
significantly
different.

8.5.1.2
Assessing
Method/
Matrix
Interactions
(
Breslow­
Day
Test)

In
order
to
assess
whether
there
are
false
positive
and
false
negative
rate
differences
between
methods,
it
is
also
necessary
to
establish
if
there
is
a
matrix
effect
on
these
parameters.
The
effect
of
matrices
on
false
positive
and
false
negative
rate
differences
between
methods
is
assessed
using
the
Breslow­
Day
test.
(
Reference
10.7).
The
Breslow­
Day
test
is
used
to
test
whether
there
is
an
interaction
between
matrix
and
method
in
terms
of
the
likelihood
of
a
false
positive
result
(
for
specificity)
and
in
terms
of
the
likelihood
of
a
false
negative
result
(
for
sensitivity).
If
a
significant
interaction
is
found
between
method
and
matrix,
then
the
chi­
square
tests
for
a
difference
between
methods
for
that
attribute
should
be
done
separately
for
each
matrix.
Otherwise,
separate
chi­
square
tests
for
each
matrix
are
not
necessary,
and
a
single
chisquare
test
can
be
run
using
the
data
from
all
matrices.

8.5.1.3
Method
Comparability
Conclusions
A
decision
on
whether
the
ATP
or
new
method
is
comparable
to
the
EPA­
approved
reference
method
will
generally
be
made
based
on
the
results
of
the
false
positive
and
false
negative
rate
comparisons.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
5
Generally,
the
decision
on
acceptability
of
the
new
method
should
be
made
based
on
the
chi­
square
test
using
data
from
all
matrices.
However,
if
the
results
of
the
Breslow­
Day
test
indicate
that
there
is
a
significant
interaction
between
method
and
matrix
for
false
positive
and/
or
false
negative
rates,
then
further
review
should
be
made
for
those
matrices
which
yielded
a
higher
false
positive
and/
or
false
negative
rate
for
the
proposed
method.

If
the
results
of
the
chi­
square
tests
indicate
that
the
false
positive
and
false
negative
rates
of
the
ATP
or
new
method
are
not
significantly
different
from
the
EPA
reference
method,
this
generally
will
be
interpreted
as
not
having
enough
evidence
to
conclude
that
the
performance
of
the
ATP
or
new
method
is
worse
than
the
EPA­
approved
reference
method.
However,
if
the
results
of
the
chi­
square
test
indicate
that
the
false
positive
and/
or
false
negative
rates
of
the
new
method
are
significantly
less
than
that
of
the
EPA­
approved
reference
method,
this
will
generally
be
interpreted
as
worse
performance
and
would
lead
to
rejection
of
the
ATP
or
new
method.

8.5.2
Quantitative
Methods
8.5.2.1
Testing
for
Normality
Many
of
the
statistical
analyses
used
to
assess
differences
in
recovery
and
precision
between
methods
require
certain
assumptions
about
the
data
be
met.
Because
the
validity
of
these
assumptions
affect
which
statistical
tests
will
be
used
to
assess
recovery
and
precision
differences,
testing
these
assumptions
is
a
prerequisite
as
a
first
step.
There
are
two
assumptions
that
should
be
evaluated
prior
to
comparing
the
two
methods
statistically.
One
assumption
is
that
the
variability
of
the
replicates
is
constant
for
each
matrix
and
method.
This
assumption
is
discussed
in
Section
8.5.2.2.
The
other
main
assumption
that
should
be
met
is
that
the
data
follow
a
normal
distribution.

An
assessment
of
normality
can
be
done
either
graphically
or
with
statistical
tests,
or
both.
A
normal
distribution
looks
like
a
bell
curve
(
i.
e.,
symmetric
around
the
mean).
A
graphical
assessment
can
be
done
using
a
histogram,
stem­
and­
leaf
or
Normal
probability
plot.
Appropriate
statistical
tests
include
the
Shapiro­
Wilk
test,
D'Agostino
test,
and
Kolmogorov­
Smirnov
test.
These
tests
are
computation
intensive,
and
it
may
be
necessary
to
use
software
(
e.
g.,
SAS)
to
run
them.
The
Shapiro­
Wilk
test
is
generally
inappropriate
for
testing
the
normality
assumption
of
a
data
set
with
greater
than
50
results.

For
environmental
data
(
especially
field
data,
though
sometimes
spiked
data
as
well),
the
distribution
of
results
will
often
be
positively
skewed
(
i.
e.,
with
a
few
unusually
high
results).
A
common
corrective
action
of
this
that
can
be
utilized
is
the
logarithmic
transformation,
base
e
(
natural
log).
If
the
data
are
positively
skewed,
this
transformation
should
be
attempted,
assuming
that
none
of
the
results
are
negative
or
equal
to
zero.
When
zero­
valued
results
are
present
in
a
skewed
data
set,
a
common
approach
is
to
add
a
small
constant
to
every
result
prior
to
transformation.
However,
the
arbitrary
choice
of
this
constant
will
have
a
large
effect
on
the
transformed
data
(
the
log
of
1
and
the
log
of
0.1
are
very
different,
for
example),
and
this
approach
is
not
recommended.
The
use
of
non­
parametric
tests,
discussed
later
in
this
guidance,
are
considered
to
be
the
appropriate
alternatives.

If
no
zero­
valued
results
are
present
in
a
positively
skewed
data
set,
then
the
log­
transformed
data
should
be
tested
for
normality.
If
the
log­
transformed
data
do
follow
a
Normal
distribution,
then
all
analyses
should
be
done
using
the
log­
transformed
results.

8.5.2.2
Evaluating
Precision
The
decision
of
what
statistical
test
to
use
to
evaluate
precision
will
generally
be
made
on
the
results
of
the
normality
test
described
in
Section
8.5.2.1.
If
the
assumption
of
normality
is
met,
then
the
F­
test
for
differences
in
variance
should
be
used.
If
the
assumption
of
normality
is
not
met,
a
non­
parametric
test
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
6
such
as
the
Conover
Squared­
Rank
test
should
be
used.
The
F­
test
is
based
on
comparisons
of
variances
(
i.
e.,
the
squared
standard
deviations).
The
Conover
Squared­
Rank
test
is
based
on
ranking
absolute
deviations
from
the
mean.
Figure
8­
1
provides
a
summary
of
the
procedure
for
evaluating
precision.

Prior
to
testing
whether
there
is
a
precision
difference
between
methods,
the
precision
of
the
different
matrices
should
be
compared
for
each
method.

If
the
results
of
the
F­
test
indicate
that
variances
differ
significantly
by
matrix,
or
if
the
results
of
the
Conover
Squared­
Rank
test
indicate
that
absolute
deviations
from
the
mean
differ
significantly
by
matrix,
then
comparisons
of
precision
between
methods
should
be
done
separately
for
each
matrix.
Otherwise,
a
single
comparison
of
method
precision
can
be
done
using
the
data
from
all
matrices.
If
the
results
of
the
F­
test
indicate
that
there
is
not
enough
evidence
to
conclude
that
the
variances
differ
significantly
by
matrix,
then
the
pooled
within­
matrix
variance
(
i.
e.,
the
average
of
the
matrix
variances
for
the
given
method)
should
be
calculated
for
each
method.
The
F­
test
should
then
be
used
to
compare
the
pooled
within­
matrix
variance
for
the
two
methods.
If
the
Conover
Squared­
Rank
test
was
used
and
there
was
not
enough
evidence
to
conclude
that
the
mean
absolute
deviation
differed
significantly
between
matrices,
then
the
Conover
Squared­
Rank
test
should
be
used
to
compare
the
mean
absolute
deviations
from
the
overall
method
mean
for
the
two
methods.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
7
Compare
absolute
deviations
from
method
mean
for
two
methods
using
Conover
squared
rank
test.
Run
test
for
normality
Data
follow
Normal
distribution
Data
do
not
follow
Normal
distribution
Compare
variances
between
matrices
for
each
method
using
F­
test.

Are
all
matrices
equal?
Yes
No
Pool
variances
over
matrices
for
each
method.
Compare
pooled
variances
for
two
methods
using
the
F­
test.
Compare
variances
between
methods
separately
for
each
matrix
using
the
F­
test.

Is
the
new
method
at
least
as
precise
for
at
least
80%
of
matrices?

Evaluate
Recovery
Yes
Failure
No
Is
the
new
method
significantly
less
precise?

No
Yes
Evaluate
Recovery
Failure
Is
the
new
method
significantly
less
precise?

No
Failure
Yes
Evaluate
Recovery
Figure
8­
1.
Evaluating
Precision
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
8
8.5.2.3
Evaluating
Recovery
Comparisons
of
mean
recovery
of
the
different
methods
should
be
done
using
either
an
analysis
of
variance
(
ANOVA)
model
or
non­
parametric
test
such
as
the
Wilcoxon­
Mann­
Whitney
(
WMW)
test.
If
the
assumption
of
normality
is
not
met,
or
if
the
precision
differs
between
methods
or
matrices
based
on
the
precision
evaluation
described
in
the
previous
section,
the
WMW
test
should
be
used.
If
all
assumptions
are
met,
the
ANOVA
model
should
be
used.
Figure
8­
2
provides
a
summary
of
the
procedure
for
evaluating
recovery.

If
an
ANOVA
model
is
used,
method­
by­
matrix
interactions
should
be
tested
for
significance
first
using
the
corresponding
ANOVA
F­
test.
If
the
method­
by­
matrix
interaction
is
significant,
it
would
mean
that
the
difference
between
methods
is
not
the
same
for
each
matrix.
In
this
case,
no
conclusion
regarding
an
overall
difference
between
the
recovery
of
methods
can
be
made.
If
the
interaction
is
not
significant,
then
the
test
for
a
significant
difference
between
methods
can
be
run
in
the
ANOVA
model,
using
the
F­
test
for
a
significant
method
main
effect.

If
the
WMW
test
is
used,
between­
matrix
and
between­
laboratory
variability
cannot
be
separated
from
replicate
variability.
Therefore,
significant
interactions
between
method
and
laboratory
or
matrix
cannot
be
tested
when
the
WMW
test
is
used.
Instead
the
WMW
model
only
tests
for
an
overall
difference
between
methods.
The
model
can
be
run
separately
for
each
matrix,
but
the
diminished
power
will
limit
the
value
of
this
approach.
It
is
therefore
recommended
that
a
single
WMW
test
be
run
comparing
method
recoveries,
without
stratifying
by
or
controlling
for
matrix
type.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
9
Evaluate
false
positive
and
false
negative
rates.
Is
recovery
significantly
lower
for
the
new
method?
Run
test
for
normality
Data
do
not
follow
Normal
distribution
Failure
Run
ANOVA
model
testing
for
method
difference,
controlling
for
matrix.
No
interaction
Interaction
No
Yes
Run
single
WMW
test
to
compare
method
recovery,
disregarding
matrices.

Is
recovery
significantly
lower
for
the
new
method?

Failure
No
Yes
Evaluate
false
positive
and
false
negative
rates.
Evaluate
false
positive
and
false
negative
rates.

Failure
No
Yes
Run
ANOVA
model
testing
for
method
difference
separately
for
each
matrix.
Count
number
of
matrices
where
the
new
method
is
equal
to
or
significantly
better.

Is
recovery
significantly
lower
for
the
new
method
for
at
least
20%
of
all
matricies?
Data
follow
Normal
distribution
Using
analysis
of
variance
(
ANOVA),
test
for
method/
matrix
interaction.
Figure
8.2
Evaluating
Recovery
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
10
8.5.2.4
False
Positive
Rates,
False
Negative
Rates,
Sensitivity,
and
Specificity
Comparisons
of
false
positive
rates,
false
negative
rates,
sensitivity,
and
specificity
should
be
conducted
according
to
the
methods
described
in
Sections
8.5.1.1
and
8.5.1.2.

8.5.2.5
Method
Comparability
Conclusions
If
there
is
not
enough
evidence
to
conclude
that
there
is
a
significant
interaction
between
method
and
matrix
for
recovery,
or
if
there
is
not
enough
evidence
to
conclude
that
there
is
a
statistically
significant
difference
in
precision
between
matrices
for
either
method,
then
it
can
be
concluded
that
any
such
differences
of
the
method
will
be
consistent.
Therefore,
lack
of
sufficient
evidence
to
conclude
a
significant
difference
between
methods
will
generally
be
interpreted
as
equal
or
better
performance,
and
might
lead
to
acceptance
of
the
ATP
or
new
method,
pending
review
of
false
positive
and
false
negative
rates.
However,
a
statistically
significant
test
(
worse)
statistic
will
be
interpreted
as
worse
performance
of
the
ATP
or
new
method,
and
would
generally
lead
to
rejection
of
the
new
method.

In
cases
where
significant
interactions
between
matrix
and
method
are
found
when
assessing
recovery,
or
in
cases
where
differences
in
precision
between
matrices
for
at
least
one
method
(
and
therefore
a
comparison
of
method
precision
could
not
be
done
over
all
matrices),
some
judgment
will
be
necessary
in
deciding
whether
the
proposed
method
should
be
deemed
acceptable.
The
decision
should
be
based
on
the
attribute
(
i.
e.,
recovery
or
precision)
for
each
matrix.
As
a
general
rule,
if
there
was
not
enough
evidence
to
conclude
that
the
new
method
was
similar
or
better
than
the
EPA­
approved
reference
method
for
that
attribute
for
at
least
80%
of
the
matrices
used
in
the
study,
then
the
new
method
can
generally
be
recommended
for
approval,
pending
review
of
false
positive
and
false
negative
rates.

If
there
is
not
enough
evidence
to
conclude
that
the
new
method
is
worse
than
the
EPA­
approved
reference
method
for
at
least
80%
of
the
matrices
for
both
precision
and
recovery,
the
false
negative
rate
and
false
positive
rate
should
next
be
compared,
based
on
the
methods
described
in
sections
8.5.1.1
and
8.5.1.2.
If
the
results
of
the
chi­
square
test
indicate
that
there
is
not
enough
evidence
to
conclude
that
the
false
negative
rate
or
false
positive
rate
of
the
ATP
or
new
method
is
worse
than
that
of
the
EPAapproved
reference
method,
this
will
be
interpreted
as
equal
or
better
performance
and
might
lead
to
acceptance
of
the
new
methods.
However,
if
the
results
of
the
chi­
square
test
indicate
that
the
false
negative
rate
or
false
positive
rate
of
the
new
method
is
significantly
greater
than
that
of
the
EPAapproved
reference
method,
this
will
generally
be
interpreted
as
worse
performance
for
100%
of
the
matrices
used
in
the
study,
and
would
lead
to
rejection
of
the
ATP
or
new
method.

For
example,
suppose
side­
by­
side
testing
is
done
in
one
laboratory
for
ten
different
matrices.
For
recovery,
there
is
not
enough
evidence
to
conclude
that
the
new
method
is
significantly
worse
than
the
EPA­
approved
reference
method
for
eight
of
the
ten
matrices.
For
precision,
there
is
not
enough
evidence
to
conclude
a
significant
matrix
effect,
and
there
is
not
enough
evidence
to
conclude
a
significant
difference
between
methods.
There
is
not
enough
evidence
to
conclude
the
false
negative
rate
or
false
positive
rate
of
the
ATP
or
new
method
is
worse
than
the
EPA­
approved
reference
method.
Under
these
circumstances,
the
ATP
or
new
method
may
be
recommended
for
approval.
However,
if
there
is
not
enough
evidence
to
conclude
that
the
new
method
is
not
worse
than
the
approved
method
for
only
seven
of
ten
matrices
for
recovery,
then
the
ATP
or
new
method
will
not,
generally,
be
recommended
for
approval.

8.5.3
QC
Acceptance
Criteria­
Based
Comparison
Studies
For
methods
with
QC
acceptance
criteria,
necessary
calculations
and
QC
criteria
are
typically
provided
in
the
method.
Generally,
QC
criteria
will
include
both
recovery
and
precision
specifications.
Recovery
criteria
take
the
form
of
a
recovery
interval
for
either
a
single
result
or
mean
of
multiple
results.
Precision
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
11
criteria
will
generally
be
either
a
maximum
standard
deviation,
RSD,
or
RPD.
Details
on
calculations
that
may
be
necessary
for
the
comparisons
are
described
below.

8.5.3.1
Recovery
Recovery
results
will
generally
be
necessary
for
data
spiked
into
both
reagent
water
(
IPR,
IDC,
etc.)
and
source
water
(
MS,
MSD,
etc.).
For
reagent
water,
percent
recovery
should
be
calculated
as
follows:

%
Recovery
=
Result
*
100
%
Spike
where
Result
is
the
amount
recovered
from
the
sample
after
spiking,
and
Spike
is
the
estimated
amount
spiked
into
the
sample.

For
source
water,
percent
recovery
should
be
calculated
as
follows:

%
Recovery
=
Result
­
Background
*
100
%
Spike
where
Result
is
the
amount
recovered
from
the
sample
after
spiking,
Spike
is
the
estimated
amount
spiked
into
the
sample,
and
Background
is
the
estimated
background
amount
measured
in
the
sample
prior
to
spiking.

For
some
QC
criteria,
it
may
be
necessary
to
calculate
the
mean
of
the
sample
recoveries
prior
to
comparing
the
results
to
the
criteria.
Generally,
this
will
be
specified
with
that
given
method.

8.5.3.2
Precision
Calculation
of
an
RSD,
RPD,
or
standard
deviation
is
generally
necessary
for
precision
criteria.
For
source
water
data,
the
calculation
should
be
based
on
the
recovered
amount,
rather
than
the
percent
recovery.
This
is
because
the
calculated
percentage
will
be
inflated
if
the
background
amount
is
large
compared
to
the
total
amount
recovered.
For
reagent
water­
spiked
data,
where
there
the
background
count
is
zero,
the
calculation
can
be
made
either
using
the
recovered
amount
or
the
percent
recovery.

Where
the
precision
criteria
is
to
be
based
on
two
results,
the
precision
criterion
will
generally
be
an
RPD.
The
equation
for
an
RPD
is
given
below:

RPD
=
*
Amount1
­
Amount2*
*
100
%
(
Amount1
+
Amount2)
/
2
Where
Amount1
and
Amount2
are
the
two
recovered
amounts.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
8­
12
Where
the
precision
criterion
is
to
be
based
on
more
than
two
results,
the
precision
criterion
will
generally
be
either
a
standard
deviation
or
RSD.
An
RSD
is
calculated
based
on
the
equation
below:

RSD
=
SD
*
100
%
Mean
Where
SD
is
the
standard
deviation
of
all
recovered
amounts,
and
Mean
is
the
mean
of
all
recovered
amounts.

8.5.3.3
Presence/
Absence
Criteria
QC
criteria
for
presence/
absence
methods
may
be
defined
as
false
positive
and/
or
false
negative
rates,
sensitivity
and/
or
specificity,
or
as
a
specified
proportion
of
positive
and/
or
negative
results
out
of
a
given
number
of
samples.
All
calculations
should
be
specific
to
the
given
situation
and
should
be
defined
explicitly
therein.

8.6
Method
Recommendation
and
Approval
Generally,
after
completion
of
the
technical
and
statistical
reviews
for
nationwide­
use
applications,
the
Director
of
Analytical
Methods,
Attn:
ATP
Program
Coordinator
(
see
Table
2­
1
and
Appendix
E)
will
prepare
a
recommendation
for
approval/
disapproval
of
the
ATP
or
new
method
and
notify
the
applicant
of
the
recommendation.

If
the
data
evaluation
demonstrates
that
the
applicant's
method
performs
at
least
as
well
as
the
EPAapproved
reference
method,
the
Director
of
Analytical
Methods,
Attn:
ATP
Program
Coordinator
will
generally
recommend
approval
to
the
Office
of
Ground
Water
and
Drinking
Water
(
OGWDW)
or
appropriate
office,
which
begins
the
regulation
development
process.
Regulation
development
includes
a
Federal
Register
notice
proposing
to
approve
an
ATP,
public
comment
on
the
proposed
method,
and
(
depending
on
public
comment)
a
final
rule
published
in
the
Federal
Register
that
approves
the
method.
Generally,
the
regulation
development
process
may
take
one
year
or
more.

For
limited­
use
ambient
water
or
wastewater
applications
the
Regional
Administrator
(
see
Table
2­
1
and
Appendix
E)
will
generally
prepare
a
recommendation
of
approval/
disapproval
of
the
ATP
or
new
method
and
notify
the
applicant
of
the
recommendation
after
completion
of
the
technical
and
statistical
reviews.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
9­
1
SECTION
9.0
STUDY
REPORT
Laboratories
or
other
organizations
responsible
for
developing
ATPs
or
new
methods
should
document
the
results
of
the
side­
by­
side
comparison
study
or
QC
acceptance
criteria­
based
comparison
study
in
a
formal
study
report
that
contains
the
elements
described
in
this
section.

The
information
and
supporting
data
included
in
the
study
report
should
be
sufficient
to
enable
EPA
to
evaluate
the
performance
of
the
ATP
or
new
method
and
make
a
decision
on
whether
it
is
comparable
to
the
reference
method.
The
applicant
is
responsible
for
ensuring
that
all
method­
specified
criteria
are
met
by
the
laboratory(
ies)
involved
in
the
study
and
that
the
study
report
contains
all
data
from
the
laboratory(
ies).
A
copy
of
all
comparison
study
data
should
be
maintained
at
the
participant
laboratory(
ies)
or
other
organization
responsible
for
developing
the
ATP
or
new
method.

Like
the
study
plan
developed
and
approved
by
EPA
before
the
study
was
performed,
the
study
report
contains
background
information
and
describes
the
study
design.
However,
the
study
report
also
details
the
process
and
results
of
the
study,
provides
an
analysis
and
discussion
of
the
results,
and
presents
study
conclusions.
The
approved
study
report
should
also
identify
and
discuss
any
deviations
from
the
study
plan
that
were
made
in
implementing
the
study,
and
the
study
plan
should
be
appended
to
and
referenced
in
the
study
report.
Significant
deviations
from
the
study
plan
without
prior
EPA
approval
could
result
in
the
rejection
of
the
study
data.

The
study
report
should
be
organized
into
the
following
sections:
°
Background
°
Study
Objectives
and
Design
°
Study
Implementation
°
Data
Reporting
and
Validation
°
Results
°
Data
Analysis
and
Discussion
°
Conclusions
°
Appendix
A
­
Method
°
Appendix
B
­
Study
Plan
°
Appendix
C
­
Supporting
Data
°
Appendix
D
­
Supporting
References
Details
on
the
information
that
should
be
included
in
each
of
these
sections
are
provided
below,
in
Sections
9.1
through
9.11.

9.1
Background
This
section
of
the
study
report
should
describe
the
ATP
or
new
method
that
was
tested,
and
identify
the
organization
responsible
for
developing
the
ATP
or
new
method.
The
background
section
of
the
study
report
should
include
the
following
information:
°
A
method
summary
°
The
organization,
method
number,
and
title
for
the
ATP
or
new
method
°
The
method
number
or
title
and
publication
number
(
given
in
40
CFR
parts
136,
141,
and
405
­
503)
for
the
EPA­
approved
reference
method
that
is
being
used
for
demonstrating
method
comparability
(
i.
e.,
the
reference
method)
°
A
description
of
the
nature
of
the
ATP
(
e.
g.,
alternate
media,
alternate
concentration
technique,
etc.)
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
9­
2
°
The
matrices,
matrix
types,
and/
or
media
to
which
the
ATP
or
new
method
is
believed
to
be
applicable
°
The
analyte(
s)
measured
by
the
ATP
or
new
method,
including
corresponding
CAS
Registry
or
other
identification
numbers,
when
available
9.2
Study
Objectives
and
Design
This
section
of
the
study
report
should
identify
the
overall
objectives
and
data
quality
objectives
of
the
study
and
briefly
describe
the
study
design.
This
information
should
be
consistent
with
the
study
objectives
and
design
specified
in
the
approved
study
plan.
Any
study
limitations
should
be
identified.
The
approved
study
plan
should
be
appended
to
the
study
report.

9.3
Study
Implementation
This
section
of
the
study
report
should
describe
the
methodology
and
approach
undertaken
in
the
study.
This
section
should
include
the
following
information:
°
The
organization
that
was
responsible
for
managing
the
study
°
The
laboratories,
facilities,
and
other
organizations
that
participated
in
the
study;
describe
how
participating
laboratories
were
selected;
and
explain
the
role
of
each
organization
involved
in
the
study
°
The
type
of
study
performed
(
i.
e.,
side­
by­
side
or
QC
acceptance
criteria­
based
comparison
study)
°
The
study
schedule
that
was
followed
°
A
brief
description
of
how
sample
matrices
were
chosen,
including,
for
QC
acceptance
criteria­
based
comparison
studies,
a
statement
of
compliance
with
recommendations
for
matrix
type
selection
°
A
description
of
any
preliminary
testing
conducted
prior
to
the
side­
by­
side
or
QC
acceptance
criteria­
based
comparison
study
(
e.
g.,
method
validation,
physical
and
chemical
assessment
of
the
matrices,
preliminary
range­
finding
analyses)
°
The
numbers
and
types
of
analyses
performed
by
the
participating
laboratories
°
A
description
of
how
samples
were
collected,
distributed,
and
stored
°
The
source
and
strain
of
the
organism
used
for
sample
spiking
°
If
spikes
were
quantified,
a
description
of
how
estimated
true
spike
values
were
determined
and
provide
all
supporting
data
°
The
type
of
water
used
in
the
preparation
of
sample
dilutions
if
not
specified
by
the
method
(
e.
g.,
reagent
water,
phosphate
buffered
water,
phosphate
buffered
saline,
etc.)
°
Any
problems
encountered
with
samples,
spiking
organisms,
equipment,
etc.
and
their
subsequent
resolution
°
Any
communications
with
EPA
relevant
to
the
study,
such
as
clarification
of
the
study
design
or
approved
changes
to
the
study
plan
°
Any
deviations
from
the
study
plan
and
their
impact
on
study
performance
and/
or
results
9.4
Data
Reporting
and
Validation
This
section
of
the
study
report
should
describe
the
procedures
that
were
used
to
report
and
validate
study
data.
While
EPA
generally
does
not
use
a
standard
format
for
analytical
data
submission,
a
list
of
necessary
data
elements
and
an
example
bench
sheet
may
be
found
in
Appendix
D
of
this
document.

9.5
Results
This
section
of
the
study
report
presents
the
study
results.
Results
may
be
presented
in
a
summary
table
that
lists
the
recovery
or
concentration
of
each
sample,
by
test
method,
laboratory,
and
matrix.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
9­
3
For
QC
acceptance
criteria­
based
comparison
studies,
results
should
indicate
the
QC
test
associated
with
each
sample
(
e.
g.
IPR,
method
blank,
MS/
MSD,
unspiked
matrix
sample).
Raw
data
and
example
calculations
should
be
submitted,
and
should
be
included
in
an
appendix
to
the
study
report
(
see
Section
9.10.1).

9.6
Data
Analysis
and
Discussion
This
section
of
the
study
report
provides
a
statistical
analysis
and
discussion
of
the
study
results.
Recovery,
precision,
false
positive
rates,
false
negative
rates,
specificity,
and
sensitivity,
as
appropriate,
should
be
calculated
by
test
method,
laboratory,
and
matrix,
and
summarized
in
a
tabular
format
that
includes
the
mean,
standard
deviation,
and
relative
standard
deviation.
The
discussion
should
address
any
discrepancies
between
the
results
and
comparability
guidelines,
or,
for
QC
acceptance
criteria­
based
comparison
studies,
any
discrepancies
between
the
results
and
the
QC
acceptance
criteria
of
the
EPAapproved
reference
method.

9.7
Conclusions
This
section
of
the
study
report
should
describe
the
conclusions
drawn
from
the
study
based
on
the
data
analysis
discussion.
The
section
should
contain
a
statement(
s)
regarding
achievement
of
the
study
objective(
s).

9.8
Appendix
A
­
Method
The
ATP
or
new
method,
prepared
in
accordance
with
EPA's
Guidelines
and
Format
document
(
Section
3.0
and
Reference
10.14),
should
be
appended
to
the
study
report.

9.9
Appendix
B
­
Study
Plan
The
study
plan
approved
by
EPA
(
Section
4.0)
should
be
appended
to
the
study
report.

9.10
Appendix
C
­
Supporting
Data
The
study
report
should
be
accompanied
by
raw
data,
quality
control
information,
and
example
calculations
that
support
the
summary
results
presented
in
the
report.

9.10.1
Raw
Data
Appendix
C
of
the
study
report
should
include
sufficient
raw
data
so
that
an
independent
reviewer
can
verify
each
determination
and
calculation
performed
by
the
laboratory
or
the
study
coordinator.
This
verification
consists
of
tracing
all
steps
of
the
method
to
the
final
result
reported.
The
raw
data
are,
generally,
method­
specific
and
may
include
but
are
not
limited
to
the
following:
°
Sample
numbers
or
other
identifiers
used
by
the
laboratory
°
Sample
collection
dates
and
times
°
Verification
that
method­
specified
QC
procedures
were
met
for
test
samples
and
all
associated
QC
samples
°
Analysis
dates
and
times
for
all
steps
in
the
method
°
Sample
volume
°
Any
measurements
of
ancillary
parameters
(
i.
e.,
temperature,
pH,
turbidity,
percent
solids,
etc.)
°
Results
for
all
intermediate
steps
in
the
method
°
Preliminary
data
steps
to
determine
the
final
result
°
Final
result
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
9­
4
°
If
appropriate,
quantitation
reports,
data
system
outputs,
and
other
data
to
link
the
raw
data
to
the
results
reported
°
Laboratory
bench
sheets
and
copies
of
all
pertinent
logbook
pages
for
all
sample
preparation
and
cleanup
steps,
and
for
all
other
parts
of
the
determination
°
Temperature
logs
for
waterbaths,
incubators,
refrigerators,
etc.
°
Media
preparation
information
°
If
appropriate,
direct
instrument
readouts
and
other
data
to
support
the
final
results
Raw
data
are
generally
needed
for
all
samples,
positive
and
negative
controls,
sterility
checks,
verifications,
blanks,
matrix
spikes
and
duplicates,
and
other
QC
analyses
specified
in
the
EPA­
approved
reference
method.
Data
should
be
organized
so
that
a
microbiologist
can
clearly
understand
how
the
analyses
were
performed.
The
names,
titles,
addresses,
and
telephone
numbers
of
the
analysts
who
performed
the
analyses
and
of
the
quality
assurance
officer
who
verified
the
analyses
should
be
provided.
For
instruments
involving
data
systems,
raw
data
on
magnetic
tape
or
disk
should
be
made
available
on
request.

9.10.2
Electronic
Data
Reporting
In
addition
to
the
hard
copy
raw
data,
applicants
should
also
submit
data
in
electronic
format
(
Excel
spreadsheet,
or
equivalent)
so
that
EPA
can
to
create
a
database
of
study
results.
EPA
anticipates
that
this
database
will
facilitate
automated
review
and
statistical
analysis
of
study
results.
The
information
included
in
electronic
format
may
include:
laboratory,
analyst,
method,
sample
type,
sample
number,
date
and
time
of
analysis,
volume
analyzed,
replicate
number,
raw
data,
and
calculated
results.
The
applicant
should
discuss
an
appropriate
electronic
format
with
EPA
prior
to
data
submission.

9.10.3
Example
Calculations
Generally,
the
study
report
should
provide
example
calculations
that
will
allow
the
data
reviewer
to
determine
how
the
laboratory
used
the
raw
data
to
arrive
at
the
final
results.
Useful
examples
include
both
detected
analytes
and
undetected
analytes.
If
the
laboratory
or
the
method
employs
a
standardized
reporting
level
for
undetected
analytes,
this
should
be
made
clear
in
the
example,
as
should
adjustments
for
sample
volume,
etc.

9.11
Appendix
D
­
Supporting
References
Hard
copies
of
all
references
and
supporting
documentation
for
the
ATP
or
new
method
should
be
attached
to
the
study
report
as
an
appendix.
The
list
of
references
may
contain
links
to
web
sites,
or
documents
available
on­
line.
However,
a
hard
copy
should
be
submitted
with
the
final
study
report.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
10­
1
SECTION
10.0
REFERENCES
10.1
AOAC.
1999.
Qualitative
and
Quantitative
Microbiology
Guidelines
for
Methods
Validation,
Journal
of
AOAC
International,
Vol.
82,
No.
2.

10.2
APHA.
1998.
Standard
Methods
for
the
Examination
of
Water
and
Wastewater.
20th
Edition.
American
Public
Health
Association.
1015
15th
Street,
NW,
Washington,
DC
20005.

10.3
APHA.
1995.
Standard
Methods
for
the
Examination
of
Water
and
Wastewater.
19th
Edition.
American
Public
Health
Association.
1015
15th
Street,
NW,
Washington,
DC
20005.

10.4
APHA.
1992.
Standard
Methods
for
the
Examination
of
Water
and
Wastewater.
18th
Edition.
American
Public
Health
Association.
1015
15th
Street,
NW,
Washington,
DC
20005.

10.5
ASTM.
1999.
D4855­
91:
Standard
Practice
for
Comparing
Test
Methods,
ASTM
Standards
on
Precision
and
Bias
for
Various
Applications.
1999
Annual
Book
of
ASTM
Standards:
Water
and
Environmental
Technology,
Volume
7.02.
100
Barr
Harbor
Drive,
West
Conshohocken,
PA
19428.

10.6
ATCC.
http://
www.
atcc.
org
10.7
Fleiss,
J.
F.
Statistical
Methods
for
Rates
and
Proportions,
2nd
ed.,
John
Wiley
&
Sons,
New
York,
NY.

10.8
ISO.
2001.
CD17994.
Water
Quality
­
Criteria
for
the
Establishment
of
Equivalence
Between
Microbiological
Methods,
Final
Version,
June
15,
2001.

10.9
National
Environmental
Laboratory
Accreditation
Conference.
2001.
National
Environmental
Laboratory
Accreditation
Conference:
Quality
Systems.
Approved
May
25,
2001,
effective
July
1,
2003
unless
otherwise
noted.
Appendix
D
­
Essential
Quality
Control
Requirements,
section
D.
3,
pp
D­
15
to
D­
16.

10.10
Title
40,
Code
of
Federal
Regulations,
Sections
136.4,
136.5,
and
141.27.

10.11
USEPA.
2001.
EPA
Requirements
for
Quality
Management
Plans.
USEPA
Office
of
Environmental
Information.
EPA/
240/
B­
01/
002.

10.12
USEPA.
1999.
Environmental
Regulations
and
Technology
Control
of
Pathogens
in
Vector
Attraction
in
Sewage
Sludge.
USEPA
Office
of
Research
and
Development.
EPA/
625/
R­
92/
013,
revised
October
1999.

10.13
USEPA.
1997.
Manual
for
the
Certification
of
Laboratories
Analyzing
Drinking
Water:
Criteria
and
Procedures
Quality
Assurance,
Fourth
Edition.
USEPA
Office
of
Ground
Water
and
Drinking
Water.
EPA­
815­
B­
97­
001.

10.14
USEPA.
1996.
Guidelines
and
Format
for
Methods
to
be
Proposed
at
40
CFR
Part
136
or
Part
141.
USEPA
Office
of
Science
and
Technology.
EPA­
821­
B­
96­
003.
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
10­
2
10.15
USEPA.
1995a.
Presence/
Absence
Membrane
Filter
Methods
for
Finished
Waters,
USEPA
Protocol
for
Alternate
Test
Procedures
for
Coliform
Bacteria
in
Compliance
with
Drinking
Water
Regulations,
Version
1.2,
December
1995.
USEPA
Office
of
Research
and
Development,
Cincinnati,
OH.

10.16
USEPA.
1995b.
Quantitative
Membrane
Filter
Methods,
USEPA
Protocol
for
Alternate
Test
Procedures
for
Coliform
Bacteria
in
Compliance
with
Water
and
Wastewater
Regulations,
Version
1.0,
December
1995.
USEPA
Office
of
Research
and
Development.
Cincinnati,
OH.

10.17
USEPA.
1995c.
Presence/
Absence
Liquid
Culture
Methods
for
Finished
Waters,
USEPA
Protocol
for
Alternate
Test
Procedures
for
Coliform
Bacteria
in
Compliance
with
Drinking
Water
Regulations,
Version
1.2,
December
1995.
USEPA
Office
of
Research
and
Development.
Cincinnati,
OH.

10.18
USEPA
1989.
Memorandum:
Analytical
Methods
for
Compliance
and
Limited
Alternate
Test
Procedures
Approvals.
December
27,
1989.

10.19
USEPA.
1979.
Handbook
for
Analytical
Quality
Control
in
Water
and
Wastewater
Laboratories.
EPA­
600/
4­
79­
019.
Environmental
Monitoring
and
Support
Laboratory,
Cincinnati,
OH.
March
1979.
APPENDIX
A
GLOSSARY
APPENDIX
A:
GLOSSARY
40
CFR
part
136
 
Title
40,
part
136
of
the
Code
of
Federal
Regulations.
This
part
specifies
approved
test
procedures
for
the
analysis
of
pollutants
regulated
under
the
Clean
Water
Act.

40
CFR
part
141
 
Title
40,
part
141
of
the
Code
of
Federal
Regulations.
This
part
specifies
EPA's
National
Primary
Drinking
Water
Regulations
pursuant
to
the
Safe
Drinking
Water
Act;
Subpart
C
of
40
CFR
part
141
lists
analytical
methods
required
for
monitoring
under
the
Act.

95%
confidence
interval
 
A
statistical
level
indicating
a
95%
probability
that
the
parameter
variable
is
enclosed
within
the
given
data
interval.

Acceptable
version
 
An
acceptable
version
is
a
method
that
is
either
identical
to
the
approved
A
method
or
exercises
the
flexibility
explicitly
allowed
in
the
method.
See
"
minor
modification."

Accuracy
 
The
degree
of
agreement
between
an
observed
value
and
an
accepted
reference
value.
Accuracy
includes
random
error
(
precision)
and
systematic
error
(
recovery)
that
are
caused
by
sampling
and
analysis.

Aliquot
 
A
representative
portion
of
a
sample.

Ambient
water
 
Ambient
water
refers
to
any
fresh,
marine,
or
estuarine
surface
water
used
for
recreation;
propagation
of
fish,
shellfish,
or
wildlife;
agriculture;
industry;
navigation;
or
as
source
water
for
drinking
water
facilities.

Analysis
of
variance
(
ANOVA)
 
A
study
of
the
effect
of
a
set
of
qualitative
variables
on
a
quantitative
response
variable,
based
on
a
decomposition
of
the
variance
of
the
response
variable.

Analyte
 
The
target
organism
or
class
of
organisms
that
are
measured
by
the
method.

Analyte
of
concern
 
An
analyte
designated
by
EPA
to
adversely
affect
or
have
the
potential
to
adversely
affect
human
health,
the
environment,
aesthetics,
or
the
senses.
Analytes
of
concern
are
listed
in
approved
methods.

Approved
method
 
A
testing
procedure
(
analytical
method)
promulgated
at
40
CFR
parts
136,
141,
405­
500,
and
other
parts
of
the
CFR
that
support
EPA's
water
programs.

Average
percent
recovery
 
The
average
of
the
recovery,
expressed
as
percent.
See
recovery.

Bias
 
A
systematic
or
persistent
distortion
of
a
measurement
process
that
deprives
the
result
of
B
representativeness;
i.
e.,
the
expected
sample
measurement
is
different
than
the
sample's
true
value.
A
data
quality
indicator.
(
QAMS)

Blank
 
See
"
method
blank."

Bulk
sample
 
A
large
sample
that
is
aliquoted
into
smaller
volumes
prior
to
analyses.

Calibration
 
The
process
of
establishing
the
relationship
between
the
concentration
or
amount
C
of
material
introduced
into
an
instrument
or
measurement
process
and
the
output
signal.

Calibration
verification
 
Means
of
establishing
that
the
instrument
performance
remains
within
pre­
established
limits.
Code
of
Federal
Regulations
 
A
codification
of
the
general
and
permanent
rules
published
in
the
Federal
Register
by
the
Executive
departments
and
agencies
of
the
Federal
Government.

Comparability
test
 
See
side­
by­
side
comparison.

Confidence
interval
 
The
numerical
interval
constructed
around
a
point
estimated
of
a
population
parameter,
combined
with
a
probability
statement
(
the
confidence
coefficient)
linking
it
to
the
population's
true
parameter
value.
If
the
same
confidence
interval
construction
technique
and
assumptions
are
used
to
calculate
future
intervals,
they
will
include
the
unknown
population
parameter
with
the
same
specified
probability.
(
EMMC)

Confirmed
counts
 
Organism
counts
that
have
been
verified
to
ensure
proper
identification.

Conover
Squared­
Rank
test
 
A
nonparametric
test
for
equality
of
variability,
based
on
the
joint
squared
ranks
of
deviations
from
the
means.
(
SPRENT)

Contract
laboratory
 
Private,
academic,
or
commercial
laboratory
under
contract
to
EPA
or
other
organization
to
perform
testing.

D'Agostino
test
 
A
statistical
test
for
determining
whether
a
given
set
of
results
follow
a
normal
D
or
log­
normal
distribution.
Best
used
for
datasets
with
at
least
50
results.
(
GILBERT)

Data
quality
objective
 
Qualitative
and/
or
quantitative
statement
of
the
overall
level
of
uncertainty
that
a
decision­
maker
is
willing
to
accept
in
results
or
decisions
derived
from
environmental
data.
Data
quality
objectives
provide
the
statistical
framework
for
planning
and
managing
environmental
data
operations
consistent
with
the
data
user's
needs.
(
EMMC)

Determinative
technique
 
The
physical
and/
or
chemical
process
by
which
measurement
of
the
identity
and
concentration
of
an
analyte
is
made.

Differential
medium
 
A
solid
culture
medium
that
makes
it
easier
to
distinguish
colonies
of
the
target
organism.

Dilution/
rinse
water
blank
 
An
aliquot
of
dilution/
rinse
water
that
is
treated
exactly
like
a
sample
and
carried
through
all
portions
of
the
procedure
until
determined
to
be
negative
or
positive.
The
dilution/
rinse
water
blank
is
used
to
determine
if
the
sample
has
become
contaminated
by
the
introduction
of
a
foreign
microorganism
through
poor
technique.

Discharge
 
Generally,
any
spilling,
leaking,
pumping,
pouring,
emitting,
emptying
or
dumping
(
40
CFR
109.2;
110.1;
116.3);
also,
see
"
discharge
of
a
pollutant"
(
40
CFR
122.2);
the
medium
that
is
spilled,
leaked,
pumped,
poured,
emitted,
emptied,
or
dumped.

Discharge
of
pollutant
 
Any
addition
of
any
pollutant
or
combination
of
pollutants
to
(
1)
waters
of
the
U.
S.
from
any
point
source
or
(
2)
to
the
waters
of
the
contiguous
zone
or
the
ocean
from
any
point
source
other
than
a
vessel
or
other
floating
craft
which
is
being
used
as
a
means
of
transportation
(
40
CFR
122.2;
401.11)

Duplicate
 
A
second
sample
collected
from
the
same
sampling
point
at
the
same
time
the
original
sample
is
collected
and
analyzed
exactly
like
the
original
sample.
Duplicate
samples
can
be
used
as
a
measure
of
sample
variability.

Effluent
 
A
medium
that
flows
out
of
a
point
source,
e.
g.,
the
discharge
from
a
sewage
E
treatment
plant.
Enrichment
 
Using
a
culture
medium
to
enhance
growth
of
the
target
organism
prior
to
isolation
of
that
organism.

Explicit
flexibility
 
Modifications
that
are
explicitly
allowed
in
an
approved
method.

F
distribution
 
A
type
of
sampling
distribution
for
a
random
variable.
The
ratio
of
2
chi­
square
F
distributions,
each
divided
by
their
respective
degrees
of
freedom.

F­
test
 
In
an
Analysis
of
Variance,
a
test
for
the
equality
of
factor
level
means
(
such
as
for
different
methods)
or
for
the
presence
of
an
interaction
between
two
factors
(
such
as
method
and
matrix).
(
RICE)
(
ASTM)

Facility
 
A
plant
or
group
of
plants
within
a
single
location
that
is
regulated
under
the
CWA
and/
or
SDWA.
A
single
facility
may
have
multiple
water
supplies,
discharges,
waste
streams,
or
other
environmental
media
that
are
subject
to
compliance
monitoring.
For
example,
a
single
facility
within
the
Pulp,
Paper,
and
Paperboard
industrial
category
may
have
a
direct
discharge,
an
indirect
discharge,
and
an
in­
process
waste
stream,
all
of
which
are
subject
to
compliance
monitoring.

False
negative
 
A
target
organism
incorrectly
identified
as
a
non­
target
organism
or
not
identified
at
all
using
the
method
of
interest.

False
positive
 
A
non­
target
organism
incorrectly
identified
as
the
target
organism
using
the
method
of
interest.

False
negative
error
rate
 
The
proportion
of
target
organisms
incorrectly
identified
as
a
nontarget
organism
or
not
identified
at
all
using
the
method
of
interest,
equal
to
(
1
­
Sensitivity).
In
statistical
testing,
the
rate
at
which
one
falsely
accepts
a
statistical
hypothesis
(
such
as
that
a
difference
between
methods
does
not
exist)
based
on
a
statistical
test,
when
the
hypothesis
is
actually
false.
Abbreviated
as
$,
and
also
referred
to
as
the
Type
II
error
rate.
(
ASTM)

False
positive
error
rate
 
The
proportion
of
non­
target
organisms
incorrectly
identified
as
the
target
organism
using
the
method
of
interest,
equal
to
(
1
­
Specificity).
In
statistical
testing,
the
rate
at
which
one
falsely
rejects
a
statistical
hypothesis
(
such
as
that
a
difference
between
methods
does
not
exist)
based
on
a
statistical
test,
when
the
hypothesis
is
actually
true.
Abbreviated
as
",
and
also
referred
to
as
the
Type
I
error
rate.
(
ASTM)

Federal
Register
 
A
daily
publication
that
provides
a
uniform
system
for
publishing
Presidential
and
Federal
agency
documents.
Documents
published
in
the
Federal
Register
make
changes
to
the
CFR
to
keep
the
CFR
current.
(
OFR)
Guidelines
and
Format
 
The
document
titled
Guidelines
and
Format
for
Methods
to
be
G
Proposed
at
40
CFR
Parts
136
and
141;
available
from
the
National
Technical
Information
Service
(
NTIS),
U.
S.
Department
of
Commerce,
Springfield,
Virginia,
22161
(
703­
487­
4600)
as
NTIS
publication
PB96­
210448.

Histogram
 
A
bar
diagram
of
the
distribution
of
a
set
of
analytical
results.
The
range
of
values
H
is
categorized
into
sets
of
subintervals,
or
bins,
and
the
number
of
results
within
each
bin
are
displayed
as
the
height
of
the
bars.
(
BERRY)

Industrial
category
 
A
category
listed
in
40
CFR
parts
405­
503.
I
Industrial
subcategory
 
A
subcategory
defined
at
40
CFR
parts
405­
503.

Initial
demonstration
of
capability
 
A
test
performed
to
establish
the
ability
to
demonstrate
control
over
the
analytical
system
and
to
demonstrate
acceptable
performance.
Initial
precision
and
recovery
 
The
analysis
of
a
minimum
of
four
spiked
reagent
water
samples
under
the
same
conditions
as
will
be
used
for
analysis
of
environmental
samples.
The
IPR
is
used
to
demonstrate
that
a
laboratory
is
able
to
produce
reliable
results
with
the
method
prior
to
analysis
of
environmental
samples.

Interaction
 
The
situation
where
the
effect
of
one
variable
(
such
as
method
type)
on
a
dependent
variable
(
such
as
recovery)
is
affected
by
the
value
of
a
third
variable
(
such
as
matrix).

Interference
 
A
positive
or
negative
effect
on
a
measurement
caused
by
a
substance
other
than
the
one
being
investigated.
(
QAD)

Interlaboratory
 
Occurring
in
multiple
laboratories.

Intralaboratory
 
Occurring
within
a
single
laboratory.

J
Kolmogorov­
Smirnov
test
 
A
statistical
test
for
determining
whether
a
given
set
of
K
results
follow
a
normal
distribution,
or
any
other
specified
distribution.
(
GILBERT)

Limited
use
 
Use
of
a
method
by
a
single
regulated
entity
or
laboratory
for
analysis
of
one
or
L
more
matrix
types.

Log­
normal
 
A
distribution
of
a
random
variable
X
such
that
the
natural
logarithm
of
X
is
normally
distributed.

Log­
phase
 
Bacterial
growth
phase
in
which
the
logarithm
of
the
bacterial
biomass
increases
linearly
with
time.

Main
effect
 
Situation
where
a
variable
(
such
as
method
type)
has
a
consistent
effect
on
a
M
dependent
variable
(
such
as
recovery).

Matrix
 
The
component
or
substrate
that
contains
the
analytes
of
interest.

Matrix
effect
 
Variability
in
the
analytical
performance
of
a
method
that
can
be
attributed
to
the
type
of
sample
analyzed.

Matrix
spike
 
A
sample
prepared
by
adding
a
known
mass
of
target
analyte
to
a
specified
amount
of
a
sample
matrix
for
which
an
independent
estimate
of
target
analyte
concentration
is
available.
A
matrix
spike
is
used,
for
example,
to
determine
the
effect
of
the
matrix
on
a
method's
recovery
efficiency.
(
QAMS)

Matrix
spike
duplicate
 
A
replicate
of
the
matrix
spike
to
test
precision.
The
MS/
MSD
are
used
in
combination
to
test
the
precision
of
an
analysis.
(
QAD)

Matrix
type
 
A
sample
medium
with
common
characteristics
across
a
given
industrial
category
or
subcategory.
For
example,
C­
stage
effluents
from
chlorine
bleach
mills,
effluent
from
the
continuous
casting
subcategory
of
the
iron
and
steel
industrial
category,
POTW
sludge,
and
inprocess
streams
in
the
Atlantic
and
Gulf
Coast
Hand­
shucked
Oyster
Processing
subcategory
are
each
a
matrix
type.
For
the
purposes
of
this
initiative
all
drinking
waters
constitute
a
single
matrix
type.

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

May
not
 
This
action,
activity,
or
procedural
step
is
prohibited.
Measurement
quality
 
Critical
level
which,
if
exceeded,
is
considered
to
append
objective
additional,
and
possibly
unacceptable,
measurement
uncertainty
to
the
corresponding
data.

Method
 
A
body
of
procedures
and
techniques
for
performing
a
task
(
e.
g.
sampling,
characterization,
quantitation)
systematically
presented
in
the
order
in
which
they
are
to
be
executed.
(
QAD)

Method
blank
 
An
aliquot
of
reagent
water
or
designated
matrix
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.

Method­
defined
analyte
 
An
analyte
without
a
specific,
known
composition
where
the
analytical
result
depends
totally
on
the
measurement
procedure.

Method
modification
 
A
change
made
to
an
approved
method.

Method
validation
 
A
process
by
which
a
laboratory
or
vendor
establishes
the
performance
of
a
new
method
or
substantiates
the
performance
of
a
method
modification.

Methods
and
Criteria
 
The
document
titled:
Analysis
of
Pollutants
in
Municipal
Water
and
Industrial
Wastewater:
Test
Procedures
and
Quality
Control
Acceptance
Criteria;
available
from
the
National
Technical
Information
Service
(
NTIS),
U.
S.
Department
of
Commerce,
Springfield,
Virginia,
22161
(
703­
487­
4600)
as
NTIS
publication
PB96­
210463,
and
incorporated
by
reference
into
this
part.

Mid­
point
response
factor
 
The
response
factor
at
the
concentration
at
which
calibration
is
verified.

Minor
modification
 
A
modified
method
that
has
been
reviewed
by
EPA
and
has
been
determined
to
be
technically
equivalent
to
a
method
approved
for
use
in
compliance
monitoring.
A
minor
modification
employs
the
same
chemistry
and/
or
biological
principles
as
the
approved
method
to
determine
the
presence/
absence
or
to
quantify
the
amount
of
the
target
organism
in
a
sample.
Supporting
data
may
be
necessary
to
demonstrate
that
a
minor
modification
will
yield
results
equivalent
to
those
obtained
using
the
approved
method
but
does
not
require
approval
as
an
alternate
test
procedure
through
proposal
and
promulgation
in
the
Federal
Register.

Modified
method
 
An
approved
method
that
has
been
modified
to
change
a
front­
end
technique.
EPA
will
judge
a
modified
method
to
be:
1)
an
acceptable
version
or
minor
modification
of
a
previously
promulgated
method,
which
does
not
require
approval
as
an
ATP
or
2)
a
significantly
different
method
which
requires
an
application
for
an
ATP
approval.

Nationwide
use
 
Use
of
a
method
by
all
regulated
entities
and
laboratories
for
analysis
of
one
N
or
more
matrix
types.

Navigable
waters
 
All
waters
of
the
United
States,
including
the
territorial
seas.
(
40
CFR
110.1)

Negative
control
 
A
non­
target
organism
processed
to
ensure
the
laboratories
are
familiar
with
the
identification
of
the
target
organism
and
to
ensure
that
confirmation
test
results
are
appropriate.
New
method
 
A
method
that
employs
a
determinative
technique
for
an
analyte
of
concern
that
differs
from
determinative
techniques
employed
for
that
analyte
in
methods
previously
approved
at
40
CFR
part
136
or
141.

Nonparametric
 
A
type
of
statistical
analysis
for
which
no
assumptions
about
the
underlying
distribution
of
the
data
are
necessary.

Non­
selective
media
 
An
enrichment
media
that
allows
most
bacteria
to
grow.

Normal
probability
plot
 
A
graphical
depiction
of
the
distribution
of
a
set
of
analytical
results.
A
normal
probability
plot
is
a
scatterplot
depicting
the
observed
results
compared
to
the
expected
results
based
on
a
normal
distribution.
If
the
observed
data
follow
a
normal
distribution,
the
graph
will
display
a
line
at
a
45
degree
angle
from
the
x­
axis.
Also
known
as
a
Q­
Q
plot.

Ongoing
demonstration
of
capability
 
The
laboratory
needs
to
demonstrate
that
the
analytical
O
system
is
in
control
on
an
ongoing
basis
through
the
analysis
of
ODC
samples
(
positive
control/
positive
control
duplicate).

Ongoing
precision
and
recovery
 
A
reagent
water
sample
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
within
the
method
for
precision
and
recovery.

Other
approved
methods
 
Promulgated
methods
that
are
not
designated
as
a
reference
method,
but
continue
to
carry
the
same
regulatory
status.

Physical
phase
 
The
physical
phase
of
a
sample
matrix
(
e.
g.,
air,
water,
soil).
P
Positive
control
 
A
target
organism
that
is
analyzed
to
ensure
that
the
laboratory
is
performing
the
method
acceptably
and
that
the
media
is
providing
appropriate
results.

Power
 
The
probability
that
a
statistical
test
will
conclude
that
a
difference
(
for
example,
between
methods)
exists,
when
a
difference
truly
does
exist.
Equal
to
1
­
$.

Precision
 
The
degree
to
which
a
set
of
observations
or
measurements
of
the
same
property,
usually
obtained
under
similar
conditions,
conform
to
themselves;
a
data
quality
indicator.
Precision
is
usually
expressed
as
standard
deviation,
variance,
or
range,
in
either
absolute
or
relative
terms.

The
precision
obtainable
from
an
environmental
measurement
method
may
be
estimated
from
replicate
analyses
of
subsamples
taken
from
the
same
(
homogenous)
sample.
Generally
speaking,
the
more
carefully
one
executes
the
various
steps
of
a
method
and
controls
the
variables
affecting
the
method's
capability,
the
more
precise
will
be
the
results.
The
use
of
a
nonhomogeneous
sample
will
compound
the
precision
estimate
with
the
sample
variability.

Preparation
 
Processing
performed
on
a
sample
prior
to
analysis,
e.
g.
extraction,
concentration,
cleanup,
etc.

Presumptive
counts
 
Numbers
of
organisms
based
on
results
that
have
not
been
confirmed
or
verified.

Procedures
 
A
set
of
systematic
instructions
for
performing
an
activity.
(
QAD)
Promulgated
method
 
A
method
that
has
been
published
or
incorporated
by
reference
into
40
CFR
parts
136,
141,
405­
500,
or
other
parts
that
support
EPA's
water
programs
(
i.
e.,
an
approved
method).

Promulgation
 
Publication
of
a
final
rule
in
the
FR.

Public
water
system
(
PWS)
 
A
system
for
the
provision
to
the
public
of
piped
water
for
human
consumption,
if
such
system
has
at
least
fifteen
service
connections
or
regularly
serves
an
average
of
at
least
twenty­
five
individuals
daily
at
least
60
days
out
of
the
year.
Such
term
includes
(
1)
any
collection,
treatment,
storage,
and
distribution
facilities
under
control
of
the
operator
of
such
system
and
used
primarily
in
connection
with
such
system,
and
(
2)
any
collection
or
pretreatment
storage
facilities
not
under
such
control
which
are
used
primarily
in
connection
with
such
system.
A
public
water
system
is
either
a
"
community
water
system"
or
a
"
noncommunity
water
system."

Quality
assurance
 
An
integrated
system
of
activities
involving
planning,
quality
control,
Q
quality
assessment,
reporting,
and
quality
improvement
to
ensure
that
a
product
or
service
meets
defined
standards
of
quality
with
a
stated
level
of
confidence.
(
QAD)

Quality
control
 
The
overall
system
of
technical
activities
whose
purpose
is
to
measure
and
control
the
quality
of
a
product
or
service
so
that
it
meets
the
needs
of
users.
The
aim
is
to
provide
quality
that
is
satisfactory,
adequate,
dependable,
and
economical.
(
QAD)

QC
acceptance
criteria
 
Performance
specifications
developed
from
validation
data
and
used
to
control
the
limits
within
which
an
analytical
method
is
operated.

QC
acceptance
criteria­
based
comparison
study
 
A
study
performed
to
evaluate
the
performance
of
a
modified
method
against
the
quality
control
acceptance
criteria
of
a
reference
method.

Range
finding
 
Preliminary
analyses
conducted
to
assess
the
ambient
concentration
of
the
R
target
organism
in
a
matrix
to
be
used
in
a
study
or
preliminary
analyses
of
spiked
samples
involving
few,
if
any,
replicates,
to
assess
method
performance
to
identify
the
spike
dose
to
be
used
in
a
study.

Raw
data
 
Data
that
have
not
been
processed.

Reagent
water
 
Water
conforming
to
Specification
D
1193,
Annual
Book
of
ASTM
Standards,
or
specifications
in
Standard
Methods
9020B.
4.
d.

Recovery
 
The
total
amount
of
the
analyte
found
in
the
sample
divided
by
the
amount
of
the
analyte
added
into
the
sample
as
a
spike.

Reference
method
 
A
method
that
serves
as
a
standard
against
which
method
modifications
can
be
compared.

Regulated
entity
 
Permittees,
PWSs,
POTWs,
and
other
entities
responsible
for
compliance
with
provisions
of
the
CWA
or
SDWA.
Relative
percent
difference
(
RPD)
 
An
estimate
of
the
variability
of
two
numbers
expressed
in
relative
terms.
Calculated
as
the
absolute
value
of
the
difference
of
the
two
numbers,
divided
by
their
mean:

RPD
A
B
A
B
=
 

+
1
2
100%
*(
)
*

Equal
to
the
relative
standard
deviation
of
the
two
numbers
multiplied
by
the
square
root
of
2.

Relative
standard
deviation
(
RSD)
 
The
standard
deviation
expressed
as
a
percentage
of
the
mean
(
100F/
X);
i.
e.,
the
coefficient
of
variation.

Replicate
 
Multiple
samples
collected
from
the
same
sampling
point
at
the
same
time
and
analyzed
exactly
the
same
way.
Replicate
samples
can
be
used
as
a
measure
of
sample
variability.

Sample
matrix
 
See
"
matrix."
S
Sample
preparation
technique
 
Any
technique
in
the
analytical
process
that
precedes
the
determinative
technique,
including
all
procedures,
equipment,
solvents,
etc.
that
are
used
in
the
preparation
and
cleanup
of
a
sample
for
analysis.
Sample
preparation
techniques
do
not
include
conditions
and/
or
procedures
for
the
collection,
preservation,
shipment,
and
storage
of
the
sample.

Sensitivity
 
In
presence/
absence
testing,
sensitivity
is
the
proportion
of
target
organisms
in
the
sample
that
were
correctly
detected
by
the
method
of
interest.

Shapiro­
Wilk
test
 
A
statistical
test
for
determining
whether
a
given
set
of
results
follow
a
normal
or
log­
normal
distribution.
Best
used
for
datasets
with
at
most
50
results.
(
GILBERT)

Side­
by­
side
comparison
 
Parallel
testing
of
a
new
or
modified
method
and
a
reference
method
to
determine
whether
the
performance
of
the
new
or
modified
method
is
acceptable
compared
to
the
reference
method.

Specificity
 
In
presence/
absence
testing,
the
proportion
of
non­
target
organisms
in
the
sample
that
were
correctly
identified
as
not
being
the
target
organism
by
the
method
of
interest.

Spike
 
The
process
of
adding
a
known
amount
of
target
analyte
to
a
sample;
used
to
determine
the
recovery
efficiency
of
the
method.
(
QAD)

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

Standard
deviation
(
F)
 
The
measure
of
the
dispersion
of
observed
values
expressed
as
the
positive
square
root
of
the
sum
of
the
squares
of
the
difference
between
the
individual
values
of
a
set
and
the
arithmetic
mean
of
the
set,
divided
by
one
less
than
the
number
of
values
in
the
set.
For
a
total
of
n
numbers:

SD
n
X
Mean
i
i
n
=
 
 

=
 
1
1
2
1
(
)

Statistical
power
 
See
power.
Stem­
and­
leaf
plot
 
A
graphical
depiction
of
a
set
of
analytical
results,
that
conveys
information
about
the
shape
of
the
distribution
while
retaining
the
numerical
information.
The
stem­
and­
leaf
plot
separates
the
digits
of
the
values
as
leaves
(
the
last
digit
of
the
values)
and
stems
(
the
remaining
digit
of
the
values).
The
individual
results
are
grouped
according
to
the
stems,
and
the
leaves
are
listed
separately
in
a
format
similar
to
a
histogram.
(
RICE)

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
organisms.

Study
plan
 
A
study
design
submitted
for
EPA
review,
comment,
and
approval
prior
to
conducting
the
side­
by­
side
or
QC
acceptance
criteria­
based
method
comparability
study.
This
process
protects
the
applicant
by
providing
written
approval
of
the
study
design
before
resources
are
spent
to
conduct
the
study.
Data
from
studies
conducted
without
EPA
review
and
approval
may
not
adequately
address
the
applicant's
study
objectives.
A
detailed
procedure
for
the
ATP
or
new
method
should
be
included
as
an
attachment
to
the
study
plan.
EPA
will
evaluate
the
study
plan
to
verify
that
the
appropriate
data
quality
objectives
identified
in
this
protocol
are
defined
and
addressed.
EPA
comments
are
incorporated
into
the
study
design
and
this
review
process
is
repeated
until
EPA
has
approved
the
study
design.

Study
report
 
A
formal
report
developed
by
laboratories
or
other
organizations
responsible
for
developing
ATPs
or
new
methods
documenting
the
results
of
the
side­
by­
side
or
QC
acceptance
criteria­
based
method
comparability
study.
The
information
and
supporting
data
needed
in
the
study
report
should
be
sufficient
to
enable
EPA
to
evaluate
the
performance
of
the
ATP
or
new
method
and
make
a
decision
on
whether
it
is
comparable
or
superior
to
the
reference
method.
The
approved
study
report
should
also
identify
and
discuss
any
deviations
from
the
study
plan
that
were
made
in
implementing
the
study,
and
the
approved
study
plan
should
be
appended
to
and
referenced
in
the
study
report.

Summary
results
 
Overall
study
statistics
(
not
sample­
specific
results).

Target
organism
 
The
organism
the
method
is
designed
to
detect.
T
U
Validate
 
Reliably
assess
the
performance
(
bias
and
precision)
of
a
method
in
a
reference
V
matrix
(
such
as
reagent
water)
and
the
matrix
in
which
the
validated
method
will
be
used
(
such
as
drinking
water,
surface
water,
or
municipal
wastewater
effluent).

Validation,
single­
laboratory
 
Assessment
of
method
performance
(
see
"
validate")
in
one
laboratory.

Validation,
interlaboratory
 
Assessment
of
method
performance
(
see
"
validate")
at
multiple
laboratories.

Variance
 
A
measure
of
the
dispersion
of
a
set
of
values.
The
sum
of
the
squares
of
the
difference
between
the
individual
values
of
a
set
and
the
arithmetic
mean
of
the
set,
divided
by
one
less
than
the
number
of
values
in
the
set.
(
The
square
of
the
sample
standard
deviation)
(
QAD)
Wilcoxon­
Mann­
Whitney
(
WMW)
test
 
A
nonparametric
analysis
used
to
determine
whether
W
the
medians
from
two
levels
of
a
given
factor
(
such
as
method)
differ
from
each
other,
based
on
the
ranks
of
all
results.
(
SPRENT)

XYZ
The
above
definitions
are
referenced
to
the
following
organizations:

ASTM
ASTM
D
4855­
91.
Standard
Practice
for
Comparing
Test
Methods.

BERRY
Berry,
D.
A.
and
B.
Lindgren.
Statistics
Theory
and
Methods.
Wardsworth,
Belmonst,
CA,
1990.

EMMC
Environmental
Monitoring
Management
Council
GILBERT
Gilbert,
R.
O.
Statistical
Methods
for
Environmental
Pollution
Monitoring.
Van
Nostrand
Reinhold,
New
York,
1987.

NELAC
QS
National
Environmental
Laboratory
Accreditation
Conference,
Quality
Systems
OFR
Office
of
Federal
Register
QAD
Quality
Assurance
Division,
National
Center
for
Environmental
Research
and
Quality
Assurance,
Office
of
Research
and
Development,
USEPA
RICE
Rice,
J.
A.
Mathematical
Statistics
and
Data
Analysis,
Second
Edition.
Wadsworth,
Belmont,
CA,
1995.

SPRENT
Sprent,
P.
Applied
Nonparametric
Statistical
Methods,
Second
Edition.
Chapman
&
Hall,
London,
1993.
APPENDIX
B
ATP
APPLICATION
FORM
EPA
Office
of
Water
Alternate
Test
Procedure
or
New
Method
Preliminary
Application
Form
for
Microbiological
Analytes
Applicant
Name
and
Address:
EPA
Use
Only
ATP
Case
Number:

Date
Application
Submitted:

Type
of
Application:
Circle
appropriate
application
type
Alternate
Test
Procedure
New
Method
Method
Number
Title
of
Method
Revision
Date
EPA­
Approved
Reference
Method:

Analyte(
s):

Applicable
Matrices:
Circle
all
that
apply
Ambient
Water
Biosolids
Drinking
Water
Wastewater
Level
of
Use:
Limited
Use
Nationwide
Study
Design:
Circle
appropriate
study
design
Side­
by­
Side
Comparison
Study
QC
Acceptance
Criteria­
Based
Comparison
Study
FOR
LIMITED
USE
APPLICATIONS
ONLY
ID
Number
of
Existing
or
Pending
Permit:

Issuing
Agency:

Type
of
Permit:

Discharge
Serial
Number:

Attachments
Check
all
that
apply
G
Justification
for
ATP
G
Alternate
Test
Procedure
or
New
Method
(
in
standard
EPA
format)

G
Method
Comparison
Table
G
Study
Plan
G
Other:

Submit
Application
and
Attachments
in
Triplicate
EPA
Office
of
Water
Alternate
Test
Procedure
or
New
Method
Final
Application
Form
for
Microbiological
Analytes
Applicant
Name
and
Address:
EPA
Use
Only
ATP
Case
Number:

Date
Application
Submitted:

Type
of
Application:
Circle
appropriate
application
type
Alternate
Test
Procedure
New
Method
Method
Number
Title
of
Method
Revision
Date
EPA­
Approved
Reference
Method:

Analyte(
s):

Applicable
Matrices:
Circle
all
that
apply
Ambient
Water
Biosolids
Drinking
Water
Wastewater
Level
of
Use:
Limited
Use
Nationwide
Study
Design:
Circle
appropriate
study
design
Side­
by­
Side
Comparison
Study
QC
Acceptance
Criteria­
Based
Comparison
Study
FOR
LIMITED
USE
APPLICATIONS
ONLY
ID
Number
of
Existing
or
Pending
Permit:

Issuing
Agency:

Type
of
Permit:

Discharge
Serial
Number:

Attachments
Check
all
that
apply
G
Justification
for
ATP
G
Alternate
Test
Procedure
or
New
Method
(
in
standard
EPA
format)

G
Method
Comparison
Table
G
Study
Plan
G
Study
Report
G
Other:

Submit
Application
and
Attachments
in
Triplicate
APPENDIX
C
APPLICATION
INVENTORY
FORM
1.
Completed
application
form.

Includes
the
name
and
address
of
the
applicant;
the
date
of
submission
of
the
application;
the
method
number,
title,
and
revision
date;
the
EPA­
approved
reference
method;
the
analyte(
s)
for
which
the
ATP
or
new
method
is
proposed;
the
type
of
application;
applicable
matrices;
study
design;
level
of
use;
NPDES
permit
information,
if
applicable;
and
the
attachments
submitted
with
the
application.
Section
2.2
Appendix
C
G
2.
Justification
for
ATP
or
new
method.

Brief
justification
for
why
the
ATP
or
new
method
is
being
proposed.
Section
2.3
G
3.
Method
in
EPA
format.

Scope
and
application
Section
3.1
G
Summary
of
method
Section
3.2
G
Definitions
of
method
Section
3.3
G
Interferences
Section
3.4
G
Safety
Section
3.5
G
Equipment
and
supplies
Section
3.6
G
Reagents
and
standards
Section
3.7
G
Sample
collection,
preservation,
and
storage
Section
3.8
G
Quality
control
Section
3.9
G
Calibration
and
standardization
Section
3.10
G
Procedure
Section
3.11
G
Data
analysis
and
calculations
Section
3.12
G
Method
performance
Section
3.13
G
Pollution
prevention
Section
3.14
G
Waste
management
Section
3.15
G
References
Section
3.16
G
Tables,
diagrams,
flowcharts,
and
validation
data
Section
3.17
G
4.
Method
comparison
table.

A
two­
column
table
comparing
the
proposed
ATP
or
new
method
with
the
EPA­
approved
reference
method.
This
table
should
include
the
number
and
title
of
each
method,
the
latest
revision
date
of
the
ATP
or
new
method,
and
a
detailed
discussion
of
each
of
the
method
sections
listed
in
Section
3.0.
Each
topic
should
be
discussed
in
a
separate
row
of
the
table
and
the
applicant
should
highlight
any
differences
between
the
ATP
or
new
method
and
the
EPA­
approved
reference
method.
Section
2.5
G
5.
Study
plan.

Background
Section
4.1
G
Objectives
Section
4.2
G
Study
design
Section
4.3
G
Coordination
Section
4.4
G
Data
reporting
Section
4.5
G
6.
Study
report.

Background
Section
9.1
G
Study
objectives
and
design
Section
9.2
G
Study
implementation
Section
9.3
G
Data
reporting
and
validation
Section
9.4
G
Results
Section
9.5
G
Data
analysis
and
discussion
Section
9.6
G
Conclusions
Section
9.7
G
Appendix
A:
Method
Section
9.8
G
Appendix
B:
Approved
study
plan
Section
9.9
G
Appendix
C:
Supporting
data
Section
9.10
G
Appendix
D:
Supporting
references
Section
9.11
G
APPENDIX
D
DATA
ELEMENTS
AND
EXAMPLE
BENCH
SHEETS
Data
Elements
The
data
elements
listed
below
should
be
reported
on
the
bench
sheets
or
in
the
lab
notebook
for
each
method,
as
applicable.
EPA
will
review
the
information
during
the
data
validation
process
to
ensure
the
method­
specific
QC
measures
are
met,
as
agreed
to
in
the
approved
study
plan.
°
Laboratory
name
°
Method
number
°
Media
°
Procedure
°
Matrix
°
Sample
collection
date/
time
°
Dates
and
times
for
all
method
steps
associated
with
holding
times
or
incubation
times
°
Analyst
initials
for
each
processing
step
in
the
method
°
Presumptive
results
for
all
applicable
media
°
Confirmed/
completed
results
for
all
applicable
media
°
All
measured
volumes
°
Dilution
information
°
Final
result
per
units
of
measurement
Example
Benchsheets
Example
bench
sheets
for
the
following
EPA­
approved
reference
methods
are
included
in
this
appendix:
°
Aeromonas
(
USEPA
Method
1605)
°
Cryptosporidium
(
USEPA
Methods
1622
and
1623)
°
E.
coli
(
SM
9221F,
SM
9222G)
°
Enterococci
(
SM
9230C)
°
Fecal
coliforms
(
SM
9221E,
9222D)
°
Fecal
streptococcus
(
SM
9230B,
SM
9230C)
°
Giardia
(
USEPA
Method
1623)
°
Total
coliforms
(
SM
9221B,
SM
9222B)

Note:
Additional
example
bench
sheets
or
electronic
copies
of
the
attached
bench
sheets
are
available
upon
request.
Laboratory:
Sample
Collection
Time:

Sample
Collection
Date:
Sampling
Point:

Analyst
Initials
Read
Temp
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
Analyst
Initials
Read
Temp
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
Analyst
Initials
Read
Temp
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
24
hr
fecal
read
(
From
24
hr
LTB)

24
hr
fecal
read
(
From
48
hr
LTB)

24
hr
E.
coli
read
(
From
24
hr
LTB)

LTB
start
24
hr
E.
coli
read
(
From
48
hr
LTB)

LTB
24
hr
read
Final
tube
combination:
Total
coliforms/
100
mL:
Fecal
tube
combination:
Fecal/
100
mL:

E.
coli
tube
combination:
E.
coli/
100
mL:

Analyst
Initials
Read
Temp
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
Analyst
Initials
Read
Temp
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
BGB
48
hr
read
(
from
48
hr
LTB)
24
hr
fecal
read
(
From
24
hr
LTB)

24
hr
fecal
read
(
From
48
hr
LTB)

24
hr
E.
coli
read
(
From
24
hr
LTB)

Analyst
Initials
Read
Temp
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
24
hr
E.
coli
read
(
From
48
hr
LTB)

Final
tube
combination:
Total
coliforms/
100
mL:
Fecal
tube
combination:
Fecal/
100
mL:

E.
coli
tube
combination:
E.
coli/
100
mL:

Analyst
Initials
Read
Temp
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
Analyst
Initials
Read
Temp
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
Analyst
Initials
Read
Temp
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
24
hr
fecal
read
(
From
24
hr
LTB)

24
hr
fecal
read
(
From
48
hr
LTB)

24
hr
E.
coli
read
(
From
24
hr
LTB)

24
hr
E.
coli
read
(
From
48
hr
LTB)

Final
tube
combination:
Total
coliforms/
100
mL:
Fecal
tube
combination:
Fecal/
100
mL:

E.
coli
tube
combination:
E.
coli/
100
mL:

Comments:
24
hr
/
48
hr
BGB
read
(
From
48
hr
LTB)

LTB:
Replicate
3
BGB:
Replicate
3
LTB:
Replicate
2
24
hr
/
48
hr
LTB
read
BGB:
Replicate
2
24
hr
/
48
hr
BGB
read
(
From
24
hr
LTB)

Multiple­
Tube
Fermentation:
Total
Coliform,
Fecal
Coliform,
E.
coli
(
SM
9221B,
SM
9221E,
SM
9221F)

24
hr
/
48
hr
BGB
read
(
From
24
hr
LTB)

LTB:
Replicate
1
BGB:
Replicate
1
EC­
MUG:
Replicate
1
24
hr
/
48
hr
LTB
read
24
hr
/
48
hr
BGB
read
(
From
48
hr
LTB)

Please
enter
date
and
time
for
the
following:
LTB
48
hr
read,

BGB
24
hr
read
(
from
24
hr
LTB),

ECMUG
24
hr
read
(
from
24
hr
LTB)
EC­
MUG:
Replicate
3
24
hr
/
48
hr
BGB
read
(
From
24
hr
LTB)
EC­
MUG:
Replicate
2
BGB
48
hr
read
(
from
24
hr
LTB),

BGB
24
hr
read
(
from
48
hr
LTB),

ECMUG
24
hr
read
(
from
48
hr
LTB)
24
hr
/
48
hr
BGB
read
(
From
48
hr
LTB)

24
hr
/
48
hr
LTB
read
Laboratory:
Sample
collection
time:

Sample
collection
date:
Sampling
point:

mEndo
incubation
start
temperature
(
oC):
mEndo
incubation
start
date/
time:

mEndo
incubation
end
temperature
(
oC):
mEndo
incubation
end
date/
time:

NA­
MUG
incubation
start
temperature
(
oC):
NA­
MUG
incubation
start
date/
time:

NA­
MUG
incubation
end
temperature
(
oC):
NA­
MUG
incubation
end
date/
time:

Replicate
number
1
Analyst
initials
Sample
volume
filtered
No.
colonies
per
filter
Total
Coliforms
per
100
mL
E.
coli
per
100
mL
100
mL
10.0
mL
1.0
mL
0.1
mL
Replicate
number
2
Analyst
initials
Sample
volume
filtered
No.
colonies
per
filter
Total
Coliforms
per
100
mL
E.
coli
per
100
mL
100
mL
10.0
mL
1.0
mL
0.1
mL
Replicate
number
3
Analyst
initials
Sample
volume
filtered
No.
colonies
per
filter
Total
Coliforms
per
100
mL
E.
coli
per
100
mL
100
mL
10.0
mL
1.0
mL
0.1
mL
E.
coli
Total
coliforms
E.
coli
No.
colonies
per
filter
Membrane
Filtration:
mEndo/
NA­
MUG
(
SM
9222B/
SM
9222G)

Total
coliforms
E.
coli
No.
colonies
per
filter
No.
colonies
per
filter
Total
coliforms
Laboratory:
Sample
collection
time:

Sample
collection
date:
Sampling
point:

mFC
incubation
start
temperature
(
oC):
mFC
incubation
start
date/
time:

mFC
incubation
end
temperature
(
oC):
mFC
incubation
end
date/
time:

NA­
MUG
incubation
start
temperature
(
oC):
NA­
MUG
incubation
start
date/
time:

NA­
MUG
incubation
end
temperature
(
oC):
NA­
MUG
incubation
end
date/
time:

Replicate
number
1
Analyst
initials
Sample
volume
filtered
No.
colonies
per
filter
Fecal
coliforms
per
100
mL
E.
coli
per
100
mL
100
mL
10.0
mL
1.0
mL
0.1
mL
Replicate
number
2
Analyst
initials
Sample
volume
filtered
No.
colonies
per
filter
Fecal
coliforms
per
100
mL
E.
coli
per
100
mL
100
mL
10.0
mL
1.0
mL
0.1
mL
Replicate
number
3
Analyst
initials
Sample
volume
filtered
No.
colonies
per
filter
Fecal
coliforms
per
100
mL
E.
coli
per
100
mL
100
mL
10.0
mL
1.0
mL
0.1
mL
E.
coli
No.
colonies
per
filter
No.
colonies
per
filter
Fecal
coliforms
E.
coli
No.
colonies
per
filter
Membrane
Filtration:
mFC/
NA­
MUG
(
SM
9222D/
SM
9222G)

Fecal
coliforms
E.
coli
Fecal
coliforms
Laboratory:
Sample
collection
time:

Sample
collection
date:
Sampling
point:

mTEC
incubation
start
temperature
(
oC):
mTEC
incubation
start
date/
time:

mTEC
incubation
end
temperature
(
oC):
mTEC
incubation
end
date/
time:

Urease
substrate
incubation
start
date/
time:
Urease
substrate
incubation
end
date/
time:

Replicate
number
1
Analyst
initials
Replicate
number
2
Analyst
initials
Replicate
number
3
Analyst
initials
E.
coli
E.
coli
Sample
volume
filtered
100
mL
10.0
mL
1.0
mL
E.
coli
Membrane
Filtration:
mTEC
(
EPA
1103.1,
SM
9213D)

10.0
mL
1.0
mL
100
mL
10.0
mL
1.0
mL
0.1
mL
E.
coli
per
100
mL
Sample
volume
filtered
No.
colonies
per
filter
E.
coli
per
100
mL
E.
coli
Sample
volume
filtered
No.
colonies
per
filter
E.
coli
per
100
mL
100
mL
0.1
mL
0.1
mL
No.
colonies
per
filter
Laboratory:
Sample
collection
time:

Sample
collection
date:
Sampling
point:

modified
mTEC
incubation
start
temperature
(
oC):

modified
mTEC
incubation
end
temperature
(
oC):

Replicate
number
1
Analyst
initials
Replicate
number
2
Analyst
initials
Replicate
number
3
Analyst
initials
100
mL
E.
coli
E.
coli
0.1
mL
100
mL
10.0
mL
1.0
mL
10.0
mL
1.0
mL
E.
coli
per
100
mL
E.
coli
per
100
mL
No.
colonies
per
filter
1.0
mL
0.1
mL
10.0
mL
E.
coli
Membrane
Filtration:
Modified
mTEC
(
EPA
1603)

Sample
Volume
filtered
No.
colonies
per
filter
E.
coli
per
100
mL
modified
mTEC
incubation
start
date/
time:

modified
mTEC
incubation
end
date/
time:

E.
coli
100
mL
No.
colonies
per
filter
0.1
mL
Sample
Volume
filtered
Sample
Volume
filtered
Sample
Collection
time:

Sampling
point:
ADB
incubation
start
date/
time:

10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
24
hr
ADB
read
24
hr
BEA
read
from
24
hr
ADB
Start
temp:

48
hr
ADB
read
24
hr
BEA
read
from
48
hr
ADB
ADB
incubation
end
date/
time:

Final
tube
combination:
Fecal
streptococcus/
100
mL:

10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
Start
temp:

24
hr
ADB
read
24
hr
BEA
read
from
24
hr
ADB
48
hr
ADB
read
24
hr
BEA
read
from
48
hr
ADB
Final
tube
combination:
Fecal
streptococcus/
100
mL:
End
temp:

Start
temp:

10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
10
mL
1.0
mL
0.1
mL
0.01
mL
0.001
mL
24
hr
ADB
read
24
hr
BEA
read
from
24
hr
ADB
48
hr
ADB
read
24
hr
BEA
read
from
48
hr
ADB
End
temp:

Final
tube
combination:
Fecal
streptococcus/
100
mL:

Multiple­
Tube
Fermentation:
Fecal
streptococcus
(
SM
9230B)
BEA
incubation
end
date/
time:

(
From
48
hr
ADB)

End
temp:

BEA
incubation
start
date/
time:

(
From
24
hr
ADB)

BEA
incubation
end
date/
time:

(
From
24
hr
ADB)

BEA
incubation
start
date/
time:

(
From
48
hr
ADB)

Replicate
Number
2
BEA
Plates
Laboratory:

Sample
Collection
Date:
Analyst
Initials
Replicate
Number
1
Comments:
BEA
Plates
Comments:
Azide
Dextrose
Broth
(
ADB)

Azide
Dextrose
Broth
(
ADB)

Analyst
Initials
Analyst
Initials
Analyst
Initials
Analyst
Initials
Replicate
Number
3
Comments:
Azide
Dextrose
Broth
(
ADB)

Analyst
Initials
BEA
Plates
Laboratory:
Sample
collection
time:

Sample
collection
date:
Sampling
point:

mEnterococcus
incubation
start
temperature
(
oC):
mEnterococcus
incubation
start
date/
time:

mEnterococcus
incubation
end
temperature
(
oC):
mEnterococcus
incubation
end
date/
time:

Replicate
number
1
Analyst
initials
Replicate
number
2
Analyst
initials
Replicate
number
3
Analyst
initials
Fecal
streptococcus
Fecal
streptococcus
Fecal
streptococcus
1.0
mL
0.1
mL
Sample
volume
filtered
100
mL
10.0
mL
100
mL
10.0
mL
1.0
mL
0.1
mL
Sample
volume
filtered
0.1
mL
Sample
volume
filtered
100
mL
10.0
mL
1.0
mL
No.
colonies
per
filter
Membrane
Filtration:
Fecal
Streptococcus
(
SM
9230C)

Fecal
streptococcus
per
100
mL
Fecal
streptococcus
per
100
mL
Fecal
streptococcus
per
100
mL
No.
colonies
per
filter
No.
colonies
per
filter
Laboratory:
Sample
collection
time:

Sample
collection
date:
Sampling
point:

mE
incubation
start
temperature
(
oC):
mE
incubation
start
date/
time:

mE
incubation
end
temperature
(
oC):
mE
incubation
end
date/
time:

EIA
incubation
start
temperature
(
oC):
EIA
incubation
start
date/
time:

EIA
incubation
end
temperature
(
oC):
EIA
incubation
end
date/
time:

Replicate
number
1
Analyst
initials
Replicate
number
2
Analyst
initials
Replicate
number
3
Analyst
initials
Enterococcus
Enterococcus
Enterococcus
0.1
mL
0.1
mL
Volume
filtered
100
mL
10.0
mL
0.1
mL
Volume
filtered
1.0
mL
Enterococci
per
100
mL
100
mL
10.0
mL
1.0
mL
No.
colonies
per
filter
Membrane
Filtration:
Enterococcus
(
mE­
EIA)
(
EPA
1106.1,
SM
9230C)

Enterococci
per
100
mL
Enterococci
per
100
mL
No.
colonies
per
filter
Volume
filtered
100
mL
No.
colonies
per
filter
10.0
mL
1.0
mL
Laboratory:
Sample
collection
time:

Sample
collection
date:
Sampling
point:

mEI
incubation
start
temperature
(
oC):

mEI
incubation
end
temperature
(
oC):

Replicate
number
1
Analyst
initials
Replicate
number
2
Analyst
initials
Replicate
number
3
Analyst
initials
Enterococcus
0.1
mL
100
mL
10.0
mL
1.0
mL
0.1
mL
Enterococcus
Volume
filtered
No.
colonies
per
filter
Enterococcus
per
100
mL
100
mL
10.0
mL
Volume
filtered
100
mL
10.0
mL
1.0
mL
No.
colonies
per
filter
Enterococcus
per
100
mL
Membrane
Filtration:
Enterococcus
(
mEI)
(
EPA
1600)

Enterococcus
per
100
mL
No.
colonies
per
filter
mEI
incubation
start
date/
time:

mEI
incubation
end
date/
time:

Enterococcus
Volume
filtered
1.0
mL
0.1
mL
Laboratory
name:

Section
1.
Media
Preparation
Information
1
2
Oxidase
dry
slides
expiration
date
3
4
5
6
Section
2:
Sample
Processing
Information
7
8
9
10
11
12
13
14
Section
3:
Sample
Analysis
Information
15
16
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Batch­
specific
Cover
Sheet:
Method
1605
(
Aeromonas)
­
ADA
with
Vancomycin
Note:
Please
complete
one
sheet
per
week
of
analysis.

Sample
spiking
date
Dilution
preparation
time
(
for
IDC,
ODC,
and
MS/
MSD
samples)

Analyst
preparing
dilutions
for
IDC,
ODC,
and
MS/
MSD
samples)
Date
nutrient
agar
slants
for
positive
controls
and
matrix
spikes
were
inoculated
Kovac's
expiration
date
Date
of
nutrient
agar
slant
preparation
Time
nutrient
agar
slants
for
positive
controls
and
matrix
spikes
were
inoculated
Date
of
nutrient
agar
plate
preparation
Sample
spiking
time
Nutrient
agar
plate
incubation
start
date
/
time
Nutrient
agar
plate
read
date
/
time
Funnel
decontamination
method
Analyst
performing
filtration
Analyst
spiking
samples
ADA­
V
read
analyst
Incubator
temperature
at
read
date
Incubator
temperature
at
start
date
ADA­
V
read
date
/
time
ADA­
V
incubation
start
date
/
time
oC
oC
Dilution
preparation
date
(
for
IDC,
ODC,
and
MS/
MSD
samples)

Trehalose
incubation
start
date
/
time
Oxidase
confirmation
date
/
time
Nutrient
agar
plate
read
analyst
Tryptone
(
indole)
read
date
/
time
Trehalose
read
date
/
time
Trehalose
read
analyst
Oxidase
read
analyst
Tryptone
(
indole)
read
analyst
Tryptone
(
indole)
incubation
start
date
/
time
Laboratory
name:

Batch
identification:

Did
the
unspiked
reagent
water
sample
exhibit
the
appropriate
response?
Yes
No
Did
negative
controls
for
oxidase,
trehalose,
and
indoleexhibit
the
appropriate
responses?
Yes
No
QC
Checklist:
Method
1605
(
Aeromonas)
­
ADA
with
Vanomycin
Note:
Please
complete
one
sheet
per
week
of
analysis.
Please
circle
the
appropriate
response
and
provide
supporting
information
as
necessary.

If
no,
please
explain.
If
no,
please
list
contaminated
method
blank(
s)
and
associated
samples.
1
Yes
No
Did
all
method
blanks
(
dilution/
rinse
water)
exhibit
the
appropriate
response?

2
Yes
No
If
no,
please
explain.
Did
ADA­
V
media
sterility
check
exhibit
the
appropriate
response?

3
Did
nutrient
agar
plate
media
sterility
check
exhibit
the
appropriate
response?
Yes
No
4
Did
nutrient
agar
slant
media
sterility
check
exhibit
the
appropriate
response?
Yes
No
If
no,
please
explain.

5
Did
trehalose
media
sterility
check
exhibit
the
appropriate
response?
Yes
No
If
no,
please
explain.

If
no,
please
explain.
7
If
no,
please
explain.
No
If
no,
please
explain.
6
Did
tryptone
media
sterility
check
exhibit
the
appropriate
response?
Yes
8
Laboratory
name:

Section
1:
Sample
information
1
Sample
number:
5
Volume
(
mL)
of
spike
(
QC
samples
only):

2
Utility:
6
Volume
filtered
(
mL):

3
Sampling
point:
7
Dilution
bottle
(
for
laboratory­
prepared
QC
samples
only,
plea
D
D2
sterility
check,
direct
streak,
streak
with
filter
reagent,
finished,
dilution/
rinse
Section
2:
Sample
results
Color
Morphology*
size
(
mm)
Color
Morphology*
size
(
mm)

1
pale
yellow
1358
2­
4
mm
60
off­
white
1358
2­
4
mm
2
of
2
2
of
2
2
dark
yellow
1358
1­
3
mm
5
off­
white
1358
1­
2
mm
1
of
1
1
of
1
3
4
5
6
7
8
9
10
D1=
30
D2=
5
D3=
D4=
D5=

Sample­
specific
Data
Report
Form:

Method
1605
(
Aeromonas)
­
ADA
With
Vanomycin
ADA­
V
colony
description
(
this
section
is
optional)
No.
of
presumptive
positive
colonies
for
this
colony
type
Please
note:
It
is
important
to
record
the
number
of
colonies
for
each
presumptively
positive
morphological
type
so
that
the
final
density
of
Aeromonas
per
sample
can
be
reported
based
on
percent
confirmation
of
each
colony
type.

Nutrient
agar
colony
description
(
this
section
is
optional)
No.
oxidase
positive
per
colony
type
No.
trehalose
positive
per
colony
type
No.
Indole
positive
per
colony
type
No.
of
presumptive
colonies
submitted
to
confirmation
for
this
colony
type
8
4
QC
Analysis
or
Matrix
filtered
(
please
circle
one):
If
this
report
form
is
for
a
method
blank,

please
indicate
samples
that
are
associated
with
this
method
blank:

A
No.
presumptive
positive
colonies
for
each
colony
type
B
No.
of
each
colony
type
submitted
to
confirmation
C
How
many
submitted
colonies
per
colony
type
confirmed
(
oxidase
positive,
ferments
trehalose,
and
produces
indole)?
D1
+
D2
+
D3
+
D4
+
D5
Total
confirmed
Aeromonas
per
sample
A
*
(
C/
B)
=
D
Calculated
no.
of
confirmed
Aeromonas
per
colony
type
60
2
1
5
1
1
Section
5:
Comments
Section
3:
Calculations
(
Use
one
row
for
each
presumptive
positive
colony
color
and
morphology.
If
more
than
five
colony
types,
please
attach
another
sheet.)

*
Morphology
choices
(
list
all
that
apply)
:
(
1)
Round,
(
2)
oval,
(
3)
symmetric,
(
4)
asymmetric,
(
5)
shiny,
(
6)
dull,
(
7)
translucent,
(
8)
opaque,
(
9)
grainy,
(
10)
fuzzy,
(
11)
other
2
1
30
+
5
=
35
1
of
2
1
of
1
Laboratory
name:

Section
1:
Sample
information
1
Sample
number:
5
Volume
(
mL)
of
spike
(
QC
samples
only):

2
Utility:
6
Volume
filtered
(
mL):

3
Sampling
point:
7
Dilution
bottle
(
for
laboratory­
prepared
QC
samples
only,
please
circle
one):
D
D2
sterility
check,
direct
streak,
streak
with
filter
reagent,
finished,
dilution/
rinse
Section
2:
Sample
results
Color
Morphology*
size
(
mm)
Color
Morphology*
size
(
mm)

1
2
3
4
5
6
7
8
9
10
D1=
D2=
D3=
D4=
D5=

Sample­
specific
Data
Report
Form:

Method
1605
(
Aeromonas)
­
ADA
With
Vanomycin
No.
of
presumptive
colonies
submitted
to
confirmation
for
this
colony
type
Section
5:
Comments
Section
3:
Calculations
(
Use
one
row
for
each
presumptive
positive
colony
color
and
morphology.
If
more
than
five
colony
types,
please
attach
another
sheet.)

*
Morphology
choices
(
list
all
that
apply):
(
1)
Round,
(
2)
oval,
(
3)
symmetric,
(
4)
asymmetric,
(
5)
shiny,
(
6)
dull,
(
7)
translucent,
(
8)
opaque,
(
9)
grainy,
(
10)
fuzzy,
(
11)
other
A
No.
presumptive
positive
colonies
for
each
colony
type
B
No.
of
each
colony
type
submitted
to
confirmation
C
How
many
submitted
colonies
per
colony
type
confirmed
(
oxidase
positive,
ferments
trehalose,
and
produces
indole)?
D1
+
D2
+
D3
+
D4
+
D5
Total
confirmed
Aeromonas
per
sample
A
*
(
C/
B)
=
D
Calculated
no.
of
confirmed
Aeromonas
per
colony
type
8
4
QC
Analysis
or
Matrix
filtered
(
please
circle
one):
If
this
report
form
is
for
a
method
blank,
please
indicate
samples
that
are
associated
with
this
method
blank:

ADA­
V
colony
description
(
this
section
is
optional)
No.
of
presumptive
positive
colonies
for
this
colony
type
Please
note:
It
is
important
to
record
the
number
of
colonies
for
each
presumptively
positive
morphological
type
so
that
the
final
density
of
Aeromonas
per
sample
can
be
reported
based
on
percent
confirmation
of
each
colony
type.

Nutrient
agar
colony
description
(
this
section
is
optional)
No.
oxidase
positive
per
colony
type
No.
trehalose
positive
per
colony
type
No.
Indole
positive
per
colony
type
HV=
high
volume
IPR=
initial
precision
and
recovery
MS=
matrix
spike
June
15,
2001,
Revision
­
Draft
NTU=
nephelometric
turbidity
unit
OPR=
ongoing
precision
and
recover
PT=
performance
test
Laboratory
name:
Laboratory
ID:

Method
1622/
23
Bench
Sheet
1.
Client
sample
number
2.
Internal
laboratory
sample
ID
(
if
applicable)

3.
Date
and
time
of
sample
receipt
4.
Received
by
5.
Temperature
of
sample
and
condition
of
sample
upon
arrival
6.
Storage
location
and
storage
temperature
7.
Sample
turbidity,
in
NTU
8.
Sample
type
(
IPR,
method
blank,
field
sample,
OPR,
MS,
PT
sample)

9.
Spiking
suspension
number
(
for
IPR,
OPR,
MS,
and
PT
samples
only)

10.
Estimated
number
of
oocysts/
cysts
spiked
(
for
IPR,
OPR,
MS,
and
PT
samples
only)
Crypto
Giardia
11.
Spiking
date
and
time
12.
Sample
volume
spiked,
in
L
13.
Sample
filtration
start
date
and
time
14.
Type
of
filter
used
(
Envirochek,
Envirochek
HV,
FiltaMax,
CrypTest,
other
[
specify])
and
lot
number:

15.
Name
of
analyst
performing
filtration
16.
Sample
volume
filtered,
to
nearest
¼
L
(
do
not
include
rinse
volume)

17.
Did
filter
clog?

18.
Elution
date
and
time
(
must
be
performed
within
96
hours
of
sample
collection/
filtration)

These
steps
must
be
completed
in
one
working
day
19.
Elution
procedure:
9
wrist
shaker
9
FiltaMax
wash
station
9
stomacher
9
backflush/
sonication
20.
Name
of
analyst
performing
elution
21.
Elution
buffer:_________________________
Elution
buffer
lot
number
and
expiration
date:

22.
Concentration
procedure
(
centrifugation,
FiltaMax
concentrator,
other
[
specify])

23.
Name
of
analyst
performing
concentration
24.
Pellet
volume
after
concentration,
in
mL
25.
(
a)
Total
volume
of
resuspended
concentrate;
(
b)
volume
transferred
to
IMS
(
in
mL)
(
a)
(
b)

26.
Number
of
subsamples
processed
independently
through
the
remainder
of
the
method
27.
IMS
system
used
(
Dynal
anti­
Cryptosporidium,
Dynal
GC­
Combo,
other
[
specify])
and
lot
number
27.
Name
of
analyst
performing
IMS
procedure
28.
Slide(
s)
used
(
Meridian,
Dynal,
other
[
specify])
and
lot
number
29.
Date
and
time
sample
applied
to
slide(
s)
to
dry
(
must
be
completed
same
working
day
as
Row
18)

30.
Detection
kit
used
(
Merifluor,
AquaGlo,
Crypt­
a­
Glo,
Giardi­
a­
Glo,
other
[
specify])
and
lot
number
31.
Analyst
performing
staining
procedure
32.
Staining
completion
date
and
time
(
must
be
complete
within
72
hours
of
Row
29)

33.
Total
number
of
oocysts
and
cysts
counted
in
sample
(
sum
of
counts
in
subsamples,
if
applicable)
Crypto
Giardia
Comments:
June
15,
2001,
Revision
­
Draft
Laboratory
name:
Laboratory
ID:

Method
1622/
1623
Cryptosporidium
Report
Form
Client
sample
number:
Internal
laboratory
sample
ID
(
if
applicable):

10­
mL
subsample
ID
(
if
packed
pellet
>
0.5
mL):
Volume
examined
(
in
L)
on
this
slide:

Analyst:
Positive
staining
control
acceptable
9
YES
9
NO
Negative
staining
control
acceptable
9
YES
9
NO
Object
located
by
FA
No.
Shape
(
oval
or
round)
Size
L
x
W
(
Fm)
DAPI
­
DAPI
+
D.
I.
C.

Light
blue
internal
staining,
no
distinct
nuclei,

green
rim
(
A)
Intense
blue
internal
staining
(
B)
Number
of
nuclei
stained
sky
blue
(
C)
Empty
oocysts
(
D)
Oocysts
with
amorphous
structure
(
E)
Oocysts
with
internal
structure
(
F)

Number
of
sporozoites
1
2
3
4
5
6
7
8
9
10
Total
FA
number
from
this
slide:
Examination
completion
date:

Examination
completion
time
(
must
be
complete
within
7
days
of
staining):

DAPI
­:
Total
number
(
A):
D.
I.
C.
­
Total
number
of
empty
oocysts
(
D):

DAPI
+:
Total
number
(
B):
D.
I.
C.
­
Total
number
of
oocysts
with
amorphous
structure
(
E):

DAPI
+:
Total
number
(
C):
D.
I.
C.
­
Total
number
of
oocysts
with
internal
structure
(
F):

Total
count
DAPI
+
(
C)
that
show
structure
by
D.
I.
C.
(
F):
June
15,
2001,
Revision
­
Draft
Laboratory
name:
Laboratory
ID
(
if
applicable):

Method
1623
Giardia
Report
Form
Client
sample
number:
Internal
laboratory
sample
ID
(
if
applicable):

10­
mL
subsample
ID
(
if
packed
pellet
>
0.5
mL):
Volume
examined
(
in
L)
on
this
slide:

Analyst:
Pos.
staining
control
acceptable
9
YES
9
NO
Neg.
staining
control
acceptable
9
YES
9
NO
Object
located
by
FA
No.
Shape
(
oval
or
round)
Size
L
x
W
(
Fm)
DAPI
­
DAPI
+
D.
I.
C.

Light
blue
internal
staining,

no
distinct
nuclei,
green
rim
(
A)
Intense
blue
internal
staining
(
B)
Number
of
nuclei
stained
sky
blue
(
C)
Empty
cysts
(
D)
Cysts
with
amorphous
structure
(
E)
Cysts
with
internal
structure
(
F)

Number
of
nuclei
Median
body
Axonemes
1
2
3
4
5
6
7
8
9
10
Total
FA
number
from
this
slide:
Examination
completion
date:

Examination
completion
time
(
must
be
complete
within
7
days
of
staining):

DAPI­:
Total
number
(
A):
D.
I.
C.:
Total
number
of
empty
cysts
(
D):

DAPI+:
Total
number
(
B):
D.
I.
C.:
Total
number
of
cysts
with
amorphous
structure
(
E):

DAPI+:
Total
number
(
C):
D.
I.
C.:
Total
number
of
cysts
with
one
internal
structure
(
F):

Total
number
DAPI
+
(
C)
that
show
structure
by
D.
I.
C.
(
F):
D.
I.
C.:
Total
number
of
cysts
with
>
one
internal
structure
(
F):
APPENDIX
E
EPA
HEADQUARTERS
AND
REGIONAL
ATP
CONTACTS
Headquarters
William
Telliard
Director,
Analytical
Methods
Attn:
ATP
Program
Coordinator
Mail
Code
4303T
U.
S.
EPA
Office
of
Water,
Office
of
Science
and
Technology
1200
Pennsylvania
Avenue,
NW
Washington,
DC
20460
Region
1
Arthur
Clark
QA
Chemist
USEPA
Region
1
EQA
60
Westview
Street
Lexington,
MA
02173
Region
2
Carol
Lynes
ATP
Program
Coordinator
USEPA
Region
2
Division
of
Science
and
Monitoring
2890
Woodbridge
Avenue
(
MS­
220)
Building
10
Edison,
NJ
08837­
3679
Region
3
Charles
Jones
Regional
QA
Officer
USEPA
Region
3
Environmental
Assessment
and
Protection
Division
1650
Arch
Street,
3ES­
10
Philadelphia,
PA
19103­
2029
Region
4
Wayne
Turnbull
Chemist/
ATP
Program
Coordinator
USEPA
Region
4
Room:
SESD
960
College
Station
Road
Athens,
GA
30605­
2720
Region
5
Kenneth
Gunter
ATP
Program
Coordinator
USEPA
Region
5
77
W.
Jackson
Blvd.,
WT­
15J
Chicago,
IL
60604
Region
6
David
Stockton
USEPA
Region
6
Laboratory
Houston
Branch
10625
Fallstone
Road
(
6MD­
HI)
Houston,
TX
77099
Region
7
Doug
Brune
ATP
Program
Coordinator
USEPA
Region
7
726
Minnesota
Avenue,
ENSV/
QA
Kansas
City,
KS
66101
Region
8
Rick
Edmonds
Regional
Quality
Assurance
Officer
USEPA
Region
8
999
18th
Street
­
Suite
500
(
8TMS­
L)
Denver,
CO
80202­
2466
Region
9
Roseanne
Sakamoto
ATP
Program
Coordinator
USEPA
Region
9
75
Hawthorne
Street,
PMD­
3
San
Francisco,
CA
94105
Region
10
Bruce
Woods
QAO
USEPA
Region
10
200
Sixth
Avenue,
OEA­
095
Seattle,
WA
98101
