Microbial
Laboratory
Guidance
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule)

June
2003
Draft
Office
of
Water
(
4607)
EPA
815­
R­
03­
XXX
http://
www.
epa.
gov/
safewater/
lt2/
index.
html
June
2003
Printed
on
Recycled
Paper
Disclaimer
The
Standards
and
Risk
Management
Division,
of
the
Office
of
Ground
Water
and
Drinking
Water,
has
reviewed
and
approved
this
guidance
for
publication.
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
trade
names
or
commercial
products
does
not
constitute
endorsement
or
recommendation
for
use.

Questions
concerning
this
document
or
its
application
should
be
addressed
to:

Mary
Ann
Feige
U.
S.
EPA
Office
of
Ground
Water
and
Drinking
Water
Technical
Support
Center
Room
127
26
West
Martin
Luther
King
Drive
Cincinnati,
OH
45268­
1320
(
513)
569­
7944
(
513)
569­
7191
(
facsimile)
feige.
maryann@
epa.
gov
Draft
June
2003
TABLE
OF
CONTENTS
Section
1:
Introduction
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1
1.1
Background
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1
1.2
LT2
Rule
Microbial
Monitoring
Requirements
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2
1.3
Use
of
Cryptosporidium
Data
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3
1.3.1
Cryptosporidium
Field
Sample
Data
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3
1.3.2
Cryptosporidium
Matrix
Spike
Data
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4
1.4
Use
of
E.
coli
Data
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4
Section
2:
General
MicrobialLaboratory
Quality
Assurance
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6
2.1
Quality
Assurance
Plans
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6
2.2
Sample
Temperature
Monitoring
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8
Section
3:
Guidance
for
Cryptosporidium
Laboratories
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10
3.1
LT2
Rule
Cryptosporidium
Sample
Analysis
Requirements
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10
3.1.1
Approved
Laboratories
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10
3.1.2
Revised
Cryptosporidium
Method
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10
3.1.3
Minimum
Sample
Volume
Analysis
Requirements
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11
3.1.4
Spiking
Suspensions
Requirements
for
Spiked
Quality
Control
Samples
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12
3.1.5
Acceptable
Sample
Results
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12
3.1.6
Cryptosporidium
Oocyst
Counts
to
Report
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12
3.2
Laboratory
Quality
Assurance
Evaluation
Program
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13
3.2.1
Application
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13
3.2.2
Personnel
Qualifications
and
Training
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14
3.2.3
Initial
Proficiency
Testing
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15
3.2.4
On­
Site
Evaluation
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15
3.2.5
Ongoing
Proficiency
Testing
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15
3.2.6
Changing
Status
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16
3.2.7
Notifying
Utilities
of
Laboratory
Status
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17
3.3
Cryptosporidium
Method
Quality
Control
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17
3.3.1
Cryptosporidium
Spiking
Materials
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19
3.3.2
Initial
Precision
and
Recovery
Test
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19
3.3.3
Method
Blank
Test
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20
3.3.4
Ongoing
Precision
and
Recovery
Test
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20
3.3.5
Ongoing
Precision
and
Recovery
Control
Charts
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20
3.3.6
Quality
Control
Batches
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21
3.3.7
Holding
Time
Requirements
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21
3.3.8
Staining
Controls
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23
3.3.9
Examination
Preparation
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24
3.3.10
Ongoing
Analyst
Verification
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24
3.3.11
Proficiency
Testing
Samples
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24
3.3.12
Acceptance
Criteria
for
Receipt
of
Field
Samples
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24
3.3.13
Matrix
Spike
Samples
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25
3.3.14
QC
Guidance
for
Method
Modifications
and
Use
of
Multiple
Method
Variations
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26
3.3.15
Guidance
on
QC
Requirements
for
Different
Sample
Volumes
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28
Draft
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3.4
Sample
Collection
Procedures
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29
3.5
Sample
Processing
and
Analysis
Procedures
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29
3.6
Recordkeeping
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29
3.7
Calculations
for
EPA
Methods
1622/
1623
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32
3.7.1
Field
Sample
Calculations
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32
3.7.2
Matrix
Spike
Sample
Calculations
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34
3.7.3
OPR
Sample
Calculations
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35
3.8
Electronic
Data
Reporting
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35
3.8.1
Data
Entry/
Upload
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36
3.8.2
PWS
Data
Review
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37
3.8.3
EPA/
State
Review
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37
3.9
Data
Archiving
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37
3.9.1
Hardcopy
Data
.
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38
3.9.2
Slides
.
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38
3.10
Equipment,
Supplies,
Reagents,
and
Standards
.
.
.
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.
.
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39
3.11
Vendor
Contact
List
.
.
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39
3.11.1
Sample
Collection
and
Filtration
Supplies
.
.
.
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.
39
3.11.2
Sample
Concentration
and
Purification
Supplies
.
.
.
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40
3.11.3
Slides
and
Related
Supplies
.
.
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41
3.11.4
Antibody
Stains
.
.
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41
3.11.5
Microscope
Equipment
.
.
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42
3.11.6
Spiking
Suspensions
and
Positive
Staining
Control
Materials
.
.
.
.
.
.
.
.
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.
.
.
42
3.11.7
Other
Laboratory
Supplies
for
EPA
Method
1622/
1623
.
.
.
.
.
.
.
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.
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.
43
Section
4:
Guidance
for
E.
coli
Laboratories
.
.
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44
4.1
Laboratory
Certification
Program
.
.
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.
44
4.2
Quality
Assurance/
Quality
Control
for
E.
coli
Analyses
.
.
.
.
.
.
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.
45
4.2.1
Quality
Control
Specifications
Applicable
to
All
E.
coli
Methods
.
.
.
.
.
.
.
.
.
.
46
4.2.2
Quality
Control
Specifications
for
Most
Probable
Number
Methods
.
.
.
.
.
.
.
.
48
4.2.3
Quality
Control
Specifications
for
Membrane
Filtration
Methods
.
.
.
.
.
.
.
.
.
.
49
4.3
Sample
Collection
Procedures
.
.
.
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.
50
4.3.1
Acceptance
Criteria
for
Receipt
of
Field
Samples
.
.
.
.
.
.
.
.
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.
.
51
4.4
Sample
Volume
and
Dilution
Guidance
.
.
.
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.
.
51
4.4.1
Sample
Volume
and
Dilution
Guidance
for
Multiple­
Well
Methods
.
.
.
.
.
.
.
51
4.4.2
Sample
Volume
and
Dilution
Guidance
for
Membrane
Filtration
.
.
.
.
.
.
.
.
.
.
51
4.4.3
Sample
Volume
and
Dilution
Guidance
for
Multiple­
Tube
Methods
.
.
.
.
.
.
.
52
4.5
E.
coli
Data
Recording
and
Calculations
.
.
.
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52
4.5.1
Multiple­
Well
Data
.
.
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53
4.5.2
Membrane
Filtration
Data
.
.
.
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.
55
4.5.3
Multiple­
Tube
Data
.
.
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59
4.6
Electronic
Data
Reporting
.
.
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.
62
4.6.1
Data
Entry/
Upload
.
.
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.
63
4.6.2
PWS
Data
Review
.
.
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.
63
4.6.3
EPA/
State
Review
.
.
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.
64
4.7
Vendor
Contact
List
for
E.
coli
Methods
.
.
.
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.
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.
64
Section
5:
References
.
.
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.
66
Section
6:
Acronyms
.
.
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.
67
Draft
June
2003
TABLES
Table
1­
1.
LT2
Rule
Monitoring
Requirements
.
.
.
.
.
.
.
.
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.
3
Table
1­
2.
Bin
Classifications
.
.
.
.
.
.
.
.
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.
3
Table
3­
1.
Laboratory
QA
Program
Personnel
Qualifications
.
.
.
.
.
.
.
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.
.
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.
.
.
.
.
.
.
.
.
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.
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.
.
.
14
Table
3­
2.
Cryptosporidium
Holding
Times
for
EPA
Method
1622/
1623
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
23
Table
3­
3.
Sample
Receipt
Data
Elements
to
Record
in
the
Laboratory
for
EPA
Method
1622/
1623
Cryptosporidium
Analysis
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
31
Table
3­
4.
Primary
Data
Elements
to
Record
in
the
Laboratory
for
EPA
Method
1622/
1623
Cryptosporidium
Analysis
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
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.
.
.
.
.
.
.
31
Table
3­
5.
QC
Data
Elements
to
Record
in
the
Laboratory
for
EPA
Method
1622/
1623
Cryptosporidium
Analysis
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
32
Table
4­
1.
Approved
E.
coli
Methods
for
LT2
Rule
and
Corresponding
Drinking
Water
Certification
Program
Techniques
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
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.
.
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.
.
.
.
45
Table
4­
2.
Quality
Control
Procedures
for
E.
coli
Methods
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
46
Table
4­
3.
Positive
and
Negative
Control
Cultures
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
48
Table
4­
4.
Incubation
Time
and
Temperature
Specifications
for
MPN
Methods
.
.
.
.
.
.
.
.
.
.
.
.
.
.
49
Table
4­
5.
Incubation
Time
and
Temperature
Specifications
for
Membrane
Filter
Methods
.
.
.
.
.
50
Table
4­
6.
Minimum
Sample
ID
Information
and
General
Sample
Data
to
Record
.
.
.
.
.
.
.
.
.
.
.
.
53
Table
4­
7.
Minimum
Data
to
Record
for
Quanti­
tray
2000
®
Colilert
®
and
Colilert­
18
®
Analyses
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
53
Table
4­
8.
Minimum
Data
to
Record
for
Quanti­
Tray
®
Colilert
®
and
Colilert­
18
®
Analyses
.
.
.
55
Table
4­
9.
Minimum
Data
Elements
for
Record
for
Membrane
Filtration
Analyses
.
.
.
.
.
.
.
.
.
.
.
56
Table
4­
10.
Minimum
Data
Elements
to
Record
for
15­
Tube
MPN
Methods
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
59
Table
4­
11.
Minimum
Data
Elements
to
Record
for
15­
Tube
Fermentation
Methods
.
.
.
.
.
.
.
.
.
.
.
60
Table
4­
12.
Examples
of
Different
Combinations
of
Positive
Tubes
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
61
FIGURES
Figure
3­
1.
Process
for
Laboratory
Approval
and
Changes
in
Laboratory
Approval
Status
.
.
.
.
.
.
18
Figure
3­
2.
Ongoing
Precision
and
Recovery
Control
Chart
Example
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
21
Draft
June
2003
APPENDICES
Appendix
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
Laboratory
Biosafety
Guidelines
Appendix
B
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
EPA
Method
1622
for
Cryptosporidium
(
June
2003)

Appendix
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
EPA
Method
1623
for
Cryptosporidium
and
Giardia
(
June
2003)

Appendix
D
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Cryptosporidium
Sample
Results
Acceptability
Checklist
for
the
LT2
Rule
Appendix
E
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Cryptosporidium
Laboratory
QA
Program
Application
Appendix
F
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Cryptosporidium
Laboratory
QA
Program
Audit
Checklist
Appendix
G
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
EPA
Method
1622/
1623
Bench
Sheet
Appendix
H
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
EPA
Method
1622/
1623
Cryptosporidium
Examination
Results
Form
Appendix
I
.
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Standard
Methods
9223B:
Colilert
®
Appendix
J
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Standard
Methods
9223B:
Colilert­
18
®
Appendix
K
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Standard
Methods
mEndo/
LES­
Endo6NA­
MUG
and
mFC6NA­
MUG
Appendix
L
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EPA
Method
1103.1
Appendix
M
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EPA
Method
1603:
Modified
mTEC
Appendix
N
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EPA
Method
1604:
MI
Medium
Appendix
O
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m­
ColiBlue24
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Broth
Appendix
P
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Standard
Methods
9221B/
9221F:
LTB
6EC­
MUG
Appendix
Q
.
.
.
E.
coli
Most
Probable
Number
Sample
Results
Acceptability
Checklist
for
the
LT2
Rule
Appendix
R
.
.
.
.
.
E.
coli
Membrane
Filtration
Sample
Results
Acceptability
Checklist
for
the
LT2
Rule
Appendix
S
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E.
coli
Report
Forms
for
Colilert
 
97­
Well
Procedures
Appendix
T
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E.
coli
Report
Form
for
Colilert
 
51­
Well
Procedures
Appendix
U
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E.
coli
Report
Form
for
Stamdard
Methods
9222B/
9222G
Appendix
V
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E.
coli
Report
Form
for
Standard
Methods
9222D/
9222G
Appendix
W
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E.
coli
Report
Form
for
Standard
Methods
9213D
mTEC
Appendix
X
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E.
coli
Report
Form
for
EPA
Method
1603
modified
mTEC
Appendix
Y
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E.
coli
Report
Form
for
EPA
Method
1604
MI
Medium
Appendix
Z
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E.
coli
Report
Form
for
mColiBlue24
®
Broth
Appendix
AA
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E.
coli
Report
Form
for
Multiple­
Tube
Fermentation
Methods
Appendix
AB
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E.
coli
Report
Form
for
Multiple­
Tube
Most
Probable
Number
Methods
1
Draft
June
2003
SECTION
1:
INTRODUCTION
The
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2ESWTR
or
LT2
rule)
requires
public
water
systems
(
PWSs)
that
use
surface
water
or
groundwater
under
the
direct
influence
of
surface
water
to
monitor
their
source
water
(
influent
water
prior
to
treatment)
for
Cryptosporidium,
E.
coli,
and
turbidity
for
a
limited
period
[
40
CFR
part
141.701(
a)­(
h)].
In
support
of
the
monitoring
requirements
specified
by
the
rule,
three
documents
have
been
developed
to
provide
guidance
to
the
affected
PWSs
and
the
laboratories
that
support
them:

1.
Source
Water
Monitoring
Guidance
Manual
for
Public
Water
Systems
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).
This
guidance
manual
for
PWSs
affected
by
the
rule
provides
information
on
laboratory
contracting,
sample
collection
procedures,
and
data
evaluation
and
interpretation
advice.

2.
Microbial
Laboratory
Guidance
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule)
(
this
document).
The
goal
of
this
manual
is
to
provide
Cryptosporidium
and
E.
coli
laboratories
analyzing
samples
in
support
of
the
LT2
rule
with
guidance
and
detailed
procedures
for
all
aspects
of
microbial
analyses
under
the
rule
to
maximize
data
quality
and
consistency.

3.
User's
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule)
Data
Collection
System.
This
manual
provides
PWSs
and
laboratories
with
instructions
on
the
entry,
review,
and
approval
of
electronic
data
using
the
LT2
Data
Collection
System,
and
for
generating
reports
using
the
system.

All
of
these
manuals
are
available
at
http://
www.
epa.
gov/
safewater/
lt2/
index.
html.
Responses
to
frequently
asked
questions
(
FAQs)
on
sampling,
analysis,
and
data
reporting
questions
for
the
LT2
rule
also
are
available
on
this
website.

This
guidance
document
is
provided
to
help
implement
the
LT2
rule.
This
guidance
document
does
not,
however,
substitute
for
the
LT2
rule
or
the
analytical
methods
approved
for
use
under
the
rule.
The
material
presented
is
intended
solely
for
guidance
and
does
not
alter
any
regulatory
or
analytical
method
requirements
not
altered
by
the
LT2
rule
itself.

Sections
1
and
2
of
the
microbial
laboratory
LT2
manual
provide
LT2
background
information
and
guidance
on
issues
that
apply
to
both
Cryptosporidium
and
E.
coli
laboratories.
Section
3
provides
guidance
specific
to
Cryptosporidium
analyses
for
the
LT2
rule,
and
Section
4
provides
guidance
for
E.
coli
analyses
performed
in
support
of
the
LT2
rule.

1.1
Background
The
LT2
rule
is
a
National
Primary
Drinking
Water
Regulation
that
requires
monitoring,
reporting,
and
public
notification
requirements
for
all
PWSs
that
use
surface
water
sources.
The
rule
requires
additional
treatment
techniques
for
some
systems,
based
on
Cryptosporidium
monitoring
results
(
40
CFR
part
141.720
­
141.721).
The
LT2
rule
was
developed
to
improve
control
of
microbial
pathogens,
including
specifically
the
protozoan
Cryptosporidium,
in
drinking
water
and
to
address
risk
trade­
offs
with
disinfection
byproducts.
Section
1:
Introduction
2
Draft
June
2003
The
LT2
rule
provides
for
increased
protection
against
microbial
pathogens
in
public
water
systems
that
use
surface
water
sources.
The
rule
focuses
on
Cryptosporidium,
a
protozoan
pathogen
that
is
widespread
in
surface
waters.
EPA
is
particularly
concerned
about
Cryptosporidium
because
it
is
highly
resistant
to
inactivation
by
standard
disinfection
practices.
Ingestion
of
Cryptosporidium
oocysts
can
cause
acute
gastrointestinal
illness,
and
symptoms
in
sensitive
subpopulations
may
be
severe,
including
risk
of
mortality.
Cryptosporidium
has
been
identified
as
the
pathogenic
agent
in
a
number
of
waterborne
disease
outbreaks.

EPA
convened
a
Federal
Advisory
Committee
to
develop
recommendations
for
both
the
Stage
2
Disinfectants
and
Disinfection
Byproducts
Rule
and
the
LT2
rule.
As
recommended
by
the
Federal
Advisory
Committee,
the
LT2
rule
requires
public
water
systems
that
use
surface
water
or
ground
water
under
the
direct
influence
of
surface
water
to
monitor
their
source
water
(
influent
water
prior
to
treatment
plant)
for
Cryptosporidium,
E.
coli
and
turbidity
[
40
CFR
part
141.701
(
a)­(
h)].
These
data
would
be
used
to
determine
whether
additional
treatment
is
needed
at
PWSs
and
to
assess
whether
a
relationship
could
be
established
between
the
Cryptosporidium
and
E.
coli
levels
in
source
water.
A
summary
of
the
LT2
rule
monitoring
requirements
is
provided
in
Section
1.2.
The
use
of
Cryptosporidium
data
is
discussed
Section
1.3
and
the
use
of
E.
coli
data
is
discussed
in
Section
1.4.

1.2
LT2
Rule
Microbial
Monitoring
Requirements
Large
systems
(
PWSs
serve
a
population
of
at
least
10,000
people)
monitoring
under
the
LT2
rule
are
required
to
collect
and
analyze
source
water
samples
for
Cryptosporidium,
E.
coli
and
turbidity
for
a
minimum
of
2
years
[
40
CFR
part
141.701
(
b)].
Small
systems
(
PWSs
that
serve
fewer
than
10,000
people)
are
required
to
monitor
their
source
water
for
E.
coli
for
a
minimum
of
1
year.
A
subset
of
small
systems
would
then
be
required
to
conduct
Cryptosporidium
analyses
over
a
1­
year
period
if
they
exceed
E.
coli
trigger
levels
[
40
CFR
part
141.701
(
c)].

Monitoring
requirements
for
each
system
size,
and
the
schedule
for
each
stage
of
monitoring,
is
described
in
Table
1­
1.

Detailed
guidance
for
sample
collection
during
the
LT2
rule,
and
procedures
for
sample
collection,
documentation,
and
shipment,
are
provided
in
Source
Water
Monitoring
Guidance
Manual
for
Public
Water
Systems
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule)
(
at
http://
www.
epa.
gov/
safewater/
lt2/
index.
html).
Cryptosporidium
samples
must
be
analyzed
by
a
laboratory
approved
for
analysis
under
the
Laboratory
Quality
Assurance
Evaluation
Program
for
the
Analysis
of
Cryptosporidium
Under
the
Safe
Drinking
Water
Act
(
Section
3.2,
below)
using
EPA
Method
1622/
1623
[
40
CFR
part
141.706
(
a)].

E.
coli
samples
must
be
analyzed
using
methods
approved
under
the
LT2
rule
for
surface
water
monitoring
(
Section
4,
below)
[
40
CFR
part
141.705
(
b)].
Source
water
turbidity
must
be
measured
when
Cryptosporidium
and
E.
coli
samples
are
collected
using
methods
approved
under
the
LT2
rule
[
40
CFR
part
141.705
(
c)].
Section
1:
Introduction
3
Draft
June
2003
Table
1­
1.
LT2
Rule
Monitoring
Requirements
Public
water
system
size
Monitoring
begins
Monitoring
duration
Monitoring
parameters
and
sample
frequency
requirements
Cryptosporidium
E.
coli
Large
systems
(
serving
10,000
or
more
people)
6
months
after
promulgation
of
LT2
rule
2
yearsa
minimum
1
sample/
monthc
minimum
1
sample/
monthd
Small
systems
(
serving
fewer
than
10,000
people)
30
months
(
2
½
years)
after
promulgation
of
LT2
rule
1
yeara,
b
see
below
§
1
every
2
weeks
§
Possible
additional
monitoring
requirement
for
Cryptosporidium
If
small
systems
exceed
E.
coli
trigger
levels,
then.
.
.

Small
systems
(
serving
fewer
than
10,000
people)
48
months
(
4
years)
after
promulgation
of
LT2ESWTR
1
year
2
sample/
month
N/
A
a
PWSs
may
be
eligible
to
use
historical
(
grandfathered)
data
in
lieu
of
these
requirements
if
certain
quality
assurance
and
quality
control
criteria
are
met
b
Small
systems
may
be
required
to
monitor
for
Cryptosporidium
for
one
year,
beginning
6
months
after
completion
of
E.
coli
monitoring;
Cryptosporidium
monitoring
required
if
the
E.
coli
annual
mean
concentrations
exceed
10/
100
mL
for
systems
using
lakes/
reservoirs
or
exceed
50/
100
mL
for
systems
using
flowing
streams
c
PWSs
monitoring
for
Cryptosporidium
may
collect
more
than
1
sample
per
month
if
sampling
is
evenly
spaced
over
the
monitoring
period
d
Not
applicable
to
large,
unfiltered
systems
because
these
systems
are
not
required
to
monitor
for
E.
coli
or
turbidity
N/
A
=
Not
applicable;
no
monitoring
required
1.3
Use
of
Cryptosporidium
Data
Two
types
of
Cryptosporidium
data
are
collected
under
the
LT2
rule:
Cryptosporidium
occurrence
data
from
the
analysis
of
field
samples,
and
method
performance
data
from
the
analysis
of
matrix
spike
(
MS)
samples.
The
use
of
occurrence
data
from
field
samples
is
discussed
in
Section
1.3.1;
the
use
of
method
performance
data
from
MS
samples
is
discussed
in
Section
1.3.2.

1.3.1
Cryptosporidium
Field
Sample
Data
The
concentration
of
Cryptosporidium
oocysts
in
source
water
samples
analyzed
during
the
LT2
rule
will
be
used
to
calculate
a
mean
Cryptosporidium
concentration
for
a
PWS
and
classify
the
PWSs
into
a
treatment
requirements
"
bin"
(
40
CFR
part
141.709).
These
bin
classifications
are
provided
in
Table
1­
2.
The
treatment
bin
classification
established
for
each
PWS
will
be
used
to
determine
whether
additional
treatment
is
needed.
PWSs
in
Bin
1
are
not
required
to
implement
additional
treatment.
PWSs
in
Bins
2
­
4
will
be
required
to
implement
increasing
levels
of
treatment
and
source
water
protection
to
address
their
higher
risk
for
high
Cryptosporidium
source
water
concentrations.

Table
1­
2.
Bin
Classifications
Cryptosporidium
Bin
Concentration
Bin
Classification
Cryptosporidium
<
0.075/
L
Bin
1
0.075/
L
#
Cryptosporidium
<
1.0/
L
Bin
2
1.0/
L
#
Cryptosporidium
<
3.0/
L
Bin
3
Cryptosporidium
$
3.0/
L
Bin
4
Section
1:
Introduction
4
Draft
June
2003
The
method
used
to
average
individual
sample
concentrations
to
determine
a
PWS's
bin
classification
depends
on
the
number
of
samples
collected
and
the
length
of
the
sampling
period.
For
PWS
serving
at
least
10,000
people
bin
classification
would
be
based
on
the
following:

°
For
PWSs
that
collect
at
least
48
samples
during
the
required
monitoring
period,
the
Cryptosporidium
bin
calculation
is
equal
to
the
mean
of
all
sample
concentrations
°
For
PWSs
that
collect
at
least
24
samples,
but
not
more
than
47
samples,
during
the
required
monitoring
period,
the
Cryptosporidium
bin
calculation
is
equal
to
the
highest
average
of
all
sample
concentrations
in
any
12
consecutive
months
in
the
monitoring
period
For
PWSs
serving
fewer
than
10,000
people,
bin
classification
would
be
based
on
the
highest
running
six
month
average
because
these
systems
collect
samples
twice
a
month
for
1
year.

1.3.2
Cryptosporidium
Matrix
Spike
Data
During
LT2
rule
Cryptosporidium
monitoring,
PWSs
are
required
to
analyze,
at
a
minimum,
one
MS
sample
for
every
20
field
samples
from
their
source
water
[
40
CFR
part
141.705
(
a)
(
2)].
Details
on
MS
sample
requirements
are
provided
in
Section
3.3.13.

For
PWSs
in
the
following
categories,
two
MS
samples
will
need
to
be
analyzed
during
LT2
rule
monitoring:

°
PWSs
serving
more
than
10,000
people,
that
perform
monthly
monitoring
for
2
years,
and
that
collect
24
field
samples
°
PWSs
serving
fewer
than
10,000
people,
that
are
triggered
into
monitoring
for
1
year,
and
that
collect
24
field
samples
For
PWSs
serving
more
than
10,000
people
and
that
perform
semi­
monthly
monitoring
for
2
years
and
collect
48
or
more
samples,
3
MS
samples
will
be
analyzed.

Although
MS
sample
results
will
not
be
used
to
adjust
Cryptosporidium
recoveries
at
any
individual
source
water,
the
results
will
be
used
collectively
to
assess
overall
recovery
and
variability
for
EPA
Method
1622/
1623
in
source
water.
The
descriptive
statistics
of
the
MS
sample
results
will
be
compared
to
the
performance
of
the
methods
during
the
Information
Collection
Rule
Supplemental
Surveys
to
verify
the
assumptions
on
method
performance
upon
which
the
LT2
rule
was
based.

When
considering
the
method
performance
that
could
be
achieved
for
analysis
of
Cryptosporidium
under
the
LT2
rule,
the
Federal
Advisory
Committee
evaluated
the
results
of
EPA
Methods
1622/
1623
in
the
ICRSS,
which
involved
87
PWSs
sampling
twice
per
month
over
1
year
for
Cryptosporidium
and
other
parameters.
During
the
ICRSS,
the
mean
Cryptosporidium
recovery
and
mean
relative
standard
deviation
of
the
MS
samples
were
43%
and
49%,
respectively
(
Reference
5.1).

1.4
Use
of
E.
coli
Data
E.
coli
data
are
being
collected
by
large
systems
during
LT2
rule
monitoring
to
assess
whether
a
relationship
can
be
established
between
the
Cryptosporidium
and
E.
coli
levels
in
source
water
and
a
microbial
index
developed
to
establish
trigger
levels
for
E.
coli
that
would
indicate
high
Cryptosporidium
concentrations
in
a
source
water
(
40
CFR
part
141.701).
If
a
relationship
can
be
established,
small
systems
initially
will
be
permitted
to
monitor
for
E.
coli,
rather
than
more
expensive
Cryptosporidium
analyses.
Only
those
systems
with
E.
coli
levels
above
the
trigger
level
established
in
the
microbial
index
would
then
be
required
to
monitor
for
Cryptosporidium
to
determine
bin
placement
(
40
CFR
part
141.702).
5
Draft
June
2003
A
preliminary
index
was
developed
during
LT2
rule
development
using
data
from
the
Information
Collection
Rule
(
ICR)
and
ICRSS.
These
data
were
evaluated
for
parameters
that
could
indicate
the
likelihood
that
a
source
water
mean
Cryptosporidium
level
would
be
above
the
Bin
2
threshold
concentration
of
0.075
oocysts/
L.
Initially,
fecal
coliforms,
total
coliforms,
E.
coli,
viruses
(
ICR
only),
and
turbidity
were
assessed
for
development
of
the
microbial
index.
Data
analyses
placed
greater
emphasis
on
E.
coli
and
fecal
coliforms
because
of
the
direct
relationship
between
these
parameters
and
fecal
contamination.
E.
coli
was
determined
to
provide
the
best
performance
as
a
Cryptosporidium
indicator
with
the
available
data.
After
E.
coli
levels
were
developed
as
a
screening
tool,
turbidity
was
considered
in
an
effort
to
enhance
the
screening
tool.
However,
turbidity
was
not
found
to
improve
accuracy.

Based
on
the
data
from
the
ICR
and
ICRSS,
the
preliminary
E.
coli
trigger
levels
were
set
at
a
mean
of
50
E.
coli/
100
mL
for
flowing
stream
 
type
source
waters
and
10
E.
coli/
100
mL
for
reservoir/
lake
source
waters.
6
Draft
June
2003
SECTION
2:
GENERAL
MICROBIAL
LABORATORY
QUALITY
ASSURANCE
All
laboratories
analyzing
Cryptosporidium
and
E.
coli
samples
for
the
LT2
rule
should
adhere
to
defined
quality
assurance
(
QA)
procedures
to
ensure
that
analytical
data
generated
under
the
rule
are
scientifically
valid
and
are
of
known
and
acceptable
quality.
Detailed
quality
control
(
QC)
requirements
and
recommendations
specific
to
Cryptosporidium
and
E.
coli
analyses
are
discussed
in
Sections
3
and
4
of
this
manual,
respectively.
Two
QA
issues
that
apply
to
both
analyses
 
quality
assurance
plans
and
sample
temperature
monitoring
 
are
discussed
below,
in
Sections
2.1
and
2.2.

2.1
Quality
Assurance
Plans
As
specified
in
both
the
Lab
QA
Program
for
Cryptosporidium
laboratories
(
http://
www.
epa.
gov/
safewater/
lt2/
cla_
final.
html
and
Section
3.2
of
this
manual)
and
the
Laboratory
Certification
Manual
(
Chapter
III,
page
III­
4)
for
E.
coli
laboratories,
each
laboratory
should
operate
a
formal
QA
program
and
should
document
the
scope
of
this
program
through
a
QA
plan.

The
laboratory's
QA
plan
should
be
a
stand­
alone
document.
However,
some
information
can
be
incorporated
into
the
document
by
reference,
including
laboratory
standard
operating
procedures
(
SOPs),
analytical
methods,
and
quality
control
(
QC)
and
calibration
notebooks.
Laboratories
currently
certified
for
coliform
analysis
under
the
drinking
water
laboratory
certification
program
may
use
their
current
QA
plan,
however,
this
plan
should
be
updated
to
address
the
specific
requirements
for
LT2
rule
monitoring.
Topics
that
should
be
addressed
in
the
QA
plan
are
outlined
below.
Details
on
LT2
Cryptosporidium
requirements
are
provided
in
Section
3
of
this
manual;
details
on
LT2
E.
coli
analyses
are
provided
in
Section
4.

For
Cryptosporidium
laboratories,
this
QA
plan
should
be
available
for
review
during
a
laboratory's
onsite
audit,
as
part
of
the
EPA's
Cryptosporidium
Laboratory
Quality
Assurance
Evaluation
Program
(
Section
3.2).
For
E.
coli
laboratories,
this
QA
plan
should
be
available
for
review
during
recertification
audits
as
part
of
the
National
Primary
Drinking
Water
Regulations.

The
following
items
should
be
addressed
in
each
QA
plan:

1.
Laboratory
organization
and
responsibility
°
Include
a
chart
showing
the
laboratory
organization
and
line
authority,
including
QA
Managers
°
List
the
key
individuals
who
are
responsible
for
ensuring
the
production
of
valid
measurements
and
the
routine
assessment
of
QC
measurements
°
Specify
who
is
responsible
for
internal
audits
and
reviews
of
the
implementation
of
the
QA
plan
and
its
requirements
2.
Personnel
°
List
each
analyst's
academic
background
and
water
analysis
experience
°
List
each
analyst's
training
on
the
method
°
Describe
training
available
to
keep
personnel
up
to
date
on
methods
and
regulations
Section
2:
General
Microbial
Laboratory
Quality
Assurance
7
Draft
June
2003
3.
Facilities:
°
Arrangement
and
size
of
laboratory
°
Bench
space
°
Storage
space
°
Lighting
°
Air
system
°
Lab
reagent
water
system
°
Waste
disposal
system
°
Safety
considerations.
The
laboratory
should
address
biosafety
in
the
laboratory
when
handling
or
processing
Cryptosporidium
samples
and
organism
controls.
Guidance
on
laboratory
biosafety
for
Cryptosporidium
is
provided
in
Appendix
A
of
this
guidance
manual.

4.
Field
sampling
procedures
(
with
SOP
used
by
laboratory
or
sent
to
PWS
clients)
°
Describe
how
samples
are
collected,
sample
containers,
sample
storage,
transport
times,
and
sample
temperature
°
Describe
sample
identification
and
information
recording
system
5.
Laboratory
sample
handling
procedures
°
Describe
sample
storage
conditions
°
Describe
the
laboratory's
sample
tracking
system;
specify
procedures
used
to
maintain
the
integrity
of
all
samples,
i.
e.,
logging,
tracking
samples
from
receipt
by
laboratory
through
analysis
to
disposal
°
Describe
sample
acceptance
criteria
6.
Equipment
°
Specifications
for
each
piece
of
equipment
used
for
Cryptosporidium
and/
or
E.
coli
analyses
°
Calibration
procedures,
frequency,
standards
for
each
piece
of
equipment
used
for
Cryptosporidium
and/
or
E.
coli
analyses
°
Quality
control
records
for
each
piece
of
equipment
used
for
Cryptosporidium
and/
or
E.
coli
analyses
°
Preventative
maintenance
and
schedules,
documentation
for
each
piece
of
equipment
used
for
Cryptosporidium
and/
or
E.
coli
analyses
7.
Supplies
°
Laboratory
glassware
and
plasticware
acceptance
conditions
°
Chemicals,
reagents,
dyes
and
culture
media
acceptance
conditions
°
Chemicals,
reagents,
dyes,
and
culture
media
storage
conditions
°
Filters
acceptance
conditions
8.
Laboratory
practices
(
may
reference
SOP)
°
Preparation
of
reagent­
grade
water
°
Glassware
washing
and
preparation
°
Sterilization
procedures
9.
Analytical
procedures
°
Describe
all
reference
methods
used
°
State
that
the
analytical
methods
described
in
this
manual
will
be
followed
°
Identify
available
SOPs
10.
Quality
control
(
QC)
checks
°
Confirmation/
verification
procedures,
frequency
°
Sterility
controls
°
Replicate
analyses;
frequency
°
QC
samples,
source;
frequency
Section
2:
General
Microbial
Laboratory
Quality
Assurance
8
Draft
June
2003
°
Positive
and
negative
controls
proficiency
testing
(
PT)
samples,
source;
frequency
°
Spiked
field
samples
°
Between­
analyst
deviation
11.
Data
reduction,
verification,
validation,
and
reporting
°
Data
reduction
(
conversion
of
raw
data
to
Cryptosporidium
oocysts/
L
and/
or
E.
coli/
100
mL)
°
Procedures
to
ensure
the
accuracy
of
data
transcription
and
calculations
°
Validation
(
ensuring
that
QC
steps
associated
with
a
field
result
are
acceptable)
°
Reporting,
including
procedures
and
format
for
reporting
data
to
utilities/
EPA
12.
Corrective
actions
°
Define
the
laboratory
response
to
unacceptable
results
from
PT
or
QC
samples
and
from
internal
QC
checks
°
Identify
persons
with
responsibility
to
take
corrective
action
°
Describe
how
the
actions
taken
and
the
effectiveness
of
the
actions
taken
will
be
documented
13.
Recordkeeping
°
Describe
how
records
are
to
be
maintained
(
e.
g.,
electronically,
hard
copy,
etc.)
°
Describe
how
long
records
are
to
be
kept
°
State
where
records
are
to
be
stored
The
laboratory
QA
plan
should
be
concise,
but
responsive
to
the
above­
listed
items.
Additional
guidance
on
developing
QA
plans
is
available
in
"
Guidance
on
Quality
Assurance
Project
Plans
(
G­
5),"
(
EPA/
600/
R­
98/
018,
February
1998),
which
is
available
as
a
download
from
http://
www.
epa.
gov/
quality/
qa_
docs.
html#
noneparqt.

However,
the
goals
of
a
lab
QA
plan
in
general
are
different
from
the
goals
of
a
QAPP,
and
not
all
of
the
issues
that
should
be
addressed
for
laboratory
QA
during
the
LT2
rule
are
covered
by
this
guidance
(
i.
e.,
laboratory
sample
handling
and
record
keeping).
However,
because
the
LT2
rule
is
a
finite
study
on
Cryptosporidium
and
E.
coli
levels
in
specific
source
waters,
some
of
the
concepts
presented
in
the
QAPP
guidance
that
typically
are
not
included
in
laboratory
QA
plans
may
aid
the
laboratory
in
updating
their
QA
plan
to
address
specific
LT2
requirements.

2.2
Sample
Temperature
Monitoring
Source
water
samples
are
dynamic
environments
and,
depending
on
sample
constituents
and
environmental
conditions,
Cryptosporidium
oocysts
present
in
a
sample
can
degrade
and
E.
coli
present
in
a
sample
can
grow
or
die
off,
biasing
analytical
results.
Cryptosporidium
and
E.
coli
samples
for
LT2
rule
monitoring
that
are
not
analyzed
the
same
day
they
are
collected
must
be
maintained
below
10
°
C
to
reduce
biological
activity.
This
is
specified
in
Section
8.0
of
the
June
2003
versions
of
EPA
Method
1622/
1623
for
Cryptosporidium
samples
and
at
40
CFR
part
705
(
b)
(
1)
and
Chapter
V,
Section
6.3,
of
the
Laboratory
Certification
Manual
(
Reference
5.2)
for
E.
coli
samples.

Samples
for
all
analyses
should
remain
above
freezing
at
all
times.
This
is
a
requirement
in
Section
8.0
of
the
June
2003
versions
of
EPA
Method
1622/
1623.
Although
not
a
significant
concern
for
10­
L
water
samples,
this
is
a
real
concern
for
Cryptosporidium
filters
and
120­
or
250­
mL
E.
coli
samples
that
are
shipped
off­
site
with
coolant
materials,
such
as
wet
ice,
blue
ice,
or
gel
packs.
E.
coli
holding
time
studies
performed
in
support
of
the
LT2
rule
(
Reference
5.4)
demonstrated
that
E.
coli
samples
can
freeze
under
these
conditions
if
samples
are
not
packed
properly.
Section
2:
General
Microbial
Laboratory
Quality
Assurance
9
Draft
June
2003
The
sample
collection
protocols
procedures
discussed
in
the
Source
Water
Monitoring
Guidance
Manual
for
Public
Water
Systems
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule)
provide
sample
packing
procedures
for
E.
coli
and
Cryptosporidium
samples.
Utility
personnel
should
follow
these
procedures
to
ensure
that
samples
remain
at
acceptable
temperatures
during
shipment.

Because
Cryptosporidium
samples
collected
for
the
LT2
rule
must
meet
the
QC
criteria
in
the
methods
[
40
CFR
part
705
(
a)
(
3)],
and
because
these
QC
criteria
include
receipt
of
samples
at
<
10
°
C
and
not
frozen,
laboratories
must
reject
LT2
Cryptosporidium
samples
that
were
not
collected
the
same
day
they
were
received,
and
are
received
at
>
10
°
C
or
frozen
(
this
is
discussed
further
in
Section
3.3.12
in
this
manual).
In
these
cases,
the
PWS
must
re­
collect
and
re­
ship
the
sample.

Several
options
are
available
to
measure
sample
temperature
upon
receipt
at
the
laboratory
and,
in
some
cases,
during
shipment:

°
Temperature
sample.
One
option,
for
Cryptosporidium
filtered
samples
(
not
for
10­
L
bulk
samples)
and
E.
coli
120­
and
250­
mL
samples,
is
for
the
PWS
to
fill
a
small,
inexpensive
sample
bottle
with
water
and
pack
this
"
temperature
sample"
next
to
the
field
sample.
The
temperature
of
this
extra
sample
volume
is
measured
upon
receipt
to
estimate
the
temperature
of
the
field
sample.
Temperature
sample
bottles
are
not
appropriate
for
use
with
bulk
samples
because
of
the
potential
effect
that
the
difference
in
sample
volume
may
have
in
temperature
equilibration
in
the
sample
cooler.
Example
product:
Cole
Parmer
cat.
no.
U­
06252­
20.

°
Thermometer
vial.
A
similar
option
is
to
use
a
thermometer
that
is
securely
housed
in
a
liquid­
filled
vial.
Unlike
temperature
samples,
the
laboratory
does
not
need
to
perform
an
additional
step
to
monitor
the
temperature
of
the
vial
upon
receipt,
but
instead
just
reads
the
thermometer.
Example
product:
Eagle­
Picher
Sentry
Temperature
Vial
3TR­
40CS­
F
or
3TR­
40CS.

°
iButton.
Another
option
for
measuring
the
sample
temperature
during
shipment
and
upon
receipt
is
a
Thermocron
®
iButton.
An
iButton
is
a
small,
waterproof
device
that
contains
a
computer
chip
to
record
temperature
at
different
time
intervals.
The
information
is
then
downloaded
from
the
iButton
onto
a
computer.
The
iButton
should
be
placed
in
a
temperature
sample
in
the
cooler,
rather
than
placed
directly
in
the
cooler,
where
it
may
be
affected
by
close
contact
with
the
coolant.
Information
on
Thermocron
®
iButtons
is
available
from
http://
www.
ibutton.
com/.
Distributors
include
http://
www.
pointsix.
com/,
http://
www.
rdsdistributing.
com,
and
http://
www.
scigiene.
com/.

°
Stick­
on
temperature
strips.
Another
option
is
for
the
laboratory
to
apply
a
stick­
on
temperature
strip
to
the
outside
of
the
sample
container
upon
receipt
at
the
laboratory.
This
option
does
not
measure
temperature
as
precisely
as
the
other
options,
but
still
mitigates
the
risk
of
sample
contamination
while
providing
an
indication
of
sample
temperature
to
verify
that
the
sample
temperature
is
acceptable.
Example
product:
Cole
Parmer
cat.
no.
U­
90316­
00.

As
with
other
laboratory
equipment,
all
temperature
measurement
devices
should
be
calibrated
routinely
to
ensure
accurate
measurements.
See
the
EPA
Lab
Certification
Manual
(
Reference
5.2)
for
more
information.
10
Draft
June
2003
SECTION
3:
GUIDANCE
FOR
CRYPTOSPORIDIUM
LABORATORIES
Cryptosporidium
analyses
conducted
in
support
of
the
LT2
rule
must
be
performed
using
EPA
Method
1622
or
EPA
Method
1623
[
40
CFR
part
141.705
(
a)].
Guidance
on
the
use
of
these
methods
during
the
LT2
rule
are
provided
in
this
section
of
the
manual.

3.1
LT2
Rule
Cryptosporidium
Sample
Analysis
Requirements
LT2
rule
requirements
of
particular
significance
to
Cryptosporidium
laboratories
are
summarized
in
Sections
3.1.1
through
3.1.6,
below,
and
discussed
in
more
detail
in
the
remainder
of
Section
3.

3.1.1
Approved
Laboratories
Systems
must
have
Cryptosporidium
samples
analyzed
by
a
laboratory
that
has
passed
a
quality
assurance
(
QA)
evaluation
under
EPA's
Laboratory
Quality
Assurance
Evaluation
Program
for
Analysis
of
Cryptosporidium
in
Water
or
a
laboratory
that
has
been
certified
for
Cryptosporidium
analysis
by
an
equivalent
State
laboratory
certification
program
[
40
CFR
part
141.706
(
a)].
Details
on
the
elements
of
the
Lab
QA
Program
QA
evaluation
are
provided
in
Section
3.2.

3.1.2
Revised
Cryptosporidium
Method
EPA
has
proposed
the
use
of
the
April
2001
versions
of
EPA
Methods
1622/
1623
in
the
LT2
rule.
However
EPA
has
requested
comment
on
the
use
of
updated
versions
(
dated
June
2003)
to
consolidate
several
method­
related
changes
EPA
believes
are
necessary
to
address
LT2
rule
monitoring
requirements.
These
changes
include
the
following:

°
Increased
flexibility
in
matrix
spike
(
MS)
and
initial
precision
and
recovery
(
IPR)
requirements.
The
requirement
that
the
laboratory
must
analyze
an
MS
sample
on
the
first
sampling
event
for
a
new
PWS
has
been
changed
to
a
recommendation;
the
revised
method
allows
the
IPR
test
to
be
performed
across
four
different
days,
rather
than
restrict
analyses
to
1
day.

°
Clarification
of
some
method
procedures,
including
the
spiking
suspension
vortexing
procedure;
the
buffer
volumes
used
during
immunomagnetic
separation
(
IMS);
requiring
(
rather
than
recommending)
that
laboratories
purchase
HCl
and
NaOH
standards
at
the
normality
specified
in
the
method;
and
the
use
of
methanol
during
slide
staining
in
Section
14.2
is
per
manufacturer's
instructions.

°
Addition
of
recommendations
for
minimizing
carry­
over
of
debris
onto
microscope
slides
after
IMS
and
information
on
microscope
cleaning.

°
Clarification
of
the
actions
to
take
in
the
event
of
QC
failures
and
clarifies
that
any
positive
sample
in
a
batch
associated
with
an
unacceptable
method
blank
is
unacceptable
and
that
any
sample
in
a
batch
associated
with
an
unacceptable
ongoing
precision
and
recovery
(
OPR)
sample
is
unacceptable.

°
A
change
in
the
sample
storage
and
shipping
temperature
to
"<
10
°
C,
and
not
frozen,"
for
samples
that
are
not
processed
the
day
they
are
collected,
and
provides
additional
guidance
on
sample
storage
and
Section
3:
Guidance
for
Cryptosporidium
Laboratories
11
Draft
June
2003
shipping
procedures
based
on
time
of
day
of
collection.
The
revision
includes
suggested
options
for
monitoring
sample
temperature
during
shipment
and/
or
upon
receipt
at
the
laboratory.

°
Addition
of
the
requirement
of
examination
using
differential
interference
contrast
(
DIC)
microscopy
to
the
analyst
verification
procedure.

°
Addition
of
an
approved
method
modification
using
the
Pall
Gelman
Envirochek
HV
filter.
This
approval
was
based
on
an
interlaboratory
validation
study
demonstrating
that
three
laboratories,
each
analyzing
reagent
water
and
a
different
source
water,
met
all
method
acceptance
criteria
for
Cryptosporidium
(
but
not
Giardia,
however,
individual
laboratories
are
permitted
to
demonstrate
acceptable
performance
in
their
laboratory).

°
Incorporation
of
detailed
procedures
for
concentrating
samples
using
and
IDEXX
Filta­
Max
 
foam
filter.
(
A
method
modifications
using
this
filter
already
is
approved
by
EPA
in
the
April
2001
version
of
the
methods.)

°
Addition
of
BTF
EasySeed
 
irradiated
oocysts
and
cysts
as
acceptable
materials
for
spiking
routine
QC
samples.
EPA
approved
the
use
of
EasySeed
 
based
on
side­
by­
side
comparison
tests
of
method
recoveries
using
EasySeed
 
and
live,
untreated
organisms.

°
Removal
of
the
Whatman
Nuclepore
CrypTest
 
cartridge
filter.
Although
a
method
modification
using
this
filter
was
approved
by
EPA
in
the
April
2001
versions
of
the
methods,
the
filter
is
no
longer
available
from
the
manufacturer,
and
so
is
no
longer
an
option
for
sample
filtration.

The
changes
in
the
June
2003
draft
revisions
of
EPA
Methods
1622
and
1623
reflect
method­
related
clarifications,
modifications,
and
additions
that
EPA
believes
should
be
addressed
for
LT2
rule
Cryptosporidium
monitoring.
Alternatively,
these
issues
could
be
addressed
through
regulatory
requirements
in
the
final
LT2
rule,
however,
EPA
believes
that
addressing
these
issues
through
a
single
source
in
updated
versions
of
EPA
Methods
1622
and
1623
(
which
could
be
approved
in
the
final
rule)
may
be
more
straightforward
and
easier
for
systems
and
laboratories
to
follow
than
addressing
them
in
multiple
sources
(
i.
e.,
existing
methods,
the
final
rule,
and
laboratory
guidance).

This
draft
manual
assumes
that
the
June
2003
versions
of
EPA
Methods
1622
and
1623
will
be
used
for
LT2
rule
monitoring.
The
June
2003
versions
of
these
methods
are
included
as
Appendix
B
and
Appendix
C
of
this
guidance
manual.
If
EPA
determines
that
the
updated
methods
will
not
be
used,
based
on
comments
to
the
proposed
rule,
then
the
rule
may
be
updated
to
include
critical
clarifications
as
requirements
and
this
guidance
document
will
be
updated
to
include
non­
critical
clarifications
as
guidance.

3.1.3
Minimum
Sample
Volume
Analysis
Requirements
Under
LT2
rule
Cryptosporidium
sample
volume
requirements
[
40
CFR
part
141.705
(
a)
(
1)],
PWSs
are
required
to
analyze,
at
a
minimum,
either:

°
10
L
of
sample,
or
°
2
mL
of
packed
pellet
volume,
or
°
As
much
volume
as
two
filters
can
accommodate
before
clogging
(
this
condition
applies
only
to
filters
that
have
been
approved
by
EPA
for
nationwide
use
with
EPA
Method
1622/
1623
 
the
Pall
Gelman
Envirochek
 
and
Envirochek
 
HV
filters,
or
the
IDEXX
FiltaMax
 
foam
filter)

The
LT2
rule
sample
volume
analysis
requirement
of
10
L
(
rather
than
10.0
or
10.00
L)
accommodates
the
potential
for
imprecisely
filled
sample
containers
or
filters.
Sample
volumes
should
be
rounded
to
the
Section
3:
Guidance
for
Cryptosporidium
Laboratories
12
Draft
June
2003
nearest
whole
liter
to
determine
compliance
(
e.
g.,
9.5
L
would
be
rounded
to
10
L,
and
would
meet
rule
requirements).

Systems
may
analyze
larger
volumes
than
10
L,
and
larger
volumes
analyzed
should
increase
analytical
sensitivity
(
detection
limit),
provided
method
performance
is
acceptable.
EPA
encourages
systems
to
analyze
similar
sample
volumes
throughout
the
monitoring
period.
However,
data
sets
including
different
samples
volumes
will
be
accepted,
provided
the
system
analyzes
the
minimum
sample
volume
requirements
noted
above.

Additional
guidance
on
sample
volume
and
sample
collection
issues
is
provided
in
the
Source
Water
Monitoring
Guidance
Manual
for
Public
Water
Systems
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule),
available
for
download
from
http://
www.
epa.
gov/
safewater/
lt2/
index.
html.

3.1.4
Spiking
Suspensions
Requirements
for
Spiked
Quality
Control
Samples
Flow
cytometer
 
counted
spiking
suspensions
must
be
used
for
ongoing
precision
and
recovery
(
OPR)
and
matrix
spike
(
MS)
samples
[
40
CFR
part
141.705
(
a)(
3)].
The
use
of
flow
cytometer
 
counted
spiking
suspensions
is
a
recommendation
in
EPA
Method
1622/
1623,
but
a
requirement
for
the
LT2
rule.
Spiking
suspensions
are
discussed
in
more
detail
in
Section
3.3.1,
below.

3.1.5
Acceptable
Sample
Results
Cryptosporidium
sample
results
reported
under
the
LT2
rule
must
meet
the
quality
control
(
QC)
requirements
specified
in
EPA
Method
1622/
1623
[
40
CFR
part
141.705
(
a)(
3)].
These
requirements
include,
but
are
not
limited
to,
sample
temperature
requirements,
minimum
frequencies
for
ongoing
precision
and
recovery
(
OPR),
method
blank,
and
matrix
spike
samples;
acceptable
OPR
and
method
blank
results;
holding
time
requirements;
and
staining
control
frequency
and
results.
A
checklist
for
these
requirements
is
provided
as
Appendix
D.
Guidance
on
implementing
Cryptosporidium
method
QC
requirements
is
provided
in
Section
3.3,
below.
These
requirements
are
based
on
the
June
2003
versions
of
EPA
Methods
1622/
1623,
the
use
of
which
EPA
has
requested
comment
on
in
the
LT2
rule
proposal.

3.1.6
Cryptosporidium
Oocyst
Counts
to
Report
Sample
examination
using
EPA
Method
1622/
1623
includes
an
immunofluorescence
assay
using
fluorescein
isothiocyanate
(
FITC)
as
the
primary
antibody
stain,
4',
6­
diamidino­
2­
phenylindole
(
DAPI)
staining
to
detect
nuclei,
and
differential
interference
contrast
microscopy
(
DIC)
to
detect
internal
structures
[
40
CFR
part
141.705
(
a)(
4)].
Cryptosporidium
oocysts
to
be
reported
using
EPA
Method
1622/
1623
are
defined
as
the
following:

1.
Those
determined
by
brilliant
apple
green
fluorescence
under
UV
light,
size
(
4
to
6
µ
m),
and
shape
(
round
to
oval)

2.
Excluding
atypical
organisms
specifically
identified
as
other
microbial
organisms
by
FITC
and
DIC
(
for
example,
those
possessing
spikes,
stalks,
appendages,
pores,
one
or
two
large
nuclei
filling
the
cell,
red
fluorescing
chloroplasts,
crystals,
spores,
etc.)

The
oocyst
counts
for
a
sample,
based
on
the
above
definition,
and
the
sample
volume
analyzed,
based
on
the
calculations
specified
in
Section
3.7,
will
be
used
to
calculate
the
oocyst
concentration
for
each
sample
during
the
LT2
rule.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
13
Draft
June
2003
3.2
Laboratory
Quality
Assurance
Evaluation
Program
To
improve
Cryptosporidium
data
quality
and
consistency
during
LT2
rule
monitoring,
EPA
requires
that
only
qualified
laboratories
analyze
Cryptosporidium
samples.
A
laboratory's
qualifications
are
determined
through
the
Laboratory
Quality
Assurance
Evaluation
Program
for
Analysis
of
Cryptosporidium
Under
the
Safe
Drinking
Water
Act
(
Lab
QA
Program)
(
67
FR
9731,
March
4,
2002)
[
40
CFR
part
141.706
(
a)].
If
laboratories
are
certified
for
Cryptosporidium
analyses
under
equivalent
State
laboratory
certification
programs,
EPA
plans
to
include
these
laboratories
in
the
list
of
approved
laboratories
posted
at
http://
www.
epa.
gov/
safewater/
lt2/
aprvlabs.
html.

The
objectives
of
the
Lab
QA
Program
are
to
evaluate
laboratories'
capacity
and
competency
to
reliably
measure
for
the
occurrence
of
Cryptosporidium
in
surface
water
using
EPA
Method
1622/
1623.
Each
laboratory
participating
in
the
program
will
be
required
to
complete
the
following
steps
to
be
qualified
through
this
program:

°
Complete
an
application
(
including
a
self­
evaluation)

°
Perform
initial
proficiency
testing
(
IPT)

°
Participate
in
an
on­
site
evaluation
°
Perform
ongoing
proficiency
testing
(
OPT)
every
four
months
Information
on
the
Laboratory
QA
Program
is
available
at
http://
www.
epa.
gov/
safewater/
lt2/
index.
html
and
summarized
below,
in
Sections
3.2.1
­
3.2.7.

3.2.1
Application
Applications
for
the
program
(
Appendix
E)
are
available
on
the
website
and
also
may
be
requested
from
the
following
address:

EPA's
Laboratory
Quality
Assurance
Evaluation
Program
Coordinator
c/
o
DynCorp/
CSC
Biology
Studies
Group
6101
Stevenson
Avenue
Alexandria,
VA
22304
EPA
reviews
each
application
to
verify
that
the
laboratory
has
submitted
the
following
information:

°
A
completed
self­
evaluation
checklist
°
Resumes
of
laboratory
personnel
°
Standard
operating
procedures
for
each
method
version
°
Initial
demonstration
of
capability
(
IDC)
data,
which
consist
of
the
following:
°
Acceptable
initial
precision
and
recovery
(
IPR)
test
results
°
Acceptable
method
blank
result
run
with
IPR
test
°
Acceptable
matrix
spike/
matrix
spike
duplicate
(
MS/
MSD)
results
with
results
from
the
unspiked
matrix
sample
analyzed
at
the
same
time
°
Table
of
contents
from
the
laboratory's
quality
assurance
plan
°
Documentation
of
personnel
training
and
number
of
samples
analyzed
and
duration
of
time
using
the
method
°
Example
of
client
data
reporting
form
Section
3:
Guidance
for
Cryptosporidium
Laboratories
14
Draft
June
2003
Completed
applications
should
be
submitted
to
EPA's
Laboratory
Quality
Assurance
Evaluation
Program
Coordinator,
c/
o
DynCorp/
CSC
Biology
Studies
Group,
at
the
address
listed
above.

3.2.2
Personnel
Qualifications
and
Training
Laboratory
personnel
must
meet
the
qualifications
of
the
Lab
QA
Program
in
order
to
analyze
Cryptosporidium
samples
for
LT2
rule
monitoring.
Personnel
qualifications
for
the
Lab
QA
Program
are
provided
in
Table
3­
1.
Each
laboratory
must
have
at
least
one
principal
analyst.

Table
3­
1.
Laboratory
QA
Program
Personnel
Qualifications
Position
Education
Experience
with
Crypto
and
IFA
Microscopy
Experience
Using
Method
1622/
1623
Number
of
Samples
Analyzed
Using
Method
1622/
1623
Principal
Analyst
BS/
BA
in
Microbiology
or
closely
related
field
1
year
continuous
6
months
100
(
50
if
approved
as
an
analyst
during
Information
Collection
Rule
[
ICR])

Analyst
2
years
college
in
Microbiology
or
equivalent
6
months
continuous
3
months
50
(
25
if
approved
as
an
analyst
during
ICR)

Technician
No
minimum
requirement
No
minimum
required
3
months
with
specific
parts
of
procedure
performing
50
(
25
if
approved
as
an
analyst
during
ICR)

During
the
on­
site
evaluation
(
Section
3.2.4),
laboratory
records
will
be
evaluated
to
verify
that
the
personnel
performing
EPA
Method
1622/
1623
analyses
meet
the
requirements
of
the
Lab
QA
Program.
For
new
staff
that
are
added
after
the
on­
site
evaluation,
the
laboratory
should
send
a
letter
to
EPA
providing
the
following
information
on
the
new
staff
member:

°
Education
°
Number
of
samples
analyzed
using
EPA
Method
1622/
1623
°
Number
of
months
of
experience
°
Verification
that
analyst
training
followed
the
laboratory's
training
SOP
In
addition
to
meeting
the
qualifications
of
the
Lab
QA
Program,
the
following
steps
should
be
completed
by
new
personnel
as
part
of
their
training
prior
to
analyzing
samples
for
LT2
(
this
should
be
specified
in
the
laboratory's
training
SOP):

°
Review
laboratory
SOPs
for
analysis
of
samples
using
Method
1622/
1623
°
Observation
of
an
experienced
analyst
performing
the
method
°
Performance
of
the
method
while
being
observed
by
an
experienced
analyst
°
Acceptable
performance
of
a
set
of
IPR
samples
using
blind
spikes
If
EPA
determines
that
a
laboratory
is
using
personnel
to
analyze
LT2
samples
who
do
not
meet
the
qualifications
established
in
the
Lab
QA
Program,
the
laboratory
will
be
put
on
conditional
status
(
Section
3.2.6).
The
laboratory
must
discontinue
the
use
of
the
personnel
in
question
until
they
have
met
the
qualifications
of
the
Lab
QA
Program,
and
must
submit
a
signed
letter
to
EPA
confirming
that
this
action
has
been
taken,
to
be
removed
from
conditional
status.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
15
Draft
June
2003
3.2.3
Initial
Proficiency
Testing
After
the
laboratory's
application
has
been
reviewed
and
accepted,
EPA
will
send
the
laboratory
a
set
of
eight
initial
proficiency
testing
(
IPT)
samples,
which
consist
of
a
suspension
of
Cryptosporidium
oocysts
in
a
concentrated
matrix.
Laboratories
will
resuspend
these
spikes
in
reagent
water
to
produce
simulated
source
water
samples,
and
analyze
the
samples
using
the
version
of
EPA
Method
1622/
1623
that
the
laboratory
plans
to
use
for
routine
Cryptosporidium
analyses.
If
a
laboratory
wishes
to
be
evaluated
for
more
than
one
version
of
the
method,
the
laboratory
will
receive
a
set
of
eight
PT
samples
for
each
version.

Laboratory
IPT
data
will
be
evaluated
against
the
mean
recovery
and
precision
(
as
relative
standard
deviation
[
RSD])
criteria
that
EPA
has
established
for
IPT
samples.
Laboratories
have
two
opportunities
to
pass
the
IPT
test.
If
a
laboratory
fails
two
times,
it
will
not
be
eligible
for
another
set
until
after
the
laboratory
staff
has
received
additional
training
in
performing
the
method
and
reapplied
to
the
program
(
discussed
further
in
Section
3.2.6).

3.2.4
On­
Site
Evaluation
Each
laboratory
that
passes
the
IPT
will
next
participate
in
an
on­
site
evaluation,
which
consists
of
two
concurrently
performed
assessments:
a
data
and
QA
evaluation
and
a
technical
evaluation.

3.2.4.1
Data
and
QA
Evaluation
During
the
data
and
QA
evaluation,
laboratory
documentation
will
be
evaluated
to
verify
compliance
with
QA
program
requirements.
The
evaluation
will
cover
the
following:

°
Equipment
and
personnel
records
°
Data
recording
procedures,
based
on
field
sample
data
and
quality
control
sample
data
°
Quality
control
test
frequency
and
acceptability
°
Quality
assurance
plans
°
Standard
operating
procedures
To
ensure
consistency
and
thoroughness
for
all
audits,
the
data
auditor
uses
a
detailed
checklist
(
Appendix
F)
to
evaluate
specific
factors
under
each
of
these
categories.

To
prepare
for
the
on­
site
evaluation,
the
laboratory
should
use
the
checklists
provided
with
the
program
application
to
perform
a
self­
audit.

3.2.4.2
Technical
Evaluation
During
the
technical
evaluation,
laboratory
sample
processing
and
analysis
using
EPA
Method
1622/
1623
will
be
evaluated.
The
laboratory
will
be
assessed
on
its
capabilities
including
the
following:

°
Sample
processing
and
analyses
°
Microscopy
To
ensure
consistency
and
thoroughness
for
all
audits,
the
technical
auditor
uses
a
detailed
checklist
(
Appendix
F)
to
evaluate
specific
factors
under
each
of
these
categories.

3.2.5
Ongoing
Proficiency
Testing
Laboratories
that
meet
the
program
performance
criteria
will
also
receive
a
set
of
three
ongoing
proficiency
testing
(
OPT)
samples
approximately
every
four
months
that
must
be
analyzed
in
the
same
Section
3:
Guidance
for
Cryptosporidium
Laboratories
16
Draft
June
2003
manner
as
the
IPT
samples.
EPA
will
evaluate
the
precision
and
recovery
data
for
OPT
samples
to
determine
if
the
laboratory
continues
to
meet
the
performance
criteria
of
the
Laboratory
QA
Program.

3.2.6
Changing
Status
Laboratories
will
be
approved
after
they
have
submitted
an
acceptable
application,
passed
the
IPT
and
passed
the
on­
site
evaluation.
Laboratories
must
be
approved
in
order
to
analyze
samples
under
LT2.
To
maintain
approval
the
laboratory
must
successfully
analyze
a
set
of
OPT
samples
once
every
fourth
month.
Details
are
provided
below
on
what
happens
when
a
laboratory
fails
to
meet
the
program
requirements.
The
process
for
laboratory
approval
and
changes
in
approval
status
is
outlined
in
Figure
3­
1.

Laboratories
that
fail
two
rounds
of
IPT
samples
must
do
the
following
to
be
eligible
to
receive
a
third
set
of
IPT
samples:

°
Receive
additional
training
°
Analyze
25
additional
practice
samples
°
Repeat
their
IPR
analysis
and
submit
the
results
to
EPA
The
additional
practice
samples
should
include
method
blanks,
OPR
samples,
and
at
least
50%
matrix
samples
including
multiple
matrix
spike
and
matrix
spike
duplicate
samples.
Additional
training
can
be
received
at
EPA's
Technical
Support
Center
in
Cincinnati
or
another
approved
laboratory.
Laboratories
that
only
have
a
few
analysts/
technicians
may
want
to
send
all
their
staff
involved
in
the
method
for
training.
For
laboratories
that
cannot
send
their
entire
staff
for
training,
they
may
elect
to
send
the
analysts
normally
responsible
for
training
of
other
analysts/
technicians
in
their
laboratory.
These
analysts
can
then
pass
along
the
information
they
received
during
training
at
EPA
to
all
of
their
staff.

If
the
laboratory
does
not
pass
the
third
IPT
following
additional
training,
they
will
be
removed
from
consideration
for
the
program
for
6
months,
during
which
time
they
must
analyze
an
additional
50
practice
samples
and
repeat
their
IPR
and
submit
the
results
to
EPA.
The
additional
practice
samples
should
include
method
blanks,
OPR
samples,
and
at
least
50%
matrix
samples
including
multiple
matrix
spike
and
matrix
spike
duplicate
samples.
After
satisfying
these
requirements
the
laboratory
may
re­
apply
for
ONE
more
set
of
IPT
samples.
If
the
laboratory
fails
this
final
IPT,
then
they
will
not
be
allowed
to
participate
in
analysis
of
samples
for
Cryptosporidium
using
EPA
Method
1622/
1623
under
the
LT2
rule.

If
a
laboratory
fails
to
meet
the
precision
or
recovery
criteria
for
a
set
of
OPT
samples,
the
laboratory
will
be
shipped
a
second
set
of
samples.
The
laboratory's
status
changes
to
"
conditional"
but
they
are
still
permitted
to
analyze
samples
under
LT2.

°
If
the
laboratory's
next
set
of
OPT
data
are
acceptable,
the
laboratory
will
be
returned
to
"
approved"
status.

°
If
a
laboratory
fails
the
next
set
of
OPT
samples
(
two
sets
of
OPTs
in
a
row),
the
laboratory
will
be
disapproved
for
analysis
of
LT2
samples
and
must
get
additional
training.
The
laboratory
must
consult
with
EPA
regarding
the
level
and
type
of
training
required,
and
must
submit
to
EPA
proof
of
this
training
upon
completion.
If
the
laboratory's
training
is
acceptable,
the
laboratory
must
repeat
the
IPT
test
before
being
re­
approved.

If
the
laboratory
is
disapproved
a
second
time,
the
laboratory
will
no
longer
be
allowed
to
participate
in
the
program.
Actions
taken
at
this
point
are
discussed
in
Section
3.2.7.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
17
Draft
June
2003
3.2.7
Notifying
Utilities
of
Laboratory
Status
Two
actions
are
taken
when
a
laboratory's
status
changes:

°
The
laboratories
must
notify
clients
°
EPA
will
post
updates
on
http://
www.
epa.
gov/
safewater/
lt2/
index.
html
When
a
laboratory
receives
notice
that
they
have
been
"
disapproved,"
the
laboratory
must
notify
their
clients
of
their
status
change
within
2
weeks
and
work
with
their
clients
to
finish
up
analysis
of
samples
that
they
have
in
house.
After
the
2­
week
period
the
laboratory
will
no
longer
be
able
to
analyze
samples
under
LT2
until
they
have
been
reapproved.
Laboratories
should
have
a
plan
in
place
to
divert
samples
to
another
laboratory
in
the
event
that
this
situation
arises.

All
samples
being
processed
by
the
laboratory
at
the
time
of
the
disapproval
are
considered
acceptable,
provided
all
QC
and
holding
time
requirements
(
detailed
in
EPA
Methods
1622
and
1623
and
Section
3.3
and
summarized
in
Appendix
D)
are
met.
Analysis
of
these
samples
should
be
completed
by
the
laboratory.
However,
no
new
LT2
samples
may
be
processed;
this
includes
samples
that
have
been
received
by
the
laboratory,
but
that
have
not
been
filtered
(
for
bulk
samples)
or
eluted
(
for
field­
filtered
samples).
Any
new
samples
processed
after
the
laboratory
has
been
disapproved
will
not
be
considered
acceptable
for
monitoring
under
the
LT2
rule.

3.3
Cryptosporidium
Method
Quality
Control
During
the
LT2
rule,
Cryptosporidium
samples
must
meet
the
quality
control
(
QC)
requirements
listed
in
EPA
Methods
1622/
1623
[
40
CFR
part
141.705
(
a)
(
3)].
The
requirements
discussed
in
this
guidance
manual
are
based
on
the
June
2003
versions
of
EPA
Methods
1622/
1623,
the
use
of
which
EPA
has
requested
comment
on
in
the
LT2
rule
proposal
(
see
Section
3.1.2
for
details).

Sections
3.3.1
through
3.3.14
provide
guidance
on
the
implementation
of
the
QC
requirements
specified
in
the
June
2003
version
of
EPA
Method
1622/
1623.
Routine
QC
requirements
that
must
be
verified
internally
by
the
laboratory
before
reporting
LT2
rule
monitoring
results
are
called
out
below,
and
included
in
checklist
format
as
Appendix
D.
This
guidance
is
provided
to
help
implement
the
QC
requirements
in
the
methods
and
does
not
substitute
for,
or
alter,
the
method
requirements.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
18
Draft
June
2003
Submit
program
application
Analyze
IPT
Samples
and
Submit
Data
to
EPA
Submit
missing
or
incomplete
information
On­
site
evaluation
of
laboratory
Analyze
2nd
set
of
IPT
samples
DId
IPTs
pass?
Lab
must
do
the
following:
1.
Receive
training
on
deficient
areas
2.
Analyze
25
samples
3.
Repeat
IPR
analysis
Analyze
3rd
set
of
IPT
samples
Lab
must
wait
6
months
to
receive
4th
set
of
IPTs
and
do
the
following:
1.
Analyze
50
additional
samples
2.
Repeat
IPR
analysis
No
Step
1:

Submit
Program
Application
Respond
to
any
deficiencies
cited
in
EPA
audit
report
Lab
is
approved
for
Lab
QA
Program
and
analysis
of
samples
under
LT2
rule
Receive
audit
report
from
EPA
Yes
Provide
additional
information
to
EPA
Analyze
OPT
samples
every
4
months
Status
changes
to
"
Conditional"
Lab
is
"
Disapproved,"
and
must
receive
additional
training
and
analyze
IPT
samples
Analyze
4th
set
of
IPT
samples
Laboratory
can
no
longer
participate
in
Lab
QA
Program
No
Step
2.

Analyze
Initial
Performance
Testing
Samples
(
IPTs)

Retain
"
Approved"
status
Step
3.

On­
Site
Evaluation
Step
4.

Analyze
Ongoing
Performance
Testing
Samples
(
OPTs)
No
No
No
Yes
No
DId
IPTs
pass?

DId
IPTs
pass?
Yes
Yes
Yes
Is
application
acceptable?

DId
IPTs
pass?

Yes
Are
deficiencies
cited?

Are
responses
acceptable
?
Yes
No
Did
OPTs
pass?

Did
OPTs
pass?
Analyze
next
set
of
OPT
samples
Laboratory
can
no
longer
participate
in
Lab
QA
Program
DId
IPTs
pass?

Yes
Yes
No
No
No
Yes
Figure
3­
1.
Process
for
Laboratory
Approval
and
Changes
in
Laboratory
Approval
Status
Section
3:
Guidance
for
Cryptosporidium
Laboratories
19
Draft
June
2003
3.3.1
Cryptosporidium
Spiking
Materials
During
LT2
Cryptosporidium
monitoring,
laboratories
must
analyze
samples
spiked
with
Cryptosporidium
parvum
oocysts
to
assess
ongoing
laboratory
and
method
performance,
per
method
QC
requirements.
These
ongoing
spiked
sample
analyses
include
ongoing
precision
and
recovery
samples
(
OPRs)
(
Section
3.3.4)
and
matrix
spike
samples
(
Section
3.3.13).
Flow
cytometer
 
counted
spiking
suspensions
must
be
used
for
these
QC
samples
[
40
CFR
part
141.705
(
a)
(
3)].

Sources
of
flow
cytometer
 
counted
Cryptosporidium
spiking
suspensions
for
use
with
routine,
spiked
Cryptosporidium
QC
samples
include
the
following:

1.
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.

2.
BioTechnology
Frontiers
(
BTF)
www.
biotechnologyfrontiers.
com
Unit
1
35­
41
Waterloo
Road
North
Ryde
NSW
Australia
Fax:
+
61
2
9889
1805
Email:
contact@
biotechnologyfrontiers.
com
BTF
prepares
and
distributes
Cryptosporidium
parvum
oocysts
and
Giardia
intestinalis
cysts
that
have
been
irradiated
to
inactivate
the
organisms.
Note:
Irradiated,
flow
cytometer
 
counted
spiking
suspensions
may
be
used
for
routine
laboratory
QC
samples,
including
initial
precision
and
recovery
(
IPR)
samples,
ongoing
precision
and
recovery
(
OPR)
samples,
and
matrix
spike
(
MS)
samples.
Per
EPA
Method
1622/
1623,
Irradiated
organisms
may
not
be
used
for
interlaboratory
validation
studies
performed
to
seek
nationwide
approval
of
modified
versions
of
the
methods.

Flow­
cytometer­
counted
spiking
suspensions
used
for
spiked
quality
control
(
QC)
samples
must
be
used
within
the
expiration
date
noted
on
the
suspension.
The
laboratory
should
spike
samples
according
to
the
procedures
provided
in
Section
11.4
of
EPA
Method
1622/
1623
or
according
to
the
procedures
provided
by
the
spiking
suspension
vendor.

3.3.2
Initial
Precision
and
Recovery
Test
The
initial
precision
and
recovery
(
IPR)
test
required
by
EPA
Method
1622/
1623
consists
of
four
reagent
water
samples
spiked
with
100
to
500
oocysts
and
is
used
to
demonstrate
initial
acceptable
performance
with
the
method.
Section
9
of
EPA
Method
1622/
1623
also
requires
the
IPR
to
be
performed
for
each
method
modification
(
additional
guidance
on
QC
when
using
multiple
method
variations
is
provided
in
Section
3.3.14).

The
results
of
the
four
analyses
are
used
to
calculate
the
average
percent
recovery
and
the
relative
standard
deviation
(
RSD)
of
the
recoveries
for
Cryptosporidium
(
Section
3.7).
For
EPA
Method
1622/
1623,
the
mean
Cryptosporidium
recovery
must
be
in
the
range
of
24%
to
100%
and
the
RSD
of
the
four
recoveries
must
be
less
than
55%.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
20
Draft
June
2003
3.3.3
Method
Blank
Test
The
method
blank
test
required
by
EPA
Method
1622/
1623
consists
of
analysis
of
an
unspiked
reagent
water
sample
to
demonstrate
freedom
from
contamination.
Per
the
method,
one
method
blank
sample
must
be
analyzed
each
week
or
every
20
field
and
matrix
spike
samples,
whichever
is
more
frequent.
If
more
than
one
method
variation
will
be
used
for
filtration
and/
or
separation
of
samples,
a
separate
method
blank
may
be
required
for
each
variation
(
see
Section
3.3.14).

If
one
or
more
Cryptosporidium
oocysts
(
as
defined
in
Section
3.1.6)
are
found
in
a
blank,
analysis
of
additional
samples
is
halted
until
the
source
of
contamination
is
eliminated
and
a
blank
shows
no
evidence
of
contamination.

LT2
rule
requirement:
For
each
method
blank,
oocysts
must
not
be
detected
[
40
CFR
part
141.705
(
a)
(
3)].

3.3.4
Ongoing
Precision
and
Recovery
Test
The
ongoing
precision
and
recovery
(
OPR)
in
EPA
Method
1622/
1623
entails
analysis
of
a
reagent
water
sample
spiked
with
100
to
500
oocysts
to
demonstrate
ongoing
acceptable
performance.
One
OPR
sample
must
be
analyzed
each
week
or
every
20
field
and
MS
samples,
whichever
is
more
frequent.
If
more
than
one
method
variation
will
be
used
for
filtration
and/
or
separation
of
samples,
a
separate
method
blank
may
be
required
for
each
variation
(
see
Section
3.3.14).

OPR
samples
should
be
analyzed
before
any
field
samples
in
a
batch
are
processed
to
verify
acceptable
performance.
OPR
Cryptosporidium
recovery
must
be
in
the
range
of
11%
to
100%
to
be
considered
acceptable.

LT2
rule
requirement:
Ongoing
precision
and
recovery
results
must
be
11%
to
100%
[
40
CFR
part
141.705
(
a)
(
3)].

3.3.5
Ongoing
Precision
and
Recovery
Control
Charts
As
noted
in
Section
9.7.5
of
the
June
2003
version
of
EPA
Method
1622/
1623,
laboratories
should
maintain
a
control
chart
of
OPR
recoveries,
graphically
displaying
the
results
of
continuing
performance.
The
control
chart
should
be
developed
using
the
most
recent
20
to
30
test
results.

The
control
chart
is
developed
by
plotting
percent
recovery
of
each
OPR
sample
over
time.
Based
on
the
mean
of
the
recoveries
on
the
chart,
the
upper
and
lower
control
limits
should
be
established
as
follows:

°
Upper
control
limit
=
0
+
2
standard
deviations
°
Lower
control
limit
=
0
­
2
standard
deviations
An
example
of
an
OPR
control
chart
is
provided
in
Figure
3­
2.
After
each
5
to
10
new
recovery
measurements,
new
control
limits
should
be
recalculated
using
the
most
recent
20
to
30
data
points.
These
calculated
control
limits
should
not
exceed
the
OPR
criteria
of
11%
to
100%.
Control
charts
can
be
used
to
track
the
laboratories
performance
and
determine
if
any
trends
in
recovery
are
occurring.
If
recovery
measurements
fall
below
the
lower
control
limit
or
above
100%,
laboratories
should
take
corrective
Section
3:
Guidance
for
Cryptosporidium
Laboratories
21
Draft
June
2003
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%

01/
04/
2001
02/
01/
2001
03/
01/
2001
03/
29/
2001
04/
26/
2001
05/
24/
2001
06/
21/
2001
07/
19/
2001
OPR
Recovery
(%)

Recovery
Mean
+
2
Standard
Deviations
Mean
­
2
Standard
Deviations
action,
investigating
potential
causes
of
the
outlying
result.
The
troubleshooting
guidance
for
OPR
failures
provided
in
Section
9.7.5
of
EPA
Method
1622/
1623
also
is
useful
for
investigating
the
cause
of
acceptable,
but
outlying,
OPR
measurements
identified
through
the
use
of
control
charts.

3.3.6
Quality
Control
Batches
As
noted
in
Appendix
D
and
Sections
3.3.3
and
3.3.4,
all
LT2
Cryptosporidium
samples
must
be
associated
with
an
acceptable
OPR
and
method
blank
sample
[
40
CFR
part
141.705
(
a)
(
3)].
LT2
samples
are
associated
with
QC
samples
through
a
"
QC
batch."
A
QC
batch
consists
of
an
OPR
and
method
blank
and
the
maximum
of
20
field
and
MS
samples
that
are
eluted,
concentrated,
and
purified
in
the
same
week
as
the
OPR
and
method
blank
samples.
If
more
than
20
field
and
MS
samples
are
processed
in
a
week,
the
OPR
and
method
blank
samples
are
associated
with
the
field
and
MS
samples
eluted,
concentrated,
and
purified
in
the
same
time
period.

Figure
3­
2.
Ongoing
Precision
and
Recovery
Control
Chart
Example
3.3.7
Holding
Time
Requirements
During
Cryptosporidium
analyses
for
the
LT2
rule,
sample
processing
should
be
completed
as
soon
as
possible
by
the
laboratory.
The
laboratory
should
complete
sample
filtration
(
if
sample
is
received
in
bulk),
elution,
concentration,
purification,
and
staining
the
day
the
sample
is
received
wherever
possible.
However,
the
laboratory
is
permitted
to
split
up
the
sample
processing
steps
if
processing
a
sample
completely
in
one
day
is
not
possible.
If
this
is
necessary,
sample
processing
can
be
halted
after
filtration,
application
of
the
purified
sample
onto
the
slide,
or
staining.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
22
Draft
June
2003
The
following
holding
times
must
be
met
for
samples
analyzed
by
EPA
Method
1622/
1623
during
the
LT2
rule:

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

°
Sample
elution,
concentration,
and
purification.
The
laboratory
must
complete
the
elution,
concentration,
purification,
and
application
of
the
sample
to
the
slide
in
one
work
day.
It
is
critical
that
these
steps
be
completed
in
one
work
day
to
minimize
the
time
that
any
target
organisms
present
in
the
sample
sit
in
eluate
or
concentrated
matrix.
This
process
ends
with
the
application
of
the
purified
sample
on
the
slide
for
drying.

°
Staining.
The
sample
must
be
stained
within
72
hours
of
application
of
the
purified
sample
to
the
slide.

°
Examination.
Although
immunofluorescence
assay
(
FA)
and
4',
6­
diamidino­
2­
phenylindole
(
DAPI)
and
differential
interference
contrast
(
DIC)
microscopy
examination
and
confirmation
ideally
should
be
performed
immediately
after
staining
is
complete,
laboratories
have
up
to
7
days
from
completion
of
sample
staining
to
complete
the
examination
and
confirmation
of
samples.
However,
if
fading/
diffusion
of
FITC
or
DAPI
staining
is
noticed,
the
laboratory
must
reduce
this
holding
time.
In
addition,
the
laboratory
may
adjust
the
concentration
of
the
DAPI
staining
solution
so
that
fading/
diffusion
does
not
occur.
The
laboratory
also
should
evaluate
the
use
of
another
mounting
medium
(
one
alternative
is
provided
in
Section
3.9.2,
below).

LT2
rule
requirement:
Each
sample
must
meet
the
QC
criteria
for
the
methods
[
40
CFR
part
141.705
(
a)
(
3)].
Per
EPA
Method
1622/
1623,
samples
must
be
processed
or
examined
within
each
of
the
holding
times
specified
by
A
breakdown
of
the
holding
times
for
each
set
of
steps
is
provided
in
Table
3­
2.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
23
Draft
June
2003
Table
3­
2.
Cryptosporidium
Holding
Times
for
EPA
Method
1622/
1623
Sample
Processing
Step
Maximum
Allowable
Time
between
Breaks
(
Samples
Should
be
Processed
As
Soon
As
Possible)

Collection
Filtration
'
Up
to
96
hours
are
permitted
between
sample
collection
(
if
shipped
to
the
laboratory
as
a
bulk
sample)
or
filtration
(
if
filtered
in
the
field)
and
initiation
of
elution
Elution
These
steps
must
be
completed
in
1
working
day
Concentration
Purification
Application
of
purified
sample
to
slide
Drying
of
sample
'
Up
to
72
hours
are
permitted
from
application
of
the
purified
sample
to
the
slide
to
staining
Staining
'
Up
to
7
days
are
permitted
between
sample
staining
and
examination
Examination
3.3.8
Staining
Controls
Positive
staining
controls
are
used
to
verify
that
the
FITC
and
DAPI
stains
are
fluorescing
appropriately.
Positive
staining
controls
are
prepared
by
applying
positive
antigen
or
200
to
400
intact
oocysts
to
a
slide
and
staining
the
slide
with
the
same
reagents
and
staining
procedure
used
to
stain
field
samples.
The
analyst
examines
several
fields
of
view
to
verify
that
the
stain
is
fluorescing
at
the
appropriate
intensity
and
uniformity.
Control
slides
and
sample
slides
should
be
read
on
the
same
day.
If
sample
slides
from
the
same
staining
batch
are
read
over
multiple
days,
the
control
slide
must
be
rechecked
each
day
before
examination
of
the
sample
slides.
If
the
laboratory
has
a
large
batch
of
slides
that
will
be
examined
over
several
days,
and
is
concerned
that
a
single
positive
control
may
fade,
due
to
multiple
examinations,
the
laboratory
should
prepare
multiple
control
slides
with
the
batch
of
field
slides
and
alternate
between
the
positive
controls
when
performing
the
positive
control
check.

Negative
staining
controls
are
used
to
verify
that
no
oocysts
or
interfering
particulates
are
present.
Negative
staining
controls
are
prepared
by
staining
and
examining
a
slide
with
phosphate
buffered
saline
solution.

The
analyst
must
indicate
on
each
sample
examination
results
form
whether
the
positive
staining
control
and
negative
staining
control
were
acceptable.

LT2
rule
requirement:
Each
sample
must
meet
the
QC
criteria
for
the
methods
[
40
CFR
part
141.705
(
a)
(
3)].
Per
EPA
Method
1622/
1623,
positive
and
negative
staining
controls
must
be
acceptable
(
Section
15.2.1)
Section
3:
Guidance
for
Cryptosporidium
Laboratories
24
Draft
June
2003
3.3.9
Examination
Preparation
To
help
the
analyst
identify
the
target
analyte
during
field
sample
slide
examination,
each
analyst
must
characterize
a
minimum
of
three
Cryptosporidium
oocysts
on
the
positive
staining
control
slide
before
examining
field
sample
slides.
This
characterization
must
be
performed
by
each
analyst
during
each
microscope
examination
session.
FITC
examination
must
be
conducted
at
a
minimum
of
200X
total
magnification,
DAPI
examination
must
be
conducted
at
a
minimum
of
400X,
and
DIC
examination
and
size
measurements
must
be
conducted
at
a
minimum
of
1000X.

Size,
shape,
and
DIC
and
DAPI
characteristics
of
the
three
Cryptosporidium
oocysts
must
be
recorded
by
the
analyst
in
a
microscope
log.

3.3.10
Ongoing
Analyst
Verification
Analyst
verifications
are
ongoing
comparisons
of
slide
counts
among
multiple
analysts
in
a
laboratory
to
assess
and
maintain
consistency
in
slide
examination
among
analysts.
At
least
monthly
when
microscopic
examinations
are
being
performed,
the
laboratory
shall
prepare
a
slide
containing
40
to
100
oocysts.
More
than
50%
of
the
oocysts
must
be
DAPI
positive
and
undamaged
under
DIC.

For
laboratories
with
multiple
analysts,
each
analyst
shall
determine
the
total
number
of
oocysts
and
cysts
and
the
number
that
are
DAPI
positive
and
DAPI
negative
for
the
entire
slide.
For
10
oocysts
and
10
cysts,
each
analyst
shall
determine
the
number
of
nuclei
by
DAPI
and
the
DIC
category
(
empty,
containing
amorphous
structures,
or
containing
identifiable
internal
structures)
of
each.
The
total
number
and
the
number
of
DAPI
positive
and
DAPI
negative
oocysts
and
cysts
determined
by
each
analyst
(
Section
10.5.2.)
must
be
within
±
10%
of
each
other.
If
the
number
is
not
within
this
range,
the
analysts
must
identify
the
source
of
any
variability
between
analysts'
examination
criteria,
prepare
a
new
slide,
and
repeat
the
performance
verification.
Differences
in
the
number
of
nuclei
by
DAPI
and
in
DIC
categorizations
among
analysts
must
be
discussed
and
resolved,
and
these
resolutions
must
be
documented.

Laboratories
with
only
one
analyst
should
maintain
a
protozoa
library
and
compare
the
results
of
slide
examinations
to
photographs
of
oocysts
and
cysts
and
interfering
organisms
to
verify
that
examination
results
are
consistent
with
these
references.
These
laboratories
also
should
perform
repetitive
counts
of
a
single
verification
slide
for
FITC
and
DAPI.
These
laboratories
should
also
coordinate
with
other
laboratories
to
share
slides
and
compare
counts.

3.3.11
Proficiency
Testing
Samples
As
part
of
the
Laboratory
QA
Program,
laboratories
must
successfully
analyze
IPT
samples
initially,
and
OPT
samples
approximately
every
4
months
when
provided
by
EPA.
Proficiency
testing
samples
required
as
part
of
the
Laboratory
QA
Program
are
described
in
Sections
3.2.2
and
3.2.5.

3.3.12
Acceptance
Criteria
for
Receipt
of
Field
Samples
Cryptosporidium
samples
for
LT2
rule
monitoring
that
are
not
analyzed
the
same
day
they
are
collected
must
be
maintained
below
10
°
C
and
not
allowed
to
freeze
to
reduce
biological
activity.
This
is
specified
in
Section
8.0
of
the
June
2003
versions
of
EPA
Method
1622/
1623.
Because
Cryptosporidium
samples
collected
for
the
LT2
rule
must
meet
the
QC
criteria
in
the
methods
[
40
CFR
part
705
(
a)
(
3)],
and
because
these
QC
criteria
include
receipt
of
samples
at
<
10
°
C
and
not
frozen,
laboratories
must
reject
LT2
Cryptosporidium
samples
received
at
>
10
°
C
or
frozen.
In
these
cases,
the
PWS
must
re­
collect
and
re­
ship
the
sample.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
25
Draft
June
2003
Several
options
are
available
to
measure
sample
temperature
upon
receipt
at
the
laboratory
and,
in
some
cases,
during
shipment,
are
provided
in
Section
2.2.

LT2
rule
requirement:
Each
sample
must
meet
the
QC
criteria
for
the
methods
[
40
CFR
part
141.705
(
a)
(
3)].
Per
EPA
Method
1622/
1623,
samples
not
processed
on
the
day
of
collection
must
be
received
at
the
laboratory
at
<
10
°
C
and
not
frozen
(
Section
8.1)

3.3.13
Matrix
Spike
Samples
The
matrix
spike
(
MS)
in
EPA
Method
1622/
1623
(
Section
9.5
of
the
June
2003
version)
entails
analysis
of
a
separate
field
aliquot
spiked
with
100
to
500
oocysts
to
determine
the
effect
of
the
matrix
on
the
method's
oocyst
recovery.
During
LT2
Cryptosporidium
monitoring,
PWSs
are
required,
at
a
minimum,
to
analyze
one
MS
sample
for
every
20
field
samples
from
their
source
water.
However,
matrix
spike
samples
may
be
analyzed
more
frequently
to
better
characterize
method
performance
in
the
matrix.

Based
on
this
requirement,
the
following
PWS
categories
must
analyze
at
least
two
MS
samples
during
LT2
rule
monitoring:

°
PWSs
serving
more
than
10,000
people
that
perform
monthly
monitoring
for
2
years
and
collect
24
field
samples
°
PWSs
serving
fewer
than
10,000
people
that
are
triggered
into
monitoring
for
1
year
and
collect
24
field
samples
For
PWSs
serving
greater
than
10,000
people
that
perform
semi­
monthly
or
more
frequent
monitoring
for
2
years
and
collect
48
or
more
samples,
a
minimum
three
MS
samples
must
be
analyzed.

LT2
rule
requirements:
(
1)
The
MS
and
field
sample
must
be
collected
from
the
same
sampling
location
by
splitting
the
sample
stream
or
collecting
the
samples
sequentially.
(
2)
The
volume
of
the
MS
sample
analyzed
must
be
within
10%
of
the
volume
of
the
field
sample
analyzed.
(
3)
The
MS
and
field
sample
must
be
analyzed
by
the
same
procedure
[
40
CFR
part
141.705
(
a)
(
2)
(
i)].

3.3.13.1
Matrix
Spike
Frequency
For
all
PWSs,
the
first
MS
sample
should
be
collected
and
analyzed
during
the
first
sampling
event
under
the
monitoring
program
and
at
least
12
months
must
elapse
between
the
first
and
last
MS
sample,
per
EPA
Method
1622/
1623
(
Section
9.1.8).
The
PWS
should
evaluate
the
MS
recoveries,
as
well
as
other
attributes
of
sample
processing
and
examination,
and
work
with
the
laboratory
to
determine
whether
sample
filtration
and
processing
procedures
are
working
acceptably,
or
need
to
be
re­
evaluated.

If
it
is
not
possible
to
analyze
an
MS
sample
for
the
first
sampling
event
due
to
laboratory
sample
processing
burden
or
other
reasons,
the
first
MS
sample
should
be
analyzed
as
soon
as
possible
to
identify
potential
method
performance
issues
with
the
matrix.
The
requirement
that
at
least
12
months
must
elapse
between
the
first
and
last
MS
sample
still
applies.
For
example,
if
a
PWS
that
is
monitoring
monthly
for
24
months
is
unable
to
process
an
MS
sample
until
the
8th
sampling
event,
due
to
laboratory
sample
processing
load,
the
second
MS
sample
can
be
processed
no
earlier
than
the
20th
sampling
event.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
26
Draft
June
2003
3.3.13.2
Matrix
Spikes
for
Field­
Filtered
Samples
Matrix
spike
samples
must
be
spiked
and
filtered
in
the
laboratory.
PWSs
that
field­
filter
10­
L
sample
may
field
filter
the
monitoring
sample
for
a
scheduled
sampling
event
during
which
an
MS
is
collected,
but
must
collect
and
ship
the
MS
sample
in
bulk
to
the
laboratory
for
spiking,
filtering,
and
analysis.

PWSs
that
field­
filter
>
10­
L
samples
should
follow
the
same
procedure.
As
noted
above,
the
volume
of
the
MS
sample
analyzed
must
be
within
10%
of
the
volume
of
the
associated
field
sample.
However,
if
shipping
the
entire
bulk
sample
is
cost­
prohibitive,
the
PWS
is
permitted
to
filter
all
but
10
L
of
the
MS
sample
in
the
field,
and
ship
the
filtered
sample
and
the
remaining
10
L
of
source
water
to
the
laboratory
to
have
the
laboratory
spike
the
remaining
10
L
of
water
and
filter
it
through
the
filter
used
to
collect
the
balance
of
the
sample
in
the
field
[
40
CFR
part
141.705
(
a)(
2)(
ii)].

3.3.13.3
Matrix
Spikes
Across
Multiple
Method
Variations
Used
for
the
Same
Public
Water
System
During
the
LT2
rule,
each
PWS
must
perform
MS
analyses
at
a
frequency
of
1
MS
per
20
field
samples,
regardless
of
whether
the
analytical
procedure
is
changed
during
the
course
of
the
analysis
of
the
20
field
samples
(
such
as
changing
from
the
use
of
one
approved
filter
to
another
approved
filter).
EPA
recommends,
but
does
not
require,
that
a
PWS
evaluate
method
performance
using
an
additional
MS
sample
if
the
PWS
changes
the
filter
used.

3.3.14
QC
Guidance
for
Method
Modifications
and
Use
of
Multiple
Method
Variations
EPA
Methods
1622/
1623
are
performance­
based
methods
and,
therefore,
allow
method
modifications
and
the
use
of
different
method
variations
(
including
the
original
method
and
modifications
of
this
method)
if
a
laboratory
can
meet
applicable
QC
criteria
(
EPA
Method
1622/
1623
[
Section
9.1.2])
.
A
method
variation
is
the
complete
set
of
sample
processing
components
(
including
the
filter,
IMS,
and
stain)
and
sample
processing
procedures
(
including
filtration,
concentration,
purification,
and
staining)
used
to
process
a
bulk
water
sample
for
examination.
The
use
of
different
sample
processing
components
(
such
as
different
filters)
or
substantively
different
sample
processing
procedures
(
such
as
additional
rinses
and
transfers
to
reduce
carryover
after
IMS)
are
considered
to
be
different
method
variations.
The
following
are
considered
to
be
different
method
variations
of
EPA
Method
1622/
1623;
this
list
is
not
exhaustive,
and
serves
only
to
provide
examples
of
different
method
variations:

°
Standard
Envirochek
 
filtration
procedure
using
elution,
centrifugation,
IMS,
and
IFA
°
Envirochek
 
HV
filtration
procedure
using
elution,
centrifugation,
IMS,
and
IFA
°
Standard
Filta­
Max
 
procedure
using
elution
tube
and
concentrator
tube,
IMS,
and
IFA
°
Filta­
Max
 
procedure
using
stomacher,
concentrator
tube,
IMS,
and
IFA
°
Filta­
Max
 
procedure
using
stomacher,
centrifugation,
IMS,
and
IFA
°
Filta­
Max
 
procedure
using
elution
tube
and
centrifugation,
IMS,
and
IFA
°
Direct
centrifugation,
IMS,
and
IFA
°
The
use
of
different
IMS
kits
°
The
use
of
different
antibody
staining
kits
°
The
use
of
additional
rinses
and
transfers
to
reduce
carryover
from
IMS
to
the
slide
°
The
use
of
multiple
filter
membranes
in
the
Filta­
Max
 
concentrator
tube
Section
3:
Guidance
for
Cryptosporidium
Laboratories
27
Draft
June
2003
Guidance
on
requirements
for
initial
and
ongoing
demonstrations
of
acceptable
laboratory
performance
for
different
method
variations
is
provided
in
Sections
3.3.14.1
through
3.1.14.3,
below.

3.3.14.1
Using
Only
One
Method
Variation
Per
EPA
Method
1622/
1623
(
Section
9.1.2)
and
the
Lab
QA
Program,
if
a
laboratory
uses
the
same
method
variation
for
all
samples,
then
the
laboratory
is
only
required
to
demonstrate
acceptable
initial
laboratory
performance
(
through
the
IPR
and
IPT
tests)
once
and
demonstrate
acceptable
ongoing
laboratory
performance
(
through
the
OPR,
method
blank,
and
OPT
tests)
using
that
method
variation.

3.3.14.2
Switching
from
One
Method
Variation
to
Another
Per
EPA
Method
1622/
1623
(
Section
9.1.2)
and
the
Lab
QA
Program,
if
a
laboratory
intends
to
switch
completely
from
one
method
variation
to
another,
then
the
laboratory
must
demonstrate
acceptable
initial
laboratory
performance
(
through
the
IPR
and
IPT
tests)
using
the
new
method
variation
before
implementing
this
procedure
for
the
analysis
of
field
samples.
If
the
laboratory
demonstrates
acceptable
initial
laboratory
performance
and
implements
the
new
method
variation
and
discontinues
use
of
the
old
method
variation,
then
the
laboratory
must
demonstrate
acceptable
ongoing
laboratory
performance
(
through
the
OPR,
method
blank,
and
OPT
tests)
using
the
new
variation.

3.3.14.3
Using
Multiple
Method
Variations
Per
EPA
Method
1622/
1623
(
Section
9.1.2)
and
the
Lab
QA
Program,
if
a
laboratory
intends
to
use
multiple
method
variations
concurrently,
then
the
laboratory
must
demonstrate
acceptable
initial
laboratory
performance
(
through
the
IPR
and
IPT
tests)
using
each
method
variation
before
implementing
this
procedure
for
the
analysis
of
LT2
rule
samples.
If
the
laboratory
demonstrates
acceptable
initial
laboratory
performance
and
implements
multiple
method
variations,
then
the
laboratory
must
demonstrate
acceptable
ongoing
laboratory
performance
(
through
the
OPR,
method
blank,
and
OPT
tests)
using
each
method
variation
if
the
method
variations
differ
through
the
use
of
different
filters
or
IMS
systems.

The
laboratory
is
not
required
to
demonstrate
acceptable
ongoing
laboratory
performance
using
each
method
variation
if
the
method
variations
differ
through
the
use
of
different
antibody
stains,
the
use
of
additional
rinses
and
transfers
for
some
samples
to
reduce
carryover
from
IMS
onto
the
slide,
or
multiple
membrane
filters
in
the
Filta­
Max
 
particle
concentrator
for
some
samples.

°
If
a
laboratory
alternates
among
more
than
one
antibody
staining
kit,
the
laboratory
must
perform
positive
and
negative
staining
controls
for
each
antibody
kit
for
each
batch
of
slides
to
which
the
kit
is
used
and
must
alternate
between
the
kits
for
ongoing
demonstrations
of
acceptable
laboratory
performance.
MS
samples
should
be
processed
using
the
same
method
variation
as
the
associated
field
sample,
regardless
of
the
method
variation
used
to
demonstrate
ongoing
acceptable
laboratory
performance.

°
If
the
laboratory
uses
additional
rinses
and
transfers
for
some
samples
to
reduce
carryover
from
IMS
onto
the
slide,
the
laboratory
must
use
this
procedure
(
which
may
reduce
recoveries)
to
demonstrate
acceptable
ongoing
laboratory
performance.
MS
samples
should
be
processed
using
the
same
method
variation
as
the
associated
field
sample,
regardless
of
the
method
variation
used
to
demonstrate
ongoing
acceptable
laboratory
performance.

°
If
the
laboratory
uses
multiple
membrane
filters
in
the
Filta­
Max
 
particle
concentrator
for
some
samples,
the
laboratory
must
use
this
procedure
(
which
may
reduce
recoveries)
to
demonstrate
acceptable
ongoing
laboratory
performance.
MS
samples
should
be
processed
using
the
same
method
variation
as
the
associated
field
sample,
regardless
of
the
method
variation
used
to
demonstrate
ongoing
acceptable
laboratory
performance.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
28
Draft
June
2003
3.3.15
Guidance
on
QC
Requirements
for
Different
Sample
Volumes
A
laboratory
with
multiple
PWS
clients
representing
a
range
of
sample
volumes
is
not
responsible
for
performing
QC
tests
at
all
of
the
volumes.
However,
if
the
laboratory
does
analyze
both
10­
L
and
50­
L
sample
volumes
for
clients
 
or
any
volumes
in
between
 
then
the
laboratory
should
demonstrate
acceptable
performance
in
a
manner
representative
of
the
sample
volumes
they
process.
Guidance
on
initial
and
ongoing
demonstrations
of
acceptable
laboratory
performance
is
provided
below,
in
Sections
3.3.15.1
through
3.3.15.3.

3.3.15.1
Guidance
on
Initial
Precision
and
Recovery
Tests
for
Different
Sample
Volumes
A
laboratory
with
multiple
PWS
clients
representing
a
range
of
sample
volumes
should
successfully
perform
the
IPR
and
method
blank
test,
as
well
as
successfully
analyze
matrix
spike/
matrix
spike
duplicate
(
MS/
MSD)
and
initial
proficiency
testing
(
IPT)
samples
(
for
the
Lab
QA
Program's
initial
demonstration
of
capability
[
IDC]),
at
the
largest
(
most
challenging)
volume.
The
laboratory
should
demonstrate
acceptable
performance
for
these
tests
using
spikes
of
no
greater
than
500
oocysts
(
however,
demonstrating
acceptable
performance
at
spikes
of
100
­
200
is
preferable).

3.3.15.2
Guidance
on
Ongoing
Precision
and
Recovery
Tests
and
Method
Blank
Tests
for
Different
Sample
Volumes
A
laboratory
with
multiple
PWS
clients
representing
a
range
of
sample
volumes
is
not
responsible
for
performing
ongoing
precision
and
recovery
(
OPR)
tests
for
each
of
the
volumes.
The
following
approaches
should
be
used,
depending
on
whether
the
laboratory
analyzes
more
than
20
PWS
samples
per
week:

°
A
laboratory
that
processes
different
sample
volumes
and
more
than
20
PWS
samples
per
week
 
­
and
that
would
necessarily
be
required
to
process
more
than
one
set
of
OPR
and
method
blank
samples
each
week
 
­
should
demonstrate
ongoing
acceptable
performance
at
both
extremes
of
the
volume
spectrum
by
performing
one
OPR
and
method
blank
at
a
volume
consistent
with
the
highest
sample
volume
submitted
by
clients
(
e.
g.
50
L)
and
performing
a
second
OPR
and
method
blank
at
a
volume
consistent
with
the
lowest
sample
volume
submitted
by
clients
(
but
not
less
than
10
L).
If
more
than
40
field
samples
are
processed
in
a
week,
and
more
than
two
QC
batches
are
required,
the
laboratory
may
use
the
volume
at
either
extreme
or
a
volume
in
between
the
two
extremes.
The
laboratory
should
work
with
their
PWS
clients
to
attempt
to
schedule
clients
with
different
sample
volume
sizes
for
different
periods
during
the
week,
so
the
field
samples
can
be
batched
with
QC
samples
of
comparable
volume.

°
Laboratories
that
process
fewer
than
20
PWS
samples
per
week
only
need
to
analyze
one
set
of
QC
samples
per
week,
even
if
the
laboratory
processes
samples
of
various
volumes.
However,
the
laboratory
should
perform
the
OPR
and
method
blank
test
using
the
volume
most
representative
of
the
anticipated
sample
volumes
of
the
LT2
rule
samples
to
be
analyzed
that
week.
The
laboratory
also
should
work
with
their
PWS
clients
to
schedule
clients
with
different
sample
volume
sizes
for
different
weeks,
so
the
field
samples
can
be
batched
with
QC
samples
of
comparable
volume.

3.3.15.3
Guidance
on
Ongoing
Proficiency
Tests
for
Different
Sample
Volumes
Laboratories
with
multiple
PWS
clients
representing
a
range
of
sample
volumes
are
not
required
to
analyze
OPT
samples
at
each
sample
volume.
The
laboratory
should
notify
EPA
of
the
sample
volume
most
representative
of
the
LT2
samples
processed
and
perform
the
OPT
test
using
this
volume.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
29
Draft
June
2003
3.4
Sample
Collection
Procedures
Several
options
are
available
to
the
PWS
in
collecting
untreated
surface
water
samples
for
Cryptosporidium
analysis,
including
the
following.

°
Collection
of
bulk
water
samples
for
shipment
to
the
laboratory
for
filtration
and
analysis.

°
On­
site
filtration
of
water
samples
from
pressurized
or
unpressurized
sources
using
the
Pall
Gelman
Envirochek
or
Envirochek
HV
capsule
filter.

°
On­
site
filtration
of
water
samples
from
pressurized
or
unpressurized
sources
using
the
IDEXX
Filta­
Max
foam
filter.

Detailed
procedures
for
each
of
these
options,
as
well
as
packing
and
shipping
the
samples
from
the
PWS
to
the
Cryptosporidium
analysis
laboratory,
are
provided
as
appendices
in
the
Source
Water
Monitoring
Guidance
Manual
for
Public
Water
Systems
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).

As
noted
in
the
PWS
guidance
manual,
EPA
strongly
recommends
that
the
laboratory
and
PWS
conduct
at
least
one
practice
sampling
and
analysis
event
prior
to
starting
official
LT2
monitoring.
Based
on
previous
experiences
in
the
Information
Collection
Rule
(
ICR)
and
ICR
Supplemental
Surveys,
unanticipated
problems
are
often
encountered
during
the
first
sampling
event,
but
are
addressed
in
subsequent
events.
Rather
than
risking
sampling
problems
during
official
LT2
monitoring,
the
PWS
and
laboratory
should
conduct
at
least
one
sampling
event
prior
to
monitoring
to
identify
and
resolve
any
problems.

3.5
Sample
Processing
and
Analysis
Procedures
Although
EPA
has
proposed
the
use
of
the
April
2001
versions
of
EPA
Methods
1622/
1623
in
the
LT2
rule,
EPA
has
requested
comment
on
the
use
of
updated
versions
(
dated
June
2003)
to
consolidate
several
method­
related
changes
EPA
believes
are
necessary
to
address
LT2
rule
monitoring
requirements.
These
changes
are
summarized
in
Section
3.1.2.

This
draft
manual
assumes
that
the
June
2003
versions
of
EPA
Methods
1622
and
1623
will
be
used
for
LT2
rule
monitoring.
The
June
2003
versions
of
these
methods
are
included
as
Appendix
B
and
Appendix
C
of
this
guidance
manual.

3.6
Recordkeeping
An
acceptable
record
keeping
system
provides
information
on
sample
collection
and
preservation,
analytical
methods,
raw
data,
calculations,
reported
results,
and
a
record
of
persons
responsible
for
sampling
and
analyses.
For
EPA
Methods
1622
and
1623,
data
should
be
provided
on
bench
sheets
(
Appendix
G)
and
slide
examination
results
forms
(
Appendix
H).
Original
data,
including
microscope
examination
counts
and
notes,
must
be
recorded
on
these
forms.

Data
should
be
recorded
in
ink
and
a
single
line
drawn
through
any
change
with
an
initialed,
dated
correction
entered
next
to
it.
Data
files
may
also
be
microfiche
or
electronic.
Electronic
data
should
be
backed
up
by
a
protected
tape
or
disk
or
hard
copy.
Microbiological
analyses
records
must
be
kept
for
at
least
5
years.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
30
Draft
June
2003
As
laboratories
perform
Cryptosporidium
analyses
during
the
LT2
rule,
the
following
data
recording
practices
should
be
followed:

°
Maintain
sample
identification
information,
including
sample
collection
and
receipt
dates
and
conditions
°
Record
all
raw
data
(
primary
measurements)
used
to
calculate
final
concentrations
of
oocysts/
L
for
each
sample
°
Record
the
date
and
time
of
each
method
step
associated
with
a
holding
time
to
verify
that
all
method
holding
times
have
been
met
°
Record
the
name
of
the
analyst
performing
each
method
step
to
verify
that
only
qualified
technicians
and
analysts
are
performing
the
method
The
minimum
data
elements
that
should
be
recorded
for
Cryptosporidium
samples
during
the
LT2
are
discussed
in
detail
below.
These
data
elements
are
critical
to
ensuring
that
final
sample
concentrations
can
be
verified
using
primary
data,
and
are
necessary
to
demonstrate
that
all
method­
specified
holding
times
were
met.
Standardized
bench
sheets
and
examination
results
forms
are
available
for
download
on
the
LT2
website
http://
www.
epa.
gov/
safewater/
lt2/
index.
html.

Sampling
records
provided
by
the
PWS
with
the
sample
should
include
the
following
information,
at
a
minimum:

°
Public
water
system
name
and
ID
number*

°
Facility
name
and
number*

°
Date
and
start/
stop
times
of
collection*

°
Sampler's
name
and
phone
number
(
or
alternate
contact
for
laboratory
if
problems
are
encountered)

°
Start
and
stop
times
of
collection
(
if
the
sample
was
filtered
in
the
field)

°
Volume
filtered
information
(
if
the
sample
was
filtered
in
the
field)

°
Whether
the
filter
clogged
(
if
the
sample
was
filtered
in
the
field)

°
Analyses
requested
(
e.
g.
routine
field
sample
analysis
or
field
sample
+
MS
analysis)

*
Note:
The
combination
of
these
three
elements
is
the
unique
sample
number
used
to
identify
the
LT2
sample
from
sample
collection,
analysis,
reporting,
and
use.

Detailed
guidance
on
sample
collection
data
recording,
as
well
as
forms
and
sample
collection
and
shipping
procedures
can
be
found
in
the
Source
Water
Monitoring
Guidance
Manual
for
Public
Water
Systems
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).
This
manual
is
available
for
download
from
http://
www.
epa.
gov/
safewater/
lt2/
index.
html.

Upon
receipt
of
the
sample
at
the
laboratory,
laboratory
personnel
should
record,
at
a
minimum,
the
information
in
Table
3­
3.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
31
Draft
June
2003
Table
3­
3.
Sample
Receipt
Data
Elements
to
Record
in
the
Laboratory
for
EPA
Method
1622/
1623
Cryptosporidium
Analysis
Public
water
system
name
and
ID
Facility
name
and
ID
Sample
collection
point
name
and
ID
Date
of
sample
collection
Date
and
time
of
sample
receipt
by
laboratory
Name
of
laboratory
person
receiving
the
sample
Temperature
of
sample
upon
receipt
Any
deficiencies
(
deficiencies
may
include
but
are
not
limited
to:
exceeded
sample
holding
time,
transport
temperature
exceeded
10
°
C,
or
sample
leaked
during
transport)

Laboratories
analyzing
samples
for
Cryptosporidium
using
EPA
Method
1622/
1623
in
support
of
the
LT2
rule
should
record
the
primary
elements
required
to
calculate
the
final
concentrations
and
percent
recoveries
for
matrix
spike
and
ongoing
precision
and
recovery
samples.
These
primary
data
elements
are
provided
in
Table
3­
4,
and
should
be
recorded
on
the
EPA
Method
1622/
1623
bench
sheet
(
Appendix
G).

Table
3­
4.
Primary
Data
Elements
to
Record
in
the
Laboratory
for
EPA
Method
1622/
1623
Cryptosporidium
Analysis
Estimated
number
of
oocysts
spiked
(
MS
and
OPR
samples),
based
on
information
provided
by
the
flow­
cytometry
laboratory
with
the
spiking
suspension
Samples
volume
spike,
in
L
(
MS
and
OPT
samples)

Sample
volume
filtered,
to
nearest
1/
4
L
Number
of
filters
used
(
if
the
filter
clogged)

Pellet
volume
after
concentration,
to
the
nearest
0.1
mL
Total
volume
of
resuspended
concentrate,
in
mL
Volume
of
the
resuspended
concentrate
transferred
to
IMS,
in
mL
Number
of
subsamples
analyzed
Total
number
of
oocysts
detected
in
the
sample
To
determine
that
all
method
QC
requirements
were
met
and
that
the
samples
was
analyzed
by
qualified
personnel
according
to
the
requirements
of
the
Laboratory
QA
Program
for
the
Analysis
of
Cryptosporidium
the
laboratory
should
record
the
elements
in
Table
3­
5.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
32
Draft
June
2003
Table
3­
5.
QC
Data
Elements
to
Record
in
the
Laboratory
for
EPA
Method
1622/
1623
Cryptosporidium
Analysis
Elution
date
and
time
(
must
be
within
96
hours
of
sample
collection)

Slide
preparation
date
and
time
(
must
be
completed
in
same
working
day
as
elution)

Sample
staining
date
and
time
(
must
be
completed
within
72
hours
of
slide
preparation)

Sample
examination
date
and
time
(
must
be
completed
within
7
days
of
sample
staining)

Analyst
performing
filtration
Analyst
performing
elution
Analyst
performing
IMS
Analyst
performing
sample
staining
Analyst
performing
sample
examination
Results
of
the
positive
and
negative
staining
controls
The
laboratory
should
also
record
any
additional
information
that
will
support
the
results
obtained
or
allow
problems
with
sample
results
and
laboratory
performance
to
be
identified.
This
additional
information
could
include
the
following:

°
Information
on
the
version
of
EPA
Method
1622/
1623
used
to
perform
the
analysis
including;
filter
type,
elution
procedure,
concentration
procedure,
IMS
system
used,
detection
kit
used,
and
source
of
oocysts
for
spiking
suspensions
°
Lot
numbers
of
reagents
and
materials
used
during
the
analysis,
including
the
filter,
elution
buffer,
IMS
system,
detection
kit,
and
spiking
suspension
°
FITC,
DAPI,
and
DIC
information
of
all
oocysts
detected
in
the
field
samples
using
the
examination
results
form
This
information
should
be
recorded
on
the
EPA
Method
1622/
1623
bench
sheet
(
Appendix
G)
and
slide
examination
results
form
(
Appendix
H),
as
appropriate.

Size,
shape,
and
DIC
and
DAPI
characteristics
of
the
three
Cryptosporidium
oocysts
for
ongoing
analyst
verification
(
Section
3.3.10)
must
be
recorded
by
the
analyst
on
a
microscope
log.

3.7
Calculations
for
EPA
Methods
1622/
1623
During
LT2
rule
monitoring,
field
sample
results
must
be
reported
in
oocysts/
L
and
MS
recoveries
must
be
calculated
and
reported.
In
addition,
laboratories
must
calculate
recoveries
for
other
QC
samples
[
40
CFR
part
141.705
(
a)].
Calculations
for
EPA
Method
1622/
1623
field
and
QC
samples
are
provided
below,
in
Sections
3.7.1
through
3.7.3.

3.7.1
Field
Sample
Calculations
To
calculate
the
concentration
of
Cryptosporidium
in
your
field
sample,
reported
as
oocysts/
L,
the
following
information
is
needed:

°
Number
of
oocysts
detected
in
the
sample
(
recorded
as
a
primary
measurement
from
the
examination
results
form)

°
Volume
analyzed
Section
3:
Guidance
for
Cryptosporidium
Laboratories
33
Draft
June
2003
Using
these
two
data
elements,
the
final
concentration
is
calculated
as:

final
concentration
(
oocysts/
L)
=
oocysts
detected
in
the
sample
volume
analyzed
(
L)

If
100%
of
the
sample
volume
filtered
is
examined,
then
the
volume
analyzed
equals
the
volume
filtered.
This
applies
whether
one
filter
or
more
than
one
filter
was
used;
if
more
than
one
filter
was
used,
and
all
of
the
volume
filtered
through
the
multiple
filters
is
processed
through
the
remainder
of
the
method,
then
the
volume
examined
is
simply
the
sum
of
the
volumes
filtered
through
each
of
the
filters
used.

If
<
100%
of
the
volume
filtered
was
processed
through
the
remainder
of
the
method,
then
additional
calculations
are
needed
to
determine
the
volume
analyzed.
This
is
discussed
in
Section
3.7.1.1.

3.7.1.1
Determining
Volume
Analyzed
when
Less
than
100%
of
Sample
Was
Examined
When
<
100%
of
the
sample
filtered
is
processed
through
the
remainder
of
the
method
and
examined
(
such
as
when
the
volume
filtered
yields
>
2
mL
of
packed
pellet
volume
after
centrifugation),
then
the
volume
analyzed
must
be
determined
using
the
following
equations
to
determine
the
percentage
of
the
sample
that
was
examined.

percent
examined
=
total
volume
of
resuspended
concentrate
transferred
to
IMS
(
mL)
(
see
Section
3.7.1.2)

total
volume
of
resuspended
concentrate
produced
(
mL)

volume
analyzed
(
L)
=
percent
examined
×
sample
volume
filtered
(
L)

3.7.1.2
Determining
the
Volume
of
Resuspended
Concentrate
to
Use
for
Packed
Pellets
>
0.5
mL
Packed
pellets
with
a
volume
>
0.5
mL
must
be
divided
into
subsamples.
Use
the
formula
below
to
determine
the
total
volume
of
resuspension
required
in
the
centrifuge
tube
before
separating
the
concentrate
into
two
or
more
subsamples
and
transferring
to
IMS.

total
volume
of
resuspended
concentrate
(
mL)
required
=
pellet
volume
(
mL)
after
centrifugation
×
5
mL
0.5
mL
3.7.1.3
Example
Calculation
Example.
A
10­
L
field
sample
was
filtered
and
processed,
producing
a
packed
pellet
volume
of
2.7
mL.
The
laboratory
transferred
20
mL
of
the
total
resuspended
concentrate
to
IMS
and
examination.
The
laboratory
detected
20
oocysts
during
examination.
The
following
calculations
were
performed
to
determine
the
volume
analyzed
and
final
concentration.

total
volume
of
resuspended
concentrate
(
mL)
required
=
2.7
mL
×
5
mL
=
27
mL
0.5
mL
percent
examined
=
20
mL
=
0.74
(
74%)
27
mL
Section
3:
Guidance
for
Cryptosporidium
Laboratories
34
Draft
June
2003
volume
analyzed
(
L)
=
0.74
×
10
L
=
7.4
L
final
concentration
(
oocysts/
L)
=
20
oocysts
=
2.7
oocysts/
L
7.4
L
3.7.2
Matrix
Spike
Sample
Calculations
To
determine
the
percent
recovery
for
a
matrix
spike
(
MS)
sample
the
following
information
is
needed:

°
The
number
of
oocysts
detected
in
the
MS
sample
°
The
estimated
number
of
oocysts
spiked
into
the
MS
sample
°
The
number
of
oocysts
detected
in
the
unspiked
field
sample
(
to
correct
for
background
concentration)

percent
recovery
=
oocysts
counted
in
MS
sample
­
oocysts
counted
in
unspiked
field
sample
×
100%
oocysts
spiked
into
MS
sample
If
both
a
matrix
spike
(
MS)
and
a
matrix
spike
duplicate
(
MSD)
are
analyzed,
then
the
mean
recovery
and
relative
percent
difference
(
RPD)
should
be
calculated
and
compared
to
the
acceptance
criteria
in
Section
21.0,
Tables
3
and
4
of
the
June
2003
version
of
EPA
Method
1623.
The
percent
recovery
for
each
sample
should
be
calculated
as
described
above
to
determine
the
mean
recovery.

To
calculate
the
mean
percent
recovery,
calculate
the
percent
recovery
for
each
sample,
as
described
above,
then
use
the
following
formula:

mean
percent
recovery
=
percent
recovery
of
MS
sample
+
percent
recovery
of
MSD
sample
2
To
calculate
the
RPD,
the
absolute
value
(
without
sign)
of
the
difference
between
the
number
of
oocysts
detected
in
the
MS
and
MSD
is
divided
by
the
mean
of
the
oocysts
detected
in
both
samples
to
yield
a
percentage
of
the
difference.

RPD
=
*
oocysts
detected
in
MS
­
oocysts
detected
in
MSD*
×
100%
((
oocysts
detected
in
MS
+
oocysts
detected
in
MSD)/
2)

Example.
The
laboratory
prepared
both
the
MS
and
MSD
by
spiking
two
10­
L
samples
with
100
oocysts
each.
For
both
the
MS
and
MSD,
the
entire
10­
L
sample
was
filtered
and
100%
of
the
sample
was
examined.
The
laboratory
detected
45
oocysts
in
the
MS
sample
and
50
oocysts
in
the
MSD.
In
the
unspiked
field
sample
only
2
oocysts
were
detected.
To
determine
the
percent
recovery
for
each
sample
and
the
mean
recovery
and
relative
percent
difference
of
the
MS
and
MSD,
the
following
calculations
were
performed.

MS
percent
recovery
=
45
oocysts
­
2
oocysts
×
100%
=
43%
100
oocysts
Section
3:
Guidance
for
Cryptosporidium
Laboratories
35
Draft
June
2003
MSD
percent
recovery
=
50
oocysts
­
2
oocysts
×
100%
=
48%
100
oocysts
mean
recovery
=
43%
+
48%
×
100%
=
45.5%
2
RPD
=
*
45
oocysts
­
50
oocysts*
×
100%
=
10.5%
((
45
oocysts
+
50
oocysts)
/
2)

3.7.3
OPR
Sample
Calculations
The
percent
recovery
of
an
OPR
sample
is
calculated
using
the
following
formula:

percent
recovery
=
oocysts
detected
×
100%
oocysts
spiked
Example:
The
laboratory
prepared
the
OPR
sample
by
spiking
50
L
with
150
oocysts.
The
entire
sample
was
filtered
and
examined.
The
laboratory
detected
76
oocysts.

percent
recovery
=
76
oocysts
×
100%
=
50.7%
150
oocysts
OPR
recoveries
are
compared
to
the
limits
for
ongoing
recovery
in
Table
3
and
4
in
Section
21.0
of
the
June
2003
version
of
EPA
Method
1623.
These
recoveries
are
tracked
over
time
using
control
charts
to
assess
precision,
as
discussed
in
Section
3.3.5,
above.

3.8
Electronic
Data
Reporting
During
the
LT2
rule,
laboratories
will
report
Cryptosporidium
data
to
their
PWS
clients
electronically
through
EPA's
LT2
Data
Collection
System.
The
LT2
Data
Collection
System
is
a
web­
based
application
that
allows
laboratory
users
to
enter
or
upload
data,
then
electronically
"
release"
the
data
to
the
PWS
for
review,
approval,
and
submission
to
EPA
and
the
State.
Although
ownership
of
the
data
resides
with
the
PWS
throughout
this
process,
the
LT2
Data
Collection
System
increases
the
ease
and
efficiency
of
the
data
entry
and
transfer
process
from
one
party
to
another
by
transferring
the
ability
to
access
the
data
from
the
laboratory
to
the
PWS
to
EPA
and
the
State,
and
ensuring
that
data
cannot
be
viewed
or
changed
by
unauthorized
parties.
A
summary
of
the
data
entry,
review,
and
transfer
process
through
the
LT2
Data
Collection
System
is
provided
in
Table
3­
6,
below.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
36
Draft
June
2003
Table
3­
6.
LT2
Data
Collection
System
Data
Entry,
Review,
and
Transfer
Process
Laboratory
actions
°
Laboratory
posts
analytical
results
to
the
LT2
Data
Collection
System
°
LT2
Data
Collection
System
reduces
data
and
checks
data
for
completeness
and
compliance
with
LT2
rule
requirements
°
Laboratory
Principal
Analyst
confirms
that
data
meet
quality
control
requirements
°
Laboratory
"
releases"
results
electronically
to
the
PWS
for
review
°
Laboratory
user
cannot
edit
data
after
it
is
released
to
the
PWS
EPA
does
not
have
access
to
data
PWS
actions
°
PWS
cannot
edit
data
­
only
review
data
and
either
return
to
laboratory
to
resolve
errors
or
submit
to
EPA
°
PWS
reviews
electronic
data
through
LT2
Data
Collection
System
°
PWS
"
releases"
data
back
to
the
laboratory
if
questions
°
If
no
questions,
PWS
submits
data
to
EPA
as
"
approved"
or
"
contested"
(
indicating
that
samples
have
been
correctly
analyzed,
but
that
the
PWS
contends
are
not
valid
for
use
in
LT2
binning)

EPA
and
State
actions
°
EPA
and
State
users
cannot
edit
data
­
only
review
data
°
EPA
and
State
review
data
through
LT2
Data
Collection
System
The
data
reporting
process
is
discussed
in
more
detail
below,
in
Sections
7.2.1
through
7.2.3,
and
discussed
in
detail
in
the
Users'
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule)
Data
Collection
System.
The
LT2
data
system
users'
guide
also
provides
detailed
information
on
the
PWS
user
registration
process.
Information
on
the
LT2
Data
Collection
System,
as
well
as
a
downloadable
users'
manual,
is
available
at
http://
www.
epa.
gov/
safewater/
lt2/
index.
html.

3.8.1
Data
Entry/
Upload
The
analyst
or
another
laboratory
staff
member
enters
a
subset
of
the
data
recorded
at
the
bench
(
Section
7.1)
into
the
LT2
Data
Collection
System,
either
by
entering
the
data
using
web
forms
or
by
uploading
data
in
XML
format.
Per
40
CFR
part
141.707(
e)(
1),
this
information
includes
the
following:

°
PWS
ID
°
Facility
ID
°
Sample
collection
point
°
Sample
collection
date
°
Sample
type
(
field
or
MS)

°
Sample
volume
filtered
(
L),
to
nearest
¼
L
°
Was
100%
of
filtered
volume
examined?

°
Number
of
oocysts
counted
°
For
samples
in
which
less
than
10
L
is
filtered
or
less
than
100%
of
the
sample
volume
is
examined,
the
laboratory
also
must
enter
or
upload
the
number
of
filters
used
and
the
packed
pellet
volume
Section
3:
Guidance
for
Cryptosporidium
Laboratories
37
Draft
June
2003
°
For
samples
in
which
less
than
100%
of
sample
volume
is
examined,
the
laboratory
also
must
report
the
volume
of
resuspended
concentrate
and
volume
of
this
resuspension
processed
through
immunomagnetic
separation
°
For
matrix
spike
samples,
the
laboratory
also
must
report
the
sample
volume
spiked
and
estimated
number
of
oocysts;
these
data
are
not
required
for
field
samples
The
laboratory
must
verify
that
all
holding
times
and
other
QC
requirements
were
met.

After
the
information
has
been
entered
or
uploaded
into
the
system,
the
system
will
reduce
the
data
to
yield
final
sample
results,
in
oocysts/
L,
verify
that
LT2
rule
Cryptosporidium
sample
volume
analysis
requirements
were
met
for
samples
in
which
less
than
10
L
were
analyzed,
and
calculate
MS
recoveries.

The
laboratory's
Primary
Analyst
under
the
Lab
QA
Program
is
responsible
for
verifying
the
quality
and
accuracy
of
all
sample
results
in
the
laboratory,
and
is
required
to
review
and
approve
the
results
before
they
are
submitted
to
the
PWS
for
review.
If
inaccuracies
or
other
problems
are
identified,
the
primary
analyst
discusses
the
sample
information
with
the
analyst
or
data
entry
staff
and
resolves
the
issues
before
the
data
are
approved
for
PWS
review.

If
no
inaccuracies
or
other
issues
are
identified,
the
Primary
Analyst
approves
the
reported
data
for
"
release"
to
the
PWS
for
review
(
EPA
does
not
receive
the
data
at
this
point).
When
the
data
are
approved,
the
rights
to
the
data
are
transferred
electronically
by
the
system
to
the
PWS,
and
the
data
can
no
longer
be
changed
by
the
laboratory.

3.8.2
PWS
Data
Review
After
the
laboratory
has
released
Cryptosporidium
data
electronically
to
the
PWS
using
the
LT2
Data
Collection
System,
the
PWS
will
review
the
results.
The
PWS
user
cannot
edit
the
data,
but
if
the
PWS
has
an
issue
with
the
sample
result,
such
as
if
the
PWS
believes
that
the
sample
collection
point
ID
or
collection
date
is
incorrect,
the
PWS
can
release
the
results
back
to
the
laboratory
for
issue
resolution.
In
addition
to
noting
the
reason
in
the
LT2
Data
Collection
System
for
the
return
of
the
data
to
the
laboratory,
you
also
should
contact
the
laboratory
verbally
to
discuss
the
issue.

If
the
PWS
determines
that
the
data
are
accurate,
the
PWS
releases
the
results
to
EPA
(
and
the
State,
if
applicable)
as
"
approved"
results.
If
the
PWS
determines
that
the
data
are
accurate,
but
believes
that
the
data
are
not
valid
for
LT2
binning
purposes,
the
PWS
can
release
the
results
to
EPA
and
the
State
as
"
contested."
Contested
samples
are
those
that
have
been
correctly
analyzed,
but
that
you
contend
are
not
valid
for
use
in
LT2
binning,
and
have
submitted
to
EPA
for
evaluation.

3.8.3
EPA/
State
Review
After
the
PWS
has
released
the
results
as
approved
or
contested,
they
are
available
to
EPA
and
State
users
to
review
through
the
LT2
Data
Collection
System.
EPA
and
State
users
cannot
edit
the
data.

3.9
Data
Archiving
he
PWS
is
required
to
archive
all
original,
hardcopy
quality
control
data
associated
with
LT2
sample
analyses
for
36
months
after
the
end
of
the
second
round
of
Cryptosporidium
monitoring
(
which
is
scheduled
to
take
place
6
years
after
the
start
of
the
first
round
[
40
CFR
part
141.731(
a)].
Although
it
is
the
PWS's
responsibility
to
meet
LT2
rule
data
storage
requirements
for
compliance
monitoring
samples,
including
MS
samples,
the
PWS
may
designate
this
responsibility
to
the
laboratory.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
38
Draft
June
2003
3.9.1
Hardcopy
Data
The
following
data
should
be
archived:

°
Bench
sheets
and
examination
results
forms
for
all
LT2
monitoring
samples,
including
both
field
samples
and
MS
samples
°
Bench
sheets
and
examination
results
forms
for
all
OPR
samples
and
method
blank
samples,
and
records
of
the
compliance
monitoring
samples
associated
with
each
OPR
sample
and
blank
sample
°
Spike
enumeration
information
received
from
Cryptosporidium
spiking
suspension
vendors
°
Bench
sheets
and
examination
results
forms
for
all
OPT
samples
As
part
of
the
Lab
QA
Program,
the
laboratory
also
must
maintain
the
same
documentation
for
their
IPR
and
IPT
data
for
each
method
variation
used
for
LT2
samples.

3.9.2
Slides
Although
not
required,
laboratories
also
may
want
to
archive
slides
and/
or
take
photographs
of
slides
to
maintain
for
clients.
Slides
should
be
stored
in
a
humid
chamber
in
the
dark
at
0
°
C
to
10
°
C.

As
an
alternate
to
the
DABCO/
glycerol
mounting
medium
currently
specified
in
EPA
Method
1622
and
1623,
laboratories
may
wish
to
evaluate
the
use
of
the
elvanol
mounting
medium,
which
hardens,
and
may
be
useful
for
archiving
slides.
Reagents
for
the
mounting
medium
include
the
following:

°
8.0
g
elvanol
 
ICN
Biomedicals
cat.
no.
151937,
Aurora,
Ohio,
or
equivalent
°
48.0
g
(
40
mL)
glycerol
°
10%
NaN3
°
DABCO
 
Sigma
Chemical
Co.
cat
no.
D­
2522,
or
equivalent
°
Tris
buffer
 
Dissolve
1.2
g
Tris
(
Fisher
cat.
no.
BP152)
in
95
mL
reagent
water,
adjust
pH
to
8.5
with
1
N
HCl
To
prepare
the
medium,
use
the
following
procedure:

°
Add
48.0
g
(
40
mL)
glycerol
to
8.0
g
elvanol
and
stir.

°
Add
49.0
mL
of
reagent
water
and
1.0
mL
10%
NaN3
and
stir.
Let
stand
4
hours
at
room
temperature.

°
Add
DABCO
in
Tris
buffer
(
4.75
g
of
DABCO
in
100
mL
Tris
buffer,
adjusted
to
pH
8.5
with
conc.
HCl)
and
stir.

°
Place
mixture
in
a
boiling
water
bath
until
the
mixture
becomes
homogenous.

°
Centrifuge
mixture
at
2000XG
for
10
minutes.
Centrifugation
of
entire
mixture
in
one
tube
is
preferable.

°
Dispense
3­
to
5­
mL
aliquots
of
the
mixture
into
tubes
and
store
at
0
°
C
to
10
°
C.

If
the
mounting
medium
sets
up
in
the
tube
during
storage,
re­
heat
in
boiling
water
bath
or
microwave
for
a
short
time
to
restore
mixture
to
liquid
state.

Make
sure
to
test
any
new
reagent
first
on
QC
samples
to
verify
that
the
mounting
medium
performs
properly
before
using
the
medium
on
any
monitoring
samples.
Section
3:
Guidance
for
Cryptosporidium
Laboratories
39
Draft
June
2003
3.10
Equipment,
Supplies,
Reagents,
and
Standards
See
Sections
6
and
7
of
EPA
Method
1622
(
Appendix
B)
or
EPA
Method
1623
(
Appendix
C)
for
details
on
the
materials
needed
to
perform
the
Cryptosporidium
analyses
specified
in
the
methods.
Detailed
information
on
vendors
for
these
materials
is
provided
in
Section
3.11.

3.11
Vendor
Contact
List
3.11.1
Sample
Collection
and
Filtration
Supplies
Alamo
Water
Treatment
13700
Highway
90
West
San
Antonio,
TX
78245
Phone:
(
800)
659­
8400
ext.
2
Fax:
(
800)
659­
8402
E­
mail:
scsales@
alamowater.
com
BDH/
Merck
www.
merckeurolab.
ltd.
uk
Merck
Eurolab
Ltd.
UK
Headquarters
Laboratory
Supplies
Merck
House,
Poole,
Dorset
BH15
1TD
Sales:
Phone:
01202
669700
Fax:
01202
665599
E­
mail:
info@
merckeurolab.
ltd.
uk
Technical
Support:
Phone:
01202
669700
Fax:
01202
665599
E­
mail:
info@
merckeurolab.
ltd.
uk
Bertram
Controls
Corporation
LLC
5
Eastview
Drive
Farmington,
CT
06032
Sales
and
Technical
Support:
Phone:
(
800)
243­
2340
Fax:
(
800)
243­
7166
E­
mail:
techsupport@
BertramContols.
com
Cole­
Parmer
www.
coleparmer.
com
625
East
Bunker
Court
Vernon
Hills,
IL
60061
Sales:
Phone:
(
800)
323­
4340
Fax:
(
847)
247­
2929
E­
mail:
sales@
coleparmer.
com
Technical
Support:
Phone:
(
800)
323­
4340
ext.
122
Fax:
(
847)
327­
2987
E­
mail:
techinfo@
coleparmer.
com
E.
Clark
and
Associates
www.
devicelink.
com/
company98
10
Brent
Drive
Hudson,
MA
01749
Sales
and
Technical
Support:
Phone:
(
800)
253­
2497
Fax:
(
978)
568­
0060
Email:
mail@
clarksol.
com
Grainger
www.
grainger.
com
Contact
sales
rep.
for
local
address
and
more
contact
info.
Sales:
Phone:
(
888)
361­
8649
Technical
Support:
Phone:
(
800)
535­
5400
Section
3:
Guidance
for
Cryptosporidium
Laboratories
40
Draft
June
2003
Sample
Collection
and
Filtration
Supplies
(
Continued)

IDEXX
www.
idexx.
com
Sales:
Phone:
(
800)
321­
0207
ext.
1
Fax:
(
207)
856­
0630
Technical
Support:
Phone:
(
800)
321­
0207
ext.
2
Fax:
(
207)
856­
0630
E­
mail:
water@
idexx.
com
Pall
Gelman
Sciences
www.
pall.
com/
gelman
600
South
Wagner
Road
Ann
Arbor,
MI
48103
Sales:
Phone:
(
800)
521­
1520
ext.
2
Fax:
(
734)
913­
6495
Technical
Support:
Phone:
(
800)
521­
1520
ext.
3
Fax:
(
734)
913­
6495
PPG
Industries
www.
ppg.
com
One
PPG
Place
Pittsburgh,
PA
15272
Sales
and
Technical
Support:
Phone:
(
412)
434­
3131
Tygon
www.
tygon.
com
Saint­
Gobain
Performance
Plastics
Corp.
Distributor:
Ryan
Herco.
Products
1501
Waco
St.
Richmond,
VA
23244
Sales:
Phone:
(
804)
672­
1158
Fax:
(
804)
672­
0878
Technical
Support:
Saint­
Gobain
Phone:
(
800)
798­
1539
Fax:
(
800)
488­
9466
Email:
linda.
d.
anderson@
saint­
gobain.
com
3.11.2
Sample
Concentration
and
Purification
Supplies
Dynal,
Inc.
www.
dynal.
net
5
Delaware
Drive
Lake
Success,
NY
11042
Sales:
Phone:
(
800)
638­
9416
ext.
1
Fax:
(
516)
326­
3298
E­
mail:
uscustserv@
dynalbiotech.
com
Technical
Support:
Phone:
(
800)
638­
9416
ext.
2
Fax:
(
516)
326­
3298
E­
mail:
ustechserv@
dynalbiotech.
com
International
Equipment
Company
www.
labcentrifuge.
com
300
Second
Avenue
Needham
Heights,
MA
02492
Sales
and
Technical
Support:
Phone:
(
800)
841­
1113
ext.
9723
Fax:
(
781)
444­
6743
E­
mail:
pat.
webb@
thermoiec.
com
Section
3:
Guidance
for
Cryptosporidium
Laboratories
41
Draft
June
2003
3.11.3
Slides
and
Related
Supplies
Dynal,
Inc.
www.
dynal.
net
5
Delaware
Drive
Lake
Success,
NY
11042
Sales:
Phone:
(
800)
638­
9416
ext.
1
Fax:
(
516)
326­
3298
E­
mail:
uscustserv@
dynalbiotech.
com
Technical
Support:
Phone:
(
800)
638­
9416
ext.
2
Fax:
(
516)
326­
3298
E­
mail:
ustechserv@
dynalbiotech.
com
Hauser
Scientific
www.
deltasci.
com
Distributed
by
Delta
Scientific
1979
Stout
Drive,
Bldg.
5
Ivyland,
PA
38974
Sales
and
Technical
Support:
Phone:
(
800)
428­
9400
Fax:
(
800)
428­
3271
E­
mail:
dsirep@
aol.
com
Meridian
Diagnostics,
Inc.
3471
River
Hills
Drive
Cincinnati,
OH
45244
Sales:
Phone:
(
800)
543­
1980
Fax:
(
513)
271­
0124
Technical
Support:
Phone:
(
800)
343­
3858
Fax:
(
513)
271­
0124
PGC
Scientifics
www.
pgcscientifics.
com
P.
O.
Box
7277
Gaithersburg,
MD
20898­
7277
Sales
and
Technical
Support:
Phone:
(
800)
424­
3300
Fax:
(
800)
662­
1112
E­
mail:
cs@
pgcscientifics.
com
Waterborne,
Inc.
www.
waterborneinc.
com
6047
Hurst
Street
New
Orleans,
LA
70118­
6129
Sales
and
Technical
Support:
Phone:
(
504)
895­
3338
Fax:
(
504)
895­
3338
E­
mail:
custserv@
waterborneinc.
com
3.11.4
Antibody
Stains
BioTechnology
Frontiers
(
BTF)
www.
biotechnologyfrontiers.
com
Unit
1
35­
41
Waterloo
Road
North
Ryde
NSW
Australia
Fax:
+
61
2
9889
1805
Email:
contact@
biotechnologyfrontiers.
com
Meridian
Diagnostics,
Inc.
3471
River
Hills
Drive
Cincinnati,
OH
45244
Sales:
Phone:
(
800)
543­
1980
Fax:
(
513)
271­
0124
Technical
Support:
Phone:
(
800)
343­
3858
Fax:
(
513)
271­
0124
Waterborne,
Inc.
www.
waterborneinc.
com
6047
Hurst
Street
New
Orleans,
LA
70118­
6129
Sales
and
Technical
Support:
Phone:
(
504)
895­
3338
Fax:
(
504)
895­
3338
E­
mail:
custserv@
waterborneinc.
com
Section
3:
Guidance
for
Cryptosporidium
Laboratories
42
Draft
June
2003
3.11.5
Microscope
Equipment
Chroma
Technology
Corp.
www.
chroma.
com
74
Cotton
Mill
Hill
Brattleboro,
VT
05301
Sales:
Phone:
(
800)
824­
7662
Fax:
(
802)
257­
9400
Technical
Support:
Phone:
(
800)
824­
7662
Fax:
(
802)
257­
9400
Nikon
www.
nikonusa.
com
NIKON
1300
Walt
Whitman
Road
Melville,
NY
11747
Customer
Information:
Phone:
(
800)
52­
NIKON
Email:
Available
through
website
Olympus
www.
olympusamerica.
com
Olympus
America
INC.
2
Corporate
Center
Drive,
Melville,
NY
11747­
3157,
U.
S.
A.
Product
Support:
Phone:
(
800)
446­
5967
Email:
Available
through
website
Zeiss
www.
zeiss.
com
Carl
Zeiss
Microimaging,
Inc.
One
Zeiss
Drive
Thornwood,
N.
Y.
10594
Sales
and
Technical
Support:
Phone:
(
800)
233­
2343
Fax:
(
914)
684­
7446
E­
mail:
micro@
zeiss.
com
3.11.6
Spiking
Suspensions
and
Positive
Staining
Control
Materials
BioTechnology
Frontiers
(
BTF)
www.
biotechnologyfrontiers.
com
Unit
1
35­
41
Waterloo
Road
North
Ryde
NSW
Australia
Fax:
+
61
2
9889
1805
Email:
contact@
biotechnologyfrontiers.
com
Hyperion
Research,
Ltd.
1008
Allowance
Avenue,
SE
Medicine
Hat,
Alberta
TIA
3G8
Canada
Sales
and
Technical
Support:
Peter
Wallis
Phone:
(
403)
529­
0847
Fax:
(
403)
529­
0852
E­
mail:
hyperion@
telusplanet.
net
Waterborne,
Inc.
www.
waterborneinc.
com
6047
Hurst
Street
New
Orleans,
LA
70118­
6129
Sales
and
Technical
Support:
Phone:
(
504)
895­
3338
Fax:
(
504)
895­
3338
E­
mail:
custserv@
waterborneinc.
com
Sterling
Parasitology
Laboratory,
University
of
Arizona,
Tucson
Building
90,
Rm.
202
Tucson,
AZ
85721
Sales
and
Technical
Support:
Marilyn
Marshall
Phone:
(
520)
621­
4433
Fax:
(
520)
621­
3588
E­
mail:
marshalm@
u.
arizona.
edu
Wisconsin
State
Laboratory
of
Hygiene,
Flow
Cytometry
Unit
2601
Agriculture
Drive
Madison,
WI
53718
Sales
and
Technical
Support:
Phone:
(
608)
224­
6260
Fax:
(
608)
224­
6213
Section
3:
Guidance
for
Cryptosporidium
Laboratories
43
Draft
June
2003
3.11.7
Other
Laboratory
Supplies
for
EPA
Method
1622/
1623
Fisher
Scientific
www.
fishersci.
com
9999
Veterans
Memorial
Drive
Houston,
TX
77038
Sales:
Phone:
(
800)
766­
7000
Fax:
(
800)
926­
1166
Technical
Support:
Phone:
(
800)
766­
7000
Fax:
(
800)
926­
1166
Sigma
www.
sigma­
aldrich.
com
P.
O.
Box
14508
St.
Louis,
MO
63178
Sales:
Phone:
(
800)
325­
3010
Fax:
(
800)
325­
8070
E­
mail:
custserv@
sial.
com
Technical
Support:
Phone:
(
800)
325­
5832
Fax:
(
314)
286­
7828
E­
mail:
techserv@
sial.
com
VWR
www.
vwr.
com
05
Heron
Drive
Bridgeport,
NJ
08014
Sales:
Phone:
(
800)
932­
5000
Fax:
(
856)
467­
3336
Technical
Support:
Phone:
(
800)
932­
5000
Fax:
(
856)
467­
3336
44
Draft
June
2003
SECTION
4:
GUIDANCE
FOR
E.
COLI
LABORATORIES
Although
public
water
system
(
PWS)
treatment
requirements
will
be
determined
from
Cryptosporidium
monitoring
results,
the
Long­
Term
2
Enhanced
Surface
Water
Treatment
(
LT2)
rule
also
requires
that
large
PWSs
(
those
serving
>
10,000)
perform
E.
coli
and
turbidity
analyses
on
source
water
samples
[
40
CFR
part
141.701
(
b)].
The
Cryptosporidium,
E.
coli,
and
turbidity
data
will
be
analyzed
to
evaluate
whether
E.
coli
and/
or
turbidity
levels
can
be
used
as
reliable
indicators
of
Cryptosporidium
occurrence
in
surface
waters
used
as
source
waters
for
drinking
water
facilities.
Depending
upon
the
results
of
these
analyses,
EPA
may
permit
small
PWSs
to
use
E.
coli
monitoring,
or
a
combination
of
E.
coli
and
turbidity
monitoring,
to
determine
the
need
for
implementing
more
expensive
Cryptosporidium
monitoring
or
improved
treatment.

Cryptosporidium
analysis
requirements
and
recommendations
are
discussed
in
Section
3
of
this
guidance
manual.
Turbidity
measurement
requirements
and
recommendations
are
addressed
in
the
Source
Water
Monitoring
Guidance
Manual
for
Public
Water
Systems
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule)
(
available
for
download
from
http://
www.
epa.
gov/
safewater/
lt2/
index.
html).
E.
coli
analysis
requirements
and
recommendations
for
the
LT2
rule,
including
laboratory
certification,
quality
assurance/
quality
control
(
QA/
QC),
and
the
procedures
for
each
of
the
methods
approved
for
use
during
LT2
E.
coli
monitoring,
are
presented
below.

All
E.
coli
sample
analyses
performed
under
the
LT2
rule
must
be
quantitative
[
40
CFR
141.705
(
b)].
Presence/
absence
data
cannot
be
used
to
assess
whether
E.
coli
concentration
can
be
used
to
indicate
Cryptosporidium
occurrence
levels.

4.1
Laboratory
Certification
Program
PWS
laboratories
and
other
laboratories
performing
LT2
rule
E.
coli
analyses
must
be
certified
for
coliform
analyses
under
the
drinking
water
certification
program
for
a
similar
method
type
(
e.
g.,
membrane
filtration,
multiple­
tube,
multiple­
well)
[
40
CFR
part
141.706
(
b)].
The
drinking
water
certification
program
was
established
to
fulfill
requirements
stipulated
by
the
Safe
Drinking
Water
Act
(
SDWA)
and
the
requirements
of
the
National
Primary
Drinking
Water
Regulations
(
NPDWR)
(
40
CFR
141.28).
The
certification
program
requires
that
all
laboratories
analyzing
drinking
water
must
be
certified
by
EPA,
including
EPA's
Regional
labs,
laboratories
on
Federal
Indian
Lands,
principal
state
laboratories,
and
laboratories
that
perform
analyses
under
the
SDWA
in
states
without
primacy.
States
that
have
primary
enforcement
responsibility
(
primacy)
are
required
to
have
laboratory
facilities
available
which
have
been
certified
by
EPA
(
see
40
CFR
142.10)
or
National
Environmental
Laboratory
Accreditation
Conference
(
NELAC).
All
labs
that
perform
testing
for
compliance
purposes
must
be
certified
by
the
state
or
EPA.
Public
water
systems
serving
at
least
25
persons
or
having
at
least
15
service
connections
must
comply
with
the
SDWA
and
the
requirements
of
the
NPDWR.

The
certification
program
consists
of
annual
performance
evaluation
samples
and
on­
site
lab
audits
every
three
years
by
EPA
or
the
state.
During
laboratory
audits
EPA
or
the
state
will
evaluate
the
laboratory's
Quality
Assurance
Plan
(
QAP),
performance
on
routine
water
samples,
and
chain­
of­
custody
procedures.
The
certification
process
and
detailed
specifications
for
certification
are
described
in
the
EPA
Manual
for
the
Certification
of
Laboratories
Analyzing
Drinking
Water
Certification
Manual)
(
Reference
5.2)
(
http://
www.
epa.
gov/
safewater/
certlab/
labindex.
html)
Section
4:
E.
coli
Laboratory
Guidance
45
Draft
June
2003
Laboratories
analyzing
E.
coli
samples
for
the
LT2
rule
must
use
an
E.
coli
method
approved
for
use
under
the
rule
and
must
be
certified
under
the
drinking
water
certification
program
for
a
similar
method
type
(
e.
g.,
membrane
filtration)
corresponding
to
the
method
the
laboratory
plans
to
use
for
LT2
rule
monitoring
[
40
CFR
part
141.705
(
b)
and
141.706
(
b)].
Approved
E.
coli
methods
and
their
corresponding
drinking
water
certification
program
coliform
techniques
are
provided
in
Table
4­
1.

Table
4­
1.
Approved
E.
coli
Methods
for
LT2
Rule
and
Corresponding
Drinking
Water
Certification
Program
Techniques
E.
coli
Methods
Approved
for
LT2
Rule
Appendix
References
for
Methods
Included
in
this
Manual
Corresponding
Drinking
Water
Certification
Program
Coliform
Analysis
Technique
Standard
Methods
9223B
(
Colilert)
®
Appendix
I
Most
probable
number
(
MPN):
Multiple­
tube
and
multiple­
well
techniques
Standard
Methods
9223B
(
Colilert­
18)
®
Appendix
J
Standard
Methods
9222B/
9222G1
(
mEndo/
LES­
Endo6NA­
MUG)
Appendix
K
Membrane
filtration
technique
Standard
Methods
9222D/
9222G
(
mFC6NA­
MUG)

Standard
Methods
9213D/
EPA
Method
1103.1
(
mTEC)
Appendix
L
EPA
Method
1603
Modified
mTEC
Appendix
M
EPA
Method
1604
MI
medium
1
Appendix
N
m­
ColiBlue24
®
Broth
1
Appendix
O
Standard
Methods
9221B/
9221F
(
LTB6EC­
MUG)
Appendix
P
MPN:
Multiple­
tube
fermentation
technique
1
If
high
levels
of
non­
E.
coli
total
coliforms
interfere
with
the
ability
to
accurately
enumerate
E.
coli
despite
additional
dilutions,
an
alternate
method
should
be
used
(
i.
e,
SM
9222D/
9222G,
SM
9213D/
EPA
Method
1103.1,
EPA
Method
1603,
SM
9221B/
9221F)

4.2
Quality
Assurance/
Quality
Control
for
E.
coli
Analyses
Laboratory
QA
for
E.
coli
analyses
during
LT2
monitoring
should
meet
the
specifications
in
the
Certification
Manual
(
Reference
5.2)
and
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
20th
Edition
(
1998)
(
Standard
Methods)
(
Reference
5.3)
for
facilities,
personnel,
laboratory
equipment,
instrumentation,
and
supplies
used
for
LT2
E.
coli
analyses.
In
addition,
each
laboratory
should
have
implemented
a
written
QA
plan
describing
the
QA
program
and
QC
activities
necessary
to
meet
the
laboratory's
specific
needs.
The
program
should
address
the
following
issues:
personnel
policies,
equipment
and
instrument
specifications,
specifications
for
supplies,
analytical
methods
and
QC
measures,
standard
operating
procedures
(
SOPs),
documentation
specifications,
performance
evaluation
samples,
internal
and
external
lab
audits,
and
corrective
actions.

E.
coli
sample
results
reported
under
the
LT2
rule
should
meet
the
quality
control
(
QC)
specifications
set
forth
in
the
approved
versions
of
the
methods
listed
in
Table
4.1
above.
Section
4.2.1
describes
overall
quality
control
specifications
for
E.
coli
analyses
performed
under
the
LT2
rule.
E.
coli
QC
specifications
specific
to
membrane
filtration
methods
and
MPN
methods
are
provided
in
Sections
4.2.2
to
4.2.3.
This
guidance
is
provided
to
help
implement
the
QC
specifications
in
the
methods
and
does
not
substitute
for
or
alter
the
method
specifications.
Sample
results
that
do
not
meet
these
specifications
are
not
considered
valid,
and
cannot
be
reported
under
the
LT2
rule.
A
checklist
for
these
specifications
is
provided
as
Appendices
Q
and
R.
Section
4:
E.
coli
Laboratory
Guidance
46
Draft
June
2003
Note:
E.
coli
samples
that
do
not
meet
the
QC
specifications
set
forth
in
these
sections
are
considered
to
be
invalid,
and
may
not
be
reported
under
the
LT2
rule.

Table
4­
2.
Quality
Control
Procedures
for
E.
coli
Methods
Quality
Control
Procedure
Reference
All
E.
coli
Methods
Holding
time
and
temperature
requirements
(
Section
4.2.1.1)
LT2
Rule:
Section
IV.
K.
2
Dilution/
rinse
water
sterility
check
(
Section
4.2.1.2)
Certification
Manual:
Section
4.4.3
Media
sterility
check
(
Section
4.2.1.3)
Certification
Manual:
Section
5.1.6.4
Positive/
negative
controls
(
Section
4.2.1.4)
Certification
Manual:
Section
5.1.6.4
Media
storage
(
Section
4.2.1.5)
Certification
Manual:
Section
5.3.2.4
MPN
Methods
(
includes
multiple­
tube
and
multiple­
well
techniques)

Incubation
time
and
temperature
specifications
(
Section
4.2.2.1)
Specified
in
Method
MPN
preparation
blank
(
Section
4.2.2.2)
Recommended
Verification
(
Section
4.2.2.3)
Specified
in
Method
Membrane
Filtration
Methods
Incubation
time
and
temperature
specifications
(
Section
4.2.3.1)
Specified
in
Method
Filtration
unit
sterilization
(
Section
4.2.3.2)
Certification
Manual:
Section
4.1
Membrane
filter
preparation
blank
(
Section
4.2.3.3)
Certification
Manual:
Section
5.2.1.3
Verification
(
Section
4.2.3.4)
Specified
in
Method
4.2.1
Quality
Control
Specifications
Applicable
to
All
E.
coli
Methods
Independent
of
QC
specifications
for
the
type
of
method
used
for
analysis,
there
are
QC
measures
that
should
be
taken
by
all
laboratories
performing
E.
coli
analyses
under
the
LT2
rule
to
ensure
the
laboratory
is
in
control
during
the
analysis
of
LT2
monitoring
samples.
Quality
assurance/
quality
control
procedures
for
all
E.
coli
methods
are
summarized
in
Sections
4.2.1.1
through
4.2.1.5.

4.2.1.1
Holding
Time
and
Temperature
Requirements
for
Field
Samples
EPA
strongly
encourages
laboratories
to
analyze
samples
as
soon
as
possible
after
collection.
However,
due
to
the
need
by
some
utilities
to
ship
samples
overnight
to
an
off­
site
laboratory
for
analysis,
the
holding
time
for
E.
coli
sample
analyses
has
been
extended
to
24
hours.
Based
on
studies
to
assess
the
Section
4:
E.
coli
Laboratory
Guidance
47
Draft
June
2003
Note:
These
QC
specifications
are
meant
to
be
performed
routinely
for
all
samples,
at
the
frequency
specified
in
the
Certification
Manual.
The
QC
specifications
are
not
additional
analyses
specifically
for
LT2
E.
coli
samples.

Note:
These
QC
specifications
are
meant
to
be
performed
routinely
for
all
samples,
at
the
frequency
specified
in
the
Certification
Manual.
The
QC
specifications
are
not
additional
analyses
specifically
for
LT2
E.
coli
samples.
If
any
contamination
is
observed,
determine
the
cause,
correct,
and
reject
any
data
from
samples
associated
with:
(
1)
dilution
or
rinse
water,
or
(
2)
media.
effect
of
increased
sample
holding
time
in
E.
coli
analysis
results,
EPA
has
concluded
that
E.
coli
samples
can
be
held
for
up
to
24
hours
prior
to
analysis
without
compromising
the
data
quality
objectives
of
the
LT2
rule
(
Reference
5.4).

Samples
must
be
maintained
between
0
°
C
and
10
°
C
during
transit
[
CFR
part
141.705
(
b)
(
1)]
and
should
not
be
allowed
to
freeze.
For
samples
shipped
off­
site,
utilities
should
consider
including
a
temperature
blank
along
with
the
sample
so
that
laboratories
can
measure
the
temperature
of
the
sample
upon
receipt.
Alternately,
utilities
could
use
Thermochron
iButtons
®
or
other
measuring
devices
to
record
the
temperature
throughout
transport
(
discussed
in
Section
2.2
in
this
manual).
Additional
information
pertaining
to
temperature
monitoring
is
provided
in
the
Source
Water
Monitoring
Guidance
Manual
for
Public
Water
Systems
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).

4.2.1.2
Dilution/
Rinse
Water
Sterility
Check
QC
specifications
for
dilution/
rinse
water
checks
are
as
follows:

°
Each
batch
(
or
lot,
if
commercially
prepared)
of
dilution/
rinse
water
should
be
checked
for
sterility
by
adding
50
mL
of
water
to
50
mL
of
a
double­
strength
non­
selective
broth
(
e.
g.,
tryptic
soy,
trypticase
soy,
or
tryptose
broth).
Incubate
at
35
°
C
±
0.5
°
C,
check
for
growth
after
24
hours
and
48
hours
(
or
for
the
longest
incubation
time
specified
in
the
method),
and
record
results.
Discard
batch
if
growth
is
detected.

4.2.1.3
Media
Sterility
Check
To
test
sterility
of
newly
prepared
media
prior
to
the
analysis
of
field
samples
incubate
one
plate
per
each
media
batch
at
the
appropriate
temperature
for
24
and
48
hours
(
or
for
the
longest
incubation
time
specified
in
the
method)
and
observe
for
growth.
If
any
contamination
is
observed,
determine
the
cause,
correct,
and
reject
any
data
from
samples
tested
with
the
media.
Section
4:
E.
coli
Laboratory
Guidance
48
Draft
June
2003
Note:
Additional
QC
analyses
are
not
required
for
the
analysis
of
E.
coli
samples
under
LT2,
provided
that
laboratories
perform
required
QC
analyses
at
the
frequency
specified
in
the
Certification
Manual.
4.2.1.4
Positive/
Negative
Controls
For
each
new
lot
or
batch
of
medium,
check
the
analytical
procedures
and
integrity
of
the
medium
before
use
by
testing
with
known
positive
and
negative
control
cultures.
See
Table
4­
3
for
examples
of
test
cultures.

Laboratories
using
commercially­
prepared
media
with
manufacturer
shelf­
lives
of
greater
than
90
days
should
run
positive
and
negative
controls
each
quarter
in
addition
to
running
the
batch/
lot­
specific
controls
and
sterility
checks.
Laboratories
are
encouraged
to
perform
positive
and
negative
control
tests
each
day
that
field
samples
are
analyzed.

Table
4­
3.
Positive
and
Negative
Control
Cultures
Positive
Control
Culture
Negative
Control
Culture
E.
coli
Enterobacter
aerogenes
(
ATCC
#
13048)
1
Klebsiella
pneumoniae
(
ATCC
#
31488)
2
1
E.
aerogenes
normally
does
not
grow
at
44.5
°
C;
if
growth
occurs,
the
waterbath
or
incubator
is
not
holding
the
44.5
°
C
temperature.
The
recommended
strain
is
MUG
negative
and
is
not
thermotolerant.
2
K.
pneumoniae
is
MUG
negative
and
should
not
fluoresce
when
transferred
to
NA­
MUG.
In
addition,
this
strain
is
thermotolerant.

Positive
and
negative
controls
should
be
chosen
based
on
the
method­
specific
requirements.
For
example
if
a
44.5
°
C
waterbath
is
not
required
by
the
method,
it
is
not
necessary
to
include
Enterobacter
aerogenes
as
a
negative
control.

4.2.1.5
Media
Storage
The
following
media
storage
specifications
should
be
met
for
E.
coli
analyses:
°
Agar
plates
may
be
held
for
up
to
2
weeks
at
1
°
C
to
5
°
C
in
plastic
bags
or
containers.
Protect
media
containing
dyes
from
exposure
to
light.
°
Broth
in
loose
fitting
caps
(
e.
g.,
snap
caps)
should
be
stored
at
1
°
C
to
<
30
°
C
for
no
more
than
2
weeks
°
Broth
in
tight
fitting
caps
(
e.
g.,
screw
caps)
should
be
stored
at
1
°
C
to
<
30
°
C
for
no
longer
than
3
months
°
All
media
should
be
at
room
temperature
prior
to
use
°
Media
exhibiting
growth
or
gas
should
be
discarded
4.2.2
Quality
Control
Specifications
for
Most
Probable
Number
Methods
In
addition
to
the
overall
QC
specifications
set
forth
in
Section
4.2.1,
laboratories
using
MPN
methods
(
e.
g.,
multiple­
tube,
multiple­
well)
for
LT2
rule
E.
coli
analyses
should
meet
method­
specific
incubation
time
and
temperature
specifications
and
method­
specific
E.
coli
verification
specifications.
Section
4:
E.
coli
Laboratory
Guidance
49
Draft
June
2003
Note:
Additional
QC
analyses
are
not
required
for
the
analysis
of
E.
coli
samples
under
LT2,
provided
that
laboratories
perform
the
verifications
at
the
frequency
specified
in
the
Certification
Manual.
4.2.2.1
Incubation
Time/
Temperature
Specifications
for
MPN
Methods
The
required
incubation
times
and
temperatures
for
MPN
methods
are
provided
in
Table
4­
4.

Table
4­
4.
Incubation
Time
and
Temperature
Specifications
for
MPN
Methods
Method
Media
Incubation
Time/
Temperature
Standard
Methods
9223B
Colilert
®
24
­
28
hours
at
35
°
C
±
0.5
°
C
Coliert­
18
®
18
­
22
hours
at
35
°
C
±
0.5
°
C
Standard
Methods
9221B/
9221F
LTB
22
­
51
hours
at
35
±
0.5
°
C
EC­
MUG
22
­
26
hours
at
44.5
°
C
±
0.2
°
C
4.2.2.2
Most
Probable
Number
Preparation
Blank
EPA
recommends
that
a
volume
of
sterilized,
buffered
water
be
analyzed
exactly
like
a
field
sample
each
day
samples
are
analyzed.
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.

4.2.2.3
Verification
Verification
specifications
are
detailed
in
the
Certification
Manual
(
Reference
5.2),
Standard
Methods
(
Reference
5.3),
and
Appendices
I
and
J
of
this
document.
Verifications
should
be
performed
in
accordance
with
method­
specific
requirements.

4.2.3
Quality
Control
Specifications
for
Membrane
Filtration
Methods
In
addition
to
the
overall
QC
specification
in
Section
4.2.1,
laboratories
using
membrane
filtration
methods
should
meet
method­
specific
incubation
times
and
temperatures,
and
should
perform
additional
QC
steps
to
demonstrate
that
membrane
filtration
equipment
is
free
from
contamination
and
cross­
over.

Note:
Additional
QC
analyses
are
not
required
for
the
analysis
of
E.
coli
samples
under
LT2,
provided
that
laboratories
perform
required
QC
analyses
at
the
frequency
specified
in
the
Certification
Manual.

4.2.3.1
Incubation
Time
and
Temperature
The
required
incubation
times
and
temperatures
for
membrane
filtration
methods
are
provided
in
Table
4­
5.
Section
4:
E.
coli
Laboratory
Guidance
50
Draft
June
2003
Table
4­
5.
Incubation
Time
and
Temperature
Specifications
for
Membrane
Filter
Methods
Method
Media
Incubation
Time/
Temperature
Standard
Methods
9222B/
9222G
mENDO6NA­
MUG
22
­
26
hours
at
35
°
C
±
0.5
°
C6
4
hours
at
35
°
C
±
0.5
°
C
Les­
ENDO6NA­
MUG
22
­
26
hours
at
35
°
C
±
0.5
°
C6
4
hours
at
35
°
C
±
0.5
°
C
mFC6NA­
MUG
22
­
26
hours
at
44.5
°
C
±
0.2
°
C6
4
hours
at
35
°
C
±
0.5
°
C
Standard
Methods
9213D
mTEC
agar
2
hours
at
35
°
C
±
0.5
°
C
6
22
­
24
hours
at
44.5
°
C
±
0.2
°
C
EPA
1603
Modified
mTEC
2
hours
at
35
°
C
±
0.5
°
C
6
22
­
24
hours
at
44.5
°
C
±
0.2
°
C
EPA
1604
MI
agar
24
hours
at
35
°
C
±
0.5
°
C
Other
Membrane
Filter
Methods
m­
ColiBlue24
Broth
24
hours
at
35
°
C
±
0.5
°
C
4.2.3.2
Filtration
Unit
Sterilization
Membrane
filter
equipment
should
be
autoclaved
before
the
beginning
of
a
filtration
series.
A
filtration
series
ends
when
30
minutes
or
longer
elapses
after
a
sample
is
filtered.
Ultraviolet
(
UV)
light
(
254
nm)
may
be
used
to
sanitize
equipment
(
after
initial
autoclaving
for
sterilization),
if
all
supplies
are
presterilized
UV
light
can
also
be
used
to
reduce
bacterial
carry­
over
between
samples
during
a
filtration
series.
The
UV
lamp
should
be
tested
quarterly
with
a
UV
light
meter
or
an
agar
plate.
Appropriate
corrective
actions
should
be
taken,
if
necessary.

4.2.3.3
Membrane
Filter
Preparation
Blank
If
membrane
filtration
is
used,
an
MF
preparation
blank
is
performed
at
the
beginning
and
the
end
of
each
filtration
series
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.

4.2.3.4
Verification
Verification
specifications
are
detailed
in
the
Certification
Manual
(
Reference
5.2),
Standard
Methods
(
Reference
5.3),
and
in
Appendices
K
through
O
of
this
document.
Verifications
should
be
done
in
accordance
with
method­
specific
requirements.

Note:
Additional
verifications
are
not
required
for
the
analysis
of
E.
coli
samples
under
LT2,
provided
that
laboratories
perform
verifications
at
the
frequency
specified
in
the
Certification
Manual.

4.3
Sample
Collection
Procedures
During
the
LT2
rule,
PWSs
must
take
source
water
samples
at
a
location
prior
to
any
treatment
and
where
the
water
is
no
longer
subject
to
surface
runoff
[
40
CFR
part
141.704
(
a)].
Guidance
on
sample
collection
locations
and
detailed
procedures
for
collecting
E.
coli
samples
for
on­
site
analysis
and
for
shipment
to
an
off­
site
laboratory
for
analysis
are
provided
in
the
Source
Water
Monitoring
Guidance
Manual
for
Public
Water
Systems
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).
This
manual
is
available
for
download
from
http://
www.
epa.
gov/
safewater/
lt2/
index.
html.
Section
4:
E.
coli
Laboratory
Guidance
51
Draft
June
2003
4.3.1
Acceptance
Criteria
for
Receipt
of
Field
Samples
Source
water
samples
are
dynamic
environments
and,
depending
on
sample
constituents
and
environmental
conditions,
E.
coli
present
in
a
sample
can
grow
or
die
off,
biasing
analytical
results.
Samples
that
are
not
analyzed
immediately
after
sample
collection
during
LT2
rule
monitoring
must
be
chilled
to
reduce
biological
activity,
and
preserve
the
state
of
source
water
samples
between
collection
and
analysis.
Samples
for
E.
coli
analyses
must
be
maintained
between
0
°
C
and
10
°
C
if
they
are
shipped
[
40
CFR
141.705
(
b)(
1)].
Samples
should
not
be
allowed
to
freeze.

Given
the
importance
of
maintaining
sample
temperatures
for
E.
coli,
laboratories
approved
for
performing
E.
coli
analyses
during
the
rule
should
establish
acceptance
criteria
for
receipt
of
E.
coli
samples
transported
to
their
laboratory.
Several
options
are
available
to
measure
sample
temperature
upon
receipt
at
the
laboratory
and,
in
some
cases,
during
shipment
are
provided
in
Section
2.2.

4.4
Sample
Volume
and
Dilution
Guidance
Because
E.
coli
analyses
will
be
performed
on
source
waters,
rather
than
finished
drinking
waters,
multiple
sample/
volumes
dilutions
may
necessary
to
assess
high
E.
coli
levels.
Initially
all
PWSs
will
be
required
to
analyze
four
sample
volumes
(
100,
10,
1.0,
and
0.1
mL)
for
all
methods
except
MTF.
For
MTF
methods
the
PWS
will
be
initially
required
to
analyze
five
sample
volumes
of
10,
1.0,
0.1,
0.01,
and
0.001
mL.
Sample
volumes
may
need
to
be
adjusted
based
on
confounding
conditions
(
e.
g.,
high
turbidity,
heavy
rainfall,
etc.).

If
the
PWS
has
historical
data
demonstrating
that
E.
coli
levels
are
consistently
low,
they
may
drop
the
smaller
sample
volumes.
However,
if
the
PWS
decides
to
eliminate
any
of
the
required
volumes
due
to
historical
data,
it
should
be
noted
that
changes
in
conditions
(
e.
g.,
increased
precipitation,
seasonality,
etc.)
may
impact
water
quality.
As
a
result,
sample
volumes
may
need
to
be
adjusted
on
a
per
sampling
event
basis.
For
example,
if
there
has
been
a
substantial
rainfall
in
the
24
hours
prior
to
sample
collection
causing
runoff,
sample
volumes
would
need
to
be
adjusted
in
order
to
obtain
valid
results
and
avoid
data
that
are
above
the
analytical
range
of
the
method.
This
could
also
be
a
concern
if
the
PWS
eliminates
the
100
mL
sample
volume
due
to
historical
data
indicating
that
values
tend
to
be
very
high
and
the
E.
coli
levels
are
lower
than
anticipated,
forcing
the
PWS
to
report
data
that
are
below
the
analytical
range
of
the
method.
EPA
recommends
bracketing
the
target
sample
volume
(
i.
e.,
analyzing
a
sample
volume
above
and
below
the
sample
volume
expected
to
yield
useable
data)
to
account
for
potential
variability.

4.4.1
Sample
Volume
and
Dilution
Guidance
for
Multiple­
Well
Methods
The
analysis
of
water
samples
under
LT2
using
multiple­
well
methods
generally
requires
the
use
of
four
aliquots
of
samples
(
100,
10,
1.0,
and
0.1
mL).
Sample
volumes
may
need
to
be
adjusted
based
on
confounding
conditions
(
e.
g.,
high
turbidity,
heavy
rainfall,
etc.).
Because
a
100­
mL
volume
is
necessary
to
fill
all
of
the
wells,
it
will
be
necessary
to
add
the
aliquots
of
sample
to
sterile
reagent
water
blanks.
The
total
volume
after
the
sample
aliquot
is
added
to
the
reagent
water
blank
should
be
approximately
100
mL
(
i.
e.,
a
90­
mL
blank
should
be
used
for
the
10­
mL
sample
volume,
a
99­
mL
reagent
water
blank
should
be
used
for
the
1.0
mL
aliquot).

4.4.2
Sample
Volume
and
Dilution
Guidance
for
Membrane
Filtration
The
analysis
of
water
samples
under
LT2
requires
four
different
aliquots
of
sample
(
100,
10,
1.0,
and
0.1
mL)
for
membrane
filtration
methods.
lternate
sample
volumes
may
be
used
if
necessary.
To
ensure
adequate
distribution
of
bacteria
during
filtration,
dispense
aliquots
of
the
sample,
with
the
exception
of
the
100
mL
volume,
into
sterile
buffered
water
blanks
(
at
least
30
mL,
depending
on
the
sample
volume
Section
4:
E.
coli
Laboratory
Guidance
52
Draft
June
2003
analzyed).
Alternately,
10
mL
of
sterile
buffer
may
be
added
to
the
filter
apparatus
prior
to
adding
the
sample.
The
buffered
water
minimizes
clumping
of
the
bacteria
on
the
filter
surface.

4.4.3
Sample
Volume
and
Dilution
Guidance
for
Multiple­
Tube
Methods
The
analysis
of
water
samples
by
a
multiple­
tube
technique
(
e.
g.,
Colilert
®
in
a
multiple­
tube
format,
SM
9222B
LTB/
EC­
MUG)
under
LT2
requires
the
use
of
a
15­
tube
most
probable
number
format
(
i.
e.,
5
tubes
at
each
of
three
dilutions,
10.0
mL,
1.0
mL,
and
0.1
mL).
Five
tubes
must
be
inoculated
for
each
sample
volume.
Initially,
the
PWS
will
be
required
to
analyze
four
sample
volumes
(
10.0,
1.0,
0.1,
and
0.01
mL).
Additional
guidance
on
selection
of
dilutions
for
multiple­
tube
methods
is
available
in
Standard
Methods
9221
(
Reference
5.3).

4.5
E.
coli
Data
Recording
and
Calculations
Laboratories
performing
E.
coli
analyses
during
the
LT2
rule
must
be
certified
for
the
analytical
technique
used
for
LT2
E.
coli
analyses
under
the
drinking
water
certification
program,
and
therefore
the
data
recording
practices
required
under
the
drinking
water
certification
program
should
be
followed
[
40
CFR
part
141.706
(
b)].
These
practices
include
the
following:

°
Maintaining
sample
identification
information
(
e.
g.,
including
sample
collection
and
receipt
dates,
times,
and
conditions)

°
Recording
all
raw
data
(
i.
e.,
primary
measurements)
used
to
calculate
final
sample
concentration
for
each
sample
°
Recording
the
incubation/
read
times
for
each
method
to
verify
that
method
specifications
were
met
°
Recording
the
name
of
the
analyst
performing
the
sample
analysis
At
a
minimum,
the
data
elements
discussed
in
detail
below
should
be
recorded
for
E.
coli
samples
during
LT2
monitoring.
These
data
elements
are
critical
to
ensure
that
final
sample
results
can
be
verified
using
primary
data,
if
necessary,
and
to
demonstrate
that
sample
analyses
were
performed
within
methodspecified
holding
times
and
incubation
times.
Standardized
bench
sheets
are
available
for
download
on
the
LT2
website
(
http://
www.
epa.
gov/
safewater/
lt2/
index.
html).
Please
note
that,
while
E.
coli
laboratories
should
record
the
data
elements
discussed
in
this
section
to
comply
with
certification
manual
specifications,
not
all
of
the
recorded
data
will
be
reported
in
the
LT2
data
system.
E.
coli
data
reporting
under
the
LT2
rule
is
discussed
in
Section
4.6.

In
addition
to
data
elements,
this
section
also
provides
standardized
procedures
for
determining
E.
coli
concentration
for
LT2
samples
for
the
various
analytical
techniques
that
are
approved
for
use
under
the
rule.
Because
these
analyses
will
be
performed
on
source
waters
rather
than
finished
drinking
waters,
and
multiple
dilutions
may
be
necessary
to
assess
higher
E.
coli
levels,
it
is
critical
that
laboratories
follow
the
same
procedures
for
determining
E.
coli
sample
concentrations
to
reduce
variability
in
reporting.

Table
4­
6
specifies
the
minimum
sample
ID
information
that
must
be
recorded
for
each
E.
coli
sample,
either
on
a
sample
collection
form
used
in
the
field
and
maintained
in
the
same
file
as
the
E.
coli
laboratory
data,
or
on
the
laboratory
data
form
(
examples
of
all
forms
are
provided
Appendices
S
through
AB)
[
40
CFR
part
141.707
(
2)].
Section
4:
E.
coli
Laboratory
Guidance
53
Draft
June
2003
Table
4­
6.
Minimum
Sample
ID
Information
and
General
Sample
Data
to
Record
PWS
ID
Facility
ID
Sample
collection
point
Sample
collection
date
Analytical
method
number
Method
type
Source
type
E.
coli/
100
mL
Turbidity
Method­
specific
data
to
record
for
each
of
the
individual
method
types,
as
well
as
standardized
calculations
for
each
method
type,
are
discussed
in
Sections
4.5.1
to
4.5.3.

4.5.1
Multiple­
Well
Data
In
addition
to
the
data
elements
listed
in
Table
4­
6,
laboratories
using
Colilert
®
and
Colilert­
18
®
methods
for
E.
coli
sample
analyses
in
support
of
the
LT2
rule
should
record
the
data
elements
specific
to
multiple­
well
techniques.
Data
elements
to
be
recorded
for
the
97­
well
format
(
Quanti­
Tray
2000
®
)
are
noted
in
Table
4­
7;
data
to
be
recorded
for
the
51­
well
format
(
Quanti­
Tray
®
)
are
noted
in
Table
4­
8.
These
elements
include
the
primary
measurements
needed
to
calculate
the
E.
coli
concentration
in
the
sample
as
well
as
all
method­
required
incubation
and
read
times
needed
to
verify
that
the
sample
analyses
were
conducted
under
analytical
control.

Calculations
for
determining
the
E.
coli
concentration
using
the
Quanti­
Tray
2000
®
(
97­
well)
and
Quanti­
Tray
®
(
51­
well)
formats
are
provided
after
each
table.

4.5.1.1
Data
Elements
for
Quanti­
Tray
2000
®
(
97­
well)
Analyses
Table
4­
7.
Minimum
Data
to
Record
for
Quanti­
tray
2000
®
Colilert
®
and
Colilert­
18
®
Analyses
Primary
Measurements
mL
of
sample
added
to
tray
(
does
not
include
reagent
water
volume)

Large
wells
positive:
Total
coliform
positive
and
UV
fluorescence
Small
wells
positive:
Total
coliform
positive
and
UV
fluorescence
Holding
Time
/
Incubation
Time
Information
Incubation
start/
read
date
Incubation
start/
read
time
Incubation
start/
read
temperature
Additional
incubation
start/
read
date
Additional
incubation
start/
read
time
Section
4:
E.
coli
Laboratory
Guidance
54
Draft
June
2003
4.5.1.2
Determining
E.
coli
Concentration
Using
Colilert
®
and
Colilert­
18
®
Quanti­
Tray
2000
®
Data
A.
Select
appropriate
dilution
to
yield
countable
results.
If
multiple
dilutions
are
used,
the
tray
exhibiting
positive
wells
in
the
40%
to
80%
range
(
39
to
78
total
positive
large
and
small
wells)
should
be
used
to
determine
MPN
value.

Note:
The
analytical
result
can
be
automatically
calculated
using
the
LT2
Data
Collection
System.
See
Section
4.6
for
additional
information.

B.
Determine
MPN.
Using
the
number
of
large
positive
wells
and
small
positive
wells
from
the
appropriate
dilution,
identify
the
corresponding
MPN/
100
mL
in
the
table
provided
by
the
vendor.
Large
well
values
are
located
in
the
left
column;
small
well
values
are
located
in
the
top
row.
For
example,
if
a
100­
mL
sample
was
analyzed,
and
there
were
29
large
positive
wells
and
5
small
positive
wells,
the
corresponding
MPN
would
be
49.6
MPN/
100
mL.

C.
Adjust
for
dilution
factor.
Because
the
MPN/
100
mL
values
in
the
table
are
based
on
100­
mL
samples,
the
MPN
value
should
be
adjusted
if
less
than
100­
mL
of
sample
volume
was
analyzed.
Use
the
following
calculation
to
adjust
the
MPN
to
account
for
the
dilution:

Analytical
result
=
MPN
value
×
100
mL
of
sample
analyzed
Example:

Volume
analyzed
=
10
mL
of
sample
(
in
90
mL
of
dilution
water)
Large
wells
positive
=
39
Small
wells
positive
=
5
The
MPN
value
calculated
based
on
the
intersection
of
10
and
2
in
the
table.
MPN
=
81.3
Analytical
result
=
81.3
×
100
=
813
E.
coli
MPN/
100
mL
10
4.5.1.3
Data
Elements
for
Quanti­
Tray
®
(
51­
well)
Analyses
The
recommended
data
elements
to
record
for
Quanti­
Tray
®
(
51­
well)
analyses
are
provided
in
Table
4­
8.
Section
4:
E.
coli
Laboratory
Guidance
55
Draft
June
2003
Table
4­
8.
Minimum
Data
to
Record
for
Quanti­
Tray
®
Colilert
®
and
Colilert­
18
®
Analyses
Primary
Measurements
mL
of
sample
added
to
tray
(
does
not
include
reagent
water
volume)

Number
of
wells
positive:
Total
coliform
positive
and
UV
fluorescence
Holding
Time
/
Incubation
Time
Information
Incubation
start/
read
date
Incubation
start/
read
time
Incubation
start/
read
temperature
Additional
incubation
start/
read
date
Additional
incubation
start/
read
time
Additional
incubation
start/
read
temperature
4.5.1.4
Determining
E.
coli
Concentration
Using
Colilert
®
and
Colilert­
18
®
(
51­
well)
Data
A.
Select
appropriate
dilution.
If
multiple
dilutions
are
used,
the
tray
exhibiting
80%
positive
wells
(
41
positive
wells)
should
be
used
to
determine
MPN
value.

Note:
The
analytical
result
can
be
automatically
calculated
using
the
LT2
Data
Collection
System.
See
Section
4.6
for
additional
information.

B.
Determine
MPN.
Using
the
number
of
positive
wells
from
the
appropriate
dilution,
identify
the
corresponding
MPN/
100
mL
in
the
table
provided
by
the
vendor.
For
example,
if
a
100­
mL
sample
was
analyzed,
and
there
were
41
positive
wells,
the
corresponding
MPN
would
be
83.1
MPN/
100
mL
C.
Adjust
for
dilution
factor.
Because
the
MPN/
100
mL
values
in
the
table
are
based
on
100­
mL
samples,
the
MPN
value
should
be
adjusted
if
less
than
100­
mL
of
sample
volume
was
analyzed.
Use
the
following
calculation
to
adjust
the
MPN
to
account
for
the
dilution:

MPN
value
×
100
=
E.
coli
MPN/
100
mL
mL
sample
analyzed
Example:

Volume
analyzed
(
mL)
=
10
mL
(
in
90
mL
of
dilution
water)
Number
of
positive
wells
=
41
MPN
=
83.1
The
analytical
result
is
calculated
as
follows:

83.1
×
100
=
831
E.
coli
MPN/
100
mL
10
4.5.2
Membrane
Filtration
Data
In
addition
to
the
general
sample
data
listed
in
Table
4­
6,
laboratories
using
membrane
filtration
methods
for
E.
coli
sample
analyses
in
support
of
the
LT2
rule
should
record
the
data
elements
specific
to
this
technique.
These
are
noted
in
Table
4­
9,
and
include
the
primary
measurements
needed
to
calculate
the
E.
Section
4:
E.
coli
Laboratory
Guidance
1
Adapted
from
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
APHA,
AWWA,
WEF
(
20th
Edition)

56
Draft
June
2003
coli
concentration
in
the
sample,
as
well
as
all
method­
required
incubation
and
read
times
needed
to
verify
that
the
sample
analyses
were
conducted
under
analytical
control.

4.5.2.1
Data
Elements
for
Membrane
Filtration
Analyses
Table
4­
9.
Minimum
Data
Elements
for
Record
for
Membrane
Filtration
Analyses
Primary
Measurements
Filter
1
volume
(
mL)
(
e.
g.,
100
mL)
CFU
on
Filter
1
Filter
2
volume
(
mL)
(
e.
g.,
10
mL)
CFU
on
Filter
2
Filter
3
volume
(
mL)
(
e.
g.,
1.0
mL)
CFU
on
Filter
3
Filter
4
volume
(
mL)
(
e.
g.,
0.1
mL)
CFU
on
Filter
4
Holding
Time
/
Incubation
Time
Information
Primary
isolation
medium
(
e.
g.,
mENDO,
mFC)
incubation
start/
read
date
Primary
isolation
medium
(
e.
g.,
mENDO,
mFC)
incubation
start/
read
time
Primary
isolation
medium
(
e.
g.,
mENDO,
mFC)
incubation
start/
read
temperature
Secondary
isolation
medium
(
e.
g,
NA­
MUG)
incubation
start/
read
date
Secondary
isolation
medium
(
e.
g,
NA­
MUG)
incubation
start/
read
time
Secondary
isolation
medium
(
e.
g,
NA­
MUG)
incubation
start/
read
temperature
4.5.2.2
Determining
E.
coli
Concentrations
Using
Membrane
Filter
Data1:

A.
E.
coli
counts
should
be
determined
from
the
volume(
s)
filtered
that
yielded
20
to
80
E.
coli
colonies
(
20­
60
for
mFC­
NA­
MUG),
and
not
more
than
200
total
colonies
per
plate.
(
Guidance
for
samples
that
do
not
yield
countable
plates
is
provided
in
Sections
E
and
F)

Note:
The
analytical
result
can
be
automatically
calculated
using
the
LT2
Data
Collection
System.
See
Section
4.6
for
additional
information.

B.
If
there
are
greater
than
200
colonies
per
membrane,
even
for
the
lowest
dilution,
the
result
is
recorded
as
"
too
numerous
to
count"
(
TNTC).
These
results
cannot
be
reported
for
LT2
monitoring,
as
they
cannot
be
used
for
the
required
data
analyses.
During
the
next
sampling
event,
analyze
an
additional,
lower
dilution
volume
(
the
highest
dilution
volume
may
be
omitted)
unless
conditions
were
unusual
(
e.
g.,
heavy
rains,
flooding,
etc.)
during
the
sampling
event
yielding
TNTC
for
all
dilutions.

C.
If
colonies
are
not
sufficiently
distinct
for
accurate
counting,
the
result
is
recorded
as
"
confluent
growth"
(
CNFG).
To
prevent
CNFG
from
occurring,
smaller
sample
aliquots
should
be
filtered.
For
example,
if
sample
volumes
of
100,
10,
1
and
0.1
mL
are
analyzed
and
even
the
0.1­
mL
plate
results
in
CNFG,
then
potentially
0.01
mL
should
be
analyzed
during
the
next
sampling
event.
For
sample
volumes
less
than
1
mL,
serial
dilutions
should
be
used,
and
1­
mL
volumes
of
the
dilutions
should
be
filtered.
The
100­
mL
volume
can
be
eliminated.
Note:
If
growth
is
due
to
high
levels
of
total
coliforms
but
low
E.
coli
then
another
method
should
be
chosen
for
analyses
that
does
not
rely
on
total
coliform
determination
prior
to
or
simultaneously
with
E.
coli
determination.
Section
4:
E.
coli
Laboratory
Guidance
57
Draft
June
2003
Note:
Results
that
are
TNTC
or
CNFG
are
not
appropriate
for
LT2
microbial
data
analysis,
and
cannot
be
entered
into
the
LT2
Data
Collection
System.
These
results
should
not
be
reported.

D.
Using
the
E.
coli
counts
from
the
appropriate
dilution,
E.
coli
CFU/
100
mL
is
calculated
based
on
the
following
equation:

E.
coli
CFU
×
100
=
E.
coli
CFU/
100
mL
mL
sample
filtered
Example
1:

Filter
1
volume
=
100
mL
CFU
=
TNTC
Filter
2
volume
=
10
mL
CFU
=
40
Filter
3
volume
=
1.0
mL
CFU
=
9
Filter
4
volume
=
0.1
mL
CFU
=
0
Using
the
guidance
on
countable
colonies
in
Step
A,
the
counts
from
the
10­
mL
plate
will
be
used
to
calculate
the
E.
coli
concentration
for
the
sample:

40
E.
coli
CFU
×
100
=
400
E.
coli
CFU/
100
mL
10
mL
E.
If
no
E.
coli
colonies
are
present,
the
detection
limit
is
calculated
as
<
largest
volume
filtered
per
100
mL.

Example
2:

Filter
1
volume
(
mL)
=
100
mL
CFU
=
0
Filter
2
volume
(
mL)
=
10
mL
CFU
=
0
Filter
3
volume
(
mL)
=
1.0
mL
CFU
=
0
Detection
limit
=
100
mL
=
E.
coli
CFU/
100
mL
Largest
volume
filtered
100
mL
=
<
1
E.
coli
/
100
mL
100
mL
Section
4:
E.
coli
Laboratory
Guidance
58
Draft
June
2003
Example
3:

Filter
1
volume
(
mL)
=
100
mL
CFU
=
Lab
accident,
no
data
available
Filter
2
volume
(
mL)
=
10
mL
CFU
=
0
Filter
3
volume
(
mL)
=
1.0
mL
CFU
=
0
Calculation
of
E.
coli/
100
mL:

100
mL
=
<
10
E.
coli
CFU
/
100
mL
10
mL
F.
If
there
are
no
filters
with
E.
coli
counts
in
the
20­
80
colony
range
(
20­
60
for
mFC­
NA­
MUG),
sum
the
E.
coli
counts
on
all
filters,
divide
by
the
volume
filtered
and
report
as
number
per
100
mL.

Example
4:

Filter
1
volume
(
mL)
=
50
mL
CFU
=
15
Filter
2
volume
(
mL)
=
25
mL
CFU
=
6
Filter
3
volume
(
mL)
=
10
mL
CFU
=
0
The
analytical
result
is
calculated
as:

(
15
+
6
+
0)
×
100
=
25
E.
coli
CFU/
100
mL
(
50+
25+
10)

Example
5
Filter
1
volume
(
mL)
=
50
mL
CFU
=
105
Filter
2
volume
(
mL)
=
25
mL
CFU
=
92
Filter
3
volume
(
mL)
=
10
mL
CFU
=
85
The
analytical
result
is
calculated
as:

(
105
+
92
+
85)
×
100
=
332
E.
coli
CFU/
100
mL
(
50
+
25
+
10)

Example
6:

Filter
1
volume
(
mL)
=
100
mL
CFU
=
82
Filter
2
volume
(
mL)
=
10
mL
CFU
=
18
Filter
3
volume
(
mL)
=
1.0
mL
CFU
=
0
The
analytical
result
is
calculated
as:

(
82
+
18
+
0)
×
100
=
90
E.
coli
CFU/
100
mL
(
100
+
10
+
1)
Section
4:
E.
coli
Laboratory
Guidance
59
Draft
June
2003
Example
7
Filter
1
volume
(
mL)
=
50
mL
CFU
=
TNTC
Filter
2
volume
(
mL)
=
25
mL
CFU
=
TNTC
Filter
3
volume
(
mL)
=
10
mL
CFU
=
83
The
analytical
result
is
calculated
as:

83
×
100
=
830
E.
coli
CFU/
100
mL
10
4.5.3
Multiple­
Tube
Data
In
addition
to
the
general
sample
data
listed
in
Table
4­
6,
laboratories
using
multiple­
tube
methods
for
E.
coli
sample
analyses
in
support
of
the
LT2
rule
should
record
the
data
elements
specific
to
these
techniques.
These
data
elements
are
noted
in
Table
4­
10
for
15­
tube
most
probable
number
methods
and
Table
4­
11
for
15­
tube
multiple­
tube
fermentation
methods.
The
data
elements
include
the
primary
measurements
needed
to
calculate
the
E.
coli
concentration
in
the
sample,
as
well
as
all
method­
required
incubation
and
read
times
needed
to
verify
that
the
sample
analyses
were
conducted
under
analytical
control.

Calculations
for
determining
the
E.
coli
concentration
using
multiple
tube
formats
are
provided
in
Section
4.5.3.2.

4.5.3.1
Data
Elements
for
Multiple­
Tube
Analyses
Table
4­
10.
Minimum
Data
Elements
to
Record
for
15­
Tube
MPN
Methods
Primary
Measurements
Number
of
positive
10.0
mL
tubes:
Total
coliform
positive
and
UV
fluorescence
Number
of
positive
1.0
mL
tubes:
Total
coliform
positive
and
UV
fluorescence
Number
of
positive
0.1
mL
tubes:
Total
coliform
positive
and
UV
fluorescence
Number
of
positive
0.01
mL
tubes:
Total
coliform
positive
and
UV
fluorescence
Number
of
positive
0.001
mL
tubes:
Total
coliform
positive
and
UV
fluorescence
Holding
Time
/
Incubation
Time
Information
Incubation
start/
read
date
Incubation
start/
read
time
Incubation
start/
read
temperature
Additional
incubation
start/
read
date
Additional
incubation
start/
read
time
Additional
incubation
start/
read
temperature
Section
4:
E.
coli
Laboratory
Guidance
2Adapted
from
2001
Supplement
to
the
20th
Edition
of
Standard
Methods
9221
C:
Explanation
of
Bacterial
Density.
This
supplement
is
available
for
download
at
http://
www.
techstreet.
com/
cgi­
bin/
detail?
product_
id=
923645
(
Accessed
April
7,
2003).

60
Draft
June
2003
Table
4­
11.
Minimum
Data
Elements
to
Record
for
15­
Tube
Fermentation
Methods
Primary
Measurements
Number
of
positive
tubes
10.0
mL
Number
of
positive
tubes
1.0
mL
Number
of
positive
tubes
0.1
mL
Number
of
positive
tubes
0.01
mL
Number
of
positive
tubes
0.001
mL
Number
of
positive
tubes
0.0001
mL
Holding
Time
/
Incubation
Time
Information
LTB
incubation
start
date
LTB
incubation
start
time
LTB
incubation
start
temperature
LTB
24­
hour
incubation
read
date
LTB
24­
hour
incubation
read
time
LTB
24­
hour
incubation
read
temperature
LTB
48­
hour
incubation
read
date
LTB
48­
hour
incubation
read
time
LTB
48­
hour
incubation
read
temperature
EC­
MUG
incubation
24­
hour
read
date
(
from
24­
hour
LTB)

EC­
MUG
incubation
24­
hour
read
time
(
from
24­
hour
LTB)

EC­
MUG
incubation
24­
hour
read
temperature
(
from
24­
hour
LTB)

EC­
MUG
incubation
24­
hour
read
date
(
from
48­
hour
LTB)

EC­
MUG
incubation
24­
hour
read
time
(
from
48­
hour
LTB)

EC­
MUG
incubation
24­
hour
read
temperature
(
from
48­
hour
LTB)

4.5.3.2
Determination
of
E.
coli
Concentrations
Using
Multiple­
Tube
Methods2:

The
guidance
and
examples
for
determining
E.
coli
concentrations
using
multiple­
tube
methods
are
based
on
the
revision
of
Standard
Methods
9221C
included
in
the
2001
Supplement
to
the
20th
Edition
of
Standard
Methods,
approved
by
the
Standard
Methods
Committee
in
1999.

Note:
The
analytical
result
can
be
automatically
calculated
using
the
LT2
Data
Collection
System.
See
Section
4.6
for
additional
information.

A.
For
each
sample
volume
(
e.
g.,
10,
1,
0.1,
and
0.01
mL
or
additional
sample
volumes
as
necessary),
determine
the
number
of
positive
tubes
out
of
five.

B.
A
dilution
refers
to
the
volume
of
original
sample
that
was
inoculated
into
each
series
of
tubes.
Only
three
of
the
dilution
series
will
be
used
to
estimate
the
MPN.
The
three
selected
dilutions
are
called
Section
4:
E.
coli
Laboratory
Guidance
61
Draft
June
2003
significant
dilutions
and
are
selected
according
to
the
following
criteria.
Examples
of
significant
dilution
selections
are
provided
in
Table
4­
12,
below.

°
Choose
the
highest
dilution
(
the
most
dilute,
with
the
least
amount
of
sample)
giving
positive
results
in
all
five
tubes
inoculated
and
the
two
succeeding
higher
(
more
dilute)
dilutions.
(
Table
4­
12,
Example
A).

°
If
the
lowest
dilution
(
least
dilute)
tested
has
less
than
five
tubes
with
positive
results,
select
it
and
the
two
next
succeeding
higher
dilutions
(
Table
4­
12,
Examples
B
and
C).

°
When
a
positive
result
occurs
in
a
dilution
higher
(
more
dilute)
than
the
three
significant
dilutions
selected
according
to
the
rules
above,
change
the
selection
to
the
lowest
dilution
(
least
dilute)
that
has
less
than
five
positive
results
and
the
next
two
higher
dilutions
(
more
dilute)
(
Table
4­
12,
Example
D).

°
When
the
selection
rules
above
have
left
unselected
any
higher
dilutions
(
more
dilute)
with
positive
results,
add
those
higher­
dilution
positive
results
to
the
results
for
the
highest
selected
dilution
(
Table
4­
12,
Example
E).

°
If
there
were
not
enough
higher
dilutions
tested
to
select
three
dilutions,
then
select
the
next
lower
dilution
(
Table
4­
12,
Example
F).

C.
MPN
values
need
to
be
adjusted
based
on
the
significant
dilutions
series
selected
above.
Because
the
MPN/
100
mL
values
in
the
table
are
based
on
the
10
mL,
1
mL,
and
0.1
mL
dilution
series,
per
method
requirements,
the
MPN
value
must
be
adjusted
if
these
are
not
the
significant
dilution
series
selected.
Use
the
following
calculation
to
adjust
the
MPN
when
the
10
mL,
1
mL,
and
0.1
mL
dilution
series
are
not
the
significant
dilution
series
selected:

Analytical
result
=
MPN
value
=
E.
coli
MPN/
100
mL
#
of
mL
in
middle
dilution
Table
4­
12.
Examples
of
Different
Combinations
of
Positive
Tubes
(
Significant
Dilution
Results
Are
in
Bold
and
Underlined)

Example
Least
dilute
Most
dilute
(
Lowest)
(
Highest)
Combination
of
positives
MPN
Index
from
Standard
Methods
E.
coli/
100
mL
(
after
adjustment)
10
mL
1
mL
0.1
mL
0.01
mL
0.001
mL
A
5
5
1
0
0
5­
1­
0
33
330
B
4
5
1
0
0
4­
5­
1
48
48
C
0
0
1
0
0
0­
0­
1
1.8
1.8
D
5
4
4
1
0
4­
4­
1
40
400
E
5
4
4
0
1
4­
4­
1
40
400
F
5
5
5
5
2
5­
5­
2
540
54,000
Section
4:
E.
coli
Laboratory
Guidance
62
Draft
June
2003
Example
A:
The
significant
dilution
series
for
the
5­
1­
0
combination
of
positives
includes
the
1
mL,
0.1
mL,
and
0.01
mL
dilutions.
Since
the
10
mL,
1
mL,
and
0.1
mL
dilutions
were
not
selected,
an
adjustment
is
necessary
to
account
for
the
dilutions
selected:

Analytical
result
=
33
=
330
E.
coli
/
100
mL
0.1
Example
B:
Since
the
10
mL,
1
mL,
and
0.1
mL
dilutions
are
the
significant
dilutions,
no
adjustment
is
necessary
and
the
result
is
48
E.
coli/
100
mL.

Example
C:
Since
the
10
mL,
1
mL,
and
0.1
mL
dilutions
are
the
significant
dilutions,
no
adjustment
is
necessary
and
the
result
is
1.8
E.
coli/
100
mL.

Examples
D
and
E:
The
significant
dilution
series
for
the
4­
4­
1
combination
of
positives
includes
the
1
mL,
0.1
mL,
and
0.01
mL
dilutions.
Since
the
10
mL,
1
mL,
and
0.1
mL
dilutions
were
not
selected,
an
adjustment
is
necessary
to
account
for
the
dilutions
selected:

Analytical
result
=
40
=
400
E.
coli
/
100
mL
0.1
Example
F:
The
significant
dilution
series
for
the
5­
5­
2
combination
of
positives
includes
the
0.1
mL,
0.01
mL
and
.001
mL
dilutions.
Since
the
10
mL,
1
mL,
and
0.1
mL
dilutions
were
not
selected,
an
adjustment
is
necessary
to
account
for
the
dilutions
selected:

Analytical
result
=
540
=
54,000
E.
coli
/
100
mL
0.01
4.6
Electronic
Data
Reporting
During
the
LT2
rule,
laboratories
will
report
E.
coli
data
electronically
through
EPA's
LT2
Data
Collection
System
to
the
PWS
staff
responsible
for
approving
and
submitting
monitoring
results
to
EPA.
The
LT2
Data
Collection
System
is
a
web­
based
application
that
allows
laboratory
users
to
enter
or
upload
data,
then
electronically
"
release"
the
data
to
the
appropriate
PWS
staff
for
review,
approval,
and
submission
to
EPA
and
the
State.
Although
ownership
of
the
data
resides
with
the
PWS
throughout
this
process,
the
LT2
Data
Collection
System
increases
the
ease
and
efficiency
of
the
data
entry
and
transfer
process
from
one
party
to
another
by
transferring
the
ability
to
access
the
data
from
the
laboratory
to
the
PWS
to
EPA
and
the
State,
and
ensuring
that
data
cannot
be
viewed
or
changed
by
unauthorized
parties.
A
summary
of
the
data
entry,
review,
and
transfer
process
through
the
LT2
Data
Collection
System
for
both
Cryptosporidium
and
E.
coli
samples
is
provided
in
Table
3­
6,
in
Section
3.8,
above.

The
data
reporting
process
is
summarized
below,
in
Sections
4.6.1
through
4.6.3,
and
discussed
in
detail
in
the
Users'
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule)
Data
Collection
System.
The
LT2
data
system
users'
guide
also
provides
detailed
information
on
the
laboratory
registration
process.
Information
on
the
LT2
Data
Collection
System,
as
well
as
a
downloadable
users'
manual,
is
available
at
http://
www.
epa.
gov/
safewater/
lt2/
index.
html.
Section
4:
E.
coli
Laboratory
Guidance
63
Draft
June
2003
4.6.1
Data
Entry/
Upload
The
analyst
or
another
laboratory
staff
member
enters
a
subset
of
the
data
recorded
at
the
bench
(
Section
4.5)
into
the
LT2
Data
Collection
System
either
by
entering
the
data
using
web
forms
or
by
uploading
data
in
XML
format
[
40
CFR
part
141.707
(
2)].
This
information
includes
the
following:

°
Sample
ID
(
optional)

°
PWS
ID
°
Facility
ID
°
Sample
collection
point
ID
°
Sample
collection
date
°
Analytical
method
number
°
Method
type
°
Source
water
type
(
provided
by
PWS
on
sample
collection
form)

°
E.
coli/
100
mL
(
see
note
below)

°
Turbidity
result
(
provided
by
PWS
on
sample
collection
form)

Note:
The
laboratory
may
enter
the
final
result
for
the
E.
coli
sample
or
may
enter
the
primary
measurements
recorded
at
the
bench,
and
have
the
LT2
Data
Collection
System
automatically
calculate
the
final
E.
coli
concentration.
Because
this
information
is
specific
to
method
type
(
membrane
filtration,
multiple­
tube,
51­
well,
and
97­
well),
the
system
provides
different
entry
screens
for
each
method
type.
The
laboratory
should
verify
that
all
holding
times
and
other
QC
specifications
were
met.

The
laboratory's
official
contact
is
responsible
for
verifying
the
quality
and
accuracy
of
all
sample
results
in
the
laboratory,
and
is
required
to
review
and
approve
the
results
before
they
are
submitted
to
the
PWS
for
review.
If
inaccuracies
or
other
problems
are
identified,
the
official
contact
discusses
the
sample
information
with
the
analyst
or
data
entry
staff
and
resolves
the
issues
before
the
data
are
released
to
the
PWS
for
review.

If
no
inaccuracies
or
other
issues
are
identified,
the
laboratory's
official
contact
approves
the
data
for
"
release"
to
the
PWS
for
review
(
EPA
does
not
receive
the
data
at
this
point).
When
the
data
are
approved
by
the
laboratory,
the
rights
to
the
data
are
transferred
electronically
by
the
system
to
the
PWS,
and
the
data
can
no
longer
be
changed
by
the
laboratory.

4.6.2
PWS
Data
Review
After
the
laboratory
has
released
E.
coli
data
electronically
to
the
PWS
using
the
LT2
Data
Collection
System,
the
PWS
will
review
the
results.
The
PWS
user
cannot
edit
the
data,
but
if
the
PWS
has
an
issue
with
the
sample
result,
such
as
if
the
PWS
believes
that
the
sample
collection
point
ID
or
collection
date
is
incorrect,
the
PWS
can
release
the
results
back
to
the
laboratory
for
issue
resolution.
In
addition
to
noting
the
reason
in
the
LT2
Data
Collection
System
for
the
return
of
the
data
to
the
laboratory,
you
also
should
contact
the
laboratory
verbally
to
discuss
the
issue.

If
the
PWS
determines
that
the
data
are
accurate,
the
PWS
releases
the
results
to
EPA
(
and
the
State,
if
applicable)
as
"
approved"
results.
If
the
PWS
determines
that
the
data
are
accurate,
but
believes
that
the
data
are
not
valid
for
other
reasons,
the
PWS
can
release
the
results
as
"
contested."
Section
4:
E.
coli
Laboratory
Guidance
64
Draft
June
2003
4.6.3
EPA/
State
Review
After
the
PWS
has
released
the
results
as
approved
or
contested,
they
are
available
to
EPA
and
State
users
to
review
through
the
LT2
Data
Collection
System.
EPA
and
State
users
cannot
edit
the
data.

4.7
Vendor
Contact
List
for
E.
coli
Methods
Contact
information
for
the
vendors
for
materials
used
in
the
methods
described
in
Section
4
is
provided
below.
Footnotes
indicate
which
vendors
are
associated
with
each
method.

American
Type
Culture
Collection1
www.
atcc.
org
P.
O.
Box
1549
Manassas,
VA
20108
Sales:
Phone:
(
800)
638­
6597
Fax:
(
703)
365­
2750
Technical
Support:
Phone:
(
800)
638­
6597
E­
mail:
tech@
atcc.
org
Becton,
Dickinson
and
Company2
www.
bd.
com
1
Becton
Drive
Franklin
Lakes,
NJ
07417
Sales:
Phone:
(
800)
675­
0908
Fax:
(
410)
316­
4723
Technical
Support:
Phone:
(
800)
638­
8663
Fax:
(
410)
527­
0244
E­
mail:
rep.
technical_
services@
bd.
com
bioMérieux
6
www.
biomerieux.
com
595
Anglum
Drive
Hazelwood,
MO
63042
Sales:
Phone:
(
800)
638­
4835
Fax:
(
800)
657­
3053
Technical
Support:
Phone:
(
800)
638­
4835
Fax:
(
800)
657­
3053
Cole­
Parmer1
www.
coleparmer.
com
625
East
Bunker
Court
Vernon
Hills,
IL
60061
Sales:
Phone:
(
800)
323­
4340
Fax:
(
847)
247­
2929
E­
mail:
sales@
coleparmer.
com
Technical
Support:
Phone:
(
800)
323­
4340
ext.
122
Fax:
(
847)
327­
2987
E­
mail:
techinfo@
coleparmer.
com
Fisher
Scientific1
www.
fishersci.
com
9999
Veterans
Memorial
Drive
Houston,
TX
77038
Sales:
Phone:
(
800)
766­
7000
Fax:
(
800)
926­
1166
Technical
Support:
Phone:
(
800)
766­
7000
Fax:
(
800)
926­
1166
Hach
5
www.
hach.
com
P.
O.
Box
389
Loveland,
CO
80539
Sales:
Phone:
(
800)
227­
4224
Fax:
(
970)
669­
2932
Technical
Support:
Phone:
(
800)
227­
4224
Fax:
(
970)
669­
2932
E­
mail:
techhelp@
hach.
com
Biosynth
International
Inc.
2
www.
biosynth.
com
1665
West
Quincy
Avenue
Naperville,
IL
60540
Sales:
Phone:
(
800)
270­
2436
Fax:
(
800)
276­
2436
Technical
Support:
Phone:
(
800)
270­
2436
Fax:
(
800)
276­
2436
E­
mail:
craig@
biosynth.
com
Section
4:
E.
coli
Laboratory
Guidance
65
Draft
June
2003
Hardy
Diagnostics
2
www.
hardydiagnostics.
com
1430
West
McCoy
Lane
Santa
Maria,
CA
93455
Sales:
Phone:
(
800)
266­
2222
Fax:
(
805)
346­
2760
E­
mail:
sales@
hardydiagnostics.
com
Technical
Support:
Phone:
(
800)
266­
2222
ext
5658
Fax:
(
805)
346­
2760
IDEXX
4
www.
idexx.
com
Sales:
Phone:
(
800)
321­
0207
ext.
1
Fax:
(
207)
856­
0630
Technical
Support:
Phone:
(
800)
321­
0207
ext.
2
Fax:
(
207)
856­
0630
E­
mail:
water@
idexx.
com
Pall
Gelman
Sciences
3
www.
pall.
com/
gelman
600
South
Wagner
Road
Ann
Arbor,
MI
48103
Sales:
Phone:
(
800)
521­
1520
ext.
2
Fax:
(
734)
913­
6495
Technical
Support:
Phone:
(
800)
521­
1520
ext.
3
Fax:
(
734)
913­
6495
Oxoid
Inc.
2
www.
oxoid.
com
Suite
100,
1926
Merivale
Road
Nepean,
Ontario
K2G1E8,
Canada
Sales:
Phone:
(
613)
226­
1318
Fax:
(
613)
226­
3728
E­
mail:
oxoid@
oxoid.
com
Technical
Support:
Phone:
(
800)
521­
1520
ext.
3
Fax:
(
734)
913­
6495
E­
mail:
oxoid@
oxoid.
com
Millipore
3
www.
millipore.
com
80
Ashby
Road
Bedford,
MA
01730
Sales:
Phone:
(
800)
645­
5476
Fax:
(
781)
533­
3110
Technical
Support:
Phone:
(
800)
645­
5476
Fax:
(
781)
533­
3110
Sigma
2
www.
sigma­
aldrich.
com
P.
O.
Box
14508
St.
Louis,
MO
63178
Sales:
Phone:
(
800)
325­
3010
Fax:
(
800)
325­
8070
E­
mail:
custserv@
sial.
com
Technical
Support:
Phone:
(
800)
325­
5832
Fax:
(
314)
286­
7828
E­
mail:
techserv@
sial.
com
VWR1
www.
vwr.
com
405
Heron
Drive
Bridgeport,
NJ
08014
Sales:
Phone:
(
800)
932­
5000
Fax:
(
856)
467­
3336
Technical
Support:
Phone:
(
800)
932­
5000
Fax:
(
856)
467­
3336
1
Possible
vendors
for
all
E.
coli
methods
2
Vendors
for
methods:
9221B/
9221F,
9222B/
9222G,
9222D/
9222G,
9213D,
EPA
Method
1603,
EPA
Method
1604
3
Vendors
for
methods:
9222B/
9222G,
9222D/
9222G,
9213D,
EPA
Method
1603,
EPA
Method
1604
4
Vendor
for
methods:
9221F,
9223B
5
Vendor
for
method:
m­
ColiBlue24
broth
6
Vendor
for
API
20E
for
EPA
Method
1604
66
Draft
June
2003
SECTION
5:
REFERENCES
5.1
Connell,
Kevin,
et
al.
2000.
ICRSS
­
Building
a
Better
Protozoa
Data
Set,
J.
AWWA.
91(
10):
30
­
43.

5.2
USEPA.
1997.
Manual
for
the
Certification
of
Laboratories
Analyzing
Drinking
Water;
Criteria
and
Procedures;
Quality
Assurance:
Fourth
Edition.
EPA
815­
B­
97­
001.

5.3
APHA.
1998.
Standard
Methods
for
the
Examination
of
Water
and
Wastewater;
20th
Edition.
American
Public
Health
Association,
American
Water
Works
Association,
Washington,
D.
C.

5.4
Pope,
M.,
et
al.
2002.
Assessment
of
the
effects
of
holding
time
and
temperature
on
E.
coli
concentrations
in
surface
water
samples.
Appl.
Environ.
Micro.
(
submitted).
67
Draft
June
2003
SECTION
6:
ACRONYMS
CFU
Colony­
forming
unit
CNFG
Confluent
growth
DAPI
4,
6­
diamidino­
2­
phenylindole
DIC
Differential
interference
contrast
microscopy
EPA
U.
S.
Environmental
Protection
Agency
FA
Immunofluorescense
assay
FITC
Fluorescien
isothiocyanate
ICR
Information
Collection
Rule
IDC
Initial
demonstration
of
capability
IFA
Immunofluorescence
assay
IMS
Immunomagnetic
separation
IPR
Initial
precision
and
recovery
IPT
Initial
proficiency
test
L
Liter
LT2
rule
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
MPC
Magnetic
particle
concentrator
MPN
Most
probable
number
MS
Matrix
spike
MS/
MSD
Matrix
spike/
matrix
spike
duplicate
NA­
MUG
Nutrient
Agar
(
NA)
with
4­
methylumbelliferyl­
beta­
D­
glucuronide
(
MUG)

NELAC
National
Environmental
Laboratory
Accreditation
Conference
NPDWR
National
Primary
Drinking
Water
Regulations
OPR
Ongoing
precision
and
recovery
OPT
Ongoing
proficiency
testing
PBMS
Performance­
based
measurement
system
PT
Proficiency
testing
PWS
Public
water
system
QA
Quality
assurance
QAP
Quality
Assurance
Plan
QC
Quality
control
Section
4:
E.
coli
Laboratory
Guidance
68
Draft
June
2003
RSD
Relative
standard
deviation
SDWA
Safe
Drinking
Water
Act
SOP
Standard
operating
procedure
TNTC
Too
numerous
to
count
UV
Ultraviolet
