Source
Water
Monitoring
Guidance
Manual
for
Public
Water
Systems
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
i
Draft
June
2003
TABLE
OF
CONTENTS
Section
1:
Introduction
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
1.1
Background
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
2
1.2
Large
System
Requirements
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
2
1.3
Small
System
Requirements
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
3
1.4
Use
of
Cryptosporidium
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
5
1.4.1
Cryptosporidium
Monitoring
Sample
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
5
1.4.2
Cryptosporidium
Matrix
Spike
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
6
1.5
Use
of
E.
coli
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
7
Section
2:
Grandfathering
Cryptosporidium
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
8
2.1
General
Guidelines
for
Generating
Cryptosporidium
Data
for
Grandfathering
.
.
.
.
.
.
.
8
2.1.1
Sample
Collection
Location
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
8
2.1.2
Sample
Collection
Schedule
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
9
2.1.3
Cryptosporidium
Analytical
Methods
for
Grandfathered
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
9
2.1.4
Cryptosporidium
Laboratories
for
Grandfathered
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11
2.1.5
E.
coli
and
Turbidity
Measurements
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11
2.2
Reporting
Grandfathered
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11
2.2.1
Data
Package
Contents
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11
2.2.2
Schedule
for
Submission
of
Grandfathered
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13
2.2.3
Procedures
for
Submission
of
Grandfathered
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13
2.3
Checklists
for
Grandfathering
Cryptosporidium
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13
Section
3:
Understanding
Cryptosporidium
Analyses
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
14
3.1
Summary
of
EPA
Methods
1622
and
1623
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
14
3.2
Cryptosporidium
Laboratory
Quality
Control
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
15
3.2.1
Initial
Precision
and
Recovery
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
15
3.2.2
Method
Blank
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
16
3.2.3
Ongoing
Precision
and
Recovery
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
16
3.2.4
Holding
Time
Requirements
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
16
3.2.5
Staining
Controls
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
17
3.2.6
Proficiency
Testing
Samples
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
17
3.2.7
Matrix
Spike
Samples
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
17
3.3
Archiving
Examination
Results
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
17
Section
4:
Understanding
E.
coli
Analyses
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
18
4.1
Summary
of
LT2
Rule
E.
coli
Methods
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
18
4.1.1
Most
Probable
Number
(
MPN)
Methods
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
18
4.1.2
Membrane
Filtration
(
MF)
Methods
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
19
4.2
E.
coli
Laboratory
Quality
Control
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
21
4.2.1
Dilution/
Rinse
Water
Sterility
Check
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
21
4.2.2
Media
Sterility
Check
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
21
4.2.3
Positive/
Negative
Controls
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
21
4.2.4
Media
Storage
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
22
4.2.5
Filtration
Unit
Sterilization
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
22
ii
Draft
June
2003
4.2.6
Preparation
Blanks
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
22
4.2.7
Verification
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
22
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
23
5.1
Defining
Your
Needs
and
Developing
a
Contract
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
23
5.1.1
Client
Information
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
24
5.1.2
Sample
Information
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
24
5.1.3
Sampling
Schedules
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
26
5.1.4
Analytical
Methodology
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
26
5.1.5
Data
Deliverables
and
Other
Contract
Issues
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
28
5.2
Developing
a
Bid
Sheet
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
31
5.3
Soliciting
the
Contract
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
32
5.3.1
Approved
Laboratories
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
32
5.3.2
Primary
and
Backup
Laboratory
Contracts
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
33
5.4
Evaluating
Bids
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
33
5.4.1
Identifying
Responsive
Bidders
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
33
5.4.2
References
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
34
5.5
Communicating
with
the
Laboratory
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
34
Section
6:
Collecting
and
Shipping
Source
Water
Samples
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
35
6.1
Sample
Volumes
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
36
6.2
Sample
Collection
Location
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
37
6.2.1
Plants
That
Do
Not
Have
a
Sampling
Tap
Located
Prior
to
Any
Treatment
.
.
37
6.2.2
Plants
That
Use
Different
Water
Sources
at
the
Same
Time
.
.
.
.
.
.
.
.
.
.
.
.
.
.
37
6.2.3
Plants
That
Use
Presedimentation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
37
6.2.4
Plants
That
Use
Raw
Water
Off­
Stream
Storage
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
38
6.2.5
Plants
That
Use
Bank
Filtration
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
38
6.3
Source
Water
Monitoring
Schedule
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
38
6.4
Sample
Collection
Guidance
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
39
6.4.1
Sample
Collection
Documentation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
40
6.4.2
Cryptosporidium
Sample
Collection
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
41
6.4.3
E.
coli
Sample
Collection
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
44
6.4.4
Measuring
Turbidity
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
44
6.4.5
Monitoring
Sample
Temperature
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
46
Section
7:
Reviewing
Cryptosporidium
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
48
7.1
Cryptosporidium
Data
Recording
at
the
Laboratory
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
48
7.1.1
LT2
Sample
Collection
Form
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
48
7.1.2
Method
1622/
1623
Bench
Sheet
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
48
7.1.3
Method
1622/
1623
Cryptosporidium
Slide
Examination
Form
.
.
.
.
.
.
.
.
.
.
.
.
49
7.2
Submitting
Cryptosporidium
Data
through
the
LT2
Data
Collection
System
.
.
.
.
.
.
.
.
49
7.2.1
Data
Entry/
Upload
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
50
7.2.2
PWS
Data
Review
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
51
7.2.3
EPA/
State
Review
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
51
7.3
What
Do
the
Sample
Examination
Results
Mean?
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
51
7.3.1
Immunofluorescent
Assay
(
IFA)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
51
7.3.2
4',
6­
diamadino­
2­
phenylindole
(
DAPI)
Examination
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
52
7.3.3
Differential
Interference
Contrast
(
DIC)
Examination
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
52
7.5
Reviewing
and
Validating
Raw
Cryptosporidium
Data
(
Optional)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
52
7.5.1
Data
Completeness
Check
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
53
7.5.2
Evaluation
of
Data
Against
Method
Quality
Control
Requirements
.
.
.
.
.
.
.
.
53
iii
Draft
June
2003
7.5.3
Calculation
Verification
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
54
7.6
Data
Archiving
Requirements
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
56
Section
8:
Reviewing
E.
coli
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
57
8.1
E.
coli
Laboratory
Data
Recording
at
the
Laboratory
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
57
8.1.1
Sample
Identification
Information
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
57
8.1.2
Primary
Data
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
57
8.1.3
Sample
Processing
and
Quality
Control
Information
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
57
8.1.4
Sample
Results
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
58
8.2
Submission
of
E.
coli
Data
through
the
LT2
Data
Collection
System
.
.
.
.
.
.
.
.
.
.
.
.
.
.
58
8.2.1
Data
Entry/
Upload
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
58
8.2.2
PWS
Data
Review
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
59
8.2.3
EPA/
State
Review
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
59
8.3
Reviewing
and
Validating
E.
coli
Data
(
Optional)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
59
8.3.1
Data
Completeness
Check
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
59
8.3.2
Evaluation
of
Data
Against
Method
Quality
Control
Requirements
.
.
.
.
.
.
.
.
60
8.3.3
Calculation
Verification
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
61
8.4
Data
Archiving
Requirements
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
67
Section
9:
References
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
68
Section
10:
Acronyms
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
69
iv
Draft
June
2003
TABLES
Table
1­
1.
Timeline
for
Large
Systems
Regulated
under
the
LT2
Rule
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
3
Table
1­
2.
Timeline
for
Small
Systems
Regulated
under
the
LT2
Rule
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
4
Table
1­
3.
Bin
Classifications
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
5
Table
1­
4.
Effect
of
the
Number
of
Oocysts
on
Bin
Classification
Based
on
Mean
of
12
Samples
.
6
Table
1­
5.
Effect
of
the
Number
of
Oocysts
on
Bin
Classification
Based
on
Mean
of
48
Samples
.
6
Table
6­
1.
Summary
of
LT2
Rule
Monitoring
Requirements
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
35
Table
6­
2.
Sample
Collection
Activities
Required
for
Each
Plant
Type
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
40
Table
6­
3.
Minimum
Data
Elements
to
Record
During
Sample
Collection
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
40
Table
6­
4.
Contacts
for
Filters
Approved
for
Use
in
EPA
Method
1622/
1623
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
43
Table
7­
1.
LT2
Data
Collection
System
Data
Entry,
Review,
and
Transfer
Process
.
.
.
.
.
.
.
.
.
.
.
50
Table
8­
1.
Incubation
Times
and
Temperatures
for
Approved
E.
Coli
Methods
.
.
.
.
.
.
.
.
.
.
.
.
.
.
61
Table
8­
2.
Examples
of
Different
Combinations
of
Positive
Tubes
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
66
v
Draft
June
2003
APPENDICES
Appendix
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Checklist
for
Beginning
Grandfathered
Cryptosporidium
Monitoring
Appendix
B
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Checklist
for
Submitting
Grandfathered
Cryptosporidium
Data
Appendix
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Example
LT2
Sample
Collection
Form
Appendix
D
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Example
Bulk
Sample
Collection
Protocol
for
Cryptosporidium
Appendix
E
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Example
Envirochek
 
Field
Filtration
Protocol
for
Cryptosporidium
Appendix
F
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Example
Filta­
Max
 
Field
Filtration
Protocol
for
Cryptosporidium
Appendix
G
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Example
E.
coli
Sample
Collection
Protocol
Appendix
H
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Method
2130B
for
Turbidity
Measurement
Appendix
I
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Great
Lakes
Instrument
Method
2
for
Turbidity
Measurement
Appendix
J
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Revised
EPA
Method
180.1
for
Turbidity
Measurement
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
ground
water
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:

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

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

°
Users'
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.

This
manual
provides
guidance
on
the
following
aspects
of
the
LT2
rule:

°
Section
1:
Overview
of
the
rule's
monitoring
requirements
and
how
the
Cryptosporidium
and
E.
coli
data
collected
under
the
rule
will
be
used
°
Section
2:
Guidance
on
submitting
historical
data
("
grandfathering")

°
Section
3:
Understanding
Cryptosporidium
analyses
°
Section
4:
Understanding
E.
coli
analyses
°
Section
5:
Establishing
a
Cryptosporidium
laboratory
contract
°
Section
6:
Guidance
on
collecting
and
shipping
LT2
monitoring
samples
°
Section
7:
Reviewing
Cryptosporidium
data
°
Section
8:
Reviewing
E.
coli
data
Section
1:
Introduction
2
Draft
June
2003
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.

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.

1.2
Large
System
Requirements
Large
systems
affected
by
the
LT2
rule
include
both
filtered
and
unfiltered
systems.

°
A
large,
filtered
system
in
the
LT2
rule
is
a
system
that:

°
Uses
surface
water
or
ground
water
under
the
direct
influence
of
surface
water
°
Serves
at
least
10,000
people
°
Provides
filtration
or
is
unfiltered,
but
required
to
install
filtration
because
the
system
no
longer
meets
all
filtration
avoidance
criteria
Large,
filtered
systems
are
required
to
conduct
initial
source
water
monitoring
that
includes
Cryptosporidium,
E.
coli,
and
turbidity
sampling
[
40
CFR
part
141.701
(
b)].

°
A
large,
unfiltered
system
in
the
LT2
rule
is
a
system
that:

°
Uses
surface
water
or
ground
water
under
the
direct
influence
of
surface
water
°
Serves
at
least
10,000
people
°
Does
not
currently
provide
filtration
and
meets
all
filtration
avoidance
criteria
Large
unfiltered
systems
are
required
to
conduct
initial
source
water
monitoring
that
includes
Cryptosporidium
sampling
only
[
40
CFR
part
141.701
(
d)].

All
of
the
Cryptosporidium
sampling
requirements
and
guidance
discussed
in
this
document
apply
equally
to
both
filtered
and
unfiltered
systems
and
both
filtered
and
unfiltered
systems
that
serve
at
least
10,000
Section
1:
Introduction
3
Draft
June
2003
people
are
referred
to
as
large
systems
in
this
document.
However,
the
E.
coli
and
turbidity
guidance
in
this
document
does
not
apply
to
large
unfiltered
systems.

The
steps
required
for
LT2
rule
compliance
for
large
systems,
and
the
schedule
for
these
steps,
are
summarized
in
Table
1­
1.

Table
1­
1.
Timeline
for
Large
Systems
Regulated
under
the
LT2
Rule
Event
Schedule
Duration
Establish
contract
with
a
Cryptosporidium
laboratory
pending
approval
under
EPA's
Lab
QA
Program
(
Section
2.4.1,
below)
As
soon
as
possible
N/
A
­
single
event
Verify
that
your
utility
laboratory
is
certified
under
the
drinking
water
laboratory
certification
program
to
perform
the
technique
you
plan
to
use
for
performing
E.
coli
analyses
under
LT2a
As
soon
as
possible
N/
A
­
single
event
Submit
grandfathered
Cryptosporidium
data
package
Within
2
months
of
rule
promulgationb
Within
8
months
of
rule
promulgationc
N/
A
­
single
event
Work
with
your
Cryptosporidium
laboratory
to
establish
a
mutually
acceptable
sampling
schedule
As
soon
as
possible
after
establishing
contract
N/
A
­
single
event
Submit
sampling
schedule
through
the
LT2
Data
Collection
System
Within
3
months
of
rule
promulgation
N/
A
­
single
event
Collect
monitoring
samplesd
Beginning
6
months
after
rule
promulgation
At
least
once
per
month
for
2
yearse
Submit
monitoring
resultsd
No
later
than
10
days
after
the
end
of
the
first
month
following
the
month
that
the
sample
was
collected
(
approximately
40
to
70
days
after
sample
collection,
depending
on
when
during
the
month
the
sample
is
collected)
At
least
once
per
month
for
2
yearse
a
Not
applicable
to
large,
unfiltered
systems
because
these
systems
are
not
required
to
monitor
for
E.
coli
or
turbidity
b
PWSs
with
at
least
2
years
of
grandfathered
data
at
the
time
of
LT2
rule
promulgation
and
that
intend
to
use
these
data
in
lieu
of
monitoring
under
the
LT2
rule
c
PWSs
with
fewer
than
2
years
of
grandfathered
data
at
the
time
of
LT2
rule
promulgation,
or
that
have
at
least
2
years
of
grandfathered
data
but
intend
to
conduct
monitoring
under
the
LT2
rule
d
PWSs
may
be
eligible
to
use
historical
(
grandfathered)
data
in
lieu
of
these
requirements
if
certain
quality
assurance
and
quality
control
criteria
are
met
(
see
Section
2)
e
PWSs
monitoring
for
Cryptosporidium
may
collect
more
than
one
sample
per
month
if
sampling
is
evenly
spaced
over
the
monitoring
period
N/
A
=
Not
applicable
1.3
Small
System
Requirements
A
small
system
in
the
LT2
rule
is
a
system
that:

°
Uses
surface
water
or
ground
water
under
the
direct
influence
of
surface
water
°
Serves
fewer
than
10,000
people
°
Provides
filtration
or
is
unfiltered
but
required
to
install
filtration
because
the
system
no
longer
meets
all
filtration
avoidance
criteria
Section
1:
Introduction
4
Draft
June
2003
°
Does
not
currently
provide
filtration
and
meets
all
filtration
avoidance
criteria
These
systems
are
required
to
conduct
initial
source
water
monitoring
for
E.
coli
as
an
indicator
of
Cryptosporidium
and,
for
those
systems
exceeding
E.
coli
trigger
levels,
Cryptosporidium
monitoring
[
40
CFR
part
141.701
(
c)].

The
steps
required
for
LT2
rule
compliance
for
small
systems,
and
the
schedule
for
these
steps,
are
summarized
in
Table
1­
2.

Table
1­
2.
Timeline
for
Small
Systems
Regulated
under
the
LT2
Rule
Event
Schedule
Duration
Verify
that
your
utility
laboratory
is
certified
under
the
drinking
water
laboratory
certification
program
to
perform
the
technique
you
plan
to
use
for
perform
E.
coli
analyses
under
LT2
Prior
to
rule
promulgation
N/
A
­
single
event
Submit
sampling
schedule
through
the
LT2
Data
Collection
System
Within
27
months
of
rule
promulgation
N/
A
­
single
event
Collect
E.
coli
samples
Beginning
30
months
(
2.5
years)
after
rule
promulgation
1
year
(
2
samples
per
month)

Submit
E.
coli
monitoring
results
No
later
than
10
days
after
the
end
of
the
first
month
following
the
month
that
the
sample
was
collected
(
approximately
40
to
70
days
after
sample
collection,
depending
on
when
during
the
month
the
sample
is
collected)
At
least
once
per
month
for
1
year
Possible
additional
monitoring
requirement
for
Cryptosporidium
if
small
systems
exceed
E.
coli
trigger
levelsa
Establish
contract
with
a
Cryptosporidium
laboratory
pending
approval
under
EPA's
Lab
QA
Program
(
Section
2.4.1,
below)
As
soon
as
possible
after
you
are
notified
that
your
plant
has
exceeded
the
E.
coli
trigger
levels
N/
A
­
single
event
Submit
sampling
schedule
through
the
LT2
Data
Collection
System
Within
45
months
of
rule
promulgation
N/
A
­
single
event
Work
with
your
Cryptosporidium
laboratory
to
establish
a
mutually
acceptable
sampling
schedule
Within
2
months
of
rule
promulgation
N/
A
­
single
event
Collect
Cryptosporidium
samples
48
months
(
4
years)
after
promulgation
1
year
(
2
samples
per
month)
b
Submit
Cryptosporidium
monitoring
results
No
later
than
10
days
after
the
end
of
the
first
month
following
the
month
that
the
sample
was
collected
(
approximately
40
to
70
days
after
sample
collection,
depending
on
when
during
the
month
the
sample
is
collected)
At
least
once
per
month
for
1
year
a
Small
systems
may
be
required
to
monitor
for
Cryptosporidium
for
1
year,
beginning
6
months
after
completion
of
E.
coli
monitoring;
Cryptosporidium
monitoring
required
if
the
E.
coli
annual
mean
concentrations
exceed
10
E.
coli/
100
mL
for
systems
using
lakes/
reservoirs
or
exceed
50
E.
coli/
100
mL
for
systems
using
flowing
streams
b
PWSs
monitoring
for
Cryptosporidium
may
collect
more
than
two
samples
per
month
if
sampling
is
evenly
spaced
over
the
monitoring
period
N/
A
=
Not
applicable
Section
1:
Introduction
5
Draft
June
2003
Details
on
the
use
of
the
Cryptosporidium
and
E.
coli
data
collected
under
the
LT2
rule
are
provided
in
Sections
1.4
and
1.5.

1.4
Use
of
Cryptosporidium
Data
Two
types
of
Cryptosporidium
data
are
collected
under
the
LT2
rule:
Cryptosporidium
occurrence
data
from
the
analysis
of
monitoring
samples,
and
method
performance
data
from
the
analysis
of
matrix
spike
(
MS)
samples.
The
use
of
occurrence
data
from
monitoring
samples
is
discussed
in
Section
1.4.1;
the
use
of
method
performance
data
from
MS
samples
is
discussed
in
Section
1.4.2.

1.4.1
Cryptosporidium
Monitoring
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
3.
The
treatment
bin
classification
established
for
each
PWSs
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
greater
risk
for
high
Cryptosporidium
source
water
concentrations.

Table
1­
3.
Bin
Classifications
Cryptosporidium
Bin
Concentration
Bin
Classification
Cryptosporidium
<
0.075
oocysts/
L
Bin
1
0.075
oocysts/
L
#
Cryptosporidium
<
1.0
oocyst/
L
Bin
2
1.0
oocyst/
L
#
Cryptosporidium
<
3.0
oocysts/
L
Bin
3
Cryptosporidium
$
3.0
oocysts/
L
Bin
4
1.4.1.1
Calculating
Bin
Classifications
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
a
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
arithmetic
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
arithmetic
mean
of
all
sample
concentrations
in
any
12
consecutive
months
in
the
monitoring
period
For
PWS
serving
fewer
than
10,000
people,
and
that
monitor
for
Cryptosporidium
for
1
year,
bin
classification
would
be
based
on
the
simple
arithmetic
mean
of
all
sample
concentrations.

In
all
cases,
the
bin
concentration
is
calculated
using
individual
sample
concentrations.
These
concentrations
are
calculated
as
"
number
of
oocysts
detected
/
volume
(
in
L)
analyzed."
Individual
sample
concentrations
are
not
calculated
as
"
oocysts
detected
/
10
L,"
nor
are
bin
concentrations
calculated
as
the
"
sum
of
the
oocysts
detected
/
the
sum
of
the
volumes
analyzed."
As
a
result,
each
sample
has
an
equal
weight
on
the
final
bin
concentration.
In
cases
where
no
oocysts
are
detected,
the
number
of
oocysts
used
to
calculate
the
sample
concentration
is
"
0."
Section
1:
Introduction
6
Draft
June
2003
1.4.1.2
Number
of
Oocysts
Detected
Versus
Bin
Classification
To
better
understand
the
relationship
between
the
absolute
number
of
oocysts
detected
in
your
samples
and
the
resulting
bin
classification,
several
crosswalks
are
provided
below.
Table
1­
4
applies
to
large
plants
conducting
monthly
monitoring
over
2
years.
This
table
provides
a
crosswalk
between
the
sum
of
the
oocysts
detected
in
10­
and
50­
L
samples
during
the
highest
12­
month
period
and
the
corresponding
bin
classification.

Table
1­
4.
Effect
of
the
Number
of
Oocysts
on
Bin
Classification
Based
on
Mean
of
12
Samples
Sum
of
oocysts
found
in
12,
10­
L
samplesa
Sum
of
oocysts
found
in
12,
50­
L
samplesb
Corresponding
range
of
mean
Cryptosporidium
concentrations
Corresponding
bin
classification
From
To
0
­
8
oocysts
0
­
44
oocysts
<
0.075
oocysts/
L
1
9
­
125
oocysts
45
­
629
oocysts
0.075
oocysts/
L
<
1.0
oocyst/
L
2
126
­
365
oocysts
630
­
1829
oocysts
1.0
oocyst/
L
<
3.0
oocysts/
L
3
366
or
more
oocysts
1830
or
more
oocysts
$
3.0
oocysts/
L
4
a
Representing
the
highest
12­
month
mean;
assumes
that
10­
L
samples
are
analyzed
for
each
event
b
Representing
the
highest
12­
month
mean;
assumes
that
50­
L
samples
are
analyzed
for
each
event
Table
1­
5
applies
to
large
plants
conducting
semimonthly
monitoring
over
2
years.
This
table
provides
a
crosswalk
between
the
sum
of
the
number
of
oocysts
detected
in
samples
during
the
entire
2­
year
monitoring
period
and
the
corresponding
bin
classification.
Again,
because
this
crosswalk
is
based
on
analysis
of
exactly
10
L
or
50
L
for
all
samples,
it
may
not
apply
to
all
plants
that
monitor
for
Cryptosporidium
on
a
semimonthly
basis,
but
it
helps
put
into
perspective
the
impact
that
one
high
sample
result
may
have
on
bin
classification.

Table
1­
5.
Effect
of
the
Number
of
Oocysts
on
Bin
Classification
Based
on
Mean
of
48
Samples
Sum
of
oocysts
found
in
48,
10­
L
samplesa
Sum
of
oocysts
found
in
48,
50­
L
samplesb
Corresponding
range
of
mean
Cryptosporidium
concentrations
Corresponding
bin
classification
From
To
0
­
35
0
­
179
oocysts
<
0.075
oocysts/
L
1
36
­
503
180
­
2519
oocyst
0.075
oocysts/
L
<
1.0
oocyst/
L
2
504
­
1463
2520
­
7319
oocysts
1.0
oocyst/
L
<
3.0
oocysts/
L
3
1464
or
more
7320
or
more
oocysts
$
3.0
oocysts/
L
4
a
Assumes
that
10­
L
samples
are
analyzed
for
each
event
b
Assumes
that
50­
L
samples
are
analyzed
for
each
event
Systems
may
analyze
larger
volumes
than
10
L,
and
larger
volumes
analyzed
should
increase
analytical
sensitivity
(
detection
limit),
provided
method
performance
is
acceptable.
Because
these
tables
are
based
on
analysis
of
exactly
10
L
or
exactly
50
L
for
all
samples,
it
may
not
apply
to
all
plants
that
monitor
monthly
for
Cryptosporidium,
but
it
helps
put
into
perspective
the
impact
that
one
high
sample
result
may
have
on
bin
classification.
In
addition,
filtering
higher
volumes
may
not
result
in
the
same
high
volume
analyzed
if
the
source
is
turbid
and
the
PWS
analyzes
only
a
portion
of
the
concentrated
sample.
The
calculations
used
to
determine
the
volume
analyzed
if
less
than
the
entire
sample
volume
is
analyzed
are
discussed
in
Section
7.5.3.

1.4.2
Cryptosporidium
Matrix
Spike
Data
During
LT2
rule
Cryptosporidium
monitoring,
PWSs
are
required
to
collect
one
matrix
spike
(
MS)
sample
for
every
20
monitoring
samples
from
their
source
water,
per
the
requirements
in
EPA
Methods
Section
1:
Introduction
7
Draft
June
2003
1622/
1623
(
Section
9.1.8).
A
description
of
MS
samples
is
provided
in
Section
3.2.7
of
this
document.
For
large
systems
that
perform
monthly
monitoring
for
2
years
and
collect
24
monitoring
samples
and
for
small
systems
that
are
triggered
into
monitoring
for
1
year
and
collect
24
monitoring
samples,
two
MS
samples
will
be
analyzed.
For
large
systems
that
perform
semimonthly
or
more
frequent
monitoring
for
2
years
and
collect
48
or
more
samples,
a
minimum
of
three
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
is
based.

When
considering
the
method
performance
that
could
be
achieved
for
analysis
of
Cryptosporidium
under
the
LT2
rule,
the
Federal
Advisory
Committee
(
FACA)
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
9.1).

1.5
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.
If
a
relationship
can
be
established,
small
systems
initially
will
be
permitted
to
monitor
for
E.
coli,
rather
than
conducting
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).

A
preliminary
index
was
developed
during
development
of
the
FACA
agreement
using
data
from
the
Information
Collection
Rule
(
ICR)
and
ICRSS
(
Reference
9.2).
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.
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.
Based
on
the
data
from
the
ICR
and
ICRSS,
the
preliminary
E.
coli
trigger
levels
were
set
at
a
mean
of
10
E.
coli/
100
mL
for
reservoir/
lake­
type
source
waters
and
50
E.
coli/
100
mL
for
flowing
stream
 
type
source
waters.

These
levels
may
potentially
be
revised
based
on
the
much
larger,
more
reliable
Cryptosporidium
and
E.
coli
data
set
collected
through
LT2
rule
monitoring.
8
Draft
June
2003
SECTION
2:
GRANDFATHERING
CRYPTOSPORIDIUM
DATA
"
Grandfathered"
Cryptosporidium
data
are
results
generated
before
monitoring
under
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2ESWTR
or
LT2
rule)
starts
and
that
a
public
water
system
(
PWS)
intends
to
use
in
determining
its
bin
classification
(
Section
1.4.1)
under
the
rule.
Grandfathered
data
may
be
used
in
lieu
of,
or
in
addition
to,
results
generated
during
LT2
rule
implementation
(
40
CFR
part
141.708).
This
section
of
the
manual
is
designed
to
assist
PWSs
in
producing
grandfathered
data
that
should
be
equivalent
to
the
data
collected
during
LT2
rule
implementation
and,
therefore,
eligible
for
use
in
bin
classification.
The
final
LT2
rule
will
establish
requirements
for
reporting
and
acceptance
of
grandfathered
monitoring
results.

2.1
General
Guidelines
for
Generating
Cryptosporidium
Data
for
Grandfathering
A
PWS's
grandfathered
Cryptosporidium
data
package
should
meet
the
following
general
conditions
(
40
CFR
part
141.708):

°
Samples
were
collected
from
the
appropriate
location(
s)

°
Samples
were
representative
of
a
plant's
source
water(
s)
and
the
source
water(
s)
have
not
changed
°
Samples
were
collected
no
less
frequently
than
each
calendar
month
on
a
regular
schedule,
beginning
no
earlier
than
January
1999
(
when
EPA
Method
1622
was
first
released
as
an
interlaboratoryvalidated
method)

°
Samples
were
collected
in
equal
intervals
of
time
over
the
entire
collection
period
(
e.
g.,
weekly,
twice­
per­
month,
monthly)

°
The
data
set
includes
all
source
water
Cryptosporidium
monitoring
results
generated
during
the
grandfathered
data
monitoring
period
(
see
details
below
 
data
from
monitoring
not
directed
towards
LT2
rule
binning
will
not
be
a
component
of
the
binning
data
set)

°
Sample
volumes
of
at
least
10
L
were
analyzed
or,
in
cases
where
10
L
were
not
analyzed,
at
least
2
mL
of
packed
pellet
volume
were
analyzed
(
additional
details
below)

°
The
data
were
generated
using
the
validated
versions
of
EPA
Methods
1622
or
1623
°
The
data
are
fully
compliant
with
the
QA/
QC
criteria
specified
in
the
version
of
Method
1622
or
Method
1623
used
to
generate
the
data,
including
analysis
of
matrix
spike
(
MS)
samples
at
a
frequency
of
at
least
5%
(
1
MS
sample
for
every
20
monitoring
samples)

The
following
sections
discuss
these
recommendations
in
more
detail.

2.1.1
Sample
Collection
Location
The
sample
collection
location
requirements
are
the
same
for
LT2
rule
monitoring
and
for
grandfathered
data
and
are
discussed
in
Section
6.2.
If
the
PWS
does
not
collect
samples
as
recommended
in
Section
6.2,
the
data
may
not
be
acceptable
for
grandfathering.
Section
2:
Grandfathering
Cryptosporidium
Data
9
Draft
June
2003
2.1.2
Sample
Collection
Schedule
During
LT2
rule
monitoring,
PWSs
will
be
required
to
collect
samples
at
least
monthly
and
in
accordance
with
a
schedule
established
by
the
PWS
prior
to
initiation
of
monitoring
(
40
CFR
part
141.703).
PWSs
may
collect
samples
more
frequently
(
e.
g.,
twice­
per­
month,
weekly),
provided
the
same
frequency
is
maintained
throughout
the
monitoring
period
[
40
CFR
part
141.701
(
e)].
Sampling
for
grandfathered
data
should
follow
these
same
criteria.

EPA
recommends
that,
prior
to
initiation
of
grandfathered
monitoring,
PWSs
develop
a
schedule
listing
the
calendar
date
on
which
each
Cryptosporidium
sample
will
be
collected
and
include
this
schedule
when
submitting
the
grandfathered
data
package
to
EPA.
PWSs
that
have
begun
grandfathered
monitoring
without
establishing
a
sampling
schedule
should
develop
a
schedule
for
the
collection
of
remaining
samples.
PWSs
should
collect
samples
within
2
days
before
or
after
the
dates
indicated
in
their
sampling
schedules.
Exceptions
to
the
sampling
schedule
are
noted
as
follows:

°
If
extreme
conditions
or
situations
exist
that
may
pose
danger
to
the
sampler,
or
which
are
unforeseen
or
cannot
be
avoided
and
which
cause
the
system
to
be
unable
to
sample
in
the
required
time
frame,
the
PWS
should
sample
as
close
to
the
scheduled
date
as
feasible
and
submit
an
explanation
for
the
alternative
sampling
date
with
the
analytical
results.

°
PWSs
that
are
unable
to
report
a
valid
Cryptosporidium
analytical
result
for
a
scheduled
sampling
date
due
to
failure
to
comply
with
the
analytical
method
quality
control
standards
(
e.
g.,
sample
is
lost
or
contaminated;
laboratory
exceeds
an
analytical
method
holding
time)
should
collect
a
replacement
sample
within
14
days
of
being
notified
by
the
laboratory
that
a
result
cannot
be
reported
for
that
date.
PWSs
should
submit
an
explanation
for
the
replacement
sample
with
the
analytical
results.

Alternative
sample
collection
dates
should
be
timed
so
as
not
to
coincide
with
another
scheduled
Cryptosporidium
sample
collection
date.
Documentation
of
alternate
sample
collection,
including
the
reason,
should
be
provided
with
the
grandfathered
data
package.

Water
treatment
plants
that
use
surface
water
or
ground
water
under
the
direct
influence
(
GWUDI),
but
are
operated
only
seasonally
(
e.
g.,
during
times
of
high­
water
demand)
should
monitor
at
least
monthly
during
the
period
when
the
plant
is
in
operation.

The
Federal
Advisory
Committee
Agreement
in
Principle
(
Agreement)
for
the
LT2
rule
recommends
that
if
PWSs
collect
a
total
of
at
least
48
samples
(
regardless
of
whether
all
of
the
samples
were
collected
before
LT2
rule
promulgation
or
some
were
collected
before
and
some
after
rule
promulgation),
the
Cryptosporidium
bin
concentration
will
be
equal
to
the
arithmetic
mean
of
all
sample
concentrations
[
40
CFR
part
141.709
(
b)(
1)].
For
PWSs
that
collect
a
total
of
at
least
24
samples,
but
not
more
than
47
samples,
the
Cryptosporidium
bin
concentration
will
be
equal
to
the
highest
arithmetic
mean
of
all
sample
concentrations
in
any
12
consecutive
months
during
which
Cryptosporidium
samples
were
collected
[
40
CFR
part
141.709
(
b)(
2)].

2.1.3
Cryptosporidium
Analytical
Methods
for
Grandfathered
Data
Methods
1622
or
1623
should
be
used
for
Cryptosporidium
analyses
for
the
LT2
rule
[
40
CFR
part
141.708
(
b)(
1)].
The
following
are
EPA­
validated
versions
of
Methods
1622
and
1623
acceptable
for
monitoring
for
Cryptosporidium
before
LT2
rule
implementation:

°
Method
1623:
Cryptosporidium
and
Giardia
in
Water
by
Filtration/
IMS/
FA.
U.
S.
Environmental
Protection
Agency,
Office
of
Water.
2001.
EPA­
821­
R­
01­
025
°
Method
1622:
Cryptosporidium
in
Water
by
Filtration/
IMS/
FA.
U.
S.
Environmental
Protection
Agency,
Office
of
Water.
2001.
EPA­
821­
R­
01­
026
Section
2:
Grandfathering
Cryptosporidium
Data
10
Draft
June
2003
°
Method
1623:
Cryptosporidium
and
Giardia
in
Water
by
Filtration/
IMS/
FA.
U.
S.
Environmental
Protection
Agency,
Office
of
Water.
1999.
EPA­
821­
R­
99­
006
(
Note:
The
2001
version
of
the
method
should
be
used
to
generate
data
after
January
1,
2002.)

°
Method
1622:
Cryptosporidium
in
Water
by
Filtration/
IMS/
FA.
U.
S.
Environmental
Protection
Agency,
Office
of
Water.
1999.
EPA­
821­
R­
99­
001
(
Note:
The
2001
version
of
the
method
should
be
used
to
generate
data
after
January
1,
2002.)

The
procedures
in
EPA
Method
1622/
1623
are
performance­
based,
and
allow
for
modifications.
The
2001
versions
of
EPA
Method
1622/
1623
also
approve
for
nationwide
use
modified
versions
of
the
methods
using
the
following
components:

°
Whatman
Nuclepore
CrypTest
®
filter
°
IDEXX
Filta­
Max
 
filter
°
Waterborne
Aqua­
Glo
 
G/
C
Direct
FL
antibody
stain
°
Waterborne
Crypt­
a­
Glo
 
and
Giardi­
a­
Glo
 
antibody
stains
Since
release
of
the
2001
versions
of
Methods
1622/
1623,
EPA
also
has
approved
a
modified
version
of
the
methods
using
the
Pall
Gelman
Envirochek
 
HV
filter
and
has
approved
the
use
of
irradiated,
flow
cytometer
 
sorted
spiking
suspensions
for
routine
QC
sample
spiking.

Laboratories
that
analyze
Cryptosporidium
samples
using
other
modified
procedures,
as
allowed
under
the
performance
criteria
of
Methods
1622/
1623,
should
be
approved
to
use
the
modified
procedures
under
the
Lab
QA
Program
discussed
in
Section
2.1.4,
below,
and
in
detail
in
the
Microbial
Laboratory
Guidance
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).

Other
notable
differences
between
the
1999
and
2001
versions
of
EPA
Method
1622/
1623
include
the
following:

°
Clarified
sample
acceptance
criteria
°
Modified
capsule
filter
elution
procedure
°
Modified
concentrate
aspiration
procedure
°
Modified
IMS
acid
dissociation
procedure
°
Updated
QC
acceptance
criteria
for
initial
precision
and
recovery
(
IPR)
and
ongoing
precision
and
recovery
(
OPR)
tests
°
Addition
of
a
troubleshooting
section
for
QC
failures
°
Modified
holding
times
°
Inclusion
of
flow
cytometry
 
sorted
spiking
suspensions
(
required
for
spiked
samples
analyzed
during
LT2
monitoring)

2.1.3.1
Minimum
Sample
Volume
and
Subsampling
Analysis
The
requirements
for
sample
volume
analyses
are
the
same
for
LT2
rule
monitoring
and
for
grandfathered
data
[
40
CFR
part
141.708
(
b)(
5)].
These
requirements
are
discussed
in
Section
6.1
of
this
manual.

2.1.3.2
Analysis
of
Matrix
Spike
Samples
The
requirements
for
analysis
of
matrix
spike
(
MS)
samples
are
the
same
for
LT2
rule
monitoring
and
for
grandfathered
data
[
40
CFR
part
141.708
(
e)].
These
requirements,
and
guidance
on
MS
sample
collection,
are
discussed
in
Section
6.4.2
of
this
manual.
Section
2:
Grandfathering
Cryptosporidium
Data
11
Draft
June
2003
2.1.4
Cryptosporidium
Laboratories
for
Grandfathered
Data
EPA
has
established
the
Laboratory
Quality
Assurance
Evaluation
Program
for
the
Analysis
of
Cryptosporidium
in
Water
(
Lab
QA
Program)
to
approve
laboratories
to
perform
Cryptosporidium
analyses
under
the
LT2
rule
(
see
http://
www.
epa.
gov/
safewater/
lt2/
index.
html).
EPA
recognizes
that
some
PWSs
could
begin
generating
grandfathered
Cryptosporidium
data
prior
to
when
the
Lab
QA
Program
is
fully
implemented
(
e.
g.,
before
EPA
is
able
to
evaluate
all
laboratories
that
will
participate
in
the
program).
Consequently,
PWSs
should
ensure
that
their
grandfathered
Cryptosporidium
samples
are
analyzed
by
laboratories
that
will
be
evaluated
under
the
Lab
QA
Program
before
the
data
are
submitted
to
EPA.
Note
that
PWSs
will
not
submit
grandfathered
data
packages
until
after
the
LT2
rule
is
final,
currently
scheduled
for
mid­
or
late
2004.
Samples
analyzed
by
laboratories
that
do
not
meet
the
criteria
for
approval
under
the
LT2
rule
may
not
be
accepted
for
grandfathering.

Laboratories
should
also
participate
in
the
EPA
Protozoa
PT
Program.
EPA
does
not
expect
there
to
be
restrictions
on
the
number
of
laboratories
involved
in
the
generation
of
a
PWS's
grandfathered
data.

2.1.5
E.
coli
and
Turbidity
Measurements
The
Agreement
would
not
exclude
the
use
of
previously
collected
Cryptosporidium
data
if
E.
coli
and
turbidity
samples
are
not
collected.
However,
the
Agreement
recommends
that
PWSs
serving
at
least
10,000
people
should
collect
E.
coli
and
turbidity
samples
along
with
Cryptosporidium
samples
when
monitoring
under
the
LT2
rule.
EPA
recommends
that
PWSs
conducting
early
(
i.
e.,
grandfathered)
monitoring
collect
and
analyze
E.
coli
samples
with
each
Cryptosporidium
sample
and
measure
turbidity
during
each
sampling
event.

2.2
Reporting
Grandfathered
Data
The
final
LT2
rule
will
establish
reporting
requirements
for
grandfathered
data.
The
following
recommendations
are
intended
to
give
PWSs
an
indication
of
potential
reporting
requirements
for
consideration
when
establishing
their
grandfathered
data
monitoring
programs.

For
consideration
of
grandfathered
data,
PWSs
should
submit
to
EPA
a
complete
data
package
as
described
below.

2.2.1
Data
Package
Contents
The
grandfathered
data
package
should
include
the
following:

1.
A
signed
cover
letter
from
the
PWS
certifying
that
the
data
represent
the
plant's
current
source
water
and
that
all
source
water
Cryptosporidium
monitoring
results
collected
during
the
LT2
rule
monitoring
period
(
defined
below)
are
included
in
the
package
2.
Sample
collection
schedule
established
before
beginning
monitoring
3.
Where
applicable,
documentation
addressing
the
dates
and
reason(
s)
for
re­
sampling,
as
well
as
the
use
of
presedimentation,
off­
stream
storage,
or
bank
filtration
during
monitoring
4.
A
list
of
the
field
and
MS
samples
submitted
in
the
data
package
(
see
Section
2.2.1.1,
below,
for
details),
identified
by
sample
ID
and
collection
date
5.
Sample
results
for
all
field
and
MS
samples
(
see
Section
2.2.1.2,
below,
for
details)
and
6.
Documentation
that
all
method­
required
quality
control
requirements
were
acceptable
for
every
field
and
MS
sample
submitted
with
the
package
(
see
Section
2.2.1.3,
below,
for
details).
Section
2:
Grandfathering
Cryptosporidium
Data
12
Draft
June
2003
2.2.1.1
Sample
Results
to
be
Reported
PWSs
that
conduct
monitoring
for
grandfathering
should
submit
results
for
all
source
water
Cryptosporidium
samples
analyzed
during
the
LT2
rule
monitoring
period,
as
defined
below
(
40
CFR
part
141.707).
This
will
include
all
samples
that
were:

°
Collected
from
the
sampling
location
used
for
LT2
rule
monitoring,

°
Not
spiked,
and
°
Analyzed
using
the
laboratory's
routine
process
for
Method
1622/
1623
analyses,
including
analytical
technique
and
QA/
QC.

EPA
plans
that
the
LT2
rule
monitoring
period
for
a
specific
PWS
will
begin
with
the
collection
of
the
first
sample
submitted
for
LT2
rule
binning
and
end
with
the
collection
of
the
final
sample
submitted
for
LT2
rule
binning
(
as
long
as
a
minimum
of
2
years
of
acceptable
data
have
been
submitted).
With
the
use
of
grandfathered
data,
the
final
sample
may
be
collected
before
the
end
of
the
LT2
rule
implementation
schedule.
Sample
results
generated
after
the
last
sample
result
in
the
PWS's
data
package
would
be
considered
outside
the
PWS's
LT2
rule
monitoring
period
and
would
not
need
to
be
submitted
to
EPA
for
LT2
rule
binning
purposes.
However,
these
results
may
be
subject
to
reporting
requirements
under
other
federal
or
State
regulations.

2.2.1.2
Data
Elements
to
be
Reported
for
Each
Sample
Result
The
following
data
elements,
at
a
minimum,
must
be
submitted
for
each
Cryptosporidium
monitoring
sample
and
MS
sample
[
40
CFR
part
141.708
(
d)]:

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

°
Sample
volume
filtered
(
L),
to
nearest
¼
L
°
Number
of
oocysts
counted
°
For
samples
in
which
less
than
10
L
is
filtered
or
less
than
100%
of
the
sample
volume
is
examined,
PWSs
should
also
report
the
number
of
filters
used
and
the
packed
pellet
volume.

°
For
samples
in
which
less
than
100%
of
sample
volume
is
examined,
PWSs
should
also
report
the
volume
of
resuspended
concentrate
and
volume
of
this
resuspension
processed
through
immunomagnetic
separation.

°
For
matrix
spike
samples,
PWSs
should
also
report
the
sample
volume
spiked
and
estimated
number
of
oocysts
spiked.
These
data
are
not
applicable
to
monitoring
samples.

EPA
recommends
that
these
data
elements
be
reported
by
submitting
a
completed
sample
collection
form,
laboratory
bench
sheet,
and
Cryptosporidium
report
form
for
each
sample.
Example
bench
sheets
and
report
forms
are
provided
as
attachments
in
the
Microbial
Laboratory
Guidance
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule),
available
for
download
from
http://
www.
epa.
gov/
safewater/
lt2/
index.
html.
Sample
documentation
forms
that
are
different
from
these
examples,
but
that
contain
the
minimum
required
data
elements
listed
above,
may
be
acceptable.
Section
2:
Grandfathering
Cryptosporidium
Data
13
Draft
June
2003
2.2.1.3
Supporting
Quality
Control
Information
The
data
package
should
include
a
signed
letter
from
the
laboratory
certifying
that
all
method­
required
quality
control
elements
(
including
sample
temperature
upon
receipt,
ongoing
precision
and
recovery
and
method
blank
results,
holding
times,
and
positive
and
negative
staining
controls)
were
performed
at
the
required
frequency,
and
were
acceptable
for
every
monitoring
and
MS
sample
submitted
with
the
package
(
however,
the
actual
MS
sample
results
are
not
required
to
meet
the
methods'
MS
QC
acceptance
criteria).
The
letter
should
include
a
list
of
the
applicable
monitoring
and
MS
samples,
and
the
corresponding
OPR
and
method
blank
sample
ID
for
each.

Alternately,
the
PWS
may
include
the
bench
sheet
and
Cryptosporidium
report
form
(
or
comparable
detailed
data
reporting
forms)
for
each
OPR
and
method
blank
sample
associated
with
the
field
and
MS
samples
in
the
grandfathered
data
package.
If
this
option
is
selected,
the
letter
from
the
laboratory
still
should
certify
that
sample
temperature
upon
receipt,
holding
times,
and
positive
and
negative
staining
controls
were
acceptable
for
all
samples.
(
The
letter
is
not
necessary
if
detailed
data
reporting
forms
containing
this
information
are
submitted
for
the
field
and
MS
sample
results.)

2.2.2
Schedule
for
Submission
of
Grandfathered
Data
EPA's
current
intent
is
that
PWSs
with
at
least
2
years
of
grandfathered
data
at
the
time
of
LT2
rule
promulgation
and
that
intend
to
use
these
data
in
lieu
of
monitoring
under
the
LT2
rule
(
i.
e.,
do
NOT
intend
to
conduct
additional
monitoring)
should
submit
these
data
to
EPA
within
2
months
following
LT2
rule
promulgation
(
currently
planned
for
mid­
or
late
2004).
EPA
plans
to
notify
these
PWSs
within
4
months
following
LT2
rule
promulgation
as
to
whether
their
data
are
sufficient
for
bin
classification
[
40
CFR
part
141.708
(
f)].

PWSs
with
fewer
than
2
years
of
grandfathered
data
at
the
time
of
LT2
rule
promulgation,
or
that
have
at
least
2
years
of
grandfathered
data
but
intend
to
conduct
monitoring
under
the
LT2
rule,
should
submit
these
data
to
EPA
within
8
months
of
LT2
rule
promulgation
(
which
provides
the
systems
with
2
months
to
review
data
from
the
last
potential
historical
sampling
event).
Data
collected
when
LT2
rule
monitoring
begins
(
6
months
after
promulgation)
will
be
submitted
through
the
LT2
Data
Collection
System
[
40
CFR
part
141.708
(
g)].

Under
the
Agreement,
PWSs
should
conduct
monitoring
under
the
LT2
rule
unless
notified
in
writing
by
EPA
that
they
have
2
years
of
acceptable
data.

2.2.3
Procedures
for
Submission
of
Grandfathered
Data
EPA
does
not
intend
to
formally
accept
grandfathered
Cryptosporidium
data
until
the
LT2
rule
is
finalized.
The
final
rule
will
include
procedures
for
submission
of
grandfathered
data.

2.3
Checklists
for
Grandfathering
Cryptosporidium
Data
To
help
PWSs
interested
in
monitoring
for
Cryptosporidium
before
LT2ESWTR
apply
the
information
provided
in
this
guidance,
two
checklists
have
been
developed.
The
"
Checklist
for
Beginning
Grandfathered
Cryptosporidium
Monitoring"(
Appendix
A)
is
designed
to
be
used
by
PWSs
to
check
their
monitoring
plans
against
this
guidance
document
before
proceeding
with
monitoring.
The
"
Checklist
for
Submitting
Grandfathered
Cryptosporidium
Data"
(
Appendix
B)
is
designed
to
be
used
by
PWSs
to
check
their
data
package
against
the
information
in
this
guidance
document
before
submitting
the
data
package
to
EPA
for
review.
14
Draft
June
2003
SECTION
3:
UNDERSTANDING
CRYPTOSPORIDIUM
ANALYSES
The
LT2
rule
requires
the
use
of
EPA
Method
1622
or
EPA
Method
1623
for
Cryptosporidium
monitoring
[
40
CFR
part
141.705
(
a)].
This
section
provides
utility
personnel
unfamiliar
with
Cryptosporidium
sample
analyses
with
information
on
how
the
analyses
are
performed
and
on
the
quality
control
(
QC)
measures
the
laboratory
uses
to
verify
data
quality.

3.1
Summary
of
EPA
Methods
1622
and
1623
EPA
Methods
1622
and
1623
resulted
from
an
EPA
effort
initiated
in
1996
to
identify
new
and
innovative
technologies
for
analysis
of
source
water
samples
for
Cryptosporidium
and
Giardia.
The
methods
are
identical
in
most
respects,
generally
differing
only
in
the
addition
of
Giardia
antibodies
in
EPA
Method
1623'
s
purification
and
staining
procedures.
Both
EPA
Methods
1622
and
1623
were
subjected
to
interlaboratory
validation
studies
using
various
source
waters,
and
used
in
a
national
survey
of
87
surface
water
plants
(
the
Information
Collection
Rule
Supplemental
Surveys)
to
provide
EPA
with
a
realistic
indication
of
how
the
methods
would
perform
when
they
were
used
in
the
monitoring
study
(
Reference
9.1).

Both
EPA
Methods
1622
and
1623
also
were
developed
as
"
performance­
based"
methods.
The
methods
include
quantitative
criteria
requirements
(
minimum
recovery
and
maximum
variability)
for
initial
and
ongoing
QC
samples.
These
criteria
are
used
to
verify
acceptable
laboratory
performance
using
the
version
of
the
method
originally
validated
or
to
determine
whether
a
modified
version
of
the
method
performs
acceptably.

In
EPA
Methods
1622
and
1623,
the
following
steps
are
performed:

°
Filtration.
The
sample
is
filtered
in
the
field
or
in
the
laboratory
using
one
of
the
filters
approved
for
use
with
EPA
Methods
1622
and
1623:

°
Pall
Gelman
Envirochek
 
capsule
filter
°
Pall
Gelman
Envirochek
 
HV
capsule
filter
°
IDEXX
Filta­
Max
 
foam
filter
The
oocysts,
cysts,
and
extraneous
materials
are
retained
on
the
filter.

°
Elution.
Materials
on
the
filter
are
removed
by
elution
with
an
aqueous
buffered
salt
detergent
solution.
This
elution
process
is
performed
differently
for
each
filter:
°
For
the
Pall
Gelman
Envirochek
 
and
Envirochek
 
HV
filters,
elution
is
performed
by
filling
the
capsule
with
elution
buffer,
attaching
the
filter
to
a
"
wrist
shaker"
type
lab
shaker,
and
allowing
the
filter
to
shake
for
5
minutes
at
a
time
in
three
different
orientations.
°
For
the
IDEXX
Filta­
Max
 
filter,
the
elution
technique
differs
by
laboratory.
Some
laboratories
may
add
the
foam
filter
and
elution
buffer
to
a
manual
plunger
chamber
to
expand
the
foam
filter
and
flush
any
oocysts
out
of
the
pores
in
the
foam.
Other
laboratories
may
add
the
foam
filter
rings
and
elution
buffer
to
a
stomacher
bag
and
use
a
stomacher
to
elute
the
filter.
Section
3:
Understanding
Cryptosporidium
Analyses
15
Draft
June
2003
°
For
the
Whatman
CrypTest
®
filter,
elution
is
performed
by
adding
elution
buffer
to
the
filter
housing
and
using
sonication
and
pressurized
backwashing
to
separate
oocysts
from
the
filter
fabric.

°
Concentration.
After
the
filter
is
eluted,
the
eluate
is
centrifuged
to
concentrate
the
eluted
particles
into
a
"
packed
pellet"
at
the
bottom
of
the
centrifuge
tube.
This
packed
pellet
is
measured
by
the
laboratory
analyst.
If
the
pellet
volume
is
#
2
mL
(
and
10
L
was
filtered)
the
entire
sample
must
be
analyzed.
If
the
pellet
volume
is
>
2
mL,
only
2
mL
is
required
to
be
analyzed
under
the
LT2
rule
(
although
the
utility
may
request
that
more
be
analyzed).

°
Aspiration
and
resuspension.
The
analyst
aspirates
the
supernatant
from
the
top
of
the
packed
pellet
to
minimize
the
total
sample
volume,
and
resuspends
the
pellet
material
by
vortexing
the
sample.
The
analyst
measures
the
total
resuspended
concentrate
volume.
If
the
packed
pellet
volume
was
>
2
mL,
and
the
entire
sample
volume
will
not
be
analyzed,
only
a
portion
of
the
concentrate
volume
will
be
processed
through
the
remainder
of
the
method.
By
dividing
the
concentrate
volume
processed
through
the
remainder
of
the
method
by
the
total
concentrate
volume,
the
laboratory
can
determine
what
percent
of
the
sample
volume
filtered
was
actually
analyzed.
By
multiplying
this
percentage
by
the
sample
volume
filtered,
the
laboratory
can
determine
the
volume
analyzed.

°
Purification.
Magnetic
beads
conjugated
to
anti­
Cryptosporidium
antibodies
are
added
to
the
sample
concentrate
and
allowed
to
mix
with
the
sample,
where
they
attach
themselves
to
any
oocysts
present.
The
magnetized
oocysts
are
separated
from
the
extraneous
materials
using
a
magnet,
and
the
extraneous
materials
are
discarded.
The
magnetic
bead
complex
is
then
detached
from
the
oocysts.

°
Application
of
the
purified
sample
to
a
slide.
After
immunomagnetic
separation,
the
purified
sample
is
applied
to
a
microscope
slide.

°
Drying
the
sample.
The
sample
is
dried
to
the
slide
for
several
hours
to
several
days
to
allow
the
sample
to
be
stained
and
rinsed
without
loss
of
organisms.

°
Staining
the
sample.
Two
stains
are
added
to
the
sample
before
it
is
examined
to
help
the
analyst
identify
any
Cryptosporidium
that
may
be
present.
The
oocysts
and
cysts
are
stained
on
the
slide
with
fluorescently
labeled
monoclonal
antibodies
and
4',
6­
diamidino­
2­
phenylindole
(
DAPI).

°
Examining
the
sample.
During
microscopic
examination
of
the
slide,
three
evaluation
techniques
are
required
by
EPA
Methods
1622
and
1623
to
determine
whether
an
object
is
a
Cryptosporidium
oocyst.
(
Guidance
on
interpreting
examination
results
is
provided
in
Section
7.3.)

3.2
Cryptosporidium
Laboratory
Quality
Control
As
required
by
both
EPA
Method
1622/
1623
and
the
Laboratory
QA
Program,
laboratories
approved
to
perform
Cryptosporidium
analyses
for
the
LT2
rule
must
perform
specific
quality
control
(
QC)
steps
during
sample
analyses
to
demonstrate
that
data
are
reliable
[
40
CFR
part
141.705
(
a)(
3)].
These
QC
steps
are
described
below,
in
Sections
3.2.1
­
3.2.7.

3.2.1
Initial
Precision
and
Recovery
Test
Before
performing
field
sample
analyses
using
EPA
Methods
1622
or
1623,
the
laboratory
must
demonstrate
acceptable
performance.
This
is
demonstrated
by
the
initial
precision
and
recovery
(
IPR)
test,
which
consists
of
four
reagent
water
samples
spiked
with
100
to
500
oocysts.
The
results
of
the
four
analyses
are
used
to
calculate
the
average
percent
recovery
and
the
relative
standard
deviation
(
RSD)
of
Section
3:
Understanding
Cryptosporidium
Analyses
16
Draft
June
2003
the
recoveries
for
Cryptosporidium.
For
EPA
Methods
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%.
If
more
than
one
process
will
be
used
for
filtration
and/
or
separation
of
samples,
a
separate
set
of
IPR
samples
must
be
analyzed
for
each
process.

3.2.2
Method
Blank
Test
The
method
blank
test
in
EPA
Method
1622/
1623
consists
of
analysis
of
an
unspiked
reagent
water
sample
to
demonstrate
freedom
from
contamination.
One
method
blank
sample
must
be
analyzed
each
week
or
every
20
samples,
whichever
is
more
frequent.
If
more
than
one
process
will
be
used
for
filtration
and/
or
separation
of
samples,
a
separate
method
blank
must
be
analyzed
for
each
process.
If
one
or
more
Cryptosporidium
oocysts
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.

3.2.3
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
samples,
whichever
is
more
frequent.
If
more
than
one
process
will
be
used
for
filtration
and/
or
separation
of
samples,
a
separate
OPR
sample
must
be
analyzed
for
each
process.
OPR
samples
must
be
analyzed
before
any
monitoring
samples
are
processed
for
each
batch
to
verify
acceptable
performance.
OPR
Cryptosporidium
recovery
must
be
in
the
range
of
11%
to
100%
to
be
considered
acceptable.

3.2.4
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,
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.

The
following
holding
times
must
be
met
for
samples
analyzed
by
EPA
Methods
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,
and
purification
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
fluorescence
assay
(
FA)
and
4',
6­
diamidino­
2­
phenylindole
(
DAPI)
and
differential
interference
contrast
(
DIC)
microscopy
examination
and
confirmation
should
be
performed
immediately
after
staining
is
complete,
laboratories
have
up
to
7
days
from
completion
of
sample
staining
to
complete
the
examination
and
confirmation
of
samples.
However,
if
fading/
diffusion
of
fluorescien
isothiocyanate
(
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.
Section
3:
Understanding
Cryptosporidium
Analyses
17
Draft
June
2003
3.2.5
Staining
Controls
Positive
staining
controls
entail
staining
and
examination
of
a
slide
with
positive
antigen
or
200
to
400
intact
oocysts
to
verify
that
the
stain
is
fluorescing
appropriately.
These
controls
are
prepared
with
each
batch
of
slides
that
are
stained.
Negative
staining
controls
entail
staining
and
examining
a
slide
with
phosphate
buffered
saline
solution
to
verify
that
no
oocysts
or
interfering
particulates
are
present.

3.2.6
Proficiency
Testing
Samples
As
part
of
the
Lab
QA
Program,
laboratories
must
successfully
analyze
initial
proficiency
testing
(
IPT)
samples
initially,
and
an
ongoing
proficiency
testing
(
OPT)
samples
three
times
per
year.
These
samples
and
the
Lab
QA
Program
are
discussed
in
more
detail
in
the
Microbial
Laboratory
Guidance
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).

3.2.7
Matrix
Spike
Samples
The
matrix
spike
(
MS)
test
in
EPA
Method
1622/
1623
entails
analysis
of
a
separate
sample
aliquot
spiked
with
100
to
500
oocysts
to
determine
the
effect
of
the
matrix
on
the
method's
oocyst
recovery.

One
MS
sample
must
be
analyzed
for
every
20
samples
from
your
PWS.
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.
You
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.

EPA
Method
1622/
1623
specifies
the
following
additional
requirements
for
MS
sample
analyses:

°
The
MS
sample
volume
analyzed
must
be
within
10%
of
the
volume
analyzed
for
the
associated
field
sample.

°
The
MS
sample
must
be
analyzed
in
the
same
QC
batch
as
the
field
sample,
using
the
same
method.

°
The
MS
sample
must
be
collected
as
a
split
sample
or
immediately
before
or
after
the
associated
field
sample.

Under
the
LT2
rule,
If
the
volume
of
the
MS
sample
is
greater
than
10
L,
the
system
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
Archiving
Examination
Results
Although
not
required,
laboratories
also
can
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.
An
alternative
mounting
medium
also
may
be
used,
which
may
potentially
preserve
slides
longer.
Details
are
provided
in
the
Microbial
Laboratory
Guidance
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).
18
Draft
June
2003
SECTION
4:
UNDERSTANDING
E.
COLI
ANALYSES
As
noted
in
Section
1,
E.
coli
and
turbidity
data
generated
under
the
LT2
rule
are
used
differently
for
large
systems
than
small
systems.
E.
coli
and
turbidity
are
reported
with
Cryptosporidium
data
by
large
systems
to
enable
EPA
to
determine
whether
an
E.
coli
trigger
level
can
be
established
through
the
microbial
index.
If
a
defensible
trigger
level
can
be
established
between
E.
coli
concentrations
and
Cryptosporidium
levels,
small
systems
will
be
able
to
perform
less­
expensive
E.
coli
analyses
initially
to
determine
whether
more
expensive
Cryptosporidium
monitoring
is
even
necessary.

Although
E.
coli
data
will
not
be
used
to
determine
whether
additional
treatment
is
needed
for
large
systems,
as
Cryptosporidium
data
will,
it
is
nonetheless
critical
that
the
large
systems
generate
reliable
E.
coli
data
to
establish
relevant
trigger
levels
for
use
by
the
small
systems.
The
E.
coli
data
generated
by
small
systems
will
be
used
to
determine
whether
Cryptosporidium
monitoring
is
required,
so
it
is
critical
that
these
data
be
reliable,
as
well.

This
section
provides
utility
personnel
unfamiliar
with
E.
coli
sample
analyses
with
an
overview
of
the
methods
used
under
the
LT2
rule
and
the
quality
control
(
QC)
measures
the
laboratory
uses
to
verify
data
quality.

4.1
Summary
of
LT2
Rule
E.
coli
Methods
E.
coli
sample
analyses
performed
under
the
LT2
rule
must
be
quantitative;
presence/
absence
E.
coli
results
are
unacceptable
under
LT2.
The
methods
described
below
are
approved
for
the
analysis
of
E.
coli
samples
under
the
LT2
rule
[
40
CFR
part
141.705
(
b)].

4.1.1
Most
Probable
Number
(
MPN)
Methods
4.1.1.1
Standard
Methods
9223B:
Colilert
®
and
Colilert­
18
®
Colilert
®
and
Colilert­
18
®
tests
are
chromogenic/
fluorogenic
enzyme
substrate
tests
for
the
simultaneous
determination
of
total
coliforms
and
E.
coli
in
water.
These
tests
use
commercially
available
media
containing
the
chromogenic
substrate
ortho­
nitrophenyl­$­
D­
galactopyranoside
(
ONPG),
to
detect
total
coliforms
and
the
fluorogenic
substrate
4­
methylumbelliferyl­$­
D­
glucuronide
(
MUG),
to
detect
E.
coli.
Media
formulations
are
available
in
disposable
tubes
for
the
multiple­
tube
procedure
or
packets
for
the
multiple­
well
procedure.
Appropriate
preweighed
portions
of
media
for
mixing
and
dispensing
into
multiple­
tubes
and
wells
are
also
available.
The
use
of
commercially
prepared
media
is
required
for
quality
assurance
and
uniformity.
All
tests
must
be
conducted
in
a
format
that
provides
quantitative
results
[
40
CFR
part
141.705
(
b)].

°
Multiple­
Tube.
For
the
multiple­
tube
procedure,
a
well­
mixed
sample
and/
or
sample
dilution/
volume
is
added
to
tubes
containing
predispensed
media.
Tubes
are
then
capped
and
mixed
vigorously
to
dissolve
the
media.
Alternatively,
this
procedure
can
be
performed
by
adding
appropriate
amounts
of
substrate
media
to
a
bulk
diluted
sample
(
with
appropriate
dilutions
for
enumeration),
then
mixing
and
dispensing
into
multiple­
tubes.
A
15­
tube
MPN
should
be
used
to
obtain
quantitative
results.
The
Section
4:
Understanding
E.
coli
Analyses
19
Draft
June
2003
number
of
dilutions/
volumes
are
determined
based
on
the
type,
quality,
and
character
of
the
water
sample.

°
Multiple­
Well.
A
multiple­
well
procedure
may
be
performed
with
sterilized
disposable
packets.
The
commercially
available
Quanti­
Tray
®
or
Quanti­
Tray
®
/
2000
multiple­
well
tests
use
Colilert
®
or
Colilert­
18
®
media
to
determine
E.
coli
(
IDEXX,
1999b,
c).
In
these
tests,
the
packet
containing
media
is
added
to
a
100­
mL
sample
(
or
appropriate
dilutions
for
enumeration).
The
sample
is
then
mixed
and
poured
into
the
tray.
A
tray
sealer
separates
the
sample
into
51
wells
(
Quanti­
Tray)
or
97
wells
(
Quanti­
Tray/
2000)
and
seals
the
package.

After
the
appropriate
sample
dilutions/
volumes
are
added,
the
tubes
or
trays
are
incubated
at
35
°
C
±
0.5
°
C
for
18
h
when
using
Colilert­
18
®
or
24
h
when
using
Colilert
®
.
If
the
response
is
questionable
after
the
specified
incubation
period,
the
sample
is
incubated
for
up
to
an
additional
4
h
at
35
°
C
±
0.5
°
C
for
both
Colilert
®
tests.
Each
tube
or
well
is
then
compared
to
the
reference
color
"
comparator"
provided
with
the
media.
A
yellow
color
greater
or
equal
to
the
comparator
indicates
the
presence
of
total
coliforms
in
the
sample,
and
the
tube
or
well
is
then
checked
for
fluorescence
under
longwavelength
UV
light
(
365­
nm).
The
presence
of
fluorescence
greater
than
or
equal
to
the
comparator
is
a
positive
test
for
E.
coli.
If
water
samples
contain
humic
acid
or
colored
substances,
inoculated
tubes
or
wells
should
also
be
compared
to
a
sample
water
blank
without
Colilert
®
reagent
added.
The
concentration
in
MPN/
100
mL
is
then
calculated
from
the
number
of
positive
tubes
or
wells
using
MPN
tables
provided
by
the
manufacturer.

4.1.1.2
Standard
Methods
9221B/
9221F:
LTB
6EC­
MUG
The
multiple­
tube
fermentation
method
for
enumerating
E.
coli
in
water
uses
multiple­
tubes
and
dilutions/
volumes
in
a
two­
step
procedure
to
determine
E.
coli
concentrations.
In
the
first
step,
or
"
presumptive
phase,"
a
series
of
tubes
containing
lauryl
tryptose
broth
(
LTB)
are
inoculated
with
undiluted
samples
and/
or
dilutions/
volumes
of
the
samples
and
mixed.
Inoculated
tubes
are
incubated
for
24
±
2
h
at
35
°
C
±
0.5
°
C.
Each
tube
then
is
swirled
gently
and
examined
for
growth
(
i.
e.,
turbidity)
and
production
of
gas
in
the
inner
Durham
tube.
If
there
is
no
growth,
acid,
or
gas,
tubes
are
re­
incubated
for
24
±
2
h
at
35
°
C
±
0.5
°
C
and
re­
examined.
Production
of
growth
and
gas
within
48
±
3
h
constitutes
a
positive
presumptive
test
for
coliforms,
which
include
E.
coli.

After
enrichment
in
the
presumptive
medium,
positive
tubes
are
subjected
to
a
second
step
for
enumeration
of
E.
coli.
Presumptive
tubes
are
agitated,
and
growth
is
transferred
using
a
sterile
loop
or
applicator
stick
to
tubes
containing
EC
broth
supplemented
with
4­
methylumbelliferyl­$­
D­
glucuronide
(
MUG).
Inoculated
tubes
are
incubated
at
44.5
°
C
±
0.2
°
C
for
24
±
2
h
in
a
water
bath.
All
tubes
exhibiting
growth
and
gas
production
are
examined
for
bright
blue
fluorescence
under
long­
wavelength
UV
light
(
366­
nm)
indicating
a
positive
test
for
E.
coli.
The
density
of
E.
coli
in
MPN/
100
mL
is
then
calculated
from
the
number
of
positive
EC­
MUG
tubes,
using
MPN
tables
or
formulas.
A
15­
tube
MPN
is
required
under
the
LT2
Rule.

4.1.2
Membrane
Filtration
(
MF)
Methods
4.1.2.1
Standard
Methods
9222B/
9222G:
mEndo/
LES­
Endo6NA­
MUG
and
Standard
Methods
9222D/
9222G:
mFC6NA­
MUG
These
membrane
filter
methods
for
enumerating
E.
coli
are
two­
step
incubation
procedures.
First,
a
sample
is
filtered
through
a
0.45
:
m
filter,
then
the
filter
is
placed
on
a
pad
saturated
with
mEndo
broth
or
a
plate
containing
mEndo
or
LES­
Endo
agar
and
incubated
for
24
±
2
h
at
35
°
C
±
0.5
°
C.
Pink
to
red
colonies
with
a
metallic
(
golden­
green)
sheen
on
the
filter
are
considered
to
be
total
coliforms.
If
initial
determination
of
fecal
coliforms
is
desired,
mFC
media
can
be
substituted
for
mEndo/
LES­
Endo.
Following
initial
isolation
of
total
coliforms
(
or
fecal
coliforms),
the
filter
is
transferred
to
nutrient
agar
Section
4:
Understanding
E.
coli
Analyses
20
Draft
June
2003
containing
4­
methylumbelliferyl­$­
D­
glucuronide
(
NA­
MUG)
and
incubated
for
4
h
at
35
°
C
±
0.5
°
C.
Sheen
colonies
on
mEndo
or
blue
colonies
on
mFC
that
fluoresce
under
a
long­
wavelength
UV
light
(
366­
nm)
are
positive
for
E.
coli.
If
high
levels
of
non­
E.
coli
total
coliforms
interfere
with
the
ability
to
accurately
enumerate
E.
coli
despite
additional
dilutions,
transfer
from
mFC
or
an
alternate
method
(
e.
g.,
SM
9213D,
EPA
Method
1603)
should
be
used.

4.1.2.2
Standard
Methods
9213D:
mTEC
The
mTEC
agar
method
is
a
two­
step
procedure
that
provides
a
direct
count
of
E.
coli
in
water,
based
on
the
development
of
colonies
on
the
surface
of
a
membrane
filter
when
placed
on
a
selective
nutrient
and
substrate
media.
This
method
originally
was
developed
by
EPA
to
monitor
the
quality
of
recreational
water.
This
method
was
also
used
in
health
studies
to
develop
the
bacteriological
ambient
water
quality
criteria
for
E.
coli.
In
this
method,
a
water
sample
is
filtered
through
a
0.45:
m
membrane
filter,
the
filter
is
placed
on
mTEC
agar
(
a
selective
primary
isolation
medium),
and
the
plate
is
incubated
first
at
35
°
C
±
0.5
°
C
for
2
h
to
resuscitate
injured
or
stressed
bacteria
and
then
at
44.5
°
C
±
0.2
°
C
for
22­
24
h
in
a
water
bath.
Following
incubation,
the
filter
is
transferred
to
a
filter
pad
saturated
with
urea
substrate
medium.
After
15
minutes,
all
yellow
or
yellow­
brown
colonies
(
occasionally
yellow­
green)
are
counted
as
positive
for
E.
coli
using
a
fluorescent
lamp
and
either
a
magnifying
lens
or
a
stereoscopic
microscope.

4.1.2.3
EPA
Method
1603:
Modified
mTEC
The
modified
mTEC
agar
method
is
a
single­
step
MF
procedure
that
provides
a
direct
count
of
E.
coli
in
water
based
on
the
development
of
colonies
on
the
surface
of
a
filter
when
placed
on
selective
modified
mTEC
media.
This
is
a
modification
of
the
standard
mTEC
media
that
eliminates
bromcresol
purple
and
bromphenol
red
from
the
medium,
adds
the
chromogen
5­
bromo­
6­
chloro­
3­
indolyl­$­
D­
glucuronide
(
Magenta
Gluc),
and
eliminates
the
transfer
of
the
filter
to
a
second
substrate
medium.
In
this
method,
a
water
sample
is
filtered
through
a
0.45:
m
membrane
filter,
the
filter
is
placed
on
modified
mTEC
agar,
incubated
at
35
°
C
±
0.5
°
C
for
2
h
to
resuscitate
injured
or
stressed
bacteria,
and
then
incubated
for
22­
24
h
in
a
44.5
°
C
±
0.2
°
C
water
bath.
Following
incubation,
all
red
or
magenta
colonies
are
counted
as
E.
coli.

4.1.2.4
EPA
Method
1604:
MI
Medium
The
MI
medium
method
is
a
single­
step
membrane
filtration
procedure
used
to
simultaneously
enumerate
total
coliforms
and
E.
coli.
In
this
EPA­
developed
method,
a
water
sample
is
filtered
through
a
0.45­:
m
membrane
filter,
the
filter
is
placed
on
an
MI
agar
or
broth
plate,
and
the
medium
is
incubated
at
35
°
C
±
0.5
°
C
for
24
h.
If
high
levels
of
non­
E.
coli
total
coliforms
interfere
with
the
ability
to
accurately
enumerate
E.
coli
despite
additional
dilutions,
an
alternate
method
(
e.
g.,
SM
9213D,
EPA
Method
1603)
should
be
used.

E.
coli
colonies
exhibit
a
blue
color
and
also
may
fluoresce
under
a
long­
wavelength
UV
light
(
366­
nm).
If
desired,
the
plates
can
also
be
observed
under
long­
wavelength
UV
light
(
366­
nm)
for
the
presence
of
total
coliform
species
that
fluoresce.
Because
the
blue
color
from
the
breakdown
of
indoxyl­$­
Dglucuronide
(
IBDG)
can
mask
fluorescence,
non­
fluorescent
blue
colonies
are
included
in
the
total
coliform
count.
Water
samples
with
high
turbidity
can
clog
the
membrane
filter,
interfering
with
filtration
and
potentially
interfering
with
the
identification
of
target
colonies.
Section
4:
Understanding
E.
coli
Analyses
21
Draft
June
2003
4.1.2.5
m­
ColiBlue24
®
Broth
This
broth
method
is
a
single­
step
MF
test
for
enumerating
total
coliforms
and
E.
coli.
As
with
NA­
MUG,
modified
mTEC,
and
MI
media,
the
selective
identification
of
E.
coli
is
based
on
the
detection
of
the
$­
glucuronidase
enzyme.
The
test
medium
includes
the
chromogen
5­
bromo­
4­
chloro­
3­
indoxyl­$­
Dglucuronide
(
BCIG
or
X­
Gluc).
The
chromogen
BCIG
is
hydrolyzed
by
$­
glucuronidase,
releasing
an
insoluble
indoxyl
salt
that
causes
the
colonies
to
exhibit
a
blue
color.
M­
ColiBlue24
®
broth
is
a
commercially
available
format
of
this
method
and
contains
a
nutritive
lactose­
based
medium
containing
inhibitors
to
eliminate
the
growth
of
non­
coliforms.
With
m­
ColiBlue24
®
broth,
a
water
sample
is
filtered
through
a
0.45:
m
membrane
filter,
and
the
filter
is
transferred
to
a
plate
containing
an
absorbent
pad
saturated
with
m­
ColiBlue24
®
broth.
The
filter
is
incubated
at
35
°
C
±
0.5
°
C
for
24
h
and
examined
for
colony
growth.
The
presence
of
E.
coli
is
indicated
by
blue
colonies.
The
presence
of
total
coliforms
(
non­
E.
coli)
is
indicated
by
red
colonies.
If
enumeration
of
total
coliforms
is
desired,
blue
and
red
colonies
should
be
included
in
the
total
coliform
count.
If
high
levels
of
non­
E.
coli
total
coliforms
interfere
with
the
ability
to
accurately
enumerate
E.
coli
despite
additional
dilutions,
an
alternate
method
(
e.
g.,
SM
9213D,
EPA
Method
1603)
should
be
used.

4.2
E.
coli
Laboratory
Quality
Control
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
described
above.
Sections
4.2.1
­
4.2.7
describe
quality
control
specifications
for
E.
coli
analyses
performed
under
the
LT2
rule.
This
guidance
is
provided
to
help
summarize
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.
Additional
information
on
the
QC
specifications
is
available
in
Section
4.2
of
the
Microbial
Laboratory
Guidance
Manual
for
the
Long­
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).

4.2.1
Dilution/
Rinse
Water
Sterility
Check
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.
The
dilution/
rinse
water
batch
should
be
discarded
if
growth
is
detected.

4.2.2
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.

4.2.3
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.
Laboratories
using
commerciallyprepared
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.
Positive
and
negative
controls
should
be
chosen
based
on
the
method­
specific
requirements.
For
example
if
a
44.5
°
C
water
bath
is
not
required
by
the
method,
it
is
not
necessary
to
include
Enterobacter
aerogenes
as
a
negative
control.
Section
4:
Understanding
E.
coli
Analyses
22
Draft
June
2003
4.2.4
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
4.2.5
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.

4.2.6
Preparation
Blanks
Preparation
blanks
should
be
analyzed
to
detect
potential
contamination
of
dilution/
rinse
water
during
the
course
of
analyses.

4.2.6.1
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.

4.2.6.2
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.

4.2.7
Verification
Verification
specifications
are
detailed
in
the
Certification
Manual
(
Reference
9.3),
Standard
Methods
(
Reference
9.4),
and
Appendices
J
through
L
of
the
Microbial
Laboratory
Guidance
Manual
for
the
Long­
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).
23
Draft
June
2003
SECTION
5:
CONTRACTING
FOR
CRYPTOSPORIDIUM
LABORATORY
SERVICES
Although
many
public
water
systems
(
PWSs)
have
established
procedures
and
policies
governing
the
purchase
of
services
and
supplies,
these
procedures
seldom
lend
themselves
to
the
purchase
of
analytical
services.
This
section
provides
a
basic
framework
for
defining
the
technical
and
contractual
requirements
associated
with
purchasing
laboratory
services
for
Cryptosporidium
analyses
for
the
LT2
rule,
awarding
contracts,
and
working
with
a
contract
laboratory.

Successfully
contracting
for
Cryptosporidium
laboratory
services
for
LT2
rule
monitoring
relies
on
the
following
steps:

Step
1:
Define
the
scope
of
your
analytical
requirements
to
develop
a
detailed
contract
and
standardized
bid
sheet
Step
3:
Solicit
qualified
laboratories
Step
4:
Award
contracts
to
a
primary
laboratory
and
a
backup
laboratory
Step
5:
Work
closely
with
your
laboratory
before
monitoring
begins
and
maintain
communications
throughout
monitoring
Each
of
these
general
steps,
and
details
on
the
activities
associated
with
each,
are
discussed
in
Sections
5.1
through
5.5.

5.1
Defining
Your
Needs
and
Developing
a
Contract
The
first
step
in
developing
an
analytical
services
contract
for
Cryptosporidium
analyses
for
LT2
rule
monitoring
is
identifying
the
"
who,"
"
what,"
"
when,"
and
"
how"
of
the
project
for
your
system
(
the
"
why"
is
the
LT2
rule
itself).
A
well­
written
contract
will
address
each
of
these
issues,
as
well
as
the
administrative
issues,
such
as
laboratory
payments
and
adjustments.

The
best
way
to
ensure
that
you
get
the
data
you
need
for
LT2
rule
Cryptosporidium
monitoring
within
the
required
time
period
is
to
specify
your
requirements
in
detail
in
the
contract.
A
well­
written
contract
can
minimize
or
eliminate
many
common
problems
in
procuring
analytical
services,
and
enable
you
to
collect
reliable
and
timely
results.

Recommendations
on
the
factors
to
consider
in
defining
the
scope
of
the
services
you
need,
and
the
information
you
should
be
sure
to
include
in
your
contract
are
provided
below.
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
24
Draft
June
2003
L
Clearly
identify
in
your
contract
the
name
and
identification
number
of
your
PWS,
as
well
as
the
name(
s)
and
identification
number
of
the
facility(
ies)
for
which
samples
need
to
be
analyzed.
This
information
ultimately
will
be
used
to
identify
your
samples
in
the
LT2
Data
Collection
System,
and
the
laboratory
you
use
for
Cryptosporidium
sample
analyses
will
need
to
know
this
information.
(
Alternately,
you
can
provide
this
information
after
award
to
the
awarded
laboratory
only.)

L
Clearly
indicate
in
your
contract
the
total
number
of:
(
1)
field
samples
and
(
2)
MS
samples
that
the
laboratory
will
be
required
to
analyze.
Add
two
additional,
optional,
sample
analyses
to
be
exercised
if
"
make­
up"
samples
are
required
due
to
problems
unrelated
to
laboratory
performance.
5.1.1
Client
Information
"
Who"
defines
your
PWS
to
the
laboratories
that
you
would
like
to
submit
bids
for
the
project.
Will
you
be
contracting
for
laboratory
services
for
a
single
plant
or
will
this
contract
require
Cryptosporidium
analyses
to
fulfill
monitoring
requirements
for
multiple
plants
in
a
system?

5.1.2
Sample
Information
"
What"
describes
the
samples
to
be
analyzed.
As
noted
in
Sections
5.1.2.1
through
5.1.2.5,
this
encompasses
a
variety
of
factors,
each
of
which
needs
to
be
evaluated
and
defined
before
you
develop
your
contract.

5.1.2.1
Number
of
Samples
What
is
the
total
number
of
samples
the
laboratory
will
need
to
analyze?
This
total
includes
not
only
routine
monitoring
samples
(
field
samples),
but
also
the
matrix
spike
(
MS)
samples
(
Section
3.2.7)
that
are
required
at
a
frequency
of
1
per
20
field
samples.
Field
samples
and
MS
samples
are
considered
"
billable"
samples
(
sample
analyses
for
which
the
laboratory
will
be
paid
their
per­
sample
cost).
Internal
laboratory
quality
control
(
QC)
samples,
such
as
method
blanks
and
ongoing
precision
and
recovery
(
OPR)
samples
should
be
considered
"
unbillable"
samples
 
sample
analyses
that
are
required,
but
apply
to
multiple
PWS
clients.
Rather
than
charging
clients
for
these
samples
directly,
laboratories
typically
will
amortize
the
costs
of
these
samples
across
billable
samples.

If
a
sample
is
collected
and
sent
to
the
laboratory,
but
cannot
be
submitted
under
the
LT2
rule
because
of
a
problem
unrelated
to
laboratory
performance
(
such
as
shipping
delays
that
violate
the
sample
holding
time),
your
PWS
will
be
required
to
collect
a
"
make­
up"
sample
(
see
Section
6.3
for
details).
You
should
add,
as
an
option
to
be
exercised
at
your
direction
in
such
an
event,
two
additional
sample
analyses
to
the
total.

5.1.2.2
Type
of
Samples
Will
your
PWS
collect
and
ship
bulk
water
samples
to
the
laboratory
for
filtration
and
processing
or
will
your
PWS
filter
samples
on­
site
and
ship
the
filter
to
the
laboratory?
Shipping
and
analytical
costs
are
likely
to
be
lower
if
you
filter
your
samples
on­
site,
but
you
will
need
to
purchase
or
rent
sample
filtration
equipment
(
see
Section
6.4
for
details)
and
have
staff
trained
to
use
the
required
procedures
or
pay
for
the
laboratory
or
another
firm
to
perform
these
tasks.
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
25
Draft
June
2003
L
Clearly
specify
in
the
SOW
whether
the
laboratory
will
receive
bulk
water
samples
or
filtered
samples.
If
filtered
samples
will
be
sent,
indicate
which
filter
you
will
use
(
see
Section
5.1.4.2).

L
If
your
PWS
will
be
purchasing
filters
directly,
specify
this
in
the
contract,
so
the
laboratory
knows
not
to
include
this
in
their
per­
sample
price.

L
Clearly
indicate
in
your
contract
the
volume
you
anticipate
collecting
for
each
sample.

L
Clearly
indicate
in
your
contract
that
different
sample
prices
are
needed
for:
(
1)
full
sample
analyses,
(
2)
subsample
analyses,
and
(
3)
extra
filters
and
the
cost
of
analysis
of
the
extra
filters.
If
you
will
be
filtering
on­
site,
and
will
be
using
your
own
equipment
to
filter
the
samples,
you
should
consider
purchasing
filters
directly
from
the
vendor,
rather
than
from
the
laboratory,
to
reduce
costs.
(
Additional
information
on
filtering
samples
on­
site
and
purchasing
filters
is
provided
in
Section
6.4.2).

5.1.2.3
Anticipated
Sample
Volume
The
LT2
rule
will
require
that
at
least
10
L
be
analyzed
for
each
sample
(
with
some
exceptions
­
see
Section
6.1)
[
40
CFR
part
141.705
(
a)(
1)].
Will
your
PWS
collect
10­
L
samples
or
collect
higher­
volume
samples,
such
as
50­
L
samples?
If
your
PWS
will
be
shipping
bulk
water
samples
to
the
laboratory,
greater
sample
volumes
will
result
in
higher
shipping
costs
and
will
likely
result
in
higher
analytical
costs.
If
your
PWS
will
be
filtering
samples
on­
site,
and
shipping
filters
to
the
laboratory,
the
sample
volume
should
not
affect
shipping
or
analytical
costs,
but
the
greater
sample
volumes
filtered
may
result
in
higher
packed
pellet
volume
and
multiple
subsamples
(
Section
5.1.2.4).

5.1.2.4
Subsamples
and
Filter
Clogs
As
noted
in
Section
3.1,
additional
steps
are
required
at
the
laboratory
for
samples
that
generate
a
larger
packed
pellet
volume
than
can
be
processed
as
one
sample
through
the
method's
purification
step.
Specifically,
the
laboratory
will
need
to
process
the
packed
pellet
from
the
sample
as
two
or
more
"
subsamples"
through
the
remainder
of
the
method
(
purification,
staining,
and
examination)
to
meet
LT2
rule
sample
volume
analysis
requirements.
If
a
sample
clogs
before
10
L
have
been
filtered,
at
least
two
filters
must
be
used
to
meet
LT2
rule
sample
volume
analysis
requirements
[
40
CFR
part
141.705
(
a)(
1)].

If
the
source
water(
s)
to
be
monitored
by
your
PWS
are
characterized
by
high
turbidity,
some
of
your
samples
may
need
to
be
processed
as
multiple
subsamples
or
may
require
two
filters
to
enable
you
to
meet
LT2
rule
monitoring
requirements.
Even
if
your
source
water(
s)
typically
is
characterized
by
low
turbidity,
you
should
allow
for
the
possibility
that
some
samples
may
result
in
larger
packed
pellet
volumes
on
occasion.
By
including
this
in
the
original
contract,
you
will
avoid
changes
to
the
contract
on
short
notice
if
subsamples
are
required
during
monitoring.
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
26
Draft
June
2003
L
Clearly
specify
in
your
contract
any
services
required
in
addition
to
routine
sample
analysis.

L
Indicate
in
your
contract
the
month
that
you
plan
to
begin
monitoring
and
whether
you
will
be
monitoring
on
a
monthly
or
more
frequent
basis.
If
possible,
do
not
specify
actual
sample
collection
dates
and
days
during
the
week;
work
with
the
awarded
laboratory
to
establish
a
schedule
that
meets
your
needs
and
does
not
cause
problems
for
the
laboratory.
5.1.2.5
Extra
Services
Will
any
additional
services
be
required
of
the
laboratory
outside
of
actual
sample
analyses?
Possible
services
include:

°
Sampling
kit
rental
for
on­
site
filtration
°
Sample
shipping
containers
°
Sample
archiving
(
laboratories
can
archive
slides
and
some
can
take
photographs
of
slides
to
maintain
for
clients)

Some
of
these
services
may
be
included
in
the
sample
analysis
cost
by
some
laboratories.
Defining
the
specific
services
your
PWS
will
need,
and
specifying
these
services
clearly
in
the
contract
will
enable
the
laboratories
to
better
assess
whether
the
requested
services
are
included
in
their
routine
costs
or
are
extra,
and
respond
accordingly.

5.1.3
Sampling
Schedules
"
When"
refers
to
your
anticipated
schedule
for
shipping
samples
to
the
laboratory.
Will
your
PWS
begin
monitoring
before
implementation
of
the
LT2
rule
with
the
intent
to
grandfather
some
or
all
of
the
data
or
will
your
PWS
monitor
according
to
the
rule
schedule?

The
minimum
monitoring
frequency
for
the
LT2
rule
is
once
per
month
[
40
CFR
part
141.701
(
e)].
During
LT2
monitoring,
will
your
PWS
collect
and
ship
samples
once
per
month,
or
will
you
monitor
more
often?

If
at
all
possible,
do
not
establish
a
firm
sampling
schedule
with
specific
dates
at
this
point.
Most
of
the
laboratories
available
to
perform
Cryptosporidium
analyses
have
multiple
PWS
clients
and
need
to
evenly
distribute
their
sample
load
within
each
week
and
across
weeks
in
a
month
to
meet
holding
time
requirements.
Rather
than
dictating
a
sample
collection
schedule
to
the
laboratory
 
and
potentially
discouraging
laboratories
from
bidding
on
the
work
or
risk
violating
holding
times
during
monitoring
 
work
with
the
awarded
laboratory
to
establish
a
schedule
that
is
will
comply
with
LT2
rule
requirements
and
is
mutually
acceptable
to
your
PWS
and
the
laboratory.

5.1.4
Analytical
Methodology
"
How"
describes
the
analytical
method
that
the
laboratory
will
use.
This
involves
two
sets
of
options:
which
method
to
use
(
EPA
Method
1622
or
EPA
Method
1623)
and
which
filter
to
use,
regardless
of
method.
It
also
refers
to
the
QC
requirements
that
must
be
met
during
sample
processing
and
analysis.
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
27
Draft
June
2003
L
If
your
PWS
has
experience
with
Cryptosporidium
sampling
and
would
like
analyses
performed
using
a
specific
filter,
clearly
indicate
this
in
the
contract.
Otherwise,
do
not
specify
a
filter
type.
5.1.4.1
EPA
Method
1622
Versus
EPA
Method
1623
Will
your
PWS
monitor
for
Cryptosporidium
only
or
Cryptosporidium
and
Giardia?
Most
laboratories
analyze
samples
for
both
Cryptosporidium
and
Giardia
using
EPA
Method
1623.
If
EPA
Method
1623
is
used
by
the
laboratory
to
analyze
your
LT2
rule
samples,
only
Cryptosporidium
data
need
to
be
submitted.
If
Giardia
data
are
collected,
they
do
not
need
to
be
submitted
to
EPA.

Your
contract
should
specify
that
EPA
Method
1622
be
used
only
if
you
are
interested
in
monitoring
for
Cryptosporidium
only
(
this
method
only
detects
Cryptosporidium).
Although
reagent
costs
for
this
method
are
slightly
less
than
for
EPA
Method
1623,
actual
sample
analysis
costs
may
not
be
lower
because
laboratories
may
not
be
able
to
allocate
the
QC
sample
costs
for
this
method
across
as
many
clients.

5.1.4.2
Filter
Options
Although
EPA
validated
EPA
Method
1622
and
EPA
Method
1623
using
one
filter
type,
modified
versions
of
the
methods
using
alternate
filter
options
have
been
approved
by
EPA
since
validation.
The
following
available
filters
are
considered
acceptable
by
EPA
for
use
with
EPA
Methods
1622
and
1623:

°
Original
Pall
Gelman
Envirochek
 
capsule
filter
°
IDEXX
Filta­
Max
 
foam
filter
°
Pall
Gelman
Envirochek
 
HV
capsule
filter
Unless
your
PWS
has
experience
with
Cryptosporidium
sampling,
and
a
basis
for
requesting
a
specific
filter
type,
you
should
indicate
in
the
contract
that
all
are
acceptable.

If
your
PWS
has
experience
monitoring
for
Cryptosporidium
and
has
a
filter
preference,
you
will
need
to
indicate
this
to
the
laboratories
interested
in
bidding
on
the
project,
as
not
all
laboratories
are
approved
by
EPA
through
the
Lab
QA
Program
to
perform
all
versions
of
the
methods.

5.1.4.3
Quality
Control
Requirements
Although
EPA
Methods
1622
and
1623
specify
the
QC
requirements
that
must
be
met
during
performance
of
the
method,
your
contract
should
reiterate
that
the
following
QC
tests
must
be
performed
at
the
required
frequency
during
processing
and
analysis
of
your
samples:

°
Method
blank
test
(
Section
3.2.2)

°
Ongoing
precision
and
recovery
(
OPR)
test
(
Section
3.2.3)

°
Holding
time
requirements
(
Section
3.2.4)

°
Staining
controls
(
Section
3.2.5)

None
of
these
QC
measures
should
be
billable,
however.
As
noted
above,
in
Section
5.1.2.1,
the
costs
for
the
method
blank,
OPR,
and
staining
control
tests
should
be
amortized
by
the
laboratory
across
the
cost
of
monitoring
samples
for
all
of
their
clients.
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
28
Draft
June
2003
L
Reiterate
in
the
contract
that
method
blanks,
OPRs,
and
staining
controls
must
be
performed
at
the
frequency
required
in
the
method,
and
that
all
holding
times
must
be
met.

L
Clearly
indicate
in
your
contract
that
the
laboratory
is
required
to
enter
Cryptosporidium
monitoring
results
for
your
samples
into
the
LT2
Data
Collection
System.
5.1.5
Data
Deliverables
and
Other
Contract
Issues
In
addition
to
the
"
who,"
"
what,"
"
when,"
and
"
how"
questions
that
need
to
be
addressed
by
the
contract,
you
also
will
need
to
provide
details
on
data
delivery,
adjustments
for
lateness,
and
sample
reanalysis
cost
issues.
These
issues
are
discussed
in
Sections
5.1.5.1
through
5.1.5.5.

5.1.5.1
Data
Submission
EPA
has
developed
the
web­
based
LT2
Data
Collection
System
to
allow
laboratories
to
report
data
to
PWSs
electronically
and
allow
PWSs
to
verify
the
data
electronically
before
submitting
the
monitoring
results
to
EPA.
This
reporting
process
is
summarized
in
Section
7.2
for
Cryptosporidium
data,
and
discussed
in
detail
in
the
Users'
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule)
Data
Collection
System.
The
laboratory,
at
a
minimum,
will
need
to
submit
the
results
for
each
Cryptosporidium
monitoring
sample
to
you
electronically.
(
Although
your
PWS
also
could
enter
these
data,
based
on
hardcopy
results
from
the
laboratory,
this
is
strongly
discouraged,
as
the
potential
for
error
increases
when
personnel
unfamiliar
with
the
generation
of
the
data
for
a
sample
enter
these
data
into
the
LT2
Data
Collection
System.)

5.1.5.2
Hardcopy
Data
Deliverables
Note:
If
you
do
not
intend
to
review
all
of
the
raw
data
generated
by
the
laboratory,
this
section
is
not
relevant,
and
can
be
ignored.
If
your
PWS
does
intend
to
review
all
of
the
raw
data
associated
with
your
LT2
samples
(
discussed
in
Section
7),
you
should
request
copies
of
the
forms
used
by
the
laboratory
to
record
sample
measurements,
sample
processing
times,
and
sample
examination
results,
as
well
as
information
on
the
QC
samples
associated
with
your
monitoring
sample.
(
If
your
PWS
will
store
and
maintain
all
sample
results,
rather
than
the
laboratory,
then
the
original
forms
should
be
requested.)

Suggestions
for
the
materials
that
should
be
requested
include
the
following:

°
Sample
result
summary
sheet,
which
should
include
the
following:

°
Monitoring
sample
identification
information
°
Monitoring
sample
result,
in
oocysts/
L
°
Laboratory
quality
control
batch
associated
with
the
sample
°
ID
number
and
result
for
the
ongoing
precision
and
recovery
(
OPR)
sample
analyzed
for
this
QC
batch
°
ID
number
and
result
for
the
method
blank
sample
analyzed
for
this
QC
batch
°
LT2
sample
collection
form
initiated
by
your
utility
and
completed
with
sample
receipt
information
by
the
laboratory
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
29
Draft
June
2003
L
If
you
need
the
laboratory
to
submit
hardcopy
results
(
this
is
not
necessary,
unless
you
intend
to
review
all
of
the
raw
data),
clearly
indicate
in
your
contract
the
materials
that
are
required.
°
Method
1622/
1623
Bench
Sheet
with
raw
data
associated
with
the
monitoring
sample
(
and
MS
sample,
if
applicable)

°
Method
1622/
1623
Cryptosporidium
Slide
Examination
Form
with
raw
data
for
the
monitoring
sample
(
and
MS
sample,
if
applicable)

°
Laboratory
comments.
If
the
laboratory
provided
comments
on
the
sample
analyses
or
results
that
require
follow­
up,
contact
the
laboratory
to
discuss,
if
necessary.
Comments
may
include
any
applicable
data
qualifiers.
The
following
is
a
list
of
potential
data
qualifiers:

°
The
recovery
for
the
associated
ongoing
precision
and
recovery
(
OPR)
sample
did
not
meet
method
requirements
°
Oocysts
were
detected
in
the
method
blank
°
Positive
and
negative
staining
controls
were
not
acceptable
or
not
examined
°
Method
holding
times
were
not
met
°
Sample
arrived
at
the
laboratory
in
unacceptable
condition
5.1.5.3
Data
Turnaround
Requirements
Under
the
LT2
rule,
PWSs
are
required
to
submit
data
no
later
than
10
days
after
the
end
of
the
first
month
following
the
month
when
the
sample
is
collected
(
approximately
40
to
70
days
after
sample
collection,
depending
on
when
during
the
month
the
sample
is
collected
)
[
40
CFR
part
141.707
(
d)].
For
example,
if
a
sample
is
collected
on
March
17,
data
must
be
submitted
by
May
10.

The
required
data
turnaround
must
be
stated
clearly
in
the
contract.
This
turnaround
time
should
be
expressed
in
calendar
days
(
not
working
days),
and
should
start
from
the
sample
collection
date.
The
data
turnaround
time
calculations
should
consider
the
day
that
the
sample
is
collected
"
day
zero,"
and
the
following
day
as
"
day
one."
(
Data
turnaround
times
in
analytical
contracts
typically
start
from
the
receipt
of
the
sample
at
the
laboratory,
but
calculating
it
from
the
sample
collection
date
is
more
logical
in
this
case
because
the
LT2
rule's
data
submission
requirements
are
based
on
sample
collection
date.)

If
the
data
turnaround
time
starts
from
sample
collection,
rather
than
sample
receipt
by
the
laboratory,
this
turnaround
should
accommodate
the
potential
for
shipping
delays
that
will
be
outside
of
the
laboratory's
control.
As
a
general
rule,
the
data
turnaround
time
should
not
be
shorter
than
the
sum
of
the
maximum
holding
times
in
the
method
 
15
days.
This
includes
up
to
4
days
between
sample
collection
and
initiation
of
the
elution
step,
which
effectively
is
the
maximum
time
for
any
shipping
delay,
as
samples
received
more
than
4
days
after
collection
will
not
be
valid,
and
cannot
be
submitted
through
the
LT2
Data
Collection
System.

Using
the
15
days
allowed
for
sample
analysis
by
the
methods
(
plus
additional
time
to
compile
the
data
package
and
mail
the
results)
as
the
shortest
realistic
turnaround
time,
determine
when
you
will
actually
need
the
results.
The
same
turnaround
time
can
be
specified
for
both
submission
of
electronic
data
and
receipt
of
hardcopy
materials.
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
30
Draft
June
2003
L
Specify
in
the
contract
the
data
turnaround
requirement
for
electronic
and
hardcopy
submission
of
data.
This
turnaround
time
should
be
calculated
as
the
time
between
sample
collection
and
receipt
of
the
hardcopy
data
by
your
PWS.

L
Clearly
indicate
in
your
contract
whether
liquidated
damages
will
be
applied
to
late
data
or
other
problems,
how
these
liquidated
damages
are
calculated,
and
the
limits
and
conditions
associated
with
the
damages.
Do
not
specify
a
data
turnaround
time
shorter
than
you
really
need,
as
it
may
increase
the
per­
sample
price
quoted
by
the
laboratories.
This
turnaround
time
should
be
short
enough
to
provide
time
to
carefully
evaluate
the
results
before
they
must
be
submitted
to
EPA,
but
long
enough
that
it
does
not
unreasonably
burden
the
laboratory
and
potentially
increase
the
per­
sample
quotes
you
receive
when
you
solicit
the
project.

5.1.5.4
Liquidated
Damages
and
Penalties
You
should
consider
including
penalty
or
damage
clauses
in
your
contracts
as
incentives
to
preclude
laboratories
from
submitting
data
late
or
performing
analyses
improperly.
Due
to
the
nature
of
the
services
provided,
it
is
often
difficult
to
assess
actual
damages
caused
by
improperly
performed
analyses.
Liquidated
damages
often
are
used
in
analytical
services
contracts
in
lieu
of
actual
damages.
Liquidated
damages
typically
specify
that,
if
the
laboratory
fails
to
deliver
the
data
specified
in
the
deliverables
section
of
the
contract,
or
fails
to
perform
the
services
within
the
specified
data
turnaround
time,
the
laboratory
will
pay
a
fixed,
agreed,
price
to
compensate
the
organization
to
whom
the
services
should
have
been
delivered.
For
example,
some
EPA
contracts
for
analytical
services
specify
that
the
laboratory
will
pay,
as
fixed,
agreed,
and
liquidated
damages,
2%
of
the
analysis
price
per
calendar
day
of
delay,
to
a
maximum
reduction
of
50%
of
the
analysis
price.

If
liquidated
damages
or
penalties
are
involved,
they
should
(
1)
be
based
on
actual
damage
caused
(
in
terms
of
cost)
by
each
day
of
lateness,
(
2)
be
strong
enough
to
discourage
late
delivery,
and
(
3)
be
reasonable
enough
that
they
will
not
discourage
laboratories
from
bidding.
If
liquidated
damages
or
penalties
will
be
applied
to
meet
the
required
data
turnaround
time,
this
information
should
be
included.
The
contract
should
specify
that
the
laboratory
will
not
be
charged
with
liquidated
damages
when
the
delay
in
delivery
or
performance
arises
out
of
causes
beyond
the
control
and
without
the
fault
or
negligence
of
the
laboratory.
It
also
may
be
necessary
to
limit
damages
to
a
certain
dollar
value
or
scope.

Other
types
of
damages
that
should
be
considered,
and
may
be
included
in
the
contract,
include
costs
for
resampling
and
administrative
costs
associated
with
the
evaluation
and
processing
of
unacceptable
data
(
data
that
do
not
meet
the
requirements
specified
in
the
contract
or
the
QC
requirements
specified
in
the
analytical
method).

5.1.5.5
Re­
Analysis
Costs
Every
laboratory
periodically
produces
data
that
are
associated
with
unacceptable
QC
data
or
are
invalid
for
other
reasons.
The
contract
should
stipulate
that
the
laboratory
will
reanalyze
samples
at
no
cost
to
your
PWS
if
the
problems
are
due
to
laboratory
error.
If
the
problems
are
due
to
an
error
outside
of
the
laboratory's
control
(
such
as
the
laboratory's
rejection
of
a
sample
received
at
>
10
°
C
that
results
in
resampling
by
your
PWS),
the
laboratory
should
not
be
responsible
for
the
additional
costs
that
may
result.
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
31
Draft
June
2003
L
Clearly
indicate
in
your
contract
when
the
laboratory
would
be
required
to
bear
the
costs
of
sample
re­
analysis
costs
and
when
these
costs
will
be
borne
by
your
PWS.

L
Clearly
indicate
in
your
contract
that
your
PWS
has
the
right
to
inspect
results
and
reject
the
results
if
they
do
not
meet
contract
requirements.
The
contract
also
should
state
that
you
have
the
right
to
inspect
the
results,
and
if
they
do
not
meet
the
requirements
in
the
contract,
you
have
the
right
to
reject
the
data,
returning
them
to
the
laboratory
without
payment.
Rejection
of
data
should
be
based
on
sound
technical
review
of
the
results.
It
also
obligates
you
to
make
no
use
of
those
results
without
making
some
payment
to
the
laboratory.

5.2
Developing
a
Bid
Sheet
After
all
project
requirements
have
been
established,
you
should
develop
a
bid
sheet
to
accompany
the
analytical
requirements
summary
during
the
solicitation.
The
bid
sheet
allows
laboratories
to
submit
bids
in
the
same
format,
making
bid
evaluations
easier,
and
also
helps
to
clarify
the
project.
Development
and
use
of
a
bid
sheet
is
recommended
regardless
of
whether
your
PWS
solicits
the
project
competitively
to
multiple
laboratories,
or
is
simply
requesting
a
quote
from
a
laboratory
you
already
know
you
will
be
using,
as
it
provides
a
very
clear
vehicle
for
submitting
and
evaluating
costs.

Bid
sheets
for
analytical
services
typically
are
formatted
as
a
chart,
with
analytical
requirements
along
one
axis
and
number
of
samples
and
prices
along
the
other.

The
bid
sheet
should
include
the
following
information:

°
Project
identifier
(
e.
g.
"
LT2
Cryptosporidium
Monitoring
Sample
Analyses
for
[
PWS
name
and/
or
facility
name]")

°
Space
for
laboratory
identification
information
°
Day,
date,
and
time
(
including
time
zone)
of
the
bid
deadline
°
Bid
submission
information
(
contact
and
mailing
address,
fax
number,
and/
or
email
address)

°
Estimated
award
date
°
Laboratory
period
of
performance
(
period
of
time
during
which
the
laboratory
is
obliged
to
resolve
issues
associated
with
analysis
of
the
samples
 
generally
6
months
after
shipment
of
last
sample)

°
Required
delivery
date
(
data
turnaround
time
and
the
basis
of
its
calculation,
such
as
from
collection
of
each
sample)

°
Bid
validity
period
(
period
of
time
during
which
bid
prices
are
considered
valid
 
generally
45
days
after
the
bid
deadline;
if
the
project
is
awarded
after
the
period
you
specify,
you
must
contact
bidding
laboratories
to
determine
whether
their
bid
is
still
valid,
or
needs
to
be
revised)
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
32
Draft
June
2003
°
A
summary
of
the
analytical
requirements:

°
Method
(
e.
g.,
Cryptosporidium
and
Giardia
by
EPA
Method
1623)

°
Filter
preference,
if
any
(
this
should
not
be
specified,
unless
your
PWS
has
experience
with
Cryptosporidium,
and
a
basis
for
requesting
the
use
of
a
specific
filter;
if
you
know
that
you
will
be
field
filtering
using
a
specific
filter,
and
shipping
this
to
the
laboratory,
it
is
important
that
you
specify
this)

°
Whether
samples
will
be
shipped
as
filtered
samples
or
bulk
water
samples
°
Sample
volume
(
e.
g.,
10
L,
50
L)

°
Total
number
of
field
samples
to
be
analyzed,
plus
two
extra,
in
case
of
"
make­
up"
samples
°
Total
number
of
MS
samples
to
be
analyzed
°
Total
number
of
potential
subsamples
to
be
analyzed
(
expressed
as
"
Up
to
[
no.]
subsamples")

°
The
number
generally
should
not
exceed
three
per
sample
°
If
you
have
high­
turbidity
water,
you
may
need
to
specify
up
to
three
subsamples
for
all
of
your
field
and
MS
samples
°
If
you
have
a
low­
turbidity
water,
you
should
specify
a
minimal
number,
just
in
case
the
need
arises
(
These
costs
would
not
be
incurred
unless
subsamples
actually
need
to
be
analyzed)

°
Total
number
of
potential
extra
filters
(
in
case
one
or
more
samples
clog
during
LT2
rule
monitoring:

°
If
you
will
be
shipping
bulk
samples
to
the
laboratory,
express
this
as
"
Up
to
[
no.]
extra
filters/
elutions"

°
If
you
will
be
filtering
samples
in
the
field,
but
receiving
filters
from
the
laboratory,
express
this
as
"
Up
to
[
no.]
extra
filters"

(
These
costs
would
not
be
incurred
unless
more
than
one
filter
actually
needs
to
be
used)

°
Columns
for
laboratories
to
enter
per­
analysis
and
total
costs
5.3
Soliciting
the
Contract
Procedures
for
soliciting
and
awarding
contracts
to
perform
analytical
services
can
vary,
depending
upon
the
scope
of
the
project
and
purchasing
requirements
within
the
organization
that
is
issuing
the
contract.
At
one
end
of
the
spectrum
are
contracts
that
are
awarded
after
placing
a
single
phone
call
and
obtaining
a
quote
from
a
single
laboratory.
The
opposite
end
of
the
spectrum
are
contracts
awarded
after
a
competitive
solicitation
and
bidding
process
involving
the
distribution
of
a
detailed
project
description
and
a
formal
bid
sheet
via
fax
or
mail.

5.3.1
Approved
Laboratories
Regardless
of
whether
you
will
be
soliciting
the
project
to
multiple
laboratories
or
working
with
a
single
laboratory
(
although
a
backup
laboratory
is
strongly
recommended
 
see
below),
you
will
need
to
limit
your
laboratories
to
only
those
approved
by
EPA
through
the
Laboratory
Quality
Assurance
Evaluation
Program
for
Analysis
of
Cryptosporidium
Under
the
Safe
Drinking
Water
Act
(
Laboratory
QA
Program)
(
67
FR
9731,
March
4,
2002).
Information
on
the
Laboratory
QA
Program
is
posted
on
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
33
Draft
June
2003
http://
www.
epa.
gov/
safewater/
lt2/
index.
html
and
this
program
is
described
in
detail
in
the
Microbial
Laboratory
Guidance
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).

Briefly,
the
objectives
of
the
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
is
required
to
complete
the
following
steps
to
be
qualified
through
this
program:

°
Acceptably
perform
initial
proficiency
testing
(
IPT)
on
blind
samples
°
Participate
in
an
on­
site
evaluation
of
their
technical,
data
management,
and
quality
assurance
procedures
°
Acceptably
perform
ongoing
proficiency
testing
(
OPT)
on
blind
samples
every
four
months
To
improve
Cryptosporidium
data
quality
and
consistency
during
LT2
rule
monitoring,
EPA
requires
that
only
those
laboratories
approved
for
Cryptosporidium
analysis
under
the
Lab
QA
Program
be
used
for
LT2
rule
monitoring
analyses
[
40
CFR
part
141.706
(
a)].
A
list
of
laboratories
approved
through
the
Lab
QA
Program
is
available
from
http://
www.
epa.
gov/
safewater/
lt2/
index.
html.

5.3.2
Primary
and
Backup
Laboratory
Contracts
Because
a
laboratory's
approval
status
may
change
during
the
LT2
rule
monitoring
period,
you
should
plan
to
award
a
primary
contract
and
a
backup
contract.
If
no
performance
problems
or
other
problems
are
encountered
during
the
LT2
rule
monitoring
period
by
the
laboratory
awarded
the
primary
contract,
then
this
laboratory
would
provide
uninterrupted
sample
analysis
support
for
the
entire
monitoring
period.
However,
if
the
laboratory
encountered
performance
problems
and
was
disapproved,
or
was
otherwise
unable
to
meet
contract
requirements,
your
PWS
could
switch
sample
analyses
to
the
backup
laboratory
under
the
contract
you
established
with
this
laboratory
before
monitoring
began.

The
award
of
primary
and
backup
contracts
should
be
discussed
in
the
contract
solicitation.
All
other
things
considered
equal,
the
award
for
the
primary
contract
could
be
made
to
the
lowest
responsive,
responsible
bidder
and
the
award
for
the
backup
contract
could
be
made
to
the
second
lowest
responsive,
responsible
bidder.

5.4
Evaluating
Bids
After
the
laboratories
have
received
the
solicitation
and
submitted
their
bids,
you
must
evaluate
the
bids
to
identify
the
laboratory
that
will
be
awarded
the
analytical
services
contract.
Specific
procedures
for
evaluating
bids
may
vary,
depending
upon
the
requirements
of
your
organization,
but
the
bid
evaluation
process
generally
entails
evaluation
and
comparison
of
each
laboratory's
proposed
cost
and
capability
to
meet
the
analysis
requirements.

5.4.1
Identifying
Responsive
Bidders
You
should
consult
your
legal
department
or
purchasing
department
to
identify
any
applicable
requirements
for
evaluating
competitive
bids
within
their
organization.
At
a
minimum,
however,
you
should
review
all
bids
and
recalculate
subtotals
and
totals
to
ensure
that
the
bidding
laboratories
did
not
make
any
mathematical
errors.
In
addition,
you
should
verify
that
there
are
no
unacceptable
contingencies
associated
with
any
of
the
bids,
such
as
the
use
of
a
filter
other
than
the
filter
that
was
specified
in
the
contract
solicitation.
Either
eliminate
from
consideration
bids
from
laboratories
that
bid
with
contingencies
or
contact
the
laboratory(
ies)
to
discuss
the
bid
and
verify
that
the
laboratory
cannot
perform
the
specified
services.
Section
5:
Contracting
for
Cryptosporidium
Laboratory
Services
34
Draft
June
2003
Of
the
remaining
(
responsive)
bids,
identify
the
lowest
bidder
to
award
the
primary
contract
and
the
second
lowest
bidder
to
award
the
backup
contract.
If
additional
assessments
of
a
laboratory's
performance
or
responsibility
are
needed,
you
may
want
to
contact
references.

5.4.2
References
If
you
have
not
worked
with
a
particular
laboratory
before
and
would
like
to
verify
that
the
laboratory
will
meet
your
needs
throughout
the
monitoring
period,
you
can
ask
the
laboratory
to
provide
contacts
and
phone
numbers
of
utility
or
government
clients
for
which
the
laboratory
has
performed
Cryptosporidium
sample
analyses
or
other
comparable
services.

Questions
to
ask
the
references
include:

°
Did
the
laboratory
provide
data
by
the
required
due
date?

°
Were
the
data
provided
by
the
laboratory
of
acceptable
quality
and
compliant
with
contract
requirements?

°
Were
laboratory
personnel
easy
to
work
with
when
problems
arose
during
all
phases
of
the
project,
including
sample
scheduling,
sample
analysis,
and
data
review?
If
problems
were
noted
during
data
review,
was
the
laboratory
prompt
and
responsive
in
addressing
your
concerns?

°
Do
you
have
any
reservations
in
recommending
this
laboratory?

5.5
Communicating
with
the
Laboratory
After
the
analytical
services
contract
is
awarded,
you
should
request
from
the
laboratory
contact
information
for
the
following
roles,
and
provide
the
laboratory
with
PWS
contacts
for
the
same
roles:

°
A
technical
contact
for
analytical
questions
or
problems
°
A
sample
control
contact
for
shipping
delays
on
the
PWS
end
and
sample
receipt
problems
on
the
laboratory
end
°
An
administrative
contact
for
invoicing
and
payment
Maintaining
communications
with
the
laboratory
is
critical
to
identifying
and
resolving
problems
quickly
and
minimizing
the
need
for
resampling
and
reshipments.
At
a
minimum,
you
should
always
notify
the
laboratory
of
sample
shipments
and
confirm
that
the
laboratory
received
the
sample
on
time
and
in
acceptable
condition.

Although
most
communications
are
typically
conducted
over
the
phone,
these
communications
also
can
be
conducted
via
email,
which
has
the
added
benefit
of
providing
your
PWS
and
the
laboratory
with
a
written
record
of
sample
receipt
confirmations,
problem
notifications,
and
problem
resolutions.
35
Draft
June
2003
SECTION
6:
COLLECTING
AND
SHIPPING
SOURCE
WATER
SAMPLES
Large
systems
(
PWSs
serving
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.
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).

Monitoring
requirements
for
each
system
size
and
the
schedule
for
each
stage
of
monitoring
is
described
in
Table
6­
1.

Table
6­
1.
Summary
of
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
LT2
rule
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
(
see
Section
2)
b
Small
systems
may
be
required
to
monitor
for
Cryptosporidium
for
1
year,
beginning
6
months
after
completion
of
E.
coli
monitoring;
Cryptosporidium
monitoring
would
be
required
if
the
E.
coli
annual
mean
concentrations
exceed
10
E.
coli/
100
mL
for
systems
using
lakes/
reservoirs
or
exceed
50
E.
coli/
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
Large
unfiltered
systems
are
required
to
conduct
source
water
monitoring
that
includes
Cryptosporidium
sampling
only
N/
A
=
Not
applicable.
No
monitoring
required.
Section
6:
Collecting
and
Shipping
Source
Water
Samples
36
Draft
June
2003
6.1
Sample
Volumes
Sample
volume
guidance
is
provided
in
Section
6.1.1
for
Cryptosporidium
samples
and
Section
6.1.2
for
E.
coli
samples.

6.1.1
Cryptosporidium
Samples
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
$
##.
5
L
would
be
rounded
up
and
sample
volumes
#
##.
4
L
would
be
rounded
down.
For
example,
9.8
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.

PWSs
with
highly
turbid
water
may
be
able
to
collect
the
required
minimum
packed
pellet
volume
by
avoiding
filtration
altogether,
and
shipping
a
bulk
water
sample
to
the
laboratory
for
centrifugation.
The
laboratory
can
mix
the
sample
thoroughly
and
centrifuge
250­
mL
or
greater
aliquot
volumes
sequentially
according
to
Section
13.2
of
Method
1622/
1623,
until
2
mL
of
packed
pellet
volume
is
generated.

If
the
PWS
encounters
variable
water
quality
that
clogs
the
filter
unpredictably,
the
PWSs
should
routinely
bring
two
filters
plus
a
cubitainer
to
the
sampling
point
for
each
sampling
event:

°
If
the
water
quality
allows
a
full
10
L
to
be
filtered
without
clogging,
the
PWS
can
simply
ship
the
filter
to
the
laboratory
and
save
the
remaining
materials
for
subsequent
events.

°
If
the
first
filter
clogs
after
5
L
or
more
have
been
filtered,
and
the
volume
is
not
anticipated
to
yield
2
mL
of
packed
pellet
volume,
the
PWS
should
be
able
to
filter
the
remaining
volume
through
the
second
filter
and
ship
both
filters
to
the
laboratory
for
processing.

6.1.2
E.
coli
Samples
PWSs
should
analyze
up
to
100­
mL
of
sample
for
LT2
monitoring.
EPA
recommends
that
the
PWS
collect
and
ship
more
than
100­
mL
of
sample
to
ensure
sufficient
volume
for
sample
analysis
is
available
in
the
event
of
spillage
at
the
laboratory.
If
spillage
or
leakage
occurs
during
shipment,
there
is
an
opportunity
for
sample
contamination
to
occur
and
the
sample
should
not
be
analyzed
(
see
Section
8.3.1).
Additional
details
on
sample
collection
procedures
are
provided
in
Section
6.4.3.
The
capacity
of
sample
containers
should
be
120­
mL
(
6
oz.)
or
250­
mL
(
8
oz.)
to
allow
for
sufficient
sample
volume
and
at
least
a
1­
inch
head
space
to
facilitate
mixing
of
the
sample
by
shaking
prior
to
analysis.
Section
6:
Collecting
and
Shipping
Source
Water
Samples
37
Draft
June
2003
6.2
Sample
Collection
Location
LT2
rule
monitoring
is
intended
to
assess
the
mean
Cryptosporidium
level
in
the
influent
to
drinking
water
plants
that
treat
surface
water
or
ground
water
under
the
direct
influence
(
GWUDI)
of
surface
water.
Generally,
monitoring
is
required
for
each
plant
that
treats
a
surface
water
or
GWUDI
source.
However,
where
multiple
plants
receive
all
of
their
water
from
the
same
influent
(
e.
g.,
multiple
plants
draw
water
from
the
same
pipe),
the
same
set
of
monitoring
results
may
be
applied
to
each
plant.
E.
coli
samples
should
be
collected
at
the
same
location
as
Cryptosporidium
samples.

PWSs
are
required
to
collect
source
water
samples
for
the
LT2
rule
from
the
plant
intake
prior
to
any
treatment
[
40
CFR
part
141.704
(
a)].
Guidance
on
sampling
at
plants
where
this
may
not
be
feasible,
or
where
other
factors,
such
as
the
use
of
multiple
sources,
need
to
be
addressed,
is
provided
below,
in
Sections
6.2.1
through
6.2.5.

6.2.1
Plants
That
Do
Not
Have
a
Sampling
Tap
Located
Prior
to
Any
Treatment
Plants
in
this
situation
should
pursue
one
of
the
following
options:

°
Manually
collect
source
water
samples
as
close
to
the
intake
as
is
feasible,
at
a
similar
depth
and
distance
from
shore.

°
Establish
a
sampling
location
prior
to
treatment
°
6.2.2
Plants
That
Use
Different
Water
Sources
at
the
Same
Time
This
includes
multiple
surface
water
sources
and
blended
surface
water
and
ground
water
sources.
Plants
in
this
situation
should
pursue
one
of
the
following
options:

°
If
there
is
a
sampling
tap
where
the
sources
are
combined
prior
to
treatment,
the
sample
should
be
collected
from
the
tap.

°
Samples
can
be
manually
collected
at
each
source
near
the
intake
on
the
same
day
and
composited
into
one
sample.
The
volume
of
sample
from
each
source
should
be
weighted
according
to
the
proportion
of
that
source
used
by
the
plant.
For
example,
if
a
plant
has
two
sources
and
75%
of
the
drinking
water
is
from
Source
A
and
25%
is
from
Source
B,
then
for
a
10­
L
sample,
7.5
L
would
be
collected
from
Source
A
and
combined
with
2.5
L
collected
from
Source
B.
Compositing
of
samples
should
reflect
plant
operation
at
the
time
the
sample
is
collected
and
may
change
during
the
monitoring
period.

°
Separate
samples
can
be
manually
collected
at
each
source
near
the
intake
on
the
same
day
and
analyzed
independently.
The
results
would
then
be
used
to
calculate
a
weighted
average
of
the
analysis
results.
The
weighted
average
would
be
calculated
by
multiplying
the
analysis
result
for
each
source
by
the
fraction
of
the
source
contribution
to
total
plant
flow
at
the
time
the
samples
were
collected,
and
then
summing
these
values.
For
example,
if
a
plant
has
two
sources
and
75%
of
the
drinking
water
is
from
Source
A
and
25%
is
from
Source
B,
then
one
sample
would
be
collected
from
each
source
and
analyzed
independently.
If
the
concentration
of
oocysts
for
the
sample
from
Source
A
was
5
oocysts/
L
and
the
concentration
of
the
sample
from
Source
B
was
2
oocysts/
L,
the
final
result
for
the
plant
for
this
sampling
event
would
be
4
oocysts/
L
([
5
oocysts/
L
x
0.75]
+
[
1
oocyst/
L
x
0.25]).

6.2.3
Plants
That
Use
Presedimentation
For
these
plants,
source
water
samples
must
be
collected
after
the
presedimentation
basin
but
before
any
other
treatment
[
40
CFR
141.704
(
b)].
Use
of
presedimentation
basins
during
monitoring
should
be
consistent
with
routine
operational
practice
and
should
be
documented.
For
systems
taken
samples
after
Section
6:
Collecting
and
Shipping
Source
Water
Samples
38
Draft
June
2003
presedimentation
basin,
no
"
Microbial
Toolbox"
credits
will
be
allowed
for
presedimentation,
if
the
plant
is
classified
into
a
bin
that
requires
additional
treatment
[
40
CFR
141.726
(
a)].

6.2.4
Plants
That
Use
Raw
Water
Off­
Stream
Storage
For
these
plants,
source
water
samples
must
be
collected
after
the
off­
stream
storage
reservoir
[
CFR
141.704
(
c)].
Use
of
off­
stream
storage
during
monitoring
should
be
consistent
with
routine
operational
practice
and
should
be
documented.

6.2.5
Plants
That
Use
Bank
Filtration
The
correct
sampling
location
for
PWSs
with
plants
using
bank
filtration
differs
depending
on
whether
the
bank
filtered
water
is
treated
by
subsequent
filtration
for
compliance
with
the
Surface
Water
Treatment
Rule
(
SWTR)
[
40
CFR
141.704
(
c)].

°
PWSs
using
bank
filtered
water
that
is
treated
by
subsequent
filtration
for
compliance
with
the
SWTR
must
collect
source
water
samples
from
the
well
(
i.
e.,
after
bank
filtration)
but
before
any
other
treatment.
Use
of
bank
filtration
during
monitoring
should
be
consistent
with
routine
operational
practice
and
should
be
documented.
Systems
collecting
samples
after
a
bank
filtration
process
may
not
receive
microbial
toolbox
credit
for
the
bank
filtration
[
40
CFR
141.726
(
c)].

°
PWSs
using
bank
filtered
water
without
additional
filtration
must
take
source
water
samples
in
the
surface
water
source
(
e.
g.,
the
river).
Use
of
bank
filtration
during
monitoring
should
be
consistent
with
routine
operational
practice
and
should
be
documented.

Before
monitoring
begins,
all
plants
must
establish
a
source
water
monitoring
schedule,
as
discussed
in
Section
6.3.

6.3
Source
Water
Monitoring
Schedule
PWSs
are
required
to
collect
samples
at
least
monthly
and
in
accordance
with
a
schedule
established
by
the
PWS
prior
to
initiation
of
monitoring.
PWSs
may
collect
samples
more
frequently
(
e.
g.,
twice­
permonth
weekly),
provided
the
same
frequency
is
maintained
throughout
the
monitoring
period
[
40
CFR
part
141.701
(
e)].

Water
treatment
plants
that
use
surface
water
or
ground
water
under
the
direct
influence
(
GWUDI),
but
are
operated
only
seasonally
(
e.
g.,
during
times
of
high­
water
demand)
should
monitor
at
least
monthly
during
the
period
when
the
plant
is
in
operation.

Systems
regulated
under
the
LT2
rule
are
required
to
submit
source
water
monitoring
schedule
to
EPA
within
3
months
of
rule
promulgation
[
40
CFR
part
141.703
(
a)].
The
schedule
is
entered
using
the
scheduler
function
within
the
LT2
Data
Collection
System.
Details
on
the
use
of
the
scheduler
are
provided
in
the
Users'
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule)
Data
Collection
System.
Systems
are
required
to
collect
samples
within
2
days
before
or
after
the
dates
indicated
in
their
sampling
schedules
[
40
CFR
part
141.703
(
b)].

The
scheduler
function
will
be
available
for
PWSs
to
establish
their
LT2
monitoring
schedule
for
a
3­
month
period,
beginning
on
the
date
of
final
rule
publication.
The
use
of
a
predetermined
monthly
or
semimonthly
sampling
schedule
at
each
PWS
during
LT2
is
designed
to
capture
storm
events
and
other
factors
that
affect
water
quality
on
a
periodic
basis.
Because
a
PWS
can
potentially
bias
the
results
of
the
monitoring
by
avoiding
sample
collection
during
periods
of
low
water
quality,
the
submission
of
prescheduled
sampling
dates
will
be
used
to
assess
compliance.
Section
6:
Collecting
and
Shipping
Source
Water
Samples
39
Draft
June
2003
6.4
Sample
Scheduling
Compliance
Issues
Permissible
exceptions
to
the
sampling
schedule
are
noted
as
follows:

°
If
extreme
conditions
or
situations
exist
that
may
pose
danger
to
the
sampler,
or
which
are
unforeseen
or
cannot
be
avoided
and
which
cause
the
system
to
be
unable
to
sample
in
the
required
time
frame,
the
system
should
sample
as
close
to
the
scheduled
date
as
feasible
and
submit
an
explanation
for
the
alternative
sampling
date
to
EPA
concurrent
with
shipment
of
the
sample
to
the
laboratory.

EPA
will
evaluate
the
explanation
and
update
the
schedule
in
the
LT2
Data
Collection
System,
if
acceptable,
to
permit
the
analytical
result
to
be
submitted
through
the
system
(
results
with
sample
collection
dates
that
do
not
comply
with
the
schedule
entered
by
the
PWS
before
monitoring
began
will
be
rejected
from
the
system).

°
Systems
that
are
unable
to
report
a
valid
Cryptosporidium
analytical
result
for
a
scheduled
sampling
date
due
to
failure
to
comply
with
the
analytical
method
quality
control
requirements
(
e.
g.,
sample
is
lost
or
contaminated;
laboratory
exceeds
analytical
method
holding
time)
must
collect
a
replacement
sample
within
14
days
of
being
notified
by
the
laboratory
that
a
result
cannot
be
reported
for
that
date.
Systems
must
submit
an
explanation
for
the
replacement
sample
with
the
analytical
results.
Systems
should
collect
an
E.
coli
sample
at
the
same
time
as
the
Cryptosporidium
replacement
sample.

Alternative
sample
collection
dates
should
be
timed
so
as
not
to
coincide
with
another
scheduled
Cryptosporidium
sample
collection
date.
Documentation
of
alternate
sample
collection,
including
the
reason,
should
be
provided
with
the
grandfathered
data
package.

6.4
Sample
Collection
Guidance
Large
plants
must
begin
collecting
source
water
samples
6
months
after
rule
promulgation
and
small
plants
must
begin
30
months
after
rule
promulgation.
Because
the
LT2
monitoring
program
is
designed
to
assess
source
water
Cryptosporidium
and
E.
coli
concentrations,
not
the
concentrations
of
these
organisms
at
points
after
any
treatment,
samples
must
be
collected
prior
to
any
treatment
and
where
the
water
is
no
longer
subject
to
surface
runoff
during
LT2
monitoring
(
40
CFR
part
141.704).

During
each
of
the
scheduled
sampling
events,
several
actions
must
be
performed
in
addition
to
collecting
the
sample.
These
actions,
and
an
indication
of
which
plant
types
each
applies
to,
are
summarized
in
Table
6­
2.
Section
6:
Collecting
and
Shipping
Source
Water
Samples
40
Draft
June
2003
Table
6­
2.
Sample
Collection
Activities
Required
for
Each
Plant
Type
Action
Large
filtered
plants
Large
unfiltered
plants
All
small
plants
Small
plants
that
exceed
the
E.
coli
trigger
level
Document
sample
collection
information
T
T
T
T
Collect
Cryptosporidium
sample
T
T
T
Collect
E.
coli
sample
T
T
Measure
turbidity
T
Monitor
sample
temperature
during
sample
transport
Ta
Ta
Tb
T
a
Those
utilities
with
on­
site
Cryptosporidium
analytical
capabilities
will
not
need
to
transport
samples
unless
the
laboratory
is
not
located
in
close
proximity
to
the
sample
collection
location
b
Those
small
plants
with
on­
site
E.
coli
analytical
capabilities
will
not
need
to
transport
samples
unless
the
laboratory
is
not
located
in
close
proximity
to
the
sample
collection
location
Guidance
and
procedures
for
each
of
these
sample
collection
activities
is
provided
in
Sections
6.4.1
­
6.4.5,
below.

6.4.1
Sample
Collection
Documentation
The
information
in
Table
6­
3
should
be
recorded
during
sample
collection
to
link
the
monitoring
result
to
the
plant,
and
to
provide
information
required
for
development
of
the
microbial
index.

Table
6­
3.
Minimum
Data
Elements
to
Record
During
Sample
Collection
Sampling
Information
Required
Recommended
PWS
name
T
Public
Water
System
Identification
(
PWSID)
numbera
T
Facility
name
T
Facility
IDa
T
Sample
collection
point
name
T
Sample
collection
point
IDa
T
Sample
collection
datea
T
Source
water
typeb
T
Requested
analysis
T
Sample
collection
time
(
start
time
for
field­
filtered
samples)
T
Meter
readings
(
for
field­
filtered
samples
only)
T
Section
6:
Collecting
and
Shipping
Source
Water
Samples
41
Draft
June
2003
Sample
collection
stop
time
(
for
field­
filtered
samples
only)
T
Turbidityb
T
a
The
combination
of
these
elements
constitute
the
unique
sample
identifier
for
LT2
monitoring
samples
b
This
information
should
be
recorded
with
the
E.
coli
sample
collection
information,
as
it
will
be
entered
into
the
LT2
data
collection
system
with
the
E.
coli
sample
results,
for
use
in
reassessing
the
microbial
index.
It
does
not
need
to
be
reported
with
the
Cryptosporidium
sample
collection
information
For
samples
that
are
shipped
off­
site,
this
information
should
be
documented
on
an
LT2
sample
collection
form
(
Appendix
C),
or
similar
form
provided
by
your
contract
laboratory.
For
samples
analyzed
on­
site
by
your
utility's
laboratory,
this
information
can
be
documented
in
a
sampling
log
book
or
other
standard
form
used
by
your
utility;
the
LT2
sample
collection
form
can
also
be
used.

The
source
water
type
for
the
sample
will
be
used
to
reassess
the
relationship
between
Cryptosporidium
and
E.
coli
concentrations
(
the
microbial
index
discussed
in
Section
1.5).
Sample
collection
personnel
must
select
from
four
source
water
types
on
the
LT2
sample
collection
form:

°
Flowing
stream
(
defined
under
the
LT2
rule
as
"
a
course
of
running
water
flowing
in
a
definite
channel")

°
Reservoir/
lake
(
defined
under
the
LT2
rule
as
"
a
natural
or
man
made
basin
or
hollow
on
the
Earth's
surface
in
which
water
collects
or
is
stored
that
may
or
may
not
have
a
current
or
single
direction
of
flow")

°
Ground
water
under
the
direct
influence
(
GWUDI)
of
flowing
stream
surface
water
°
GWUDI
of
reservoir/
lake
surface
water
The
source
water
type
should
be
selected
based
on
the
type
of
source
water
that
accounts
for
the
majority
of
the
surface
water
used
as
source
water
at
the
time
of
sample
collection.
For
example,
if
the
plant
uses
a
mix
of
approximately
55%
reservoir/
lake
water
and
45%
flowing/
stream
water,
the
"
reservoir/
lake"
option
should
be
circled
on
the
LT2
sample
collection
form.

The
majority
of
source
water
for
plants
that
use
GWUDI
is
ground
water.
However,
as
noted
above,
the
selection
of
source
water
type
under
the
LT2
rule
is
based
on
the
majority
of
surface
water
used
as
source
water.
As
a
result,
the
selection
of
source
water
type
is
based
on
the
type
of
surface
water
that
accounts
for
the
majority
of
the
influence
of
the
ground
water
source.

The
turbidity
of
the
source
water
also
needs
to
be
measured.
Cryptosporidium
sample
collection
procedures
are
discussed
in
Section
6.4.2;
E.
coli
sample
collection
procedures
and
turbidity
measurement
procedures
are
discussed
in
Section
6.4.3
and
6.4.4,
respectively.

6.4.2
Cryptosporidium
Sample
Collection
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.
A
detailed
protocol
for
collecting,
packing,
and
shipping
bulk
samples
is
provided
as
Appendix
D.

°
On­
site
filtration
of
water
samples
using
the
Pall
Gelman
Envirochek
 
or
Envirochek
 
HV
capsule
filter.
A
detailed
protocol
for
filtering
samples
on­
site
from
pressurized
or
unpressurized
sources
is
provided
as
Appendix
E.

°
On­
site
filtration
of
water
samples
using
the
IDEXX
 
Filta­
Max
foam
filter.
A
detailed
protocol
for
filtering
samples
on­
site
from
pressurized
or
unpressurized
sources
is
provided
as
Appendix
F.
Section
6:
Collecting
and
Shipping
Source
Water
Samples
42
Draft
June
2003
Regardless
of
the
procedure
used
to
collect
Cryptosporidium
samples,
the
sample
must
be
eluted
from
the
filter
within
96
hours
of
sample
collection,
per
EPA
Method
1622/
1623
(
Section
8.2).
If
this
holding
time
is
violated,
the
laboratory
will
reject
the
sample,
and
your
PWS
will
be
required
to
recollect
and
reship
the
sample.

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
the
method
(
Section
8.2).

6.4.2.1
Matrix
Spike
Samples
Method
1622/
1623
requires
matrix
spike
(
MS)
samples
to
be
analyzed
at
a
frequency
of
1
MS
sample
for
every
20
monitoring
samples
from
each
plant.
This
frequency
translates
to
the
following,
for
each
plant
category:

°
For
large
PWSs
that
perform
monthly
monitoring
for
2
years
(
resulting
in
24
monitoring
samples),
2
MS
samples
must
be
collected
and
analyzed
°
For
large
PWSs
that
perform
semi­
monthly
or
more
frequent
monitoring
for
2
years
(
resulting
in
48
or
more
samples),
a
minimum
of
3
MS
samples
will
be
collected
and
analyzed
°
For
small
PWSs
that
are
triggered
into
Cryptosporidium
monitoring
and
collect
semi­
monthly
samples
for
1
year
(
resulting
in
24
samples),
2
MS
samples
must
be
collected
and
analyzed
The
MS
sample
and
the
associated
unspiked
sample
must
be
analyzed
by
the
same
procedure
and
the
MS
sample
must
be
the
same
volume
as
the
associated
monitoring
sample.
If
the
volume
of
the
MS
sample
is
greater
than
10
L,
the
system
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.

Utilities
collecting
and
shipping
bulk
water
samples
for
filtration
and
analysis
at
the
laboratory
should
split
their
sample
stream
and
collect
the
monitoring
sample
volume
and
MS
sample
volume
simultaneously.

°
The
sample
stream
should
be
split
using
flow
controllers
on
both
sides
of
the
split
to
regulate
the
pressure
difference
between
the
side
being
subjected
to
filtration
(
resulting
in
higher
pressure)
and
the
side
flowing
into
a
bulk
sample
container.
A
mixing
chamber
(
filter
housing
without
filter)
can
be
added
immediately
upstream
from
the
Y
to
aid
in
equalizing
the
distribution
of
sample
particulates
to
either
side.

°
If
splitting
the
sample
stream
is
not
practical,
the
utility
should
collect
the
MS
sample
immediately
before
or
after
the
monitoring
sample.

MS
sample
results
would
not
be
used
to
adjust
Cryptosporidium
recoveries
at
any
individual
source
water;
but
MS
results
would
be
used
collectively
to
assess
overall
recovery
and
variability
for
EPA
Method
1622/
1623
in
source
water.
No
resampling
would
be
necessary
for
MS
samples
that
do
not
meet
Method
1622/
1623
recovery
guidelines.
Section
6:
Collecting
and
Shipping
Source
Water
Samples
43
Draft
June
2003
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)].

6.4.2.2
Purchasing
Filters
If
one
of
the
field
filtration
options
is
used,
you
may
want
to
consider
purchasing
filters
in
bulk
from
the
manufacturer
(
or
the
manufacturer's
local
distributor),
as
it
may
be
cheaper
than
purchasing
the
filters
from
your
Cryptosporidium
contract
laboratory
as
part
of
the
sampling
kit.
This
approach
also
provides
your
PWS
with
a
ready
supply
of
extra
filters
on­
site,
if
a
filter
clogs
during
a
sampling
event.
Plants
wishing
to
explore
this
option
should
call
one
of
the
contacts
in
Table
6­
4.

Table
6­
4.
Contacts
for
Filters
Approved
for
Use
in
EPA
Method
1622/
1623
Pall
Life
Sciences
(
Envirochek
 
and
Envirochek
 
HV
capsule
filters)
IDEXX
(
Filta­
Max
 
foam
filters)

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
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
The
PWS
also
can
purchase
and
assemble
the
entire
sampling
kit
and
maintain
this
kit
on
site,
rather
than
shipping
it
back
and
forth
between
the
Cryptosporidium
laboratory
and
the
plant.
If
the
filters
you
use
have
associated
shelf
lives
and
storage
conditions,
ensure
that
the
filters
are
stored
according
to
the
manufacturers'
directions
and
are
not
used
past
the
specified
shelf
life.

The
components
and
part
numbers
for
the
sampling
kit
are
specified
in
the
individual
protocols
for
each
filter.
If
the
sampling
kit
is
maintained
on­
site
by
the
utility,
the
utility
should
use
disposable
materials
wherever
possible
to
mitigate
the
risk
of
cross­
contamination
between
samples
or
sampling
events,
and
must
disinfect
the
non­
disposable
sampling
equipment
between
uses
(
if
the
laboratory
provides
the
sampling
kit,
this
disinfection
step
is
performed
at
the
laboratory.)

Sampling
kit
cleaning
should
consist
of
the
following:

°
Cleaning
equipment
by
scrubbing
with
warm
detergent
solution
and
exposing
to
hypochlorite
solution
(
minimum
of
a
5%
solution
of
bleach
and
water)
for
at
least
30
minutes
at
room
temperature
°
Rinsing
the
equipment
with
reagent
water
and
placing
the
equipment
in
an
area
free
of
potential
Cryptosporidium
contamination
until
dry
6.4.2.3
Filter
Clogs
and
Highly
Turbid
Water
Samples
PWSs
with
highly
turbid
source
waters
are
likely
to
generate
larger
packed
pellet
volumes
after
centrifugation
and
to
clog
filters
than
PWSs
with
low­
turbidity
waters.
As
noted
in
Section
6.1,
at
least
2
mL
of
packed
pellet
volume
must
be
analyzed
(
for
samples
in
which
10
L
is
filtered),
or
as
much
volume
Section
6:
Collecting
and
Shipping
Source
Water
Samples
44
Draft
June
2003
as
two
filters
can
accommodate
before
clogging.
(
If
more
than
10
L
is
filtered,
then
less
of
the
packed
pellet
volume
needs
to
be
analyzed.)

PWSs
with
highly
turbid
water
may
be
able
to
collect
the
required
minimum
packed
pellet
volume
by
avoiding
filtration
altogether,
and
shipping
a
bulk
water
sample
to
the
laboratory
for
centrifuging.
The
laboratory
can
centrifuge
250­
mL
or
greater
aliquot
volumes
sequentially,
until
a
packed
pellet
volume
of
2
mL
is
generated.

6.4.3
E.
coli
Sample
Collection
For
most
large
systems,
E.
coli
analyses
will
be
conducted
on­
site,
so
samples
will
not
be
shipped
in
most
cases,
unlike
Cryptosporidium
samples.
However,
many
small
systems
will
collect
E.
coli
samples
and
ship
them
off­
site
for
analysis.
Regardless
of
whether
the
samples
are
analyzed
by
the
utility's
own
laboratory
or
by
a
commercial
laboratory,
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
the
general
coliform
analysis
technique
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
discussed
in
the
Microbial
Laboratory
Guidance
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).
Summary
information
on
these
methods
is
also
provided
in
Section
4
of
this
document.

Collect
E.
coli
samples
in
sterile,
non­
toxic,
plastic,
or
glass
containers
with
a
leak­
proof
lid.
The
capacity
of
sample
containers
should
be
120­
mL
(
6
oz.)
or
250­
mL
(
8
oz.)
to
allow
for
sufficient
sample
volume
and
at
least
a
1­
inch
head
space
to
facilitate
mixing
of
the
sample
by
shaking
prior
to
analysis
A
detailed
protocol
for
collecting
source
water
samples
for
E.
coli
analysis,
as
well
as
packing
and
shipping
guidance
for
utilities
that
transport
samples
off­
site
for
analysis,
is
provided
as
Appendix
G.

EPA
strongly
encourages
laboratories
to
analyze
samples
as
soon
as
possible
after
collection.
E.
coli
samples
must
be
analyzed
within
24
hours
of
sample
collection
[
40
CFR
part
141.705
(
b)(
1)].
Note:
This
is
a
longer
time
period
than
currently
permitted
in
Standard
Methods
and
the
Manual
for
the
Certification
of
Laboratories
Analyzing
Drinking
Water,
and
is
based
on
data
demonstrating
that
surface
water
samples
could
be
held,
chilled,
for
up
to
24
hours
and
still
yield
valid
results
(
Reference
9.5).

Samples
should
be
maintained
above
freezing
and
below
10
°
C
in
a
refrigerator
or
in
a
cooler
with
wet
ice,
blue
ice,
or
gel
packs,
etc.
Additional
guidance
on
monitoring
sample
temperature
is
available
in
Section
6.4.5
of
this
manual.

6.4.4
Measuring
Turbidity
PWSs
must
measure
the
turbidity
of
the
source
at
the
time
of
Cryptosporidium
and
E.
coli
sample
collection
during
LT2
rule
monitoring.
Turbidity
must
be
measured
by
a
party
approved
by
the
State
[
40
CFR
part
141.706
(
c)]
using
methods
for
turbidity
measurement
approved
at
40
CFR
part
141.74
[
40CFR
part
141.705
(
c)].
These
methods
include:

°
Method
2130B,
published
in
Standard
Methods
for
the
Examination
of
Water
and
Wastewater
(
19th
or
20th
Edition).
The
full
text
of
the
19th
Edition
is
provided
as
Appendix
H.

°
Great
Lakes
Instrument
(
GLI)
Method
2.
The
full
text
of
this
method
is
provided
as
Appendix
I.

°
Revised
EPA
Method
180.1,
approved
in
August
1993
in
Methods
for
the
Determination
of
Inorganic
Substances
in
Environmental
Samples
(
EPA­
600/
R­
93­
100).
The
full
text
of
this
method
is
provided
as
Appendix
J.
Section
6:
Collecting
and
Shipping
Source
Water
Samples
45
Draft
June
2003
Systems
must
use
turbidimeters
that
conform
to
one
of
the
approved
methods
for
measuring
turbidity,
such
as
Hach
Turbidimeter
1720D
with
EPA
Method
180.1,
GLI
Turbidimeter
Accu
4
with
GLI
Method
2,
or
equivalents
(
Note:
These
examples
do
not
constitute
an
endorsement
of
specific
instrumentation.
Approved
methods
provide
specifications
that
turbidimeters
must
meet,
and
conformance
of
instruments
with
these
particular
specifications
must
be
determined
prior
to
analysis.).
For
regulatory
reporting
purposes,
either
an
on­
line
or
a
benchtop
turbidimeter
may
be
used,
and
systems
must
comply
with
all
quality
control
requirements
specified
in
methods
and
regulations.
If
a
system
chooses
to
utilize
on­
line
units
for
monitoring,
the
system
must
validate
the
continuous
measurements
for
accuracy
on
a
regular
basis
using
a
protocol
approved
by
the
State
[
40
CFR
part
141.74
(
c)
(
1)].

6.4.4.1
Measuring
Sample
Turbidity
During
LT2
Monitoring
When
measuring
turbidity,
cuvettes
must
be
clear,
colorless
glass
or
plastic.
The
tube
must
be
kept
clean,
both
inside
and
out,
to
provide
accurate
readings.
If
a
sample
tube
is
scratched,
it
must
be
discarded.

°
Measuring
Sample
Turbidity
Using
SM
2130B.
Measure
turbidity
immediately
after
sample
collection
to
prevent
temperature
changes,
particle
flocculation,
and
sedimentation
from
changing
sample
characteristics.
Shake
sample
well
before
pouring
into
cuvette.
Gently
agitate
to
remove
air
bubbles
from
the
inside
of
the
sample
before
pouring
the
sample
into
cell.
Wait
until
all
the
air
bubbles
disappear
and
remove
all
moisture
from
the
outside
of
the
sample
cell
before
placing
it
into
the
instrument.
If
fogging
occurs,
warm
the
sample
by
warm
water
bath
for
a
short
time,
then
reagitate
the
sample
before
placing
it
in
the
turbidimeter.
Read
turbidity
directly
from
instrument
display.
Note:
Measurements
should
be
within
the
calibration
range.

°
Measuring
Sample
Turbidity
Using
GLI
Method
2
or
Revised
EPA
Method
180.1.
Different
procedures
should
be
followed,
depending
on
the
turbidity
of
the
sample:

°
For
turbidities
estimated
to
be
less
than
40
NTU.
Shake
the
sample
thoroughly
to
disperse
the
solids.
After
waiting
for
the
air
bubbles
to
disappear,
pour
the
sample
into
the
turbidimeter
tube
and
read
directly
from
the
instrument
scale.

°
For
turbidities
estimated
to
be
greater
than
40
NTU.
Dilute
the
sample
with
turbidity­
free
water
and
compute
the
turbidity
with
the
dilution
factor
included.

6.4.4.2
General
Quality
Control
for
Turbidity
Measurements
Utilities
performing
environmental
sample
measurements
must
be
approved
by
the
State
(
or
EPA
Region,
for
states
that
do
not
have
primacy)
under
the
drinking
water
laboratory
certification
program
[
40
CFR
part
141.706
(
c)].
Each
utility
laboratory
is
required
to
operate
a
formal
quality
control
(
QC)
program
and
to
maintain
performance
records
that
define
the
quality
of
the
data
generated.
Two
types
of
calibration
are
required
for
turbidity
measurements:

°
A
primary
suspension
standard.
The
primary
suspension
standard
should
be
used
to
calibrate
the
turbidimeter
initially
and
at
least
every
four
months
in
order
to
prevent
instrument
drift.
The
calibration
should
be
documented.
The
standards
should
be
replaced
when
they
exceed
the
expiration
date.

Acceptable
primary
suspensions
include
Formazin
(
a
recipe
for
preparation
can
be
found
at
EPA
Method
180.1
and
Standard
Method
2130B),
AMCO­
AEPA­
1
(
available
from
Advanced
Polymer
Systems),
and
Hach
StablCal
Stabilized
Formazin
Standards
(
available
from
Hach
Company).
Please
note
that
Formazin
standards
are
relatively
unstable,
particularly
at
low
concentrations.
Therefore,
dilutions
used
for
calibration
need
to
be
prepared
on
the
day
they
will
be
used.
Stock
solutions
may
be
stable
for
a
month
(
at
400
NTU)
to
1
year
(
at
4000
NTU).
Consult
an
approved
method
for
more
information.
Section
6:
Collecting
and
Shipping
Source
Water
Samples
46
Draft
June
2003
°
A
secondary
suspension
standard.
The
secondary
suspension
standard
is
used
daily
to
check
the
calibration
of
the
instrument.
The
calibration
should
be
documented,
and
should
not
vary
by
more
than
10%
from
the
initial
calibration
values
(
if
they
do
vary
by
more
than
10%,
the
system
should
be
corrected
so
that
performance
is
acceptable).
The
standards
should
be
replaced
when
they
exceed
the
expiration
date.

Acceptable
secondary
standards
include
all
primary
standards,
or
other
materials
that
are
suggested
by
instrument
manufacturers
 
such
as
sealed
sample
cells
filled
with
a
labeled
suspension
or
metal
oxide
particulates
in
a
polymer
gel,
or
a
turbid
glass
cube.
The
purpose
of
the
secondary
standard
is
to
provide
a
quick
check
of
calibration.
The
secondary
standards
should
have
a
fixed
turbidity
that
does
not
vary
from
use
to
use.

6.4.5
Monitoring
Sample
Temperature
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
9.5)
demonstrated
that
E.
coli
samples
can
freeze
under
these
conditions
if
samples
are
not
packed
properly.

The
sample
collection
protocols
procedures
in
Appendices
D,
E,
F,
and
G
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
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.

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)

The
sample
collection
protocols
discussed
in
Section
6.4.2
for
Cryptosporidium
samples
and
Section
6.4.3
for
E.
coli
samples
provide
guidance
on
packing
samples
to
maintain
appropriate
temperatures.
Utility
personnel
should
follow
these
procedures
to
ensure
that
samples
remain
at
acceptable
temperatures
during
shipment.
Section
6:
Collecting
and
Shipping
Source
Water
Samples
47
Draft
June
2003
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.

All
temperature
measurement
devices
should
be
calibrated
routinely
to
ensure
accurate
measurements.
See
the
U.
S.
EPA
Manual
for
the
Certification
of
Laboratories
Analyzing
Drinking
Water
(
Reference
9.3)
for
more
information.
48
Draft
June
2003
SECTION
7:
REVIEWING
CRYPTOSPORIDIUM
DATA
When
Cryptosporidium
samples
are
processed
and
analyzed
by
the
laboratory,
data
on
sample
measurements,
sample
processing
times,
and
slide
examination
results
are
recorded
at
the
laboratory
and
reported
to
the
PWS
through
the
LT2
Data
Collection
System
and
via
hardcopy
forms.
This
section
provides
an
overview
of
the
data
recording
and
reporting
processes
and
discusses
the
significance
of
the
examination
results
reported
by
the
laboratory.
This
section
also
provides
guidance
to
those
PWSs
interested
in
reviewing
laboratory
data.

7.1
Cryptosporidium
Data
Recording
at
the
Laboratory
The
Cryptosporidium
laboratory
records
LT2
rule
monitoring
data
using
the
following
standardized
forms:

7.1.1
LT2
Sample
Collection
Form
This
form
(
an
example
of
which
is
provided
as
Appendix
C)
is
initiated
at
the
plant
upon
sample
collection
and
is
completed
at
the
laboratory.
The
following
information
is
recorded
on
this
form
by
the
Cryptosporidium
laboratory:

°
Date
and
time
of
sample
receipt
°
Laboratory
personnel
receiving
the
sample
°
Sample
temperature
upon
receipt
°
Sample
condition
upon
receipt
Although
none
of
this
information
is
entered
into
the
LT2
data
collection
system,
it
provides
documentation
for
the
utility,
the
laboratory,
and
EPA
or
State
officials
on
sample
receipt
information
relevant
to
LT2
rule
requirements
regarding
sample
temperatures
and
sample
holding
times.

7.1.2
Method
1622/
1623
Bench
Sheet
The
laboratory
uses
the
bench
sheet
to
record
all
information
associated
with
sample
processing,
up
to,
but
not
including,
sample
examination.
Information
on
filtration
(
if
performed
in
the
laboratory),
elution,
concentration,
immunomagnetic
separation,
and
sample
staining
are
documented
on
this
form.
These
data
include:

°
Sample
ID
°
Dates
and
times
for
all
steps
associated
with
method­
required
holding
times
°
All
primary
measurements
used
to
calculate
sample
volume
analyzed,
if
less
than
100%
of
the
volume
filtered
was
analyzed.
This
information
includes
the
following:
°
The
volume
of
the
sample
after
the
concentrate
(
packed
pellet)
has
been
resuspended
°
The
volume
of
this
resuspended
concentrate
that
was
actually
analyzed
(
These
two
values
are
used
to
calculate
the
percent
of
the
sample
volume
analyzed,
if
less
than
100%
of
the
volume
filtered
was
analyzed.)
Section
7:
Reviewing
Cryptosporidium
Data
49
Draft
June
2003
°
Filter
clog
and
packed
pellet
information,
which
may
need
to
be
provided
to
demonstrate
compliance
with
LT2
rule
sample
analysis
requirements
if
less
than
10
L
was
analyzed
°
Cryptosporidium
spiking
information
for
OPR
and
MS
samples
°
Analyst
names
or
initials
for
each
step
°
Reagent
and
filter
lot
information
7.1.3
Method
1622/
1623
Cryptosporidium
Slide
Examination
Form
The
laboratory
uses
the
slide
examination
form
to
document
detailed
information
on
slide
examination.
This
information
includes
the
following:
°
Sample
ID
°
Date
and
time
the
examination
was
completed
°
Positive
and
negative
staining
control
results
°
Detailed
information
on
the
characteristics
of
each
object
on
the
slide
that
the
analyst
determined
was
a
Cryptosporidium
oocyst,
including
the
following:
°
Size
of
the
oocyst
°
Shape
of
the
oocyst
°
Whether
the
DAPI
stain
applied
to
the
sample
revealed
the
presence
of
nuclei,
and,
if
so,
how
many
were
observed
by
the
analyst
°
Whether
the
analyst
observed
internal
structures
during
DIC
examination
7.2
Submitting
Cryptosporidium
Data
through
the
LT2
Data
Collection
System
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
7­
1,
below.
Section
7:
Reviewing
Cryptosporidium
Data
50
Draft
June
2003
Table
7­
1.
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.

7.2.1
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.
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.

°
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.
Section
7:
Reviewing
Cryptosporidium
Data
51
Draft
June
2003
°
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
(
see
Section
6.1),
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.

7.2.2
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.

7.2.3
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.

7.3
What
Do
the
Sample
Examination
Results
Mean?

As
noted
in
Section
3.1,
the
laboratory
applies
two
stains
to
a
sample
slide,
and
then
examines
the
sample
using
three
different
techniques
to
determine
whether
objects
that
cannot
be
ruled
out
as
Cryptosporidium
oocysts
are
on
a
sample
slide.
A
description
of
these
stains
and
techniques
 
and
how
each
is
used
to
evaluate
objects
examined
by
the
analyst,
is
provided
below.

7.3.1
Immunofluorescent
Assay
(
IFA)

One
of
the
two
stains
added
to
the
sample
before
examination
is
a
fluorescent
antibody
stain
that
reacts
with
Cryptosporidium.
The
antibodies
in
this
stain,
which
exhibit
an
intense
apple­
green
fluorescence
Section
7:
Reviewing
Cryptosporidium
Data
52
Draft
June
2003
when
the
slide
is
examined
using
ultraviolet
light,
will
attach
to
Cryptosporidium
oocysts
that
may
be
present
in
the
sample.
During
IFA,
the
analyst
scans
the
entire
well
at
relatively
low
magnification
(
200X)
for
apple­
green
fluorescing
objects
the
size
and
shape
of
oocysts.
If
such
an
object
is
located,
the
analyst
proceeds
to
the
next
step
in
the
examination
process.
The
analyst
cannot
conclude
at
this
stage
that
an
apple­
green
fluorescing
organism
the
size
and
shape
of
a
Cryptosporidium
oocyst
is
indeed
an
oocyst
because
the
object
may
be
another
organism
that
has
cross­
reacted
with
the
antibody
stain.
Additional
examination
procedures
are
used
to
determine
whether
this
is
the
case.

7.3.2
4',
6­
diamadino­
2­
phenylindole
(
DAPI)
Examination
The
second
stain
added
to
the
sample
before
examination
is
DAPI,
a
dye
that
interacts
with
nucleic
acids
and
stains
nuclei
that
may
be
present
within
the
oocyst.
The
DAPI
stain
fluoresces
when
the
slide
is
examined
using
ultraviolet
light.
During
the
DAPI
examination,
the
analyst
observes
the
object
at
medium
magnification
(
400X)
to
determine
whether
it
contains
stained
nuclei.
Cryptosporidium
oocysts
contain
four
nuclei.

Although
looking
for
four
nuclei
during
DAPI
examination,
if
the
object
has
less
than
four
nuclei,
the
analyst
cannot
rule
out
the
possibility
that
the
organism
is
a
Cryptosporidium
oocyst.
For
example,
if
less
than
four
stained
nuclei
are
observed,
the
object
may
actually
have
four
nuclei,
but
some
may
not
be
visible
in
the
plane
of
focus.
Similarly,
objects
in
which
no
stained
nuclei
are
observed
may
be
organisms
other
than
Cryptosporidium,
may
be
dead
Cryptosporidium
oocysts,
or
may
even
be
live
oocysts
that
are
resistant
to
DAPI
staining.

The
DAPI
examination
is
one
of
several
tools
for
the
analyst
to
use
to
determine
whether
an
object
is
an
oocyst.
The
analyst
cannot
conclude
whether
the
object
is
an
oocyst
based
on
this
examination
alone,
nor
can
the
analyst
conclude,
based
on
negative
results,
that
the
organism
is
non­
infectious.
As
a
result,
the
analyst
must
proceed
to
the
next
step
in
the
examination
process,
even
if
less
than
four
nuclei
are
observed.

7.3.3
Differential
Interference
Contrast
(
DIC)
Examination
The
third
evaluation
performed
by
the
analyst
is
an
examination
of
the
object
at
high
magnification
(
1000X).
Using
DIC,
the
analyst
looks
at
the
object's
external
or
internal
morphological
characteristics
(
this
does
not
require
the
use
of
a
stain).
The
analyst
looks
for
characteristics
atypical
of
Cryptosporidium
oocysts
(
e.
g.,
spikes,
stalks,
appendages,
pores,
one
or
two
large
nuclei
filling
the
cell,
crystals,
spores,
etc.).
If
atypical
structures
are
not
observed,
and
the
object
cannot
be
ruled
out
as
an
oocyst
based
on
the
results
of
the
IFA
and
the
DAPI
examination,
the
analyst
reports
this
object
as
a
Cryptosporidium
oocyst.

Based
on
the
DIC
examination,
the
size
of
the
object
is
determined
and
compared
to
the
acceptable
range
for
the
target
organism.
If
the
size
and
shape
of
the
object
is
within
the
acceptable
range,
the
analyst
records
the
size
and
shape
and
characterizes
the
Cryptosporidium
oocyst
in
one
of
three
ways:
(
1)
an
oocyst
with
internal
structures,
i.
e.,
those
having
recognizable
structures
consistent
with
Cryptosporidium,
(
2)
an
oocyst
with
amorphous
structures,
or
(
3)
an
empty
oocyst.
Assignment
of
these
characterizations
is
dependent
on
analyst
judgement
and
none
of
these
characterizations
is
a
direct
indicator
of
whether
oocysts
are
viable
and
infectious.

7.5
Reviewing
and
Validating
Raw
Cryptosporidium
Data
(
Optional)

If
your
PWS
plans
to
review
the
raw
data
generated
by
the
laboratory,
you
should
request
from
the
laboratory
the
hardcopy
data
needed
to
verify
the
electronic
results
(
see
Section
5.1.5).
However,
this
step
is
not
required.
However,
for
those
PWSs
interested
in
taking
this
extra
step,
Sections
7.5.1
through
7.5.3
provide
guidance
on
how
to
review
and
validate
hardcopy
data
and
verify
accuracy.
Section
7:
Reviewing
Cryptosporidium
Data
53
Draft
June
2003
7.5.1
Data
Completeness
Check
Upon
receipt
of
the
hardcopy
sample
results
for
a
monitoring
sample,
verify
that
the
laboratory
has
submitted
the
following
materials:

°
Sample
result
summary
sheet,
which
should
include
the
following:

°
Monitoring
sample
identification
information
°
Monitoring
sample
result,
in
oocysts/
L
°
Laboratory
quality
control
batch
associated
with
the
sample
°
Result
for
the
ongoing
precision
and
recovery
(
OPR)
sample
analyzed
for
this
QC
batch
°
Result
for
the
method
blank
sample
analyzed
for
this
QC
batch
°
LT2
sample
collection
form
initiated
by
your
utility
and
completed
with
sample
receipt
information
by
the
laboratory
°
Method
1622/
1623
Bench
Sheet
with
raw
data
associated
with
the
monitoring
sample
(
and
MS
sample,
if
applicable)

°
Method
1622/
1623
Cryptosporidium
Slide
Examination
Form
with
raw
data
for
the
monitoring
sample
(
and
MS
sample,
if
applicable)

°
Laboratory
comments.
If
the
laboratory
provided
comments
on
the
sample
analyses
or
results
that
require
follow­
up,
contact
the
laboratory
to
discuss,
if
necessary.
Comments
may
include
any
applicable
data
qualifiers.
The
following
is
a
list
of
potential
data
qualifiers:

°
The
recovery
for
the
associated
ongoing
precision
and
recovery
(
OPR)
sample
did
not
met
method
requirements
°
Oocysts
were
detected
in
the
method
blank
°
Positive
and
negative
staining
controls
were
not
acceptable
or
not
examined
°
Method
holding
times
were
not
met
°
Sample
arrived
at
the
laboratory
in
unacceptable
condition
Any
of
the
above
data
qualifiers
would
result
in
the
sample
being
considered
invalid
for
LT2
use
and
the
laboratory
should
not
report
the
results
for
the
sample
to
EPA.
The
PWS
may
be
required
to
resample.

If
forms
are
missing,
incomplete,
or
incorrect,
contact
the
laboratory
immediately
to
discuss
and
request
resubmission
of
the
missing
forms
and/
or
spreadsheets.

7.5.2
Evaluation
of
Data
Against
Method
Quality
Control
Requirements
To
verify
that
the
laboratory
analyzed
your
monitoring
sample
within
the
analytical
controls
specified
by
the
method,
check
the
following
information:

°
Sample
condition
upon
receipt.
Verify
on
the
completed
LT2
sample
collection
form
that
your
sample
was
received
in
acceptable
condition
(
not
leaking,
etc.),
and
at
a
temperature
between
0
°
C
and
10
°
C,
and
not
frozen.

°
Method
blank.
Verify
that
the
laboratory
analyzed
a
method
blank
with
the
monitoring
sample's
QC
batch
and
confirm
that
the
method
blank
did
not
contain
any
oocysts.
Section
7:
Reviewing
Cryptosporidium
Data
54
Draft
June
2003
°
Ongoing
precision
and
recovery
sample.
Verify
that
the
laboratory
analyzed
an
OPR
sample
with
the
monitoring
sample's
QC
batch
and
that
the
OPR
sample
recovery
was
between
11%
and
100%,
as
required
by
EPA
Methods
1622
and
1623.

°
Holding
times.
Using
the
sample
collection
date
and
time
on
the
LT2
data
collection
form
and
the
dates
and
times
of
the
method
steps
recorded
by
the
laboratory
on
the
Method
1622/
1623
bench
sheet
and
report
form
for
the
monitoring
sample,
verify
the
following:

°
The
laboratory
began
elution
no
more
than
96
hours
from
sample
collection
°
The
laboratory
performed
the
elution,
concentration,
purification,
and
slide
preparation
(
application
of
the
purified
sample
to
the
slide)
within
1
working
day
(
the
date
of
the
elution
step
should
be
the
same
as
the
date
of
the
slide
preparation
step)

°
The
laboratory
stained
the
slide
within
72
hours
of
application
of
the
purified
sample
to
the
slide
(
generally,
the
date
next
to
the
slide
staining
step
should
be
no
more
than
3
days
later
than
the
date
next
to
the
slide
preparation
step)

°
The
laboratory
examined
the
slide
within
7
days
of
staining
(
the
examination
date
should
be
no
more
than
7
days
from
the
slide
staining
date)

°
Positive
and
negative
staining
controls.
Based
on
the
information
at
the
top
of
the
Method
1622/
1623
Cryptosporidium
reporting
form,
verify
that
the
laboratory
performed
positive
and
negative
staining
controls,
and
that
the
results
of
these
controls
were
acceptable.

7.5.3
Calculation
Verification
The
laboratory
does
not
directly
report
the
final
concentration
of
oocysts/
L
in
the
sample
to
EPA.
Instead,
they
report
a
series
of
primary
measurements
that
are
used
by
the
LT2
data
system
to
automatically
calculate
the
final
concentration.
The
volume
filtered,
the
total
volume
of
resuspended
concentrate,
and
the
volume
transferred
to
IMS
are
used
to
determine
the
volume
analyzed.
The
laboratory
also
records
the
total
count
of
oocysts
detected,
which
is
divided
by
the
volume
analyzed
to
determine
the
final
concentration
of
oocysts/
L.
Although
the
final
results
are
automatically
calculated
by
the
LT2
data
collection
system
using
the
primary
measurements
supplied
by
the
laboratory,
the
plant
still
may
wish
to
verify
them.
Guidance
on
recalculating
and
verifying
final
results
using
primary
measurements
is
provided
below.

7.5.3.1
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
Using
these
two
data
elements,
the
final
concentration
is
calculated
as:

final
concentration
=
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
Section
7:
Reviewing
Cryptosporidium
Data
55
Draft
June
2003
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
below.

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
(
see
Section
7.1.2)

total
volume
of
resuspended
concentrate
produced
volume
analyzed
(
L)
=
percent
examined
×
sample
volume
filtered
Determining
the
Volume
of
Resuspended
Concentrate
to
Use
for
Packed
Pellets
>
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
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
volume
analyzed
(
L)
=
0.74
×
10
L
=
7.4
L
final
concentration
(
oocysts/
L)
=
20
oocysts
=
2.7
oocysts/
L
7.4
L
7.5.3.2
Matrix
Spike
Sample
Calculations
For
matrix
spike
(
MS)
samples,
the
laboratory
records
all
of
the
same
information
that
is
recorded
for
field
samples,
in
addition
to
information
specific
to
matrix
spike
samples.
The
sample
volume
spiked
and
estimated
number
of
oocysts
spiked
into
the
sample
are
used
to
calculate
the
concentration
of
spiked
Section
7:
Reviewing
Cryptosporidium
Data
56
Draft
June
2003
organisms
in
the
sample.
To
correct
for
background
concentration,
the
number
of
organisms
detected
in
the
unspiked
field
sample
is
subtracted
from
the
number
of
oocysts
detected
in
the
MS
sample.

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
true
value
of
the
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
7.6
Data
Archiving
Requirements
LT2
rule
monitoring
data
must
keep
monitoring
results
until
36
months
after
source
water
monitoring
has
been
completed.
Although
it
is
the
PWS's
responsibility
to
meet
LT2
rule
data
storage
requirements
for
compliance
monitoring
samples,
the
PWS
may
designate
this
responsibility
to
the
laboratory.

Although
not
required,
laboratories
also
can
archive
slides
and/
or
take
photographs
of
slides
to
maintain
for
clients.
As
noted
in
Section
5.1.2.5,
this
may
be
considered
an
extra
service
and
result
in
extra
costs,
as
these
steps
may
not
be
routinely
performed
by
the
laboratory.
Slides
should
be
stored
in
a
humid
chamber
in
the
dark
at
0
°
C
to
10
°
C.
An
alternative
mounting
medium
also
may
be
used,
which
may
potentially
preserve
slides
longer.
Details
are
provided
in
the
Microbial
Laboratory
Guidance
Manual
for
the
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
(
LT2
Rule).
57
Draft
June
2003
SECTION
8:
REVIEWING
E.
COLI
DATA
When
E.
coli
samples
are
processed
and
analyzed
by
the
laboratory,
data
on
sample
measurements,
sample
processing
times,
and
slide
examination
results
are
recorded
at
the
laboratory
and
reported
to
the
PWS
through
the
LT2
Data
Collection
System.
This
section
provides
an
overview
of
the
data
recording
and
reporting
processes
and
provides
guidance
on
how
to
review
the
data
you
receive
from
the
laboratory.

8.1
E.
coli
Laboratory
Data
Recording
at
the
Laboratory
The
laboratories
performing
E.
coli
analyses
during
the
LT2
rule
record
the
following
general
types
of
information:

°
Sample
identification
information
°
All
primary
measurements
used
to
calculate
the
final
E.
coli
concentration
for
each
sample
°
The
incubation
start
and
read
times
for
each
method
to
verify
that
method
requirements
were
met
°
The
name
of
the
analyst
performing
the
sample
analysis
°
Quality
control
(
QC)
analysis
results
(
e.
g.,
positive/
negative
controls,
blanks,
etc.)

8.1.1
Sample
Identification
Information
Sample
identification
information
is
used
to
track
the
sample
through
sample
collection,
analysis,
and
data
reporting.
At
a
minimum,
the
laboratory
records
the
sample
ID,
the
target
parameter
(
E.
coli),
and
the
method
being
used
(
e.
g.,
Membrane
Filtration:
SM
9222D/
SM
9222G).

8.1.2
Primary
Data
The
laboratory
records
all
primary
measurements
needed
to
calculate
the
final
concentration
of
E.
coli
per
100
mL.
Primary
measurements
for
membrane
filtration
methods
will
include
the
volumes
filtered
and
the
plate
counts
for
each
volume
filtered.
The
multiple­
well
and
multiple­
tube
formats
will
include
the
volumes
or
dilutions
of
samples
analyzed
and
the
number
of
positive
wells
or
tubes
per
each
volume
analyzed.

8.1.3
Sample
Processing
and
Quality
Control
Information
The
laboratory
records
information
on
the
bench
sheet
on
how
they
processed
and
analyzed
the
sample,
including
incubation
start/
end
date
and
times
and
temperature,
and
the
analyst
performing
each
step
of
the
method.
The
lot
numbers
of
reagents,
media,
and
materials
used
to
process
the
sample
and
the
results
of
QC
analyses
should
be
recorded
in
a
media
log
book
or
QC
checklist.
In
addition
to
being
used
to
resolve
questions
regarding
validity
of
results,
this
information
may
be
used
by
the
laboratory
to
determine
the
source
of
any
problems
the
laboratory
is
having
with
method
performance.
Section
8:
Reviewing
E.
coli
Data
58
Draft
June
2003
8.1.4
Sample
Results
The
final
E.
coli
concentration
for
field
samples
will
be
reported
as
CFU/
100
mL
or
MPN/
100
mL
depending
on
the
method
used
for
analysis.
If
no
E.
coli
are
detected
in
the
sample,
a
low
censored
value
based
on
the
volume
of
sample
analyzed
must
be
reported
(
e.
g.
<
1CFU
/
100
mL
or
<
1.8
MPN/
100
mL).
E.
coli
concentration
will
never
be
reported
as
a
zero.

8.2
Submission
of
E.
coli
Data
through
the
LT2
Data
Collection
System
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
7­
1,
in
Section
7.2,
above.

The
data
reporting
process
is
summarized
below,
in
Sections
8.2.1
through
8.2.3,
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
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.

8.2.1
Data
Entry/
Upload
The
analyst
or
another
laboratory
staff
member
enters
a
subset
of
the
data
recorded
at
the
bench
(
Section
8.1)
into
the
LT2
Data
Collection
System
either
by
entering
the
data
using
web
forms
or
by
uploading
data
in
XML
format.
This
information
includes
the
following:

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

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

°
E.
coli/
100
mL
(
see
note
below)

Note:
The
laboratory
may
then
enter
the
final
result
for
the
sample
that
was
calculated
at
the
laboratory
or
may
enter
the
primary
measurements
recorded
at
the
bench,
and
have
the
LT2
Data
Collection
System
automatically
calculate
the
final
sample
concentration.
Because
this
information
is
specific
to
method
type
(
membrane
filtration,
multiple
tube
fermentation,
51­
well,
and
97­
well),
the
system
provides
Section
8:
Reviewing
E.
coli
Data
59
Draft
June
2003
different
entry
screens
for
each
method
type.
The
laboratory
staff
entering
the
data
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
approved
for
PWS
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,
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.

8.2.2
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."

8.2.3
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.

8.3
Reviewing
and
Validating
E.
coli
Data
(
Optional)

If
the
PWS
staff
responsible
for
submitting
data
to
EPA
plans
to
review
the
raw
data
generated
by
the
laboratory,
the
original,
hardcopy
records
(
whether
generated
by
an
in­
house
laboratory
or
a
contract
laboratory)
should
be
compared
to
the
electronic
results.
However,
this
step
is
not
required.
Sections
8.3.1
through
8.3.3
provides
guidance
on
how
to
review
and
validate
the
hardcopy
data
and
verify
accuracy.

8.3.1
Data
Completeness
Check
Upon
receipt
of
hardcopy
sample
results
for
a
monitoring
sample,
verify
that
the
following
information
is
included:

°
Sample
result
summary
sheet,
which
should
include
the
following:

°
Monitoring
sample
identification
information
°
Monitoring
sample
result,
in
E.
coli/
100
mL
°
Laboratory
quality
control
checklist
(
or
other
verification
from
the
laboratory
that
all
QC
specifications
were
met)
Section
8:
Reviewing
E.
coli
Data
60
Draft
June
2003
°
LT2
sample
collection
form
initiated
at
the
time
of
sample
collection
and
completed
with
sample
receipt
information
by
the
laboratory
°
E.
coli
Method
Bench
Sheet
completed
by
the
laboratory
with
primary
sample
processing
and
analysis
data
associated
with
the
monitoring
sample
°
Laboratory
comments.
If
the
laboratory
provided
comments
on
the
sample
analyses
or
results
that
require
follow­
up,
contact
the
laboratory
to
discuss,
if
necessary.
Comments
may
include
any
applicable
data
qualifiers.
The
following
is
a
list
of
potential
data
qualifiers:

°
Sample
arrived
at
the
laboratory
in
unacceptable
condition
(
i.
e.,
leaking)

°
Sample
holding
time
exceeded
°
Sample
holding
temperature
not
within
acceptable
range
°
Unacceptable
blank
sample
result
°
Unacceptable
positive
or
negative
control
result
°
Media
sterility
checks
were
not
acceptable
°
Method
incubation
times
or
temperatures
were
not
within
acceptable
range
°
Membrane
filtration:
Too
much
sediment
on
the
filter
°
Membrane
filtration:
Confluent
growth
of
non­
target
organism
°
Membrane
filtration:
Colonies
too
numerous
to
count
(
TNTC)

°
Membrane
filtration:
Pre­
or
post­
filtration
series
sterility
check
not
acceptable
(
e.
g.,
contamination
with
E.
coli
organism)

°
Quanti­
Tray
®
was
damaged
or
leaked
°
Sample
was
not
distributed
to
all
wells
in
Quanti­
Tray
®
°
All
rows
of
tubes
were
not
inoculated
°
Positive
presumptive
tubes
were
not
transferred
into
the
appropriate
confirmatory
medium
Any
of
the
above
data
qualifiers
would
result
in
the
sample
being
considered
invalid
for
LT2
use
and
the
results
for
the
sample
should
not
be
entered
into
the
LT2
data
collection
system.
If
the
laboratory
enters
the
results
into
the
LT2
data
collection
system,
the
PWS
should
not
submit
the
results
to
EPA.

If
forms
are
missing,
incomplete,
or
incorrect,
contact
the
laboratory
immediately
to
discuss
and
request
resubmission
of
the
missing
forms
and/
or
spreadsheets.

8.3.2
Evaluation
of
Data
Against
Method
Quality
Control
Requirements
To
verify
that
the
laboratory
analyzed
your
monitoring
sample
within
the
analytical
controls
specified
by
the
method,
check
the
following
information:

°
Sample
condition
upon
receipt.
If
the
sample
was
shipped
to
the
laboratory,
verify
on
the
completed
LT2
sample
collection
form
that
your
sample
was
received
in
acceptable
condition
(
e.
g.,
not
leaking,
etc.),
and
at
a
temperature
below
10
°
C,
but
not
frozen.

°
QC
samples
associated
field
samples.
The
frequency
of
analysis
of
quality
control
samples
including
method
blanks,
positive
and
negative
controls,
etc.
varies
according
to
method
requirements
Section
8:
Reviewing
E.
coli
Data
61
Draft
June
2003
and
specifications
in
the
Certification
Manual.
Verify
that
the
required
QC
samples
were
run
with
the
field
sample.
A
summary
of
these
QC
specifications
is
provided
in
Section
4.2
of
this
document.

°
Holding
time.
Using
the
sample
collection
date
and
time
on
the
LT2
data
collection
form
and
the
date
and
time
of
the
first
method
step
recorded
by
the
laboratory
on
the
E.
coli
method
bench
sheet,
verify
that
the
laboratory
began
sample
analysis
within
24
hours
of
sample
collection.

°
Incubation
times
and
temperatures.
Using
the
dates
and
times
of
the
method
steps
recorded
by
the
laboratory
on
the
E.
coli
method
bench
sheet,
verify
that
the
method­
specified
incubation
times
and
temperatures,
specified
in
Table
8­
1
were
met.

Table
8­
1.
Incubation
Times
and
Temperatures
for
Approved
E.
Coli
Methods
Method
Media
Incubation
Time/
Temperature
Standard
Methods
9223B
Colilert
®
24
to
28
hours
at
35
°
C
±
0.5
°
C
Colilert­
18
®
18
to
22
hours
at
35
°
C
±
0.5
°
C
Standard
Methods
9221B/
F
LTB
24
±
2
and
48
±
3
hours
at
35
°
C
±
0.5
°
C
EC­
MUG
24
±
2
hours
at
44.5
°
C
±
0.2
°
C
Standard
Methods
9222B/
9222G
mENDO
!
NA­
MUG
24
±
2
hours
at
35
°
C
±
0.5
°
C
!
4
hours
at
35
°
C
±
0.5
°
C
LES­
ENDO
!
NA­
MUG
24
±
2
hours
at
35
°
C
±
0.5
°
C
!
4
hours
at
35
°
C
±
0.5
°
C
Standard
Methods
9222D/
9222G
mFC
!
NA­
MUG
24
±
2
hours
at
44.5
°
C
±
0.2
°
C
!
4
hours
at
35
°
C
±
0.5
°
C
Standard
Methods
9213D
mTEC
agar
2
hours
at
35
°
C
±
0.5
°
C
!
22
to
24
hours
at
44.5
°
C
±
0.2
°
C
EPA
1603
Modified
mTEC
2
hours
at
35
°
C
±
0.5
°
C
!
22
to
24
hours
at
44.5
°
C
±
0.2
°
C
EPA
1604
MI
medium
24
hours
at
35
°
C
±
0.5
°
C
Other
Membrane
Filter
Method
m­
ColiBlue24
®
®
Broth
24
hours
at
35
°
C
±
0.5
°
C
8.3.3
Calculation
Verification
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
8.3.3.1
through
8.3.3.4.

8.3.3.1
Calculations
for
Determining
the
E.
coli
Concentration
Using
the
Colilert
®
Quanti­
Tray
2000
®
(
97­
well)

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.

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.
Section
8:
Reviewing
E.
coli
Data
62
Draft
June
2003
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
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
8.3.3.2
Calculations
for
Determining
the
E.
coli
Concentration
Using
the
Colilert
®
Quanti­
Tray
51
®
(
51­
well)

A.
Select
appropriate
dilution.
If
multiple
dilutions
are
used,
the
tray
exhibiting
positive
wells
around
the
80%
range
(
41
positive
wells)
should
be
used
to
determine
MPN
value.

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
26
positive
wells,
the
corresponding
MPN
would
be
36.4
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
Section
8:
Reviewing
E.
coli
Data
63
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.
8.3.3.3
Calculations
for
determining
the
E.
coli
concentration
using
membrane
filter
data
(
adapted
from
Reference
9.4)

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
8.2.1
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
plates
results
in
CNFG,
then
potentially
0.01
mL
should
be
analyzed
during
the
next
sampling
event.
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.

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.
Section
8:
Reviewing
E.
coli
Data
64
Draft
June
2003
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
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)
Section
8:
Reviewing
E.
coli
Data
65
Draft
June
2003
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)

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
8.3.3.4
Calculation
of
E.
coli
Concentrations
Using
Multiple­
Tube
Methods
(
adapted
from
Reference
9.6):

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
8.2.1
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
significant
dilutions
and
are
selected
according
to
the
following
criteria.
Examples
of
significant
dilution
selections
are
provided
in
Table
8­
2,
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
8­
2,
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
8­
2,
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
8­
2,
Example
D).
Section
8:
Reviewing
E.
coli
Data
66
Draft
June
2003
°
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
8­
2,
Example
E).

°
If
there
were
not
enough
higher
dilutions
tested
to
select
three
dilutions,
then
select
the
next
lower
dilution
(
Table
8­
2,
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
8­
2.
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
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:
Section
8:
Reviewing
E.
coli
Data
67
Draft
June
2003
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
0.001
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
8.4
Data
Archiving
Requirements
Under
the
LT2
rule,
monitoring
data
must
keep
until
36
months
after
source
water
monitoring
has
been
completed
[
40
CFR
part
141.731
(
a)].
Although
it
is
the
PWS's
responsibility
to
meet
LT2
rule
data
storage
requirements
for
compliance
monitoring
samples,
the
PWS
may
designate
this
responsibility
to
the
laboratory.
68
Draft
June
2003
SECTION
9:
REFERENCES
9.1
Connell,
Kevin,
et
al.
2000.
ICRSS
­
Building
a
Better
Protozoa
Data
Set,
J.
AWWA.
91(
10):
30
­
43.

9.2
Pope,
Misty,
et
al.
2003.
"
Using
E.
coli
To
Indicate
Source
Water
Susceptibility
to
High
Concentrations
of
Cryptosporidium,"
in
Information
Collection
Rule
Data
Analysis.
AWWARF,
Denver,
CO.

9.3
USEPA.
1997.
Manual
for
the
Certification
of
Laboratories
Analyzing
Drinking
Water;
Criteria
and
Procedures;
Quality
Assurance:
Fourth
Edition.
EPA
815­
B­
97­
001.

9.4
APHA.
1998.
Standard
Methods
for
the
Examination
of
Water
and
Wastewater;
20th
Edition.
American
Public
Health
Association,
American
Water
Works
Association,
Washington,
D.
C.

9.5
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).

9.6
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/
cgibin
detail?
product_
id=
923645.
69
Draft
June
2003
SECTION
10:
ACRONYMS
CFU
Colony­
forming
unit
CNFG
Confluent
growth
DAPI
4,
6­
diamidino­
2­
phenylindole
DIC
Differential
interference
contrast
EPA
U.
S.
Environmental
Protection
Agency
FA
Immunofluorescense
assay
FITC
Fluorescien
isothiocyanate
GWUDI
Ground
water
under
the
direct
influence
of
surface
water
ICR
Information
Collection
Rule
IFA
Immunofluorescence
assay
IMS
Immunomagnetic
separation
IPR
Initial
precision
and
recovery
IPT
Initial
proficiency
testing
L
Liter
LT2
rule
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
LT2ESWTR
Long
Term
2
Enhanced
Surface
Water
Treatment
Rule
MPN
Most
probable
number
MS
Matrix
spike
MS/
MSD
Matrix
spike/
matrix
spike
duplicate
µ
m
Micrometer
NA­
MUG
Nutrient
agar
(
NA)
with
4­
methylumbelliferyl­
beta­
D­
glucuronide
(
MUG)

nm
Nanometer
NPDWR
National
Primary
Drinking
Water
Regulations
NTU
Nephelometric
turbidity
unit
OPR
Ongoing
precision
and
recovery
OPT
Ongoing
proficiency
testing
PBMS
Performance­
based
measurement
system
PT
Proficiency
testing
PWS
Public
water
system
QA
Quality
assurance
Section
10:
Acronyms
70
Draft
June
2003
QAP
Quality
assurance
plan
QC
Quality
control
RSD
Relative
standard
deviation
SDWA
Safe
Drinking
Water
Act
TNTC
Too
numerous
to
count
UV
Ultraviolet
