Supplement 2 to the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water

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All characterization (DAPI and DIC) and size measurements must be
determined using 1000X total magnification and reported to the nearest 0.5 |im
[Section 15.2, Note {Section 15.2, Note)].

The microscopy segment of EPA Methods 1623 and 1623.1 requires proper
alignment and adjustment of sophisticated optics. Without proper alignment and
adjustment the microscope will not function at optimum efficiency and reliable
identification and enumeration of oocysts/cysts will not be possible.
Accordingly, it is important that all aspects of the microscope from the light
sources to the oculars be properly adjusted [Section 10.3.1.1 (Section 10.3)].
The laboratory should have a service agreement to service the microscope
annually in order to ensure the microscope is in alignment. Analysts should be
able to make minor adjustments that may be required for use of microscope.

Microscope adjustments (epifluorescent mercury bulb, transmitted bulb,
interpupillary distance and oculars, ocular adjustment for microscopes without
binocular capability) must be performed consistent with [Sections 10.3.2, 10.3.3
and 10.3.4 (Section 10.7, Appendix B.l andB.2)].

The microscope must be equipped with at least a 20X scanning objective and
100X oil immersion objectives [Section 6.9.1 (Section 6.7.1)].

The microscope must have an ocular micrometer or imaging software to
measure oocysts/cysts. The laboratory must have a stage micrometer available
to calibrate the ocular micrometer or imaging software. The ocular micrometer
should be calibrated for each objective used. The calibration procedures must be
followed when the microscope is first used and when an objective is replaced
[Sections 6.9.land 10.3.5 (Section 6.7.1 and Appendix B. 3)]. The calibration
information must be available at the corresponding microscope.

3.22.10	Kohler illumination must be established for the 100X oil objective for DIC
examination of morphological characteristics of oocysts/cysts. If more than one
objective is used for DIC microscopy, Kohler illumination should be
reestablished for the new objective lens each time the objective is changed. The
Kohler illumination procedure must be followed each time an analyst uses the
microscope [Section 10.3.6 (Section 10.8)].

3.22.11	The microscope should be cleaned after each period of use following the
procedure in [Section 10.4 (Section 10.9)] or using an equivalent procedure.

QC 3.22.12 The laboratory should maintain a log of the number of hours the UV bulb has

been used. Alternatively, a lamp hour meter may be used. Mercury bulbs should
not be used longer than they have been rated [Section 10.3.2.11 (Appendix
B.l.11)].

3.22.5
QC 3.22.6

3.22.7

3.22.8
QC 3.22.9

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4. General Laboratory Practices

Laboratories seeking certification should adhere to the laboratory practices described in

EPA Methods 1623 and/or 1623.1. Additional information is available in the attached

Checklists A, B, and C.

4.1	Laboratory Safety

4.1.1	Achievement of a safe workplace is the responsibility of the organization, the
laboratory manger, the supervisory personnel and the employees themselves.
All laboratory employees should make every effort to protect themselves and
their fellow employees by adhering to the health and safety program that has
been developed and documented specifically for their laboratory. Additional
information is available in Standard Methods Section 1090, [Sections 5.0 and
20.0 (Sections 5.0 and 20.0)].

4.1.2	Health and safety policies and procedures should be posted and/or readily
available to all personnel as part of the laboratory's SOPs.

4.1.3	The laboratory should disinfect bench surfaces before and after analysis.

4.1.4	The laboratory should provide and document training of all applicable personnel
in the proper use of personal protective equipment (PPE), precautionary
measures, and control of chemical and biological hazards. Safety training
requires a concerted effort by the laboratory and should be conducted on a
routine basis by competent and qualified individuals to be effective.

4.1.5	Laboratory operations that generate aerosols should be conducted in a biological
safety cabinet.

4.1.6	Personal protective clothing/equipment should not be worn in non-laboratory
areas. Gloves must be changed whenever they are contaminated. Laboratory
coats, which have been contaminated with hazardous chemicals/biological
materials, should be removed. Laboratory benches should be disinfected before
and after analyses and after a spill with a hard surface disinfectant capable of
inactivating oocyst/cysts.

4.1.7	Laboratory should record not only accidents, but also "near misses" to permit
full evaluation of the effectiveness of the health and safety program.

4.1.8	The laboratory should establish periodic reviews of the plan with documentation
of the recommendations and corrective measures implemented.

4.2	Chemicals/Reagents

QC 4.2.1 All chemicals and reagents used should meet the requirements specified in

Section 7.0 of EPAMethods 1623 and/or 1623.1. If not specified, "analytical

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reagent grade" or American Chemical Society (ACS) grade chemicals or better
should be used. All chemicals should be dated when received, opened, and
discarded within the expiration date.

QC 4.2.2 All reagents must be prepared using reagent water [Section 7.3 (Section 7.3)].

All reagents should be clearly labeled with the identity of the reagent(s), date of
preparation, expiration date, and technician's initials. All reagents should be
stored consistent with Section 7.0 of EPA Methods 1623 and/orl623.1.

4.3 Reagent Water

4.3.1 Only reagent water meeting the following acceptance criteria must be used for
preparing reagents:

Parameter

Limits

Frequency

Conductivity

>0.5 megohms or <2
|imhos/cm (|i si em ens/cm) at
25°C

Monthly

Pb, Cd, Cr, Cu, Ni, Zn

Not greater than 0.05 mg/L
per contaminant. Collectively
not greater than 0.1 mg/L

Annually

Total Chlorine Residual

<0.1 mg/L

Monthly

Heterotrophic Plate
Count2

< 500 CFU/mL or MPN <
500/mL3

Monthly

1DPD (N.N-diethyl-p-phenylenediamine) Method shoulc

be used

2Pour Plate Method (Standard Methods 9215B) or IDEXX SimPlate® Method

3Colony Forming Units (CFU) or Most Probable Number (MPN)

4.3.2 Additional information is available in Standard Methods 1080 and 9020.

QC 4.3.3 The reagent water still or DI unit should be maintained according to the
manufacturer's instructions. Maintenance should be documented.

4.4 Glassware Washing

4.4.1	All glassware and plastic ware should be thoroughly cleaned with a detergent
designed for laboratory use. Glassware and plastic ware should be cleansed with
hot water and detergent, rinsed with hot water to remove all traces of residual
detergent, and rinsed with distilled, deionized, or reagent water as the final
rinse. All contaminated laboratory glassware should be autoclaved or
chemically sterilized before cleaning.

4.4.2	The dishwasher influent and rinse water plumbing system should be made of
stainless steel or other nontoxic material.

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4.5 Quality Assurance

4.5.1	To ensure that analytical data generated under the rule are technically valid,
legally defensible, and of known and acceptable quality, each laboratory must
operate a formal QA program and document the scope of the program through a
QAplan, as specified in EPA Method 1623 andl623.1 Section9.1. All
laboratory activities including, but not limited to sampling, analytical methods,
QC checks, instrument operation, data generation, data validation, corrective
action procedures, and recording keeping should be described in the QA plan.

4.5.2	The QA plan should contain a laboratory organization chart or staff listing
which identifies staff organization and responsibilities, including QA manager
and lab director. All laboratory personnel should be familiar with the contents
of the QA plan. It is the responsibility of the laboratory QA manager to ensure
the QA plan is current. The laboratory QA manager should be independent from
the laboratory management and should have a working knowledge of the
statistics involved in QC of laboratory analyses and a basic understanding of the
methods the laboratory uses. Ideally, this person should have a staff position
reporting directly to upper management, not a line position.

4.5.3	The laboratory QA plan should contain a schedule of all preventative
maintenance for equipment.

4.5.4	The laboratory QA plan should be a separately prepared document (stand alone
document). However, some information can be incorporated by reference,
including laboratory SOPs, analytical methods, QC information, and applicable
literature. The QA plan should address the specific analytical/QC requirements
of EPA Method 1623 or 1623.1 and any compliance monitoring. The QA plan
should address the items listed in Chapter III of the Manual for the Certification
of Laboratories Analyzing Drinking Water (5th Edition) and Section 2.1 of the
Microbial Laboratory Guidance Manual for the Final Long Term 2 Enhanced
Surface Water Treatment Rule (Lab Guidance Manual). See

https://nepis.epa.gov/Exe/ZyPDF.cgi?Dockev=2000UDF3.txt. The QA Plan
should be updated at least annually. The QA plan should be reviewed as a part
of the on-site audit.

4.5.5	Detailed QC requirements specific to Cryptosporidium analyses are discussed in
Section 9 of EPA Methods 1623 and 1623.1 and further recommendations are
discussed in Section 3 of the Lab Guidance Manual. All laboratories analyzing
drinking water compliance samples must adhere to all required QC procedures
specified in the approved methods. The Lab Guidance Manual is available for
download from

https://nepis.epa.gov/Exe/ZyPDF.cgi?Dockev=2000UDF3.txt.

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5.

Analytical Methodology

5.1	General

A laboratory must be certified for all analytical methods that it uses for
compliance purposes.

The laboratory is required to maintain records of modifications made to EPA
Method 1623 or 1623.1 [Section 9.1.2.2 {Section 9.3)]. The documentation must
address the items listed in [Section 9.1.2.2 (Section 9.3)] and include the results
from the following QC tests comparing the modified method to EPA Method
1623 or 1623.1 acceptance criteria listed in Tables 3 and 4 of each Method.

•	IPR [Section 9.4 (Section 9.5)]

•	OPR [Section 9.7 {Section 9.8)]

•	MS/MSD [Section 9.5 {Section 9.6)]

•	MB [Section 9.6 {Section 9.7)]

Additional guidance is given in EPA Method 1623 Tables 3 and 4 and Tables 2,
3, and 4 for EPA Method 1623.1.

5.2	IMS Procedure

5.2.1	Laboratories should follow EPA Method 1623 or 1623.1 or manufacturer's
instructions for oocyst/cyst capture and dissociation of beads/oocyst/cyst
complex [Section 13.3 {Section 13.3)].

5.2.2	The total volume transferred should not be reduced to less than 5 mL above the
packed pellet or volume stated by manufacturer of filtration/concentration
device [Section 13.2 {Section 13.2)].

5.2.3	The maximum amount of pellet that should be processed through an individual
IMS reaction is 0.5 mL. If the packed pellet is greater than 0.5 mL, the pellet
must be sub-sampled as described in [Section 13.2.4 {Section 13.2.3)]. The
volume of the packed pellet can be estimated by comparison to a set of pellet
standards. Pellet standards may be prepared for long-term usage by measuring
appropriate amounts of colored glycerol, sand, or colored floral arranging gel.
Pellet standards should range from 0.1 mL to 0.5 mL, in 0.1 mL increments and
0.5 to 3.0 mL in 0.5 mL increments based on the pellet sizes typically observed.

5.2.4	Two acid dissociations are required [Section 13.3.3, Note {Section 13.3.3,

Note)].

5.2.5	The volumes of IMS reagents listed in the manufacturer's instructions and
Methods 1623 and 1623.1 are method requirements [{Section 13.3.1, Note)].

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5.1.1
QC 5.1.2

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5.3	Staining Procedure

5.3.1	If a laboratory has more than one option specified for slide drying, the criteria
determining when each option is performed should be listed in the staining SOP.

5.3.2	Application of stain to slides should be consistent with manufacturer's
instructions [Section 14.2 (,Section 14.2)\.

5.3.3	Incubation of slides should be in accordance with EPA Method 1623/1623.1 or
manufacturer's instructions [Section 14.3 (Section 14.3)~\.

5.3.4	Application of wash buffer to slides should be in accordance with EPA Method
1623/1623.1 or manufacturer's instructions [Sections 7.6, 14.5 and 14.7
{Sections 7.8, 14.5, and 14.7)\.

5.3.5	Laboratories must prepare positive and negative staining controls [Section
14.1 {Section 14.1)\.

5.3.6	Slides that are not read immediately after staining should be stored in a humid
chamber in the dark at 1°C to 10°C until read [Section 14.10 {Section 14.10)].

5.4	Sample Examination

5.4.1	Compliance sample analyses must be performed by a certified laboratory and
meet the QC requirements specified in EPA Methods 1623 or 1623.1. These
requirements include, but are not limited to, sample temperature requirements,
minimum frequencies for OPR, MB, MS samples; acceptable OPR and MB
results; holding time requirements; and acceptable staining control results and
frequency.

5.4.2	Slide wells should be scanned in a systematic fashion similar to that portrayed
in [Section 21.0, Figure 4 {Section 21.0, Figure 3)].

5.4.3	All slides should demonstrate clear contrast between the levels of background
fluorescence and the organisms when observed using FITC or DAPI.

QC 5.4.4 All slide examination results should be recorded for Cryptosporidium oocysts
and Giardia cysts. Examples of both a bench sheet and slide examination form
are attached. All organisms that meet the criteria specified in [Sections 15.2.2
and 15.2.3 {Sections 15.2.2 and 75.2.3)] less atypical organisms must be
reported. Atypical organisms identified by DIC or DAPI, i.e., possessing spikes,
stalks, appendages, pores, one or two large nuclei filling the cell, red fluorescing
organelles, and/or spores, should be noted on the slide examination form.

5.4.5 All sample examinations must be performed with the required magnification as
noted below.

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QC 5.4.6 Each analyst must characterize a minimum of three Cryptosporidium oocysts
and three Giardia cysts on the positive staining control slide before examining
field sample slides. This characterization must be performed by each analyst
during each microscopic examination session. FITC examination must be
conducted at a minimum of 200X total magnification, DAPI examination must
be conducted at a minimum of 400X total magnification, and DIC examination
and size measurements must be conducted at a minimum of 1000X total
magnification. Size, shape (morphology), and DIC and DAPI characteristics of
three Cryptosporidium oocysts and three Giardia cysts must be recorded by the
analyst on a microscope log. The analyst also must indicate on each sample
examination form whether the positive staining control was acceptable [Section
15.2. \.\(Section 15.2.1.1)1

QC 5.4.7 Negative staining controls should not contain any oocysts or cysts [Section
15.2.1.2 (Section 15.2.1.2)]. The analyst must indicate on each sample
examination form whether the negative staining control was acceptable.

5.4.8 The analyst must not proceed to field sample examination until the negative

staining control demonstrates acceptable results and the positive staining control
contains oocysts/cysts within the expected range, and the appropriate
fluorescence for both FA and DAPI [Sections 15.2.1 and 15.2.1.3 (Sections
15.2.1 and 15.2.1.3)]. Corrective actions should be listed in the QA plan or
SOPs if the positive and/or negative staining controls are not acceptable.

5.4.9 Sample Examination - Cryptosporidium

QC	5.4.9.1 A positive result is a Cryptosporidium oocyst which exhibits all of the

following: 1) typical FA fluorescence, 2) typical size and shape, 3)
nothing atypical on DAPI, and 4) nothing atypical on DIC microscopy
[(Section 21.0, Figure -/)], Each positive result must be characterized
and assigned to one of the DAPI and DIC categories described below
[Section 15.2.2.4 (Section 15.2.2.1)].

5.4.9.2 FITC - The analyst must scan the entire well at a minimum of 200X
total magnification, for apple-green fluorescing oocyst shapes. If
brilliant apple green fluorescing ovoid or spherical objects (4 to 6 |im
in diameter) are observed with brightly highlighted edges, the
magnification must be increased and the microscope switched to the
UV filter block for DAPI [Section 15.2.2.2 (Section 15.2.2.3)] and
then to DIC [Section 15.2.2.3 (Section 15.2.2.4 )].

QC	5.4.9.3 DAPI- The analyst must use a minimum of 400X total magnification.

Using the UV filter block, each object must be categorized with one of
the following characteristics;

(a) Light blue internal staining (no distinct nuclei) with green rim

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(b)	Intense blue staining

(c)	Up to four distinct sky blue nuclei

Slides must be examined for atypical DAPI fluorescence, e.g., more
than four stained nuclei, size of stained nuclei, and wall structure and
color. If atypical structures are not observed, the analyst must
categorize each object meeting the criteria defined as a positive result
defined in [Section 15.2.2.4 (Section 15.2.2.7)] and record oocysts in
category (a) as DAPI-negative; record oocysts in categories (b) and (c)
as DAPI-positive.

QC	5.4.9.4 DIC- The analyst must use a minimum of 1000X total magnification

(oil immersion lens). Using DIC the analyst should look for external or
internal morphological characteristics atypical of Cryptosporidium
oocysts (spikes, stalks, appendages, pores, one or two large nuclei
filling the cell, crystals, spores etc.). If atypical structures are not
observed, each of the apple green fluorescing objects must be
categorized as follows:

(a)	An empty Cryptosporidium oocyst

(b)	A Cryptosporidium oocyst with amorphous structure

(c)	A Cryptosporidium oocyst with internal structure (one to four
sporozoites/oocyst)

Using 1000X total magnification the analyst must record the shape,
measurements (to the nearest 0.5 |im), and number of sporozoites (if
applicable) for each apple green fluorescing object meeting the size
and shape characteristics.

5.4.10 Sample Examination - Giardia

QC	5.4.10.1 A positive result is a Giardia cyst which exhibits all of the following:

1) typical FA fluorescence, 2) typical size and shape, 3) nothing
atypical on DAPI, and 4) nothing atypical on DIC microscopy
[(Section 21.0, Figure -/)], Each positive result should be characterized
and assigned to one of the DAPI and DIC categories described below
[Section 15.2.3.4 (Sections 15.2.3.7)].

5.4.10.2 FITC- The analyst should use a minimum of 200X total magnification
to scan the entire well for apple-green fluorescing cyst shapes. If
brilliant apple green fluorescing round to ovoid objects (8-18 |im long
by 5-15 |im wide) are observed with brightly highlighted edges, the
magnification should be increased and the microscope switched to the
UV filter block for DAPI [Section 15.2.3.2 (,Section 75.2.3.3)]and then
to DIC [Section 15.2.3.3 (,Section 15.2.3.4)].

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QC	5.4.10.3 DAPI- The analyst should use a minimum of 400X total

magnification. Using the UV filter block, each object should be
categorized with one or more of the following characteristics:

(a)	Light blue internal staining (no distinct nuclei) and a green rim

(b)	Intense blue internal staining

(c)	Up to four sky blue nuclei

Slides should be examined for atypical DAPI fluorescence, e.g., more
than four stained nuclei, size of stained nuclei, and wall structure and
color. If atypical structures are not observed, the analyst should
categorize each object meeting criteria defined as a positive result in
[Section 15.2.3.4 (Section 15.2.3.1)] and record cysts in category (a) as
DAPI negative; record cysts in categories (b) and (c) as DAPI positive.

QC	5.4.10.4 DIC- The analyst should use a minimum of 1000X total magnification

(oil immersion lens). Using DIC microscopy the analyst should look
for external or internal morphological characteristics atypical of
Giardia cysts (e.g., spikes, stalks, appendages, pores, one or two large
nuclei filling the cell, crystals, spores, etc.). If atypical structures are
not observed, the analyst should categorize each apple green
fluorescing object as one of the following based on DIC examination:

(a)	An empty Giardia cyst

(b)	A Giardia cyst with amorphous structure

(c)	A Giardia cyst with one type of internal structure (nuclei, median
body, or axonemes), or

(d)	A Giardia cyst with more than one type of internal structure

Using 1000X total magnification the analyst should record the shape,
measurements (to the nearest 0.5 |im), the number of nuclei and
presence of median body or axonemes (if applicable) for each apple
green fluorescing object meeting the size and shape characteristics.

5.4.11 It is strongly recommended that positive and interfering organisms detected in
field samples be documented by photography.

QC 5.4.12 The date and time of sample examination along with the analyst's name should
be recorded on the examination form [Sections 15.2.4 and 15.2.6 (Section 15.2.4
and 15.2.6)\.

QC 5.4.13 Cryptosporidium and Giardia concentrations should be reported as oocysts/L
and cysts/L respectively [Section 15.2.5 (Section 75.2.5)]. If no oocysts or
cysts, as defined in [Sections 15.2.2.4 and 15.2.3.4 (Sections 15.2.2.1 and
15.2.3.1)] are detected, report zero organisms.

VTI-20

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6. Sample Collection, Handling, and Preservation
6.1 Sample Collection

6.1.1	Laboratories should ensure proper collection of samples by providing SOP and
chain of custody.

6.1.2	Several options are available for public water systems (PWSs) for collection of
untreated surface water samples for Cryptosporidium analysis which are as
follows:

•	On site filtration of water samples from unpressurized or pressurized
sources using the Pall Envirochek™ or Envirochek™ HV filter.

•	On site filtration of water samples from pressurized or unpressurized
sources using the IDEXX Filta-Max®foam filter

•	Collection of bulk water samples for shipment to the laboratory for filtration
and analyses.

Detailed procedures/information for each of these options and packing and
shipping information are available in the Source Water Monitoring Guidance
Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water
Treatment Rule (Source Water Monitoring Guidance Manual). See
https://nepis. epa.gov/Exe/ZyPDF. cgi?Dockey=2000CZCJ.txt.

6.1.3	PWSs should collect source water compliance samples from the plant intake
prior to chemical treatment, unless certified by the State to collect the source
water sample after chemical treatment. Systems that recycle filter backwash
water should collect source water samples prior to the point of filter backwash
water addition.

6.1.4	The use of multiple sources during monitoring should be consistent with routine
operational practice. If there is a tap prior to treatment where sources are
combined the sample can be collected at this location. If not, the PWS may
collect samples from each source prior to treatment and composite into one
sample or collect samples from each source prior to treatment, analyze
separately, and calculate a weighted average of the analytical results.

6.1.5	For LT2 Rule compliant samples, there may be instances where a replacement
sample is necessary, i.e., the PWS is unable to report a valid Cryptosporidium
result for a scheduled sampling date. Possible situations may include method
holding time exceeded, sample volume requirements not met, QC samples fail
acceptance criteria etc. The PWS should submit an explanation for the delayed
sampling date to the EPA/State concurrent with the shipment of the replacement
sample to the laboratory. The system should collect a replacement sample as
close to the required date as feasible but within 21 days of being notified by the
laboratory that a result cannot be reported for that date.

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6.1.6	A laboratory with multiple PWS clients representing a range of sample volumes
is not responsible for performing QC tests with all volumes. However, if the
laboratory does analyze both 10 L and 50 L sample volumes for clients then the
laboratory should demonstrate acceptable performance in a manner
representative of the sample volumes they process. Guidance on initial and
ongoing demonstrations of acceptable laboratory performance is provided in
Sections 3.3.14.1 through 3.3.14.3 of the Lab Guidance Manual.

6.1.7	Sample collection and/or filtration systems should be well maintained and
cleaned appropriately following use. The filtration system should be able to
maintain a seal during use with no leaks.

Sample Volume Analysis Requirements

6.2.1	For LT2 Rule compliant samples, the following minimum sample volume
requirements must be met:

•	10 L of sample or

•	2 mL of packed pellet volume or

•	As much as two filters can accommodate before clogging (this is applicable
only to filters that have been EPA approved for nationwide use).

6.2.2	Systems may analyze larger volumes than 10 L, provided the laboratory has
demonstrated acceptable performance on initial and ongoing spiked reagent
water and source water samples [Section 9.1.2.1.1 (Section 9.2)]. For LT2 Rule
compliant monitoring, it is preferred that PWSs analyze similar volumes
throughout the monitoring period. However, data sets including different sample
volumes are acceptable provided the system analyzes the minimum sample
volumes.

6.2.3	MS samples must be collected from the same location as the field sample as
split samples or as samples sequentially collected immediately after one
another. The MS sample volume analyzed must be within 10% of the volume
analyzed for the field sample [Section 9.5.1 (Section 9.6.2)].

Preservation/Sample Temperature Monitoring

6.3.1	Cryptosporidium oocysts present in samples can degrade, biasing analytical
results. Cryptosporidium samples must be stored and maintained between 1°C
and 10°C to reduce biological activity [Section 8 (Section 5)].

6.3.2	Samples for all analyses must remain above freezing at all times. Although this
may not pose a significant problem with 10 L water samples, this may be a
concern for Cryptosporidium filters that are shipped with coolant materials such
as wet ice, blue ice, dry ice, or gel packs.

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6.3.3	Per EPA Method 1623 and 1623.1, laboratories must reject samples that are
received frozen or >20°C unless the sample was collected the same day it was
received. In general, same day refers to atypical 8-hour work day [Section 8.1.3
{Section 8.1.3)].

6.3.4	Several options to measure sample temperature are available such as
temperature control samples/vials, iButton, stick on temperature strips, and
infrared thermometers. Temperature measurement devices should be calibrated
routinely to ensure accurate measurements [Section 8.1.4 (Section 8.1.4)~\.

Holding Time Requirements

6.4.1	Cryptosporidium samples should be analyzed as soon as possible. The
laboratory should complete sample filtration, elution, concentration, purification
and staining the day the sample is received whenever possible. If it is not
possible to complete sample analyses in one day the sample processing can be
halted after filtration, application of the purified sample onto the slide or
staining. The bullets below summarize the maximum holding times for
Cryptosporidium samples analyzed with EPA Method 1623 and 1623.1 [Section
8.2 and Table 1 {Section 8.2 and Table 5)].

•	4 days (96 hours) between collection/filtration and elution

•	1-working day between elution and application of sample to the slide

•	3 days (72 hours) between application of sample to slide and staining

•	7 days (168 hours) between staining and completion of sample examination

6.4.2	Explanation of holding time requirements for samples analyzed by EPA Method
1623 and 1623.1:

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

•	Sample elution, concentration, and purification. The laboratory must
complete the elution, concentration, purification, and application of the
sample to the well slide in one work day. This process ends with the
application of the purified sample to the slide for drying. The laboratory
should follow the stain manufacturer's instructions for slide drying. The
slides must be completely dried before staining and stored to maintain the
dried state until stained.

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

•	Examination. Although FA, DAP I, and DIC microscopy examination and
verification is ideally performed immediately after staining is complete,
laboratories have up to 168 hours (7 days) from completion of sample
staining to complete the examination and verification of samples. If
fading/diffusion of FITC or DAPI staining occurs the laboratory should
reduce this holding time. Other options include adjustment of the DAPI
staining solution so that fading/diffusion does not occur [Section 14.6

VTI-23

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(,Section 14.6)] or the laboratory may evaluate the use of another mounting
medium.

6.5 Analysis of Complex Samples

6.5.1	If the sample holding time has not been exceeded and a full volume sample
cannot be filtered due to high levels of oocysts, cysts and/or interfering
organisms, substances, or materials clogging the filter or precluding acceptable
microscopic examination, an aliquot of the sample should be diluted with
reagent water and a smaller aliquot filtered. This dilution must be recorded and
reported with the results [Section 16.2 (Section 16.2)]. If the holding times have
been exceeded, the sampling site should be resampled. If this is not possible, the
results should be qualified accordingly.

6.5.2	Siliconized or low adhesion centrifuge tubes should be used for samples which
are known to adhere to the centrifuge tube walls. Additional options include
rinsing centrifuge tubes with phosphate buffered saline Tween® (PBST) elution
buffer or Sigmacote® prior to use.

7. Cryptosporidium Quality Control

7.1 EPA Method 1623 and 1623.1 QC

QC 7.1.1 Laboratories must analyze samples spiked with Cryptosporidium oocysts to
assess ongoing laboratory and method performance in accordance with EPA
Method 1623 and 1623.1 QC requirements. These ongoing spiked sample
analyses include an IPR test [Section 9.4 (Section 9.5)], and ongoing
demonstration of laboratory capability and method performance through the MS
test [Section 9.5 (Section 9.6)], the MB test [Section 9.6 (Section 9.7)], the OPR
test [Section 9.7 (Section 9.8)], staining controls [Sections 14.1 and 15.2.1
(Sections 14.1 and 15.2.1)], and analyst verification tests [Section 10.6 (Section
9.10)]. Laboratory performance is compared to established performance criteria
to determine if the results of analyses meet the performance characteristics of
[Table 3] in each method. The laboratory should spike samples according to the
procedures described in [Section 11.4 (Section 11.2)] or according to the
instructions provided by the spiking suspension vendor. EPA Method 1623.1
[Table 2] lists the required routine QC requirements.

Laboratories are to document/demonstrate a minimum Cryptosporidium
recovery of 22% prior to analysis of samples at the frequency required in
[Section 9.7 (Section 9.8)]. See Method 1623 Quality Control Acceptance
Criteria update for OPR samples in Federal Register Vol. 74, No. 36, February
25, 2009. (The OPR Quality Control Acceptance Criteria for OPR samples
using Method 1623.1 is 33% [(Table 3)].) This Federal Register notice Vol. 74,
No. 36, describes the current Lab QA Program for Cryptosporidium including
details for the basis of downgrading or suspending a laboratory's certification

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for analyses of Cryptosporidium in support of the Safe Drinking Water Act.

This information is available online at http://www.regulations.gov under Docket
ID No. EPA-HQ-OW-2002-0011.

7.1.2	QC Batch

All compliance Cryptosporidium samples must be associated with an acceptable
OPR and MB sample [Section 9.1.7 (Section 9.8.6)] through a "QC batch." A
QC batch consists of an OPR, and MB, and a maximum of 20 field and MS
samples combined that are eluted, concentrated, and purified in the same week
as the OPR and MB samples using the same reagents. A week is defined as any
168 hour (7 day) period that begins with the processing of the OPR. If more
than 20 field and MS samples are analyzed in a week, another OPR and MB
must be eluted, concentrated, and purified using the same reagents as the
additional field and MS samples [Sections 9.6 and 9.7 (,Sections 9.7 and 9.8)].
A field sample and its associated MS sample should be analyzed in the same
QC batch.

7.1.3	Cryptosporidium Spiking Materials

Sources of flow cytometer-counted Cryptosporidium spiking suspensions for
spiked QC samples include the following vendors, or equivalent:

•	Wisconsin State Laboratory of Hygiene - http://www.slh.wisc.edu/

•	Bioballs

•	Waterborne, Inc. - http://www.waterborneinc.com

The Wisconsin State Laboratory of Hygiene prepares and distributes live
Cryptosporidium oocysts and Giardia cysts that have not been treated to reduce
viability. BTF and Waterborne prepare and distribute Cryptosporidium oocysts
and Giardia cysts that have been irradiated to inactivate the organisms.

Irradiated organisms may be used for routine QC samples including IPR, OPR
and MS samples but should not be used to demonstrate acceptability through a
multi-laboratory validation study using multiple matrices.

QC 7.1.4 IPR Test

The IPR test consists of four reagent water samples each spiked with 100 to 500
oocysts. The results of the four analyses are to be used to calculate mean percent
recovery and the relative standard deviation (RSD) of the recoveries. The mean
Cryptosporidium recovery should be from 24% to 100% and the RSD of the
four recoveries should be less than or equal to 55% for Method 1623 [Table 3],
(The mean Cryptosporidium recovery should be 38 to 100 % with an RSD of
37%) for laboratories performing IPR tests using Method 1623.1 [(Table 3)]).
Characterization of the first three Cryptosporidium oocysts and three Giardia
cysts must be reported on the slide examination form for each IPR sample
[Section 9.4.5 (Section 9.5.5)].

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7.1.5

MB Test

QC	7.1.5.1 The MB test consists of analysis of an unspiked reagent water sample

to show freedom from contamination. The method requires that one
MB sample must be analyzed each week samples are analyzed or for
each QC batch, up to and including 20 samples (MS and field samples
combined), whichever is more frequent [Section 9.6 (Section 9.7)]. If
more than one method variation is used for filtration and/or another
technique which uses different apparatus and/or reagents, a separate
MB is required for each variation. However, if more than one labeling
reagent is used a separate MB is not required [Section 7.6.4, Note
{Section 7.8.4, Note and 14.0, Note)~\.

7.1.5.2	MB samples should be analyzed before any field samples in a batch
are processed to verify acceptable performance. If oocysts are detected
in a MB, the MB is unacceptable and analysis of additional samples is
halted until the source of contamination is eliminated and a blank
shows no evidence of contamination. If the repeated MB is acceptable,
field samples within the holding times can be processed.

7.1.5.3	To demonstrate freedom from contamination and check the efficacy of
the cleaning system, the MB should be processed in the same manner
as the associated OPR and field samples. The carboy used for the MB
is randomly selected from carboy stock or disposable carboys are used
for all samples.

7.1.6 OPR Test

QC	7.1.6.1 The OPR consists of 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 samples are analyzed or
for each QC batch, up to and including 20 samples (MS and field
samples combined), whichever is more frequent [Section 9.7 (Section
9.8)]. If more than one method variation is used for filtration and/or
another technique which uses different apparatus and/or reagents, a
separate OPR is required for each variation. However, if more than
one labeling reagent is used a separate OPR is not required [Section
7.6.4, Note (Section 7.8.4, Note and 14.0, Note)].

QC	7.1.6.2 OPR samples must be analyzed before any field samples in a batch are

processed to verify acceptable performance. OPR Cryptosporidium
recovery must be from 22% to 100% to be considered acceptable
(Federal Register Vol. 74, No. 36, February 25, 2009) for EPA
Method 1623. OPR Cryptosporidium recovery must be from 33 % to
100% for EPA Method 1623.1 [(Section 9.8.3)]. Characterization of

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the first three Cryptosporidium oocysts and three Giardia cysts must
be reported on the slide examination form [Section 9.7.1.2 (,Section
9.5.7.2)]. If the OPR Cryptosporidium recovery is not acceptable, no
samples may be processed. Finding the problem and repeating the
OPR to assure the analytical system is "in control," is required before
proceeding with sample analysis. If the repeated OPR is acceptable,
field samples within holding times can be processed.

QC 7.1.7 OPR Charts

7.1.7.1	Laboratories should maintain a control chart of OPR recoveries,
graphically displaying the results of continuing performance when
using Method 1623; moreover, this is a requirement for Method
1623.1 [Section 9.7.6 {Section 9.12.1 and Table 2)]. The control chart
should be developed using the most recent 20 to 30 test results.

7.1.7.2	The control chart should be developed by plotting percent recovery of
each OPR sample over time. See Figure 3-2 of The Lab Guidance
Manual for an example of an OPR control chart. Based on the mean
recoveries, upper control limit should be established as the mean + 2
standard deviations and lower control limit as the mean - 2 standard
deviations. After each 5 to 10 new recovery measurements the
laboratory should recalculate new control limits using the most recent
20 to 30 data points. If recoveries fall outside the control limits, or
declining trends are observed, laboratories should take corrective
action to investigate the potential causes of the outlying result. Trouble
shooting guidance is available in [Section 9.7.5 (Section 9.8.7)].

7.1.8 Staining Controls

QC	7.1.8.1 Positive staining controls are used to verify that the FITC and DAPI

stains are fluorescing appropriately. Positive staining controls should
be prepared by applying 200 to 400 intact oocysts to a slide and
staining the slide with the same reagents and staining procedure used
to stain field and MS sample slides. The analyst should examine
several fields to verify that the stain is fluorescing at the appropriate
intensity and uniformity. Each analyst must characterize a minimum of
3 Cryptosporidium oocysts and 3 Giardia cysts on the positive staining
control slide before examining slides from field samples [Section
15.2.1.1 (Section 15.2.1.1)]. Control slides and sample slides should be
read on the same day. If sample slides from the same staining batch are
read over multiple days, the control slide should be rechecked each
day before examination of the sample slides. If the laboratory has a
large batch of slides that will be examined over several days and is
concerned that a single positive control may fade due to multiple
examinations, the laboratory should prepare multiple control slides at

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the same time with the batch of field slides and alternate between the
positive controls when performing the positive control check.

7.1.8.2 Negative staining controls are used to verify that no oocysts or

interfering particulates are present. Negative staining controls are
prepared by staining and examining a slide with phosphate buffered
saline (PBS) solution.

QC	7.1.8.3 The analyst should indicate on each Cryptosporidium slide

examination form whether the positive staining and negative staining
control were acceptable. Each sample must meet the QC criteria for
EPA Method 1623 or 1623.1. Positive and negative staining controls
must demonstrate acceptable results [Section 15.2.1 (Section 75.2.7)].

7.1.9 Verification of Analyst Performance

7.1.9.1 Analyst verifications are ongoing comparisons of slide counts and
characterizations used to assess and maintain consistency in slide
examination among analysts. The goal is to continually refine/upgrade
microscopy skills. When microscopic examinations are being
performed, the laboratory must prepare at least monthly one or more
slides containing 40 to 200 oocysts. More than 50% of the oocysts
must be DAPI positive and undamaged under DIC. Another option is
order prepared slides from the Wisconsin State Laboratory of Hygiene,
Flow Cytometry Unit (http://www.slh.wisc.edu/) or other approved
vendor.

QC	7.1.9.2 For laboratories with multiple analysts, each analyst must determine

the DAPI category (DAPI negative, DAPI positive with intense
internal blue staining, and DAPI positive with the number of nuclei)
and the DIC category (empty, containing amorphous structures, or
internal structure characterization) of the same 10 selected oocysts. It
is recommended that the DAPI and DIC categorization of the selected
oocysts occur with all the analysts at the same time, i.e., each analyst
should determine the category independently, then the differences in
DAPI and DIC categorizations among analysts are to be discussed,
resolved, and the resolutions documented. Alternatively, oocyst
coordinates may be recorded for each analyst to locate and categorize
the oocysts at different times. Differences among analysts should still
be discussed, resolved, and documented.

7.1.9.3 Laboratories should be aware that both FITC and particularly DAPI

fluorescence may fade during analyst verification. Accordingly, DAPI
comparisons should be performed first. Repeat comparisons with new
FITC stained organisms.

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7.1.9.4	Each analyst must determine the total number of oocysts by FITC
fluorescence at 20X magnification for the entire slide. It is
recommended that this count be performed last or on a separate slide
than that used for DAPI and DIC characterization so that fading will
not influence counts. The total number of oocysts enumerated by each
analyst must be within ±10% of each other. If the number is not within
this range the analysts must identify the source of any variability
between analysts' examination/identification criteria, prepare a new
slide, and repeat the performance verification.

7.1.9.5	Laboratories with only one analyst should compare the results of slide
examinations to a "protozoa library" of photographs of oocysts and
cysts and interfering organisms to verify that examination results are
consistent with these established references. These laboratories should
perform repetitive counts of a single verification slide for FITC and
demonstrate repetitive enumerations within ±10% of each other. These
laboratories are encouraged to establish contact with other laboratories
to share slides and compare counts.

7.1.10 MS Samples

7.1.10.1	The MS sample entails analysis of an extra bulk water sample spiked
with 100 to 500 oocysts. The MS sample is processed at the same
time and using the same procedures as the associated field samples to
determine the effect of the source water matrix on recovery.

7.1.10.2	For all PWSs, the first MS sample should be collected and analyzed
during the first sampling event [Section 9.1.8 (Section 9.6.P)\. If it is
not possible to analyze an MS sample for the first sampling event, the
first MS sample should be analyzed as soon as possible to identify
potential method performance issues with the matrix. MS samples
must be analyzed once every 20 field samples but may be analyzed
more frequently than one every 20 field samples to better characterize
method performance in the matrix.

Consistent with this requirement, PWSs falling into the following
categories should analyze at least two MS samples during LT2 Rule
monitoring:

•	Large PWSs that perform monthly monitoring for two years
(resulting in 24 samples)

•	Small PWSs that are "triggered" into Cryptosporidium monitoring
and collect semi-monthly samples for one year or monthly samples
for two years (resulting in 24 samples)

For large PWSs that perform semi-monthly or more frequent
monitoring for two years (resulting in 48 or more samples), a

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minimum of three MS samples should be collected and analyzed. If a
PWS monitors more frequently or collects more than 60 samples, a
minimum of four MS samples should be analyzed.

7.1.10.3 For MS samples associated with field filtered samples, MS samples
should be collected as bulk samples and spiked in the laboratory prior
to filtration. The volume of the MS sample must be within 10% of the
volume of the associated field sample. PWSs that field filter 10 L
samples may field filter the monitoring sample but should collect and
ship the 10 L MS sample in bulk to the laboratory for spiking,
filtering, and analysis.

For PWSs that field filter > 10 L samples, all but 10 L of the MS
sample should be filtered in the field. The remaining 10 L of source
water for MS analysis should be collected in bulk and shipped to the
laboratory. The laboratory should then spike the 10 L bulk sample and
pump it through the filter containing the balance of the sample already
filtered in the field. The associated monitoring sample should be
collected as usual.

QC	7.1.10.4 Similar to OPR control charts, laboratories should assess precision of

MS recoveries. This can be accomplished by maintaining a control
chart that graphically displays the results of continuing performance. It
is recommended in Method 1623 and required for Method 1623.1 that
the precision assessment be maintained across all MS samples as well
as stratified by source. The control chart should be developed when at
least 5 MS samples have been completed [Section 9.5.1.4 (Section
9.12.2)].

The control chart should be developed by plotting percent recovery of
each MS sample versus time. Based on the mean recoveries on the
chart the upper control limit should be the mean + 2 standard
deviations and the lower control limit should be the mean - 2 standard
deviations. Control charts can be used to compare performance of
different method variations and different analysts along with
performance in different matrices. If recovery measurements fall
outside the control limits, laboratories should take corrective action to
investigate the potential causes of the outlying result.

7.2 Certified Laboratories

7.2.1 PWSs must have Cryptosporidium samples analyzed by a laboratory that has
been certified for Cryptosporidium analysis. Details on the elements of the
EPA's Laboratory QA Evaluation Program are provided in Federal Register
Vol. 74, No. 36, February 25, 2009 available online at
http://www.regulations.gov under Docket ID No. EPA-HQ-OW-2002-0011),

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Chapter III of the Manual for the Certification of Laboratories Analyzing
Drinking Water (5th Edition), and Section 3.2 of the Lab Guidance Manual. It is
suggested that each certified laboratory establish and maintain a relationship
with another certified laboratory and develop a protocol to provide backup
analyses to clients if needed.

7.2.2 Criteria for Certification Status [February 25, 2009 Federal Register Notice
available online at http://www.regulations.gov under Docket ID No. EPA-HQ-
OW-2002-0011.]

Analysis of a Proficiency Testing (PT) sample is a key component of the
certification process. Laboratories seeking certification for Cryptosporidium
analysis under the LT2 Rule should successfully participate in a PT study. The
laboratory should acquire a set of eight initial PT samples from an approved PT
provider. Laboratories should suspend these spikes in reagent water to produce
simulated source water samples, and analyze the samples using the
modifications of EPA Method 1623 or 1623.1 that the laboratory plans to use
for Cryptosporidium analyses. If a laboratory wishes to be evaluated for more
than one modification of the method, the laboratory should acquire a set of eight
PT samples for each version. Data submitted by the laboratory from the analysis
of the PT samples will be evaluated against the mean recovery and precision (as
a RSD) for the PT samples from other laboratories in the same study, and the
laboratory will be notified of their results. Failure to successfully participate in
two consecutive PT studies should result in the laboratory staff receiving
additional training to better perform the method.

Laboratories that meet the above performance criteria should acquire PT
samples approximately every four to six months. The Certification Authority
should evaluate the precision and recovery data for PT samples to determine if
the laboratory continues to meet the performance criteria of the Certification
Authority. In addition, the Certification Authority should review raw PT data
records to ensure compliance with Method 1623 or 1623.1 requirements.

8. Records and Data Reporting

8.1 Legal and Technical Defensibility

In order to have technically defensible compliance monitoring data, the laboratory should
maintain thorough and accurate records. The QA plan and SOPs should describe the
policies used by the facility to record and validate data, record retention, and storage. If
samples are expected to be part of an enforcement action, chain of custody procedures
should be implemented (See Appendix A of the Manual for the Certification of
Laboratories Analyzing Drinking Water (5th Edition)).

8.2 Recordkeeping

An effective record keeping system provides information on sample collection and
preservation, analytical methods, raw data, calculations, reported results, and a record of

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persons responsible for sampling and analyses. Laboratories using EPA Method 1623 or
1623.1 should record original data including microscopic examination counts and notes.
The data may be recorded on the recommended bench sheets and slide examination
forms. Bench sheets and slide examination forms are included in this chapter.

Data should be recorded in ink and a single line drawn through any change with initials,
dates and correction entered. Data files may also be microfiche or electronic. Electronic
data should be backed up by protected tape, disc, or hard copy. PWSs are required to
maintain records of microbiological analyses of compliance samples for five years (40
CFR141.33). A change in ownership, merger, or closure of a laboratory does not negate
this requirement. The client water system should be notified before disposing of records
so they may request copies if needed. This includes all raw data, calculations, and QC
data. If the laboratory changes its computer hardware or software, provisions should be
made for transferring old data to the new system so that the data may be retrievable
within the record retention period. Data that is expected to become part of litigation may
need to be retained longer.

8.3 Data Recording Practices

The following data recording practices should be followed with field, MS, QC, and PT
samples for all laboratories performing compliance analyses:

• Record sample identification information, including sample collection and receipt
dates and conditions

• Record all raw data (primary measurements) used to calculate final concentrations of
oocysts/L for each sample

• Record the date and time of each method step associated with a holding time to verify
that all method holding times have been met

• Record the name of the analyst performing each method step to verify that only
qualified analysts are performing the method

These data elements are critical to ensuring that final sample concentrations can be
verified using primary data and are necessary to demonstrate that all method specified
holding times are met. These records should be available for review by Certification
Officer.

8.4 Sampling Records

Sampling records provided by the PWS should include the following information:

• PWS name and ID number

• Facility name and number

• Sample collection point name and ID

• Date and start/stop times of collection

• Sampler's name and telephone number (or alternate contact for laboratory)

• Source water temperature and turbidity

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

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

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

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Detailed guidance on sample collection data recording as well as forms and sample
collection and shipping procedures can be found in the Source Water Monitoring
Guidance Manual:

https://nepis.epa.gov/Exe/ZyPDF.cgi?Dockey=2000CZCJ.txt.

8.5	Sample Receipt

Upon sample receipt at the laboratory, the following information should be recorded on a
bench sheet, log, or record book. Acceptance criteria for sample receipt should be
established and documented in a laboratory's SOPs [Sections 8.1.3 and 8.1.4 {Sections
8.1.3 and 8.1.4)~\. Laboratories should immediately notify utilities of any deficiencies
requiring a resample and document the reasons for rejection.

•	PW S name and ID

•	Facility name and ID

•	Sample collection point name and ID

•	Turbidity at the collection point taken immediately after sample collection

•	Date and time of sample collection (start and stop times if field filtered)

•	Date and time of sample receipt by the laboratory

•	Volume filtered (if sample is filtered in the field)

•	Name of laboratory person receiving the sample

•	Temperature of sample upon receipt

•	Any deficiencies (exceeded sample holding time, transport temperature exceeded
20°C or sample leaked during transport, frozen samples, etc.)

8.6	Primary Data Elements for Calculations

Laboratories analyzing compliance samples for Cryptosporidium using EPA Method
1623 or 1623.1 should record the following primary data elements required to calculate
the final concentrations and percent recoveries for MS, OPR, and PT samples. These data
should be recorded on the recommended bench sheet and slide examination form.

•	Estimated number of oocysts spiked (MS and OPR samples) based on information
provided by the flow-cytometry laboratory producing the spiking suspension

•	Sample volume spiked, in L (MS, OPR and PT samples)

•	Sample volume filtered, to nearest ]A L

•	Number of filters used (if the filter is clogged)

•	Pellet volume after concentration, to the nearest 0.1 mL

•	Total volume of the resuspended concentrate transferred to IMS, in mL

•	Number of subsamples analyzed

•	Total number of oocysts detected in the sample

8.7	Key QC Data Elements

The following data should be recorded to determine that all method QC requirements
were met and that samples were analyzed by qualified personnel consistent with the
requirements of the Laboratory QA Program for the analyses of Cryptosporidium.

•	Elution date and time (must be within 96 hours of sample collection)

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

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

•	Sample examination date and time (must be within 168 hours (7 days) of sample
staining)

•	Person (PWS or laboratory employee) performing filtration

•	Technician or analyst performing elution

•	Technician or analyst performing IMS

•	Technician or analyst preparing slide

•	Technician or analyst staining the slide

•	Analyst performing sample examination

•	Results of the positive and negative staining controls

The laboratory should also record any additional information that will support the results
or aid in identification of problems of sample analyses such as filter type, elution
procedure, IMS system used, lots numbers of reagents, detection kits, spiking
suspensions, etc. This information should be recorded on bench sheets, and slide
examination forms.

9. Calculations for EPA Method 1623 and 1623.1

9.1 The laboratory may choose to report the total oocysts and volume analyzed along with

the oocysts/L [Section 15.2.5 (Section 75.2.5)] and other data elements described in EPA
Method 1623 and 1623.1 to the PWS. The laboratory will need to calculate OPR and MS
recoveries to report to their PWS and to maintain QC control charts.

9.2 Calculating Oocyst Concentrations

The following information will be needed to calculate the concentration of
Cryptosporidium in a sample:

•	Number of oocysts in the sample (recorded from slide examination form)

•	Volume analyzed

/oocystsx oocysts detected in sample

Final concentration

^oocysts^

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 < 100% of the volume filtered was processed through the remainder of the method then
additional calculations are needed [Section 13.2.4.2 (Section 13.2.3.2)].

9.3 Determining the Volume of Resuspended Concentrate to use for Packed Pellets >0.5
mL

Packed pellets with a volume >0.5 mL must be divided into subsamples. The laboratory
should 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 [Section 13.2.4 {Section 13.2.3)].

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pellet volume (mL) after centrifugation
Total volume ofresuspended concentrate (mL)required =	——		x5 mL

9.4	MS Recovery Calculations

To determine the percent recovery for a MS sample the following information is needed
[Section 9.5.1.2 (,Section 9.6.2.2)]:

•	The number of oocysts counted in the MS sample

•	The number of oocysts spiked into the MS sample

•	The number of oocysts counted in the unspiked field sample (to correct for
background concentration)

oocysts counted in MS sample - oocysts counted in unspiked field sample

Percent recovery =	xl00%

oocysts spiked in MS sample

This calculation is based on the assumption that the same sample volume was examined
for both the field and MS samples. If the sample volumes examined are different the
laboratory should calculate the number of oocysts per L for both the field and MS
samples before calculating percent recovery. If both a MS and a MSD are analyzed then
the mean recovery and relative percent difference (RPD) should be calculated and
compared to the EPA Method 1623 orl623.1 acceptance criteria [Tables 3 and 4 (Tables
3 and 4)\.

To calculate the mean percent recovery the laboratory should calculate the percent
recovery for each sample as described above then perform the following calculation
[Section 9.5.2.2 (,Section 9.6.3.2)]:

percent recovery of MS sample + percent recovery of MSD sample
Mean percent recovery = 			

In order for the laboratory to calculate the RPD, the absolute value (without sign) of the
difference between the numbers of oocysts counted in the MS and MSD should be
divided by the mean of the oocysts counted in both samples to yield a percentage of the
difference. This calculation assumes that the same volume is analyzed for both the MS
and MSD. The laboratory should calculate the number counted per L before calculating
the RPD if sample volumes analyzed are different [Section 9.5.2.3 (Section 9.6.3.3)].

loocysts counted in MS - oocysts counted in MSDI

RPD = -J					xl00%

(oocysts counted in MS+oocysts counted in MSDJ/2

9.5	OPR Sample Calculations

The laboratory should calculate percent recovery of an OPR sample using the following
formula [Section 9.7.2 (,Section 9.8.2)]:

oocysts detected

Percent recovery =	-—— xl00%

oocysts spiked

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OPR recoveries are to be compared to the limits for ongoing recovery in EPA Method
1623 or 1623.1 (See Section 7.1.6.2 of this certification chapter). These recoveries should
be tracked/monitored over time to assess precision.

10. Data Archiving

10.1	Hardcopy Data

The following data should be archived:

•	Bench sheets and slide examination forms for all compliance monitoring samples,
including both field samples and MS samples

•	Bench sheets and slide examination forms for all OPR samples and MB samples, and
records of compliance monitoring samples associated with each OPR sample and
blank sample

•	Spike enumeration information received from Cryptosporidium spiking suspension
vendors

•	Bench sheets and slide examination forms for all ongoing PT samples

As part of the Lab QA Program the laboratory should also maintain the same
documentation for their IPR and initial PT data for each method variation used for
compliance samples.

10.2	Slides

Although not required, laboratories may wish to consider archiving slides and/or
photographing slides to maintain for clients. Slides should be stored in the dark between
1°C and 10°C and not frozen in the appropriate environment for the type of mounting
medium used.

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Acronym List

ACS	American Chemical Society

ATP	Alternate Test Procedure

DABCO	l,4-diazabicyclo[2.2.2]octane

DAPI	4',6-diamidino-2-phenylindole

DCTS	LT2 RULE/Stage 2 Data Collection and Tracking System

DIC	Differential interference contrast

DPD	N.N-diethyl-p-phenylenediamine

EPA	Environmental Protection Agency

FA	Fluorescence assay or immunofluorescence assay

FITC	Fluorescein isothiocyanate

GLP	Good Laboratory Practice

HDPE	High-density polyethylene

IDC	Initial Demonstration of Capability

IFA	Immunofluorescence assay or fluorescence assay

IMS	Immunomagnetic separation

IPR	Initial Precision and Recovery

Lab Guidance Manual	Microbial Laboratory Guidance Manual for the Final Long Term 2

Enhanced Surface Water Treatment Rule

LDPE	Low-density polyethylene

LT2 Rule	Long Term 2 Enhanced Surface Water Treatment Rule

MB	Method Blank

MPN	Most Probable Number

MS	Matrix Spike

MSD	Matrix Spike Duplicate

NIST	National Institute of Standards and Technology

OPR	Ongoing Precision and Recovery

PBS	Phosphate Buffered Saline

PBST	Phosphate Buffered Saline Tween®

PCFC	Portable Continuous Flow Centrifuge

PPE	Personal Protective Equipment

PT	Proficiency Testing

PTFE	Polytetrafluorethylene

PWS	Public Water System

QA	Quality Assurance

QC	Quality Control

RCF	Relative Centrifugal Force

RPD	Relative Percent Difference

RPM	Rotations per Minute

RSD	Relative Standard Deviation

Source Water Monitoring Source Water Monitoring Guidance Manual for Public Water

Guidance Manual	Systems for the Long Term 2 Enhanced Surface Water Treatment

Rule

SOP	Standard Operating Procedure

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Checklist Item Classification

Below is a description of the categories used to classify each item on Checklists A, B, and C.
The categories correspond to information provided in Notes 1-3 at the beginning of Chapter VII.

Recommendation: Indicates suggested improvements for analytical or data recording/QA
procedures. Recommendations do not indicate that method or program requirements were not
met. Laboratories are not required to implement recommendations but are requested to respond
in writing to each recommendation giving details on how they plan to implement the
recommendation or why they have decided against implementation.

Critical: Indicates areas that EPA believes are necessary elements of good laboratory practice
or may impact method performance. Critical items are not required by the EPA Methods but
laboratories seeking approval under the Laboratory QA Program are strongly encouraged to
implement all critical items. Laboratories should respond in writing that critical items have been
implemented and provide documentation as appropriate. If the item is not implemented, the
laboratory should provide an alternative approach for addressing data quality issues or describe
why data quality will not be affected if no action is taken.

Requirement: Indicates areas where QC requirements are specified in the EPA Method or LT2
rule and may impact data quality and/or data comparability. Laboratories seeking approval under
the Laboratory QA Program must implement all requirements and respond in writing and
provide documentation as appropriate.

Method Procedure (1623): Indicates areas where the laboratory's method procedures deviate
from the December 2005 version of Method 1622/1623 and may affect method performance.
Laboratories seeking approval under the Laboratory QA Program should provide a letter to EPA
either: (1) indicating that current procedures will be changed to eliminate method deviations or
(2) certifying with data from Tier 1 testing (Section 9.1.2.1.1 of Method 1622/1623, December
2005) that the method modification produced results equivalent or superior to results produced
by the method as written. Please note that not all method modifications can be demonstrated to
produce equivalent performance through Tier 1 testing; for some modifications the laboratory
may need to design a study or provide additional data to demonstrate equivalency and submit the
study design to EPA for approval.

Method Procedure (1623.1): Indicates areas where the laboratory's method procedures deviate
from Method 1623.1 and may affect method performance. Table 1 lists the options that can be
performed according to the procedures described in the method.

Commendation: Indicates areas where the laboratory has exceeded method or program
requirements, shown innovation, or demonstrated excellent laboratory technique. No response is
required by the laboratory.

VTI-38

-------
Method 1623/1623.1 Microscopy Visual Guide

Cryptosporidium oocyst criteria:

•	Brilliant apple-green fluorescence • Brightly highlighted edges

•	4 - 6 pm size	• Spherical to ovoid shape

•	No atypical characteristics by FA, DAPI fluorescence or DIC microscopy

Giardia cyst criteria:

•	Brilliant apple-green fluorescence • Brightly highlighted edges

•	8 - 18 pm long by 5 - 15 pm wide • Spherical to ovoid shape

•	No atypical characteristics by FA, DAPI fluorescence or DIC microscopy

FA

DAPI

DIC
image

Image
not in DIC

VTI-39

-------
Example Checklists for Onsite Evaluation of Laboratory Analyzing Drinking Water for Cryptosporidium

and Giardia Using Method 1623/1623.1

Checklist A - Method 1623/1623.1 Audit Package and Data Review
Checklist B - Laboratory SOP Review

Checklist C - Method 1623/1623.1 Technical Review - Sample Processing and Microscopy

VTI-40

-------
Method 1623/1623.1 Checklist A

Checklist A - Method 1623/1623.1 Audit Package and Data Review

Laboratory Name

Name and Affiliation of Evaluator

Date of Evaluation







Good Laboratory Practice (GLP) is generally defined as a system of management controls for the laboratories to ensure the consistency and reliability of results. Adapted
from other federal programs for the purposes of the Cryptosporidium Laboratory QA Evaluation Program, GLP includes personnel, equipment, and standard operating
procedures appropriate for the program.

Item to be Evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

1 Quality Assurance

1.1 Is documentation (e.g., resume, sample list)
available for all Method 1623/1623.1 staff?

9.1

9.1

1.

Requirement
GLP











1.1.1 Have technicians/analysts analyzed the
required number of samples using
Method 1623 or1623.1?

22.2

22.2

1.1 -1.3

Requirement
GLP











1.2 Are employee training records available and up to
date?

9.1

9.1

1.7

Critical
GLP











1.2.1 Have all analysts documented that they
have read and understood the QA Plan
and SOPs?

-

-

1.7

Critical
GLP











1.3 Is the laboratory performing analyst verification
monthly and does the lab have corrective action
procedures in place if criteria are not met?

10.6

9.10

7.1.9

Requirement











1.3.1 If the laboratory has only one analyst, is
the analyst demonstrating analyst
verification through comparison with
photo libraries or repetitive counts?

10.6.4

9.10.4

7.1.9.5

Recommendation











1.4 Does the quality assurance plan address

requirements for Cryptosporidium analysis under
LT2ESWTR?







Critical











1.5 Have acceptable initial precision and recovery
analyses been performed for each version of the
method the laboratory is using?

9.1.2.1.1

9.2

7.1.4

Requirement











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-41

-------
Method 1623/1623.1 Checklist A

Item to be Evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

1.6 Of the field/PT samples reviewed, is each field/PT
sample associated with an acceptable method
blank?

9.6.1

9.7

7.1.2
7.1.5.1

Requirement











1.6.1 Were all method blanks (MB) evaluated
without contamination?

9.6.2.1

9.7.2

7.1.5

Requirement









# MB reviewed:

1.6.2 Were the same lots of reagents (elution,
IMS, and staining) used for the method
blank and the associated field/PT
samples?

-

-

7.1.5.3

Critical











1.6.3 Is method blank analyzed prior to the
analysis of field/PT samples?

9.6

9.7

7.1.5.2

Requirement











1.7 Is each field/PT sample associated with an

acceptable ongoing precision and recovery (OPR)
sample?

9.7

9.8

7.1.2
7.1.6.1

Requirement











1.7.1 What percentage of OPR samples
evaluated met the recovery criteria?

9.7.3
Table 3
Table 4

9.8.3
Table 3
Table 4

7.1.6.2











# OPR reviewed:

1.7.2 Were the same lots of reagents (elution,
IMS, and staining) used for the OPR and
the associated field/PT samples?

-

-

7.1.6.1

Critical











1.7.3 Is OPR analyzed prior to the analysis of
field/PT samples?

9.7.1

9.8.1

7.1.6.2

Requirement











1.7.4 Does the laboratory maintain control
charts of OPR results?

9.7.6
9.1

9.8.3
9.12.1
Table 2

7.1.7

1623
Recommendation

1623.1 Requirement











1.7.5 What is the mean and relative standard
deviation (RSD), or standard deviation, of
the recoveries of the OPR samples
included in the control chart?

9.4.3
Table 3
Table 4

9.5.3
Table 3
Table 4

7.1.6.2

QC Criteria









Mean:
RSD:

1.8 Were matrix spike (MS) samples analyzed at the
minimum frequency of 1 MS per 20 (up to and
including) field samples from each source?

9.1.8

9.6.1

7.1.2
7.1.10.2

Requirement









# MS reviewed:

1.8.1 Were MS sample volumes within 10% of
their associated field samples' volumes?

9.5.1

9.6.2

7.1.10.3

Requirement











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-42

-------
Method 1623/1623.1 Checklist A

Item to be Evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

1.8.2 Were MS samples analyzed at the same
time and using the same method
variation as their associated field
samples?

Table 2

Table 2

7.1.10.1

Requirement











1.8.3 What is the mean and relative standard
deviation of the MS samples reviewed?

Table 3
Table 4
Table 5

Table 3
Table 4
Table 6

-

QC Criteria









Mean:
RSD:

1.8.4 Does the laboratory maintain control
charts of MS results?

9.5.1.4
9.1

9.6.2.3
9.12.2
Table 2

7.1.10.4

1623
Recommendation

1623.1 Requirement











1.9 Were OPR samples spiked with 100 - 500
organisms?

9.7

9.8

7.1.6.1

Requirement











1.10 Does the laboratory perform IMS controls and
maintain IMS control charts? If not, how do they
troubleshoot low recoveries?

9.7.5.3

9.8.7.3
9.13

-

Recommendation











1.11 Does the laboratory have an adequate record
system for tracking samples, including unique ID,
from collection through log-in, analysis, and data
reporting?

-

-

8.0

Critical
GLP











1.12 Is the laboratory using the Method December
2005 version of Method 1623 or Method 1623.1
for LT2 samples?







Requirement











2 Data Recording Procedures

2.1 Is shipping information complete, i.e., time/date of
sample collection, sampler's name, time/date of
sample receipt, receiver's initials, sample
condition?

8.1.3

8.1.3

8.5

Requirement











2.1.1 Were all samples evaluated received at
<20° C and not frozen?

8.1.3

8.1.3

6.3.3

Requirement











2.2 Do sample numbers on the chain of custody

match the sample numbers on the report forms?

-

-

-

Requirement











2.3 Are current Method 1623/1623.1 bench sheets
used to record sample processing data?

-

-

8.2

Recommendation











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-43

-------
Method 1623/1623.1 Checklist A

Item to be Evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

2.4 Are all primary measurements during each step
recorded, including all raw data used in
calculations?

9.1.2.2.5

9.3.5

8.0

Requirement











2.5 Technician/analyst, date, and time of elution is
recorded?

12.2.6.2.1

12.2.7.1
12.3.2.1

8.7

Requirement











2.6 Technician/analyst, date, and time of slide
preparation is recorded?

13.3.3.11

13.3.3.11

8.7

Requirement











2.7 Technician/analyst, date, and time of staining is
recorded?

14.10

14.10

8.7

Requirement











2.8 Are batch and lot numbers of reagents used in the
analysis of the sample recorded?

-

-

8.7

Critical











2.8.1 Lot number for the IMS kit is recorded?

-

-

8.7

Critical











2.8.2 Lot number of the staining kit is
recorded?

-

-

8.7

Critical











2.8.3 Lot number of the spiking suspensions is
recorded?

-

-

3.21.4
8.7

Critical











2.9 Spike value recorded for all spiked samples?

-

-

3.21.4
8.6

Requirement











2.10 Are Method 1623/1623.1 Cryptosporidium Slide
Examination forms used to record sample
examination results?

15.2

15.2

8.2

Requirement











2.11 Name of examining analyst is recorded?

15.2.6

15.2.6

8.7

Requirement











2.12 Date and time of sample examination is recorded?

15.2.4

15.2.4

8.7

Requirement











2.13 Are calculations of final concentrations and
recoveries complete and correct?

-

-

-

Requirement











2.14 Is the size of the cysts and oocysts reported to the
nearest 0.5 |jm?

15.2.2.3
15.2.3.3

15.2.2.4
15.2.3.4

5.4.9.4
5.4.10.4

Requirement











2.15 Is each reported positive organism detected in a
field sample characterized and recorded?

15.2

15.2.2.1
15.2.3.1

5.4.9.1
5.4.10.1

Requirement











2.16 Do values recorded on the data sheets match the
values reported to the client?

-

-

8.1

Requirement











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-44

-------
Method 1623/1623.1 Checklist A

Item to be Evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

2.17 Are mistakes on all forms crossed out with a
single line, initialed, and dated?

-

-

8.2

Critical











2.18 Are data always legible and recorded in pen?

-

-

8.2

Critical











2.19 Was the final report reviewed by QA manager, lab
director or an individual other than the analyst?

-

-

8.1

Critical











2.20 Do records demonstrate each analyst's

characterization of 3 oocysts and 3 cysts from
positive control for each microscopy session?

15.2.1.1

15.2.1.1

5.4.6

Requirement











2.21 Data shows that no more than 0.5 mL of pellet
was used per IMS?

13.2.4

13.2.3

5.2.3
8.6

Requirement











3 Holding Times -Method 1623.1

3.1 Is sample elution initiated within 96 hours of
sample collection or field filtration?

8.2.1
Table 1

8.2.1
Table 5

6.4
8.7

Requirement











3.2 Are sample elution, concentration, and purification
steps completed in one work day?

8.2.2
Table 1

8.2.2
Table 5

6.4
8.7

Requirement











3.3 Are slides stained within 72 hours of application of
the purified sample to the slide?

8.2.3
Table 1

8.2.3
Table 5

6.4
8.7

Requirement











3.4 Are stained slides read and confirmed within 7
days of staining? [Section 8.2.4 and Table 5]

8.2.4
Table 1

8.2.4
Table 5

6.4
8.7

Requirement











4 Spike enumeration procedures

4.1 Source of flow cytometry-enumerated spiking
suspensions.

-

11.2

-













4.2 If 50-L samples are analyzed, what positive

control procedure does the laboratory follow for
OPR and MS samples: (A) spike entire 50 L, (B)
spike and filter 10 L before filtering 40 L, or (C)
filter 40 L before spiking and filtering 10 L.

-

-

7.1.10.3













* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-45

-------
Method 1623/1623.1 Checklist A

The following items below are optional if the laboratory is NELAC certified. If the laboratory opts to provide NELAC certification, complete
Provide copy of certification.

the box

below by entering the NELAC certification number and date.

NELAC Certification Number:



Certification Date:



5 Laboratory Equipment and Supplies

5.1 Reagent-grade water testing

5.1.1 Is reagent water tested monthly for

conductivity and total chlorine residual?

-

-

4.3.1

Critical
GLP











5.1.1.1 Were the results for the above
parameters acceptable? Total
chlorine residual not greater
than 0.1 mg/L, conductivity not
greater than 2 |jmhos/cm?

-

-

4.3.1

Critical
GLP











5.1.2 Has the reagent water been tested

annually for metals - Pb, Cd, Cr, Cu, Ni,
Zn?

-

-

4.3.1

Critical
GLP











5.1.2.1 Were the results for the metals
testing acceptable; each metal
not greater than 0.05 mg/L and
collectively not greater than 0.1
mg/L?

-

-

4.3.1

Critical
GLP











5.1.3 Is reagent water tested monthly for
heterotrophic plate count?

-

-

4.3.1

Critical
GLP











5.1.3.1 Are the results for the

heterotrophic plate count
acceptable, < 500 CFU/mL?

-

-

4.3.1

Critical
GLP











5.1.4 Is still or Dl unit maintained according to
manufacturer's instructions?

-

-

4.3.3

Critical
GLP











5.2 pH meter

5.2.1 Accuracy ± 0.1 units, scale graduations,
0.1 units?

-

-

3.1.1

Critical
GLP











5.2.2 Is a record maintained for pH

measurements and calibrations?

-

-

3.1.4

Critical
GLP











5.2.3 Is pH meter standardized each use

period with pH 7, 4 or 10 standard buffers
(selection dependent upon desired pH)?

-

-

3.1.4

Critical
GLP











5.2.4 Are all pH buffers dated when received
and opened, and discarded before
expiration date?

-

-

3.1.5

Critical
GLP











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-46

-------
Method 1623/1623.1 Checklist A

5.3 Balances (top loader or pan balance)

5.3.1 Are balance calibrations verified monthly
using ASTM Class 1, Class 2 or Class 3
weights or weights traceable to Class 1,
Class 2, or Class 3 weights, or
equivalent? Non-reference weights
should be calibrated every six months
with reference weights.

-

-

3.2.2

Critical
GLP











5.3.2 Is correction data and Certificate of
Traceability available for weights?

-

-

3.2.3

Critical
GLP











5.3.3 Is preventative maintenance conducted
yearly at a minimum?

-

-

3.2.4

Recommendation
GLP











5.4 Temperature recording device

5.4.1 Are calibration of thermometers checked
annually (dial thermometers quarterly) at
the temperature used against a reference
NIST thermometer or equivalent?

8.1.4

8.1.4

3.3.2

Requirement
GLP











5.4.2 Is the sample storage refrigerator able to
maintain temperature of 1 to 10°C?

-

-

3.7.1

Critical
GLP











5.5 Micropipetters

5.5.1 Have micropipetters been calibrated
within the past year?

9.2.1

Appendix
A

3.8.2

Requirement
GLP











5.6 Centrifuge

5.6.1 Is a maintenance contract in place or

internal maintenance protocol available?

9.1

9.1

3.15.5

Critical
GLP











5.6.2 Is the centrifuge calibrated yearly?

-

-

3.15.5

Critical
GLP











5.7 Autoclave

5.7.1 Are date, contents, sterilization time and
temperature, and technician initials
recorded for each cycle?

-

-

3.5.3

Critical
GLP











5.7.2 Is a maximum registering thermometer or
continuous monitoring device used
during each autoclave cycle?

-

-

3.5.5

Critical
GLP











5.7.3 Is automatic timing mechanism checked
with stopwatch quarterly?

-

-

3.5.6

Critical
GLP











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-47

-------
Method 1623/1623.1 Checklist A

5.7.4 Are spore strips or ampules used
monthly to confirm sterilization?

-

-

3.5.5

Critical
GLP











6 Quality Assurance Manual

6.1 Does the laboratory have a formal QA laboratory
plan prepared and ready for examination?

9.1

9.1

4.5.1

Requirement











6.2 Is a laboratory organization chart or other

information available listing staff organization and
responsibilities? Does it identify the QA manager
and lab director?





4.5.2

Recommendation











6.2.1 Is the QA manager separate from the lab
director?

-

-

4.5.2

Recommendation
GLP











6.3 Does the laboratory have a schedule and/or
procedure for all preventative maintenance of
equipment?

-

-

4.5.3

GLP











Comments:

* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-48

-------
Method 1623/1623.1 Checklist B

Checklist B - Laboratory SOP Review

Laboratory Name

Name and Affiliation of Evaluator

Date of Evaluation







Good Laboratory Practice (GLP) is generally defined as a system of management controls for the laboratories to ensure the consistency and reliability of results. Adapted
from other federal programs for the purposes of the Cryptosporidium Laboratory QA Evaluation Program, GLP includes personnel, equipment, and standard operating
procedures appropriate for the program.

Item to be Evaluated

For each item, does the SOP specify:

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

1 Sample Spiking

1.1 The suspension vial is vortexed for 30 seconds or pel
manufacturer's instructions?

11.4.3.1.2

11.2.3.2

-

Method Procedure











1.2 The carboy used for the method blank is randomly
selected from carboy stock to check efficacy of
cleaning system or disposable carboys are used for
all samples?

-

-

7.1.5.3

Critical











1.3 The details of the suspension vial rinse, including
volumes?

11.4.3.1

11.2.3

-

Method Procedure











1.4 Acceptable sample spiking procedures, including
issues not noted in items 1.1 through 1.3?







Critical
GLP











2 Filtration/Elution

2.1 Envirochek HV filtration

2.1.1 The flow rate is maintained at
approximately 2 L/min?

12.2.1.2

12.2.1.2

-

Method Procedure











2.1.2 The volume filtered is measured using a
flow totalizer or calibrated carboy?

12.2.4.2

12.2.4.2

-

Requirement











2.1.3 The sample is stirred during filtration?

12.2.4.1

12.2.4.1

-

Method Procedure











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-49

-------
Method 1623/1623.1 Checklist B

Item to be Evaluated

For each item, does the SOP specify:

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

2.1.4 The details of the carboy rinse after
filtration including volume?

12.2.4.5

12.2.4.6

-

Method Procedure











2.1.5 Appropriate maintenance and cleaning
procedures?

-

-

-

Critical











2.1.6 Acceptable Envirochek® filtration

procedures, including issues not noted in
items 2.1.1 through 2.1.5?







Critical
GLP











2.2 Envirochek HV capsule filter elution

2.2.1 Measurement of the volume of the elution
buffer used or that the volume covers the
membrane?

12.2.6.2.2

12.2.8.2

-

Method Procedure











2.2.2 The speed that samples are shaken?

12.2.6.2.3

12.2.8.3

-

Method Procedure











2.2.3 The dispersant is added to the sample as
per Method 1623.1?



12.2.7

-

1623
Recommendation

1623.1 Requirement











2.2.4 The samples are shaken three times for 5
minutes each time, and each in a different
orientation?

12.2.6.2

12.2.8

-

Method Procedure











2.2.5 Procedures for filter capsule rinse and

addition of rinsate to the centrifuge bottle?

12.2.6.2.8

12.2.8.8

-

Method Procedure











2.2.6 Acceptable Envirochek® capsule filter
elution procedures, including issues not
noted in items 2.2.1 through 2.2.5?







Critical
GLP











2.3 Filta-Max filtration

2.3.1 The flow rate is maintained at <4 L per
minute for Filta-Max®?

12.3.1.1.3

12.3.1.1.3

-

Method Procedure











2.3.2 The volume filtered is measured using a
flow totalizer or calibrated carboy?

12.3.1.5.2

12.3.1.5.2

-

Requirement











2.3.3 Appropriate maintenance and cleaning
procedures? [Section 12.3.4]

12.3.4

12.3.4

-

Requirement











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-50

-------
Method 1623/1623.1 Checklist B

Item to be Evaluated

For each item, does the SOP specify:

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

2.3.4 Acceptable Filta-Max® filtration

procedures, including issues not noted in
items 2.3.1 through 2.3.3?







Critical
GLP











2.4 Filta-Max filter wash station elution

2.4.1 The use of PBST to elute the filter?

7.4.2.4

7.6.2.4

-

Method Procedure











2.4.2 The amount of PBST used for each wash?
(approx. 600 mL)

12.3.2.2

12.3.2.2

-

Method Procedure











2.4.3 The plunger is moved up and down 20
times during the first wash?

12.3.2.2.1
h

12.3.2.2.1
h

-

Method Procedure











2.4.4 The plunger is moved up and down gently
to avoid generating excess foam?

12.3.2.2.1
h

12.3.2.2.1
h

-

Method Procedure











2.4.5 That during the second wash the plunger
is moved up and down 10 times?

12.3.2.2.2
b

12.3.2.2.2
b

-

Method Procedure











2.4.6 The instructions for cleaning the wash
station between samples?

12.3.4.2

12.3.4.2

-

Requirement











2.4.7 The housing is rinsed after filter is

removed and the rinse is included in the
sample volume?

12.3.2.2.1
d

12.3.2.2.1
d

-

Method Procedure











2.4.8 Acceptable Filta-Max® filter wash station
elution procedures, including issues not
noted in items 2.4.1 through 2.4.7?







Critical
GLP











3 Concentration

3.1 Filta-Max filter sample concentration (as an alternative or in addition to Section 3.2)

3.1.1 The force of the vacuum is maintained
below 30 cm Hg?

NOTE

pg 43

NOTE
pg 34

-

Method Procedure











3.1.2 That concentration is performed after each
of the washes?

12.3.2.2.1

j

12.3.2.2.1

j

-

Method Procedure











3.1.3 The sample is concentrated so that some
liquid remains above the filter (enough to
cover the stir bar about half-way)?

12.3.3.2.1
c

12.3.3.2.1
b

-

Method Procedure











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-51

-------
Method 1623/1623.1 Checklist B

Item to be Evaluated

For each item, does the SOP specify:

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

3.1.4 The stir bar and concentration tube are
rinsed after each concentration and the
liquid added to the concentrate?

12.3.3.2

12.3.3.2

-

Requirement











3.1.5 The filter membrane is washed twice with
5 mL of PBST each time?

12.3.3.2.3

12.3.3.2.3

-

Method Procedure











3.1.6 Acceptable Filta-Max® filter sample

concentration procedures, including issues
not noted in items 3.1.1 through 3.1.5?







Critical
GLP











3.2 Envirochek HV and Filta-Max® filter sample centrifugation

3.2.1 The sample is centrifuged at 1500 x G
(maximum 2000 x G) using a swinging
bucket rotor?

13.2.1
including
NOTE

13.2.1
including
NOTE

-

Method Procedure











3.2.2 Instructions to ensure the centrifuge tubes
are properly balanced prior to
centrifugation?

-

-

3.15.4

Critical











3.2.3 The sample is centrifuged for 15 minutes
with start time beginning when centrifuge
reaches the required speed?

13.2.1

13.2.1

-

Method Procedure











3.2.4 The centrifuge is slowly decelerated at the
end without using the brake?

13.2.1

13.2.1

-

Method Procedure











3.2.5 Acceptable Envirochek® HV and Filta-
Max® filter sample centrifugation
procedures, including issues not noted in
items 3.2.1 through 3.2.4?







Critical
GLP











4 Purification and Slide Preparation

4.1 The centrifuged sample supernatant is aspirated no
lower than 5 mL of supernatant above every 0.5 mL
of the pellet or portion of 0.5 mL pellet?

13.2.2

13.2.2

13.2.3

5.2.2

5.2.3

Requirement











4.1.1 The type and internal diameter of pipette
used for aspiration of supernatant?

-

NOTE
pg 37

-

Recommendation











4.1.2 The rate of aspiration (i.e., mL/ min or
pressure of the vacuum)?

-

13.2.2

-

Recommendation











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-52

-------
Method 1623/1623.1 Checklist B

Item to be Evaluated

For each item, does the SOP specify:

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

4.2 The tube is vortexed vigorously until pellet is
completely resuspended?

13.2.3

13.2.2.1

-

Method Procedure











4.3 Appropriate procedures for dividing pellets greater
than 0.5 mL into subsamples and the analysis of
the subsamples?

13.2.4

13.2.3

-

Critical











4.4 No more than 0.5 mL of pellet is used per IMS?

13.2.4

13.2.3

5.2.3

Method Procedure











4.5 The resuspended pellet volume is quantitatively
transferred to the flat-sided tube (2 rinses) including
the determination of the rinse volumes?

13.3.2.1

13.3.2.1

-

Method Procedure











4.6 SL-Buffer A is used at room temperature or that it is
checked for precipitate before use?

NOTE
pg 47

NOTE
pg 39

3.17.2

Method Procedure











4.7 The volume of 10x SL-Buffer A is 1 mL?

13.3.1.2

13.3.1.2

5.2.5

Method Procedure











4.8 The volume of 10x SL-Buffer B is 1 mL?

13.3.1.3

13.3.1.3

5.2.5

Method Procedure











4.9 Instructions for thorough resuspension of IMS
beads prior to addition to the flat-sided tube?

13.3.2.2
13.3.2.4

13.3.2.2
13.3.2.4

-

Method Procedure











4.10 100 |jL of Cryptosporidium and Giardia beads are
used?

13.3.2.3
13.3.2.5

13.3.2.3
13.3.2.5

5.2.5

Method Procedure











4.11 The flat-sided tube is rotated at 18 rpm for 1 hour at
room temperature?

13.3.2.6

13.3.2.6

-

Method Procedure











4.12 Which magnetic concentrators, MPC®-1 or MPC®-6,
are used?







Method Procedure











4.13 The placement of the flat-sided tube in the magnet
and the rock technique and time?

13.3.2.9

13.3.2.8

13.3.2.9

-

Method Procedure











4.14 The sample is quantitatively transferred from the
flat-sided tube to the microcentrifuge tube (2 rinses)
including rinse volumes?

13.3.2.13

13.3.2.14

-

Method Procedure











4.15 The flat-sided tube is allowed to sit one minute after
each transfer to accumulate residual sample, then
the residual is transferred to microcentrifuge tube?

13.3.2.13

13.3.2.14

-

Method Procedure











4.16 The magnet is in the vertical position in the MPC®-
S?

-

13.3.2.13

-

Method Procedure











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-53

-------
Method 1623/1623.1 Checklist B

Item to be Evaluated

For each item, does the SOP specify:

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

4.17 The beads are rinsed with PBS while inside the
microcentrifuge tube?

13.3.4

13.3.2.17

-

1623
Recommendation

1623.1 Requirement











4.18 Standard NaOH (5 jiL, 1N) and standard HCI (50
jiL, 0.1 N) are used?

NOTES
pg 49-50

NOTES

pg 42

3.17.5

Requirement











4.19 The sample is vortexed vigorously for 50 seconds
immediately after the addition of acid and 30
seconds after the sample has set for 10 minutes at
room temperature?

13.3.3

13.3.3

-

Method Procedure











4.20 The magnet is in the slanted position in the MPC®-S
for dissociation steps?

-

13.3.3.6

-

Method Procedure











4.21 A second dissociation is performed?

13.3.3.10

13.3.3.10

5.2.4

Requirement











4.22 When the second dissociation is performed, the
laboratory:

A)	uses a second slide, or

B)	adds the additional volume to the original slide?

13.3.3.10

13.3.3.10
13.4.5

-

Circle one:
A B











4.23 The volume and the timing of the NaOH addition to
the wells?

13.3.3.8

13.3.3.8

-

Method Procedure











4.24 When the slides are dried (e.g., room temperature
or slide warmer), the laboratory:

A)	uses room temperature, or

B)	uses 35° to 42°C, or

C)	follows manufacturer's instructions?

13.3.3.12

13.3.3.12

-

Circle one:
ABC











4.25 If the laboratory has more than one option specified
for slide drying, are criteria included for when each
option will be used?

-

-

5.3.1

Recommendation











4.26 That positive and negative staining controls are

prepared at the same time the slides are prepared?

14.1

14.1.3

-

Requirement











4.27 Acceptable sample purification and slide

preparation procedures, including issues not noted
in items 4.1 through 4.26?







Critical
GLP











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-54

-------
Method 1623/1623.1 Checklist B

Item to be Evaluated

For each item, does the SOP specify:

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

5 Sample Staining

5.1 Which stain to use and to follow manufacturer's
instructions for FITC stain application?

14.2

14.2

5.3.2

Method Procedure











5.2 The slides are incubated in a humid chamber in the
dark at room temperature for approximately 30
minutes or per manufacturer's directions?

14.3

14.3

5.3.3

Method Procedure











5.3 The working DAPI stain is prepared the day it is
used?

7.7.2

7.9.2

3.19.2

Method Procedure











5.4 The stock DAPI is stored at 1 to 10°C in the dark?

7.7.1

7.9.1

3.19.1

Method Procedure











5.5 The volume of working DAPI applied and the
incubation time?

14.6

14.6

-

Method Procedure











5.6 The technique used to drain the excess stain from
the well and to rinse the well?

14.5

14.5

-

Method Procedure











5.7 What type and amount of mounting media used?

7.8

7.10

-

Method Procedure











5.8 That all the edges of the cover slip are sealed well
with clear fingernail polish, unless Elvanol® is
used?

14.9

14.9

-

Method Procedure











5.9 The finished slides or slides not read immediately
are stored in a humid chamber in the dark at 1° to
10°C (humid chamber not required for Elvanol®)?

14.10

14.10

5.3.6

Method Procedure











5.10 Acceptable sample staining procedures, including
issues not noted in items 5.1 through 5.9?







Critical
GLP











6 Microscope and Examination

6.1 Instructions for ocular and Kohler adjustments?

10.3.4
10.3.6

10.7

10.8

3.22.10

Requirement











6.2 That all measurements must be recorded to the
nearest 0.5 micron?

15.2.2.3
15.2.3.3

15.2.2.4
15.2.3.4

3.22.5

Requirement











6.3 Microscope cleaning procedures?

10.4

10.9

3.22.11

Requirement











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-55

-------
Method 1623/1623.1 Checklist B

Item to be Evaluated

For each item, does the SOP specify:

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

6.4 The recording of coordinates of all cysts and

oocysts on the worksheet for future reference; and
slide orientation on the microscope stage to
standardize coordinate recording?

-

-

-

Recommendation











6.5 The examination and acceptance of positive and
negative staining controls before proceeding with
examination of field samples?

15.2.1

15.2.1

5.4.6

5.4.7

Requirement











6.6 That each analyst characterizes 3 oocysts and 3
cysts on the positive staining control at each
examination session?

15.2.1.1

15.2.1.1

5.4.6

Requirement











6.7 Corrective actions if positive and/or negative
staining controls are not acceptable?

-

-

5.4.8

Recommendation











6.8 The criteria for organism identification?

15.2.2

15.2.2

15.2.3

5.4.9

5.4.10

Requirement











6.9 Every positive organism in a field sample is
characterized and recorded?

15.2

15.2.2.1
15.2.3.1

5.4.9.1
5.4.10.1

Requirement











6.10 Acceptable microscope and examination

procedures, including issues not noted in items 6.1
through 6.9?







Requirement
GLP











7 Reagents

7.1 Procedures for the preparation of all essential
chemicals and reagents?

7.0

7.0

4.2

Critical











7.2 That expiration dates are specified for all reagents
prepared by the laboratory?

-

-

4.2.2

Critical











8 Quality Assurance

8.1 Training protocol for new employees?

9.1

9.1

1.7

Requirement
GLP











8.2 Procedures for performing analyst verification?

10.6

9.10

7.1.9

Requirement
GLP











8.3 Positive and interfering organisms detected in field
samples are documented by photography?

-

-

5.4.11

Recommendation











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-56

-------
Method 1623/1623.1 Checklist B

Item to be Evaluated

For each item, does the SOP specify:

Reference*

Classification

Satisfactory

Comments/
Response Requested

1623

1623.1

Cert

Yes

No

NA

UNK

8.4 Acceptable procedures for sample collection for
field or utility personnel?

-

-

6.1

Critical
GLP











8.5 Criteria for sample acceptance and corrective
action procedures?

8.1.3

8.1.3

6.

Requirement
GLP











8.6 Method required holding times?

8.2

8.2

6.4

Requirement
GLP











8.7 Manual data recording procedures?

-

-

8.0

Critical
GLP











8.8 Procedures for checking the accuracy of data
transcriptions, including electronic data entry?

-

-

8.1

Critical
GLP











8.9 Procedures for checking the accuracy of manual
calculations?

-

-

8.1

Critical
GLP











8.10 Procedures for electronic data entry and storage?

-

-

8.2

Critical
GLP











8.11 How backup of stored data is performed?

-

-

8.2

Critical
GLP











8.12 Corrective action procedures for OPR failures?

9.7.4

9.8.5

7.1.6.2

Requirement
GLP











8.13 Corrective action procedures for method blank
contamination?

9.6.2

9.7.3

7.1.5.2

Requirement
GLP











8.14 Procedures for identifying and assessing declining
trends in recovery through review of control charts
and/or other recovery data?

-

-

7.1.7.2

Recommendation
GLP











8.15 Corrective action procedures for investigating QC
failures or declining trends in recovery?

-

-

7.1.7.2

Recommendation
GLP











8.16 Acceptable glassware washing procedures?

-

-

4.4

Critical
GLP











Comments:

* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to
the Fifth Edition of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-57

-------
Method 1623/1623.1 Checklist C

Checklist C - Method 1623/1623.1 Technical Review - Sample Processing and Microscopy

Laboratory Name

Name and Affiliation of Evaluator

Date of Evaluation







Good Laboratory Practice (GLP) is generally defined as a system of management controls for the laboratories to ensure the consistency and reliability of results. Adapted from other
federal programs for the purposes of the Cryptosporidium Laboratory QA Evaluation Program, GLP includes personnel, equipment, and standard operating procedures appropriate for
the program.

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



1 Laboratory Facilities

1.1 Does laboratory appear to have

established appropriate safety and health
practices prior to use of this method?

5.0

5.0

4.1

Critical











1.2 Do all laboratory personnel wear gloves
when handling biohazard and toxic
compounds, and change gloves before
touching other surfaces and equipment?

CO

in in

5.3

4.1.6

Critical
GLP











1.3 Does the laboratory disinfect bench
surfaces before and after analyses?

-

-

4.1.3

Critical
GLP











1.4 Does the laboratory have adequate bench
space to perform the method?

-

-

2.0

Critical
GLP











1.5 Other than the issues noted in items 1.1
through 1.4 (if any), no other facility issues
were observed?



















2 Reagents

2.1 Is reagent water used to prepare all
reagents?

7.3

7.3

4.3.1

Requirement











2.2 Are all reagents clearly labeled with
identity of reagent, date of preparation,
technician initials, and expiration date?

-

-

4.2.2

Critical
GLP











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-58

-------
Method 1623/1623.1 Checklist C

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



2.3 Are SOPs available in the work area, and
does laboratory practice reflect written
procedures?







Critical
GLP











3 Sample Spiking

Technician:

3.1 Was spike suspension vial vortexed for 30
seconds or per manufacturer's
instructions?

11.4.3.1.2

11.2.3.2

-

Method Procedure











3.2 Is the carboy used for method blank
randomly selected from carboy stock to
check efficacy of cleaning system?

-

-

7.1.5.3

Critical
GLP











3.3 Was the suspension vial adequately
rinsed?

11.4.3.1

11.2.3

-

Method Procedure











3.4 Are SOPs for sample spiking available in
the work area, and does laboratory
practice reflect written procedures?







Critical
GLP











3.5 Other than issues noted for items 3.1
through 3.4 (if any) was sample spiking
demonstrated successfully?



















4 Filtration/Elution

4.1 Envirochek HV filtration

Technician:

4.1.1 Are all components required for
sample filtration present and in
good condition?

6.1
6.2.1-6.2.2
6.3

6.1 -
6.2.8

6.1.7

Requirement
GLP











4.1.2 Is the filter assembly set up
correctly?

Figure 3a

Figure 1

-

Method Procedure
GLP











4.1.3 Is the pump adequate for needs?

6.3.3

6.2.4

-

Requirement
GLP











4.1.4 Is the appropriate flow rate
maintained (approximately 2
L/min)?

12.2.1.2

12.2.1.2

-

Method Procedure











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-59

-------
Method 1623/1623.1 Checklist C

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



4.1.5 Is the volume filtered measured
using a flow totalizer or calibrated
carboy?

12.2.4.2

12.2.4.2

-

Requirement











4.1.6 Is the system well maintained
and cleaned appropriately
following use?

4.5

4.5

6.1.7

Critical
GLP











4.1.7 Is the system able to maintain
seal during use with no leaks?

-

-

6.1.7

Requirement
GLP











4.1.8 Are SOPs for Envirochek® HV
filtration available in the work
area, and does laboratory
practice reflect written
procedures?







Critical
GLP











4.1.9 Other than issues noted for items
4.1.1 through 4.1.8 (if any) was
Envirochek HV filtration
demonstrated successfully?



















4.2 Envirochek HV capsule filter elution

Technician:

4.2.1 Is the elution buffer prepared as
per Method?

7.4.1

7.6.1

-

Method Procedure











4.2.2 Is the wrist-shaker assembly set
up correctly with arms fully
extended?

12.2.6.1.1

12.2.6.1

3.14.2

Method Procedure
GLP











4.2.3 Is the dispersant addition
performed as per Method
1623.1?

-

12.2.7

-

1623
Recommendation
1623.1 Method
Procedure











4.2.4 Is volume of elution buffer

measured to ensure the use of
one 250 mL centrifuge tube?

12.2.6.2.2

12.2.8.2

-

Method Procedure











4.2.5 Are the samples shaken at an
appropriate speed?

12.2.6.2.3

12.2.8.3

3.14.3

Method Procedure











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-60

-------
Method 1623/1623.1 Checklist C

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



4.2.6 Are the samples shaken three
times for 5 minutes each time,
and each in a different
orientation?

12.2.6.2

12.2.8

-

Method Procedure











4.2.7 Are SOPs for Envirochek® HV
capsule filter elution available in
the work area, and does
laboratory practice reflect written
procedures?







Critical
GLP











4.2.8 Other than issues noted for items
4.2.1 through 4.2.7 (if any) was
Envirochek" HV capsule filter
elution demonstrated
successfully?



















4.3 Filta-Max filtration

Technician:

4.3.1 Which filter is used - Filta-Max®
(black end caps) or Filta-Max
xpress® (red end caps)?



















4.3.2 Are all components required for
sample filtration present and in
good condition?

6.1
6.2.1
6.2.3
6.3

6.1
6.2.1-6.2.7
6.2.9

6.1.7

Requirement
GLP











4.3.3 Is the filter assembly set up
correctly?

Figure 3b

Figure 2

-

Method Procedure
GLP











4.3.4 Is appropriate flow rate

maintained of <4 L per minute for
Filta-Max®?

12.3.1.1.3

12.3.1.1.3

-

Method Procedure











4.3.5 Is the volume filtered measured
correctly using a flow meter or
calibrated carboy?

12.3.1.5.2

12.3.1.5.2

-

Requirement
GLP











4.3.6 Is system well maintained and
cleaned appropriately following
use?

12.3.4

12.3.4

6.1.7

Requirement
GLP











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-61

-------
Method 1623/1623.1 Checklist C

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



4.3.7 Is system able to maintain seal
during use with no leaks?

-

-

6.1.7

Requirement
GLP











4.3.8 Does the laboratory indicate on
the filter housing the correct
direction of flow?

12.3.1.3

12.3.1.3

-

Critical











4.3.9 Are SOPs for Filta-Max® filtration
available in the work area, and
does laboratory practice reflect
written procedures?







Critical
GLP











4.3.10 Other than issues noted for items
4.3.1 through 4.3.9 (if any) was
Filta-Max® filtration demonstrated
successfully?



















4.4 Filta-Max filter wash station elution

Technician:

4.4.1 Is an automatic or manual wash
station used?



















4.4.2 Is the filter wash station set up
correctly?

12.3.2.1

12.3.2.1

-

Requirement
GLP











4.4.3 Is residual suspension rinsed
from all containers?

12.3.2.2.1d

12.3.2.2.1d

-

Critical











4.4.4 Is PBST used to elute the filter?

7.4.2.4

7.6.2.4

-

Method Procedure











4.4.5 Is an appropriate amount of
PBST used for each wash?
(approx. 600 mL)

12.3.2.2

12.3.2.2

-

Method Procedure











4.4.6 During the first wash, is the

plunger moved up and down 20
times?

12.3.2.2.1h

12.3.2.2.1h

-

Method Procedure











4.4.7 Is the plunger moved up and

down gently to avoid generating
excess foam?

12.3.2.2.1h

12.3.2.2.1h

-

Method Procedure











4.4.8 During the second wash, is the
plunger moved up and down 10
times?

12.3.2.2.2b

12.3.2.2.2b

-

Method Procedure











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-62

-------
Method 1623/1623.1 Checklist C

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



4.4.9 If the automatic washer is used,
is the machine operating
properly?

12.3.2.1

12.3.2.1

-

Requirement











4.4.10 Is the wash station cleaned

adequately between samples?

12.3.4.2

12.3.4.2

-

Requirement
GLP











4.4.11 Are SOPs for Filta-Max® filter

wash station elution available in
the work area, and does
laboratory practice reflect written
procedures?







Critical
GLP











4.4.12 Other than issues noted for items
4.4.1 through 4.4.11 (if any) was
Filta-Max® filter wash station
elution demonstrated
successfully?



















5 Concentration



















5.1 Filta-Max filter sample concentration

Technician:

5.1.1 Is concentrator set up correctly?

12.3.3.2.1b

12.3.3.2.1a

-

Requirement
GLP











5.1.2 Is the force of the vacuum

maintained below 30 cm Hg?

NOTE
pg 43

NOTE
pg 34

-

Method Procedure











5.1.3 Is concentration performed after
each of the washes?

12.3.2.2.1j

12.3.2.2.1 j

-

Method Procedure











5.1.4 Is the sample concentrated so
that some liquid remains above
the filter (enough to cover the stir
bar about half-way)?

12.3.3.2.1c

12.3.3.2.1b

-

Method Procedure











5.1.5 Are the stir bar and concentration
tube rinsed after each
concentration and the liquid
added to the concentrate?

12.3.3.2.1c

12.3.3.2.1b

-

Requirement











5.1.6 Was the filter membrane washed
twice with 5 mL of PBST?

12.3.3.2.3

12.3.3.2.3

-

Method Procedure











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-63

-------
Method 1623/1623.1 Checklist C

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



5.1.7 Are SOPs for Filta-Max® filter

sample concentration available in
the work area, and does
laboratory practice reflect written
procedures?







Critical
GLP











5.1.8 Other than issues noted for items
5.1.1 through 5.1.7 (if any) was
Filta-Max® filter sample
concentration demonstrated
successfully?



















5.2 Envirochek HV and Filta-Max filter sample centrifugation

Technician:

5.2.1 Is the sample centrifuged at 1500
x G (maximum 2000 x G) using a
swinging bucket rotor?

13.2.1
and
NOTE
pg 46

13.2.1
and
NOTE
pg 37

-

Method Procedure
GLP











5.2.2 Are the centrifuge tubes properly
balanced prior to centrifugation?

-

13.2.1

3.15.4

Critical











5.2.3 Does lab have easily accessible
method for determining relative
centrifugal force of centrifuges?

-

-

3.15.1

Critical
GLP











5.2.4 Is the sample centrifuged for 15
minutes, with time beginning
when centrifuge reaches desired
speed?

13.2.1

13.2.1

-

Method Procedure











5.2.5 Is the centrifuge slowly

decelerated at the end without
the brake?

13.2.1

13.2.1

-

Method Procedure











5.2.6 Is the pellet volume determined?

13.2.1

13.2.1

5.2.3

Requirement











5.2.7 Is there a set of standards for
comparison of pellet size?

-

-

5.2.3

Recommendation
GLP











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-64

-------
Method 1623/1623.1 Checklist C

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



5.2.8 Are SOPs for Envirochek® and
Filta-Max® filter sample
centrifugation available in the
work area, and does laboratory
practice reflect written
procedures?







Critical
GLP











5.2.9 Other than issues noted for items
5.2.1 through 5.2.8 (if any) was
Envirochek HV or Filta-Max®
filter sample centrifugation
demonstrated successfully?



















6 Purification and Slide Preparation

Technician:

6.1 Is an approved IMS kit/manufacturer
used?

7.5

7.7.1

-

Method Procedure
GLP











6.2 Is the supernatant from the centrifuged
sample aspirated no lower than 5 mL of
supernatant above every 0.5 mL pellet or
portion of 0.5 mL pellet?

13.2.2

13.2.2

13.2.3

5.2.2

5.2.3

Requirement











6.2.1 Are the samples aspirated using
the pipette, with the documented
internal diameter, as specified in
the SOP?

-

NOTE
pg 37

-

Critical











6.2.2 Is the proper rate (mL/min) or
pressure (psi) maintained
throughout aspiration?

-

13.2.2

13.2.3

-

Method Procedure











6.3 Is the pellet vortexed a sufficient time for
resuspension?

13.2.3
13.2.4.1.3
13.2.4.2

13.2.2.1
13.2.3.1.2

13.2.3.2

-

Method Procedure











6.4 Is the resuspended pellet volume

quantitatively transferred to the flat-sided
tube (2 rinses)?

13.3.2.1

13.3.2.1

-

Method Procedure











6.5 Are the IMS beads thoroughly

resuspended prior to addition to the flat-
sided tube?

13.3.2.2
13.3.2.4

13.3.2.2
13.3.2.4

-

Method Procedure











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-65

-------
Method 1623/1623.1 Checklist C

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



6.6 Is the flat-sided tube rotated at 18 rpm for
1 hour at room temperature?

13.3.2.6

13.3.2.6

-

Method Procedure











6.7 Is the rotating mixer calibrated annually?

-

-

3.17.4

Critical
GLP











6.8 Is flat-sided tube correctly placed in

magnet and rocked through 90 degrees
about once per second?

13.3.2.7-
13.3.2.9

13.3.2.7-
13.3.2.9

-

Method Procedure











6.9 Is all the liquid removed when decanting is
performed with the magnet up?

13.3.2.11

13.3.2.11

-

Method Procedure











6.10 Is the sample quantitatively transferred
from the flat-sided tube to the
microcentrifuge tube (2 rinses)?

13.3.2.13

13.3.2.14

-

Method Procedure











6.11 Are the beads rinsed with PBS while
inside the microcentrifuge tube?

13.3.4

13.3.2.17

-

1623
Recommendation

1623.1 Requirement











6.12 Is standard NaOH (5 |jL, 1N) and standard
HCI (50 |jL, 0-1N) used?

NOTE
pg 49 & 50

NOTE
pg 42

3.17.5

Requirement
GLP











6.13 Is sample vortexed vigorously for 50

seconds immediately after the addition of
acid and 30 seconds after the sample has
set for 10 minutes at room temperature?

13.3.3.2-
13.3.3.4

13.3.3.2-
13.3.3.4

-

Method Procedure











6.14 Is a second dissociation performed?

13.3.3.10
NOTE
pg 49

13.3.3.10
NOTE
pg 41

5.2.4

Requirement











6.15 When the second dissociation is
performed, does the laboratory:

(A)	use a second slide

(B)	add the additional volume to the
original slide?

13.3.3.10

13.3.3.10
13.4.5

-

Circle one:
A B











6.16 Are the slides clearly labeled so they can
be associated with the correct sample?

13.3.3.7

13.3.3.7

-

Requirement











6.17 What type of slides is used?







GLP











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-66

-------
Method 1623/1623.1 Checklist C

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



6.18 Is slide dried at: (A) room temperature, (B)
35° to 42°C, or (C) in the refrigerator?

13.3.3.12

13.3.3.12

-

Circle one:
ABC











6.19 If the slide is warmed, is incubator or slide
warmer calibrated and labeled?

-

-

3.4

Critical
GLP











6.20 Are SOPs available in the work area for
sample purification and slide preparation,
and does laboratory practice reflect written
procedures?







Critical
GLP











6.21 Other than issues noted for items 6.1

through 6.20 (if any) was purification and
slide preparation demonstrated
successfully?



















7 Sample Staining

Technician:

7.1 What staining kit/manufacturer is used?

14.2

14.2

3.18.1

GLP











7.2 Is FITC stain applied according to
manufacturer's directions?

14.2

14.2

5.3.2

Method Procedure











7.3 Are positive and negative staining controls
performed?

14.1

14.1

5.3.5

Requirement











7.4 Are the slides incubated in a humid

chamber in the dark at room temperature
for approximately 30 minutes or per
manufacturer's directions?

14.3

14.3

5.3.3

Method Procedure











7.5 Are the labeling reagents rinsed away
properly after incubation, without
disturbing the sample?

14.5

14.5

-

Method Procedure











7.6 Was the working DAPI stain prepared the
day it was used?

7.7.2

7.9.2

3.19.2

Method Procedure











7.7 Is stock DAPI stored at 1 to 10°C in the
dark?

7.7.1

7.9.1

3.19.1

Method Procedure











7.8 Is the DAPI stain applied properly and
allowed to stand for a minimum of 1
minute?

14.6

14.6

-

Method Procedure











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-67

-------
Method 1623/1623.1 Checklist C

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



7.9 Is the DAPI stain rinsed away properly
without disturbing the sample?

14.7

14.7

-

Method Procedure











7.10 Is the mounting media applied properly?

14.8

14.8

-

Method Procedure











7.10.1 What type of mounting media is
used?

7.8

7.10

-

GLP











7.10.2 Are all the edges of the cover slip
sealed well with clear fingernail
polish, unless Elvanol® is used?

7.9
14.9

7.11
14.9

-

Method Procedure











7.11 Are the finished slides stored in a humid
chamber in the dark at 1 to 10°C (humid
chamber not required for Elvanol®)?

14.10

14.10

5.3.6

Method Procedure











7.12 Are SOPs for sample staining available in
the work area, and does laboratory
practice reflect written procedures?







Critical
GLP











7.13 Other than issues noted for items 7.1

through 7.12 (if any) was sample staining
demonstrated successfully?



















8 Microscope and Examination

8.1 Is microscope equipped with appropriate
excitation and band pass filters for
examining FITC labeled specimens as
demonstrated with lab, and auditor
provided, positive staining control?

6.9.2

6.7.2

3.22.3

Requirement
GLP











8.2 Is microscope equipped with appropriate
excitation and band pass filters for
examining DAPI labeled specimens as
demonstrated with lab, and auditor
provided, positive staining control?

6.9.3

6.7.3

3.22.3

Requirement
GLP











8.3 Does the microscope have appropriate
objectives and filters for DIC, which
change easily to and from
epifluorescence?

6.9.1

6.7.1

3.22.4

Requirement
GLP











* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-68

-------
Method 1623/1623.1 Checklist C

Item to be evaluated

Reference*

Classification

Satisfactory

Comments/
Response Requested



1623

1623.1

Cert



Yes

No

NA

UNK



8.4 Are all portions of the microscope, from
the light sources to the oculars, properly
adjusted?

10.3

10.0
Appendix B

3.22.6

Requirement











8.5 Is the DIC image appropriate for each
laboratory microscope?

-

Figure 4

Visual
Guide

Requirement











8.6 Is microscope cleaned after every
session?

10.4

10.9.8

3.22.11

Requirement
GLP











8.7 Does the microscope have a 20X
scanning objective?

6.9.1

6.7.1

3.22.8

Requirement
GLP











8.8 Does the microscope have a 100X oil
immersion objective?

6.9.1

6.7.1

3.22.8

Requirement
GLP











8.9 Is the microscope equipped with an ocular
micrometer?

6.9.1

6.7.1

3.22.9

Requirement
GLP











8.10 Is a stage micrometer available to
laboratory?

6.9.1
10.3.5

6.7.1
App. B 3

3.22.9

Requirement











8.11 Is a calibration table for 100X objective
located close to the microscope(s)?

10.3.5.7

App. B 3.7

3.22.9

Requirement











8.12 Has the mercury bulb been used less than
the maximum hours recommended by the
manufacturer?

10.3.2.11

App.B 1.11

3.22.12

Requirement











8.13 Does the laboratory have a preventative
maintenance agreement in place to
service the microscope annually?

-

-

3.22.6

Critical
GLP











8.14 Are SOPs for sample examination
available in the work area, and does
laboratory practice reflect written
procedures?







Critical
GLP











8.15 Other than issues noted for items 8.1

through 8.13 (if any) was Microscope and
Examination demonstrated successfully?



















* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-69

-------
Method 1623/1623.1 Checklist C

9 Positive Staining Control and OPR Slides

9.1 Does the laboratory's positive staining
control slide contain (oo)cysts at the
appropriate fluorescence intensity for
FITC?

15.2.1.3

15.2.1.3

5.4.8
5.4.9.2
5.4.10.2

Requirement











9.2 Does the laboratory's positive staining
control slide contain (oo)cysts at the
appropriate fluorescence intensity for
DAPI?

15.2.1.3

15.2.1.3

5.4.8
5.4.9.3
5.4.10.3

Requirement











9.3 Does the laboratory's positive staining
control slide contain an appropriate level
of background fluorescence?

-

-

5.4.3

Recommendation











9.4 Is concentration of oocysts on the positive
staining control slide appropriate?

14.1.1
15.2.1.3

14.1.1
15.2.1.3

7.1.8.1

Requirement











9.5 Does the laboratory's positive staining
control exhibit appropriate contrast and
organism features by DIC?

-

Figure 4

Visual
Guide

Requirement











9.6 Does the laboratory's OPR slide contain
(oo)cysts at the appropriate fluorescence
intensity for FITC?

15.2.2.1
15.2.3.1

15.2.2.2
15.2.3.2

5.4.9.2
5.4.10.2

Requirement











9.7 Does the laboratory's OPR slide contain
(oo)cysts at the appropriate fluorescence
intensity for DAPI?

15.2.2.2
15.2.3.2

15.2.2.3
15.2.3.3

5.4.9.3
5.4.10.3

Recommendation











9.8 Does the laboratory's OPR slide contain
an appropriate level of background
fluorescence?

-

-

5.4.3

Requirement











9.9 Does the laboratory's OPR slide exhibit
appropriate contrast and organism
features by DIC?

9.7.1.1

9.8.1.1
Figure 4

Visual
Guide

Requirement











9.10 Does the technical auditor's count of
Cryptosporidium oocysts and Giardia
cysts on the OPR slide sent by the
laboratory agree within 10% of laboratory
count?

10.6.3.1

9.10.3.1

7.1.9.4

Requirement











Comments:

* References refer to Section numbers in Method 1623 December 2005 (1623); Method 1623.1 January 2012 (1623.1); Certification Manual Chapter VII Supplement to the Fifth Edition
of the Manual for the Certification of Laboratories Analyzing Drinking Water (Cert)

November 2012

VTI-70

-------
Method 1623/1623.1 Checklist C

10 Onsite Sample Processing

Method Step

Name

Position

Demonstrated Technique
Successfully

yes/no

Spiking - (filter type)







Filtration - (filter type)







Spiking flat-sided tube, and
processing IMS control







Aspiration and transfer from 250 mL
bottle























11 Onsite Blind Spike Results

Sample

Crypto Spike
Value

Crypto Count

Crypto
Recovery (%)

Giardia Spike
Value

Giardia Count

Giardia
Recovery (%)











































12 Evaluation of Onsite Sample Processing and Blind Spike Results - Comments and Recommendations

Classification

Comments

Response Requested



















13 Was analyst microscope operation acceptable? (yes/no)





Requirement

Method 1623:10.3.4.1
Method 1623.1: 10.7.1

Requirement

Method 1623:10.3.4.2-3
Method 1623.1: 10.7.2-3

Requirement

Method 1623:10.3.6
Method 1623.1: 10.8

Name

Position

Adjust Interpupillary
Distance

Focus both eyepieces

Establish Kohler Illumination































VTI-71

-------
Method 1623/1623.1 Checklist C

14 Slide Count and Analyst Verification Results (yes/no)



Requirement

Method 1623:
10.6.3.1

Method 1623.1:
9.10.3.1

Requirement

Method 1623:
10.6.3.1

Method 1623.1:
9.10.3.1

Requirement

Method 1623:15.2
Method 1623.1: 15.2

Requirement

Method 1623: 15.2.2.3

15.2.3.3

Method 1623.1: 15.2.2.4

15.2.3.4

Requirement

Method 1623:15.2.2.3

15.2.3.3

Method 1623.1: 15.2.2.4

15.2.3.4

Analyst

Crypto Count Within
10% of Target Count

Giardia Count
Within 10% of
Target Count

Examine and Record
Characteristics

Measurement (100X)

Demonstrated Internal
Structures





































15 Evaluation of Analyst Microscopy and Examination Skills - Comments and Recommendations

Classification

Comments

Response Requested



















VTI-72

-------
Laboratory Name:



|Laboratory ID:







Method 1623/1623.1 Bench Sheet

Sample Identification Information

*

Lab Sample ID:





Turbidity (NTU):

*

PWS ID:





Person Receiving Sample:

*

Facility ID:





Temperature (°C)

g sample receipt:



*

Sample Collection Point ID:





Date of sample receipt:

*

Sample collection date & time:





Time of Sample receipt:

*

Sample type (circle one):

Initial precision and recovery (IPR) Method blank Field (monitoring) sample
Ongoing precision and recovery (OPR) Matrix spike (MS) Proficiency testing (PT)

Sample Spiking Information (for IPR, OPR, MS, and PT samples only)

*

Estimated number spiked:

Crypto

Giartiia

Spiking time:

*

Sample volume spiked (L):





Spiking date:

Spike manufacturer & ID:





Spiking analyst:

Sample Filtration

Filter type (circle one): Envirochek HV Filta-Max PCFC

Method version (circle one): 1623 1623.1



Did filter clog? (circle one):

Yes

No

Filtration time:



Filter lot number



*

Number of filter(s) used?:





Filtration date:

*

Volume filtered (L) to nearest 1/4L:





Filtration analyst:

Filter Elution (must be initiated within 96 hours of sample collection/filtration)



Elution procedure (circle one):

Wrist shaker

Filta-Max wash station

Elution Buffer lot number:



Type of Elution buffer (circle one):



NaHMP/LA-12 LA-12



PBST PCFC



Elution buffer expiration date:

Elution time:

NaHMP lot number:

Elution date:

NaHMP expiration date:

Elution analyst:





Concentration, IMS, and Slide Preparation





(must be completed on same working day that samples are eluted)





Procedure (circle one):

Centrifugation Filta-Max concentrator Other (specify)



*

Pellet volume after concentration (mL) to nearest 0.1mL:



Concentration analyst:

*

Total volume of resuspended concentrate (mL):





IMS analyst:

*

Volume of resuspended concentrate transferred to IMS (mL):



Slide preparation time:

Number of subsamples processed through entire method:

Slide preparation date:

IMS lot number:

Slide preparation analyst:



IMS system (circle one):

Dynal GC-Combo Dynal anti-Crypto Other (specify)





Slides (circle one): Meridian Dynal

Waterborne Other (specify)







Slide Staining (must be completed within 72 hours of application of sample to the slide)



Detection kit (circle one):

BTF EasyStain Merifluor

Crypt-a-glo Giardi-a-glo Aqua-glo Other (specify)

Detection kit lot number:

Staining date & time:





Number of slides for this sample:

Staining analyst:

*

Examination Results as Total FA number from all slides for sample Cryptosporidium:

Giardia:

Comments:

* = Data entered into LT2/Stage2 Data Collection and Tracking System

VTI-73

-------
Laboratory name:

Laboratory ID:

Method 1623/1623.1 Slide Examination Form

Sample ID:

Analyst:

Examination/verification completion:

(must be completed within 168 hours (7 days) of staining)
Date: Time:

Slide number:

Total number of slides for this sample:

Positive staining control acceptable & 3 oocysts and cysts characterized
with FITC, Size, Shape, DAPI and DIC ~ YES P NO

Negative staining control acceptable

~ YES ~ NO

FITC, Size, Shape, DIC and DAPI Characteristics Must Be Recorded for all Oocysts Detected in Field Sample

~ YES ~ NO

Cryptosporidium Results

Object
located
by FA
No.

Shape
(oval
or
round)

Size
L x W
(pm)

DAPI -

DAPI +

D.I.C,

Light blue internal
staining, no distinct
nuclei, green rim

Intense

blue
internal
staining

Number of
nuclei stained
sky blue

Empty
oocysts

Oocysts with
amorphous
structure

Oocysts with internal
structure

Number of sporozoites

1

















2

















3

















4

















5

















6

















7

















8

















9

















10

















Total FA number from this slide:

Analyst signature: P.A. ~

Principle Analyst (PA)Signature:

Comments:

Giardia Resu

Object
located
by FA
No.

Shape
(oval

or
round)

Size
LxW
(Mm)

DAPI -

DAPI +

D.I.C.

Light blue internal
staining, no distinct
nuclei, green rim

Intense

blue
internal
staining

Number of

nuclei
stained sky
blue

Empty
cysts

Cysts with
amorphous
structure

Cysts with internal structure

Number
of nuclei

Median
body

Axonemes

1





















2





















3





















4





















5





















6





















7





















8





















9





















10





















Total FA number from this slide:

Comments:

ts

VTI-74

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