f/EPA
United States
Environmental Protection
Agency
Office of Ground Water and
Drinking Water
EPA/814-B-95-003
June 1995
ICR Protozoan Method for
Detecting Giardia Cysts and
Cryptosporidium Oocysts in
Water by a Fluorescent
Antibody Procedure
-------
EPA/814-B-95-003
June 1995
ICR Protozoan Method for
Detecting Giardia Cysts
and Cryptosporidium
Oocysts in Water by a
Fluorescent Antibody
Procedure
United States Environmental Protection Agency
OGWDW/TSD/WSTB
26 Martin Luther King Drive
Cincinnati, OH 45268
Printed on Recycled Paper
-------
The use of manufacturer trade names in this document
does not constitute endorsement by the United States
Environmental Protection Agency.
On the cover: A classic Giardia cyst photographed
at 1,OOOX magnification showing nuclei, axonemes,
and median bodies.
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Contents
About This Document vi
Acknowledgments vii
A Note About Safety viii
Chapter 1: Introduction to the Method 1
Scope of the Method 1
Summary of Test Method 1
Significance and Use 1
Interferences 2
Education, Training and Proficiency 2
Principal Analyst/Supervisor 2
Analyst 2
Technician 3
Terminology 3
Keywords 3
End Notes 3
Chapter 2: Equipment 5
Sample Processing Apparatus 5
Sample Examination Apparatus 5
Reagents and Materials 5
Purity of Reagents 5
Preparation of Reagents 5
Purity of Water 5
Sample Collection Reagents 5
Sample Processing Reagents 5
Sample Examination Reagents 6
Sample Collection Materials 7
End Notes 7
Chapter 3: Filter Elution 9
Filter Washing 10
Handwashing 10
Stomacher Washing 11
Concentration of Particulates 13
Chapter 4: Flotation Purification 15
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CONTENTS
Chapter 5: Indirect Fluorescent Antibody Procedure 17
Determining Sample Volume per Filter 17
Preparing the Filtration Manifold 19
Membrane Filter Preparation 19
Sample Application 22
Indirect Fluorescent Antibody Staining 24
Filter Mounting 26
Chapter 6: Microscopic Examination 29
Adjustment of Interpupillary Distance and Oculars for Each Eye 29
Interpupillary Distance 29
Ocular Adjustment for Each Eye 30
Calibration of an Ocular Micrometer 32
Kohler Illumination 34
Microscopic Examination 36
End Notes 41
Chapter 7: Calculations and Reporting 43
Calculations 43
Reporting 45
Chapter 8: Water Sample Controls 47
Water Sample Negative Control 47
Water Sample Positive Control 48
Appendix A: Cleaning the Manifold and Stainless Steel Wells A - 1
Manifold A - 1
Stainless Steel Wells A - 1
Appendix B: Microscope Adjustments B - 1
Adjustment of the Epifluorescent Mercury Bulb and Transmitted Light Bulb Fila-
ment B- 1
Mercury Bulb Adjustment B - 1
Transmitted Bulb Adjustment B - 2
End Notes B - 2
Appendix C: Sample Calculations C - 1
Positive Samples C - 1
Negative Samples C - 1
Appendix D: Sample Forms D - 1
IV
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CONTENTS
Appendix E: Sampling E - 1
Sampling Apparatus Preparation and Assembly E - 1
Sampling Apparatus E - 1
Filter Holder E - 1
Label E-1
Hoses E - 1
Pump E -2
Fluid Proportioner or Proportioning Injector E - 2
Raw Water Sample Collection E - 2
Finished Water Sample Collection E - 3
End Notes E - 4
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About This Document
This bench-top guide is intended for use by laboratory
personnel analyzing water samples for the presence of
Giardia cysts and Cryptosporidium oocysts in accor-
dance with the Information Collection Requirements Rule. It
provides details of the procedure presented in the accompany-
ing video. To further associate the steps of the procedure with
the training video, this manual is illustrated with stills taken
directly from the video.
Several graphic conventions are used throughout the manual
to differentiate steps or draw attention to important informa-
tion:
A step icon is used to denote each step in the proto-
zoan monitoring protocol. These steps also are illus-
trated in the training video.
Important information, and valuable tips which are not
part of the published protocol, are denoted by this icon.
EPA recommends that laboratory analysts and technicians
watch the training video and read this manual in order to be-
come familiar with the method for detecting cysts and oocysts
by this fluorescent antibody procedure. The video concludes
with a summary of the method. Analysts and technicians also
can use the manual as a bench-top reference during the execu-
tion of the method.
VI
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Acknowledgments
This bench-top guide and the accompanying training
video were developed by the U.S. Environmental Pro-
tection Agency (EPA), Office of Water, Office of Ground
Water and Drinking Water (OGWDW), Cincinnati, Ohio. Jim
Walasek of OGWDW's Technical Support Division (TSD) was
the project manager, and provided the aerial photography used
in the video. Frank Schaefer, Ph.D. of the Office of Research
and Development's National Exposure Research Laboratory
(NERL) wrote the text of the guide. He was the principal tech-
nical advisor in the preparation of the video, and was respon-
sible for the photomicrography used in both the guide and the
video. Shay Fout, Ph.D. of NERL also served as technical
advisor on the project. The exhibits found in Appendix E were
prepared by Frederick Williams of NERL. Ken Mayo of The
Cadmus Group, Inc. in Waltham, Massachusetts edited, de-
signed, and coordinated production of the guide.
The still photos used in this guide come from the accompany-
ing training video. The video was taped at the U.S. EPA labo-
ratories in Cincinnati, Ohio. Melissa Godoy of Impact Video
Productions in Cincinnati was the producer, and Graham Spen-
cer and Steve Willis were the editors. Donna Jensen of Cadmus
acted as analyst during the taping, and Michelle Thomas, an
Oak Ridge DOE (Department of Energy) Fellow at EPA, was
the technician.
VII
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A Note About Safety
Neither this bench-top manual, nor the training video
which accompanies it, address all of the safety and
quality assurance needs of the analytical method. The
materials used are of a potentially biohazardous nature, and it
is the responsibility of the users of this method to establish
appropriate safety and health practices.
In particular, the analyst/technician must know and observe
the normal safety procedures required in a microbiology labo-
ratory while preparing, using, and disposing of sample con-
centrates, reagents and materials, and while operating steril-
ization equipment. Do not mouth pipet in any portion of
this procedure.
VIII
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Chapter 1
Introduction to the Method
Scope of the Method
This test method describes the detection and
enumeration of Giardia cysts and
Cryptosporidium oocysts in ground, sur-
face, and finished waters by a fluorescent antibody
procedure. These pathogenic intestinal protozoa
occur in domestic and wild animals as well as in
humans. The environment may become contami-
nated through direct deposit of human and animal
feces or through sewage and wastewater discharges
to receiving waters. Ingestion of water containing
these organisms may cause disease.
Results obtained by this method should be inter-
preted with extreme caution. High turbidity, as well
as turbidities less than 1 NTU, can affect the re-
covery efficiency of this method. Failure to detect
organisms of interest and/or a low detection limit
does not ensure that the water tested is pathogen-
free.
This method does not purport to address all of the
safety problems associated with its use. It is the
responsibility of the user of this method to estab-
lish appropriate safety and health practices and
determine the applicability of regulatory limitations
prior to use.
Summary of Test Method
In this test method, pathogenic intestinal protozoa
are concentrated from a large volume water sample
by retention on a yarn-wound filter. Retained par-
ticulates are eluted from the filter with an eluting
solution and are concentrated by centrifugation.
Giardia cysts and Cryptosporidium oocysts are
separated to some extent from other paniculate
debris by flotation on a Percoll-sucrose solution
with a specific gravity of 1.1. A monolayer of the
water layer/Percoll-sucrose interface is placed on
a membrane filter, indirectly stained with fluores-
cent antibody, and examined under a microscope.
Cysts and oocysts are classified according to spe-
cific criteria (immunofluorescence, size, shape, and
internal morphological characteristics), and the
results are reported in terms of the categories per
100 L. The reporting categories include cysts and
oocysts that are empty, that have amorphous struc-
ture, and that have internal structure. A sum of the
cysts and oocysts that fall into each of these cat-
egories is also reported as the total Indirect Fluo-
rescent Antibody (IFA) count.
Significance and Use
This test method will provide a quantitative indi-
cation of the level of contamination in raw and
treated drinking waters with the environmentally
resistant stages of two genera of pathogenic intes-
tinal protozoa: Giardia and Cryptosporidium.
The method will not identify the species of proto-
zoa, or the host species of origin. It cannot deter-
mine the viability status or the infectivity status
of detected cysts and oocysts.
This test method may be useful in determining the
source or sources of contamination of water sup-
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CHAPTER 1
INTRODUCTION TO THE METHOD
plies, as well as the occurrence and distribution of
protozoa in water supplies. The method also may
be useful in evaluating the effectiveness of treat-
ment practices.
Interferences
Turbidity due to inorganic and organic debris and
other organisms, can interfere with the concentra-
tion, purification and examination of the sample
for Giardia cysts and Cryptosporidium oocysts.
Inorganic and organic debris may be naturally oc-
curring (e.g., clays and algae) or may be added to
water in the treatment process (e.g., iron and alum
coagulants and polymers).
Organisms and debris that autofluoresce or dem-
onstrate nonspecific fluorescence when examined
by epifluorescent microscopy (e.g., algal and yeast
cells and Spironucleus (Hexamita) sp.1) could in-
terfere with the detection of cysts and oocysts and
contribute to false positive values.
Chlorine compounds, and perhaps other chemicals
used to disinfect or treat drinking water and waste-
water, may interfere with the visualization of in-
ternal structures of Giardia cysts and Cryptospo-
ridium oocysts.
Freezing filter samples, eluates, or concentrates
could interfere with the detection and/or identifi-
cation of cysts and oocysts originally present in
the sample.
Education, Training and
Proficiency
Minimal personnel requirements include the fol-
lowing:
Principal Analyst/Supervisor
To be qualified for approval, a laboratory must have
a principal analyst, who may also serve as a super-
visor if an additional analyst(s) is to be involved.
The principal analyst/supervisor oversees the en-
tire analysis and carries out QC performance
checks on technicians and/or other analysts. This
person must confirm all protozoan internal struc-
tures that are demonstrated at the microscope by
subordinates.
The principal analyst/supervisor must be an expe-
rienced microbiologist with at least a B.A./B.S.
degree in microbiology or a closely related field.
The principal analyst also must have at least 1 year
of continuous bench experience with immunofluo-
rescent antibody (IFA) techniques and microscopic
identification and have analyzed at least 100 water
and/or wastewater samples for Giardia and/or
Cryptosporidium. In addition, PE samples must be
analyzed using the ICR protozoan method, and the
results must fall within acceptance limits. The prin-
cipal analyst/supervisor must demonstrate accept-
able performance during an on-site evaluation.
Analyst
This person(s) performs at the bench level under
the supervision of a principal analyst/supervisor
and is involved in all aspects of the analysis, in-
cluding preparation of sampling equipment, filter
extraction, sample processing, microscopic proto-
zoan identification, and data handling. Recording
presence or absence of morphological character-
istics may be done by the analyst but must be con-
firmed by the principal analyst.
The analyst must have two years of college lecture
and laboratory course work in microbiology or a
closely related field. The analyst also must have at
least 6 months bench experience, must have at least
3 months experience with IFA techniques, and must
have successfully analyzed at least 50 water and/
or wastewater samples for Giardia and/or
Cryptosporidium. Six months of additional bench
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CHAPTER 1
INTRODUCTION TO THE METHOD
experience in the above areas may be substituted
for two years of college. In addition, PE samples
must be analyzed using the ICR protozoan method
and results must fall within acceptance limits. The
analyst must also demonstrate acceptable perfor-
mance during an on-site evaluation.
Technician
This person extracts filters and processes the
samples under the supervision of an analyst, but
does not perform microscopic protozoan detection
and identification. The technician must have at least
three months of experience in filter extraction and
processing of protozoa samples.
Terminology
• Axoneme. An internal flagellar structure which
occurs in some protozoa, e.g., Giardia,
Spironucleus, and Trichomonas.
• Cyst. A phase or a form of an organism pro-
duced either in response to environmental con-
ditions or as a normal part of the life cycle of the
organism. It is characterized by a thick and en-
vironmentally-resistant cell wall.
• Median bodies. Prominent, dark-staining,
paired organelles consisting of microtubules and
found in the posterior half of Giardia. In G.
lamblia (from humans), these structures often
have a claw-hammer shape, while in G. muris
(from mice) the median bodies are round.
• Oocyst. The encysted zygote of some Sporozoa,
e.g., Cryptosporidium. This is a phase or a form
of the organism produced either in response to
environmental conditions or as a normal part of
the life cycle of the organism. It is characterized
by a thick and environmentally resistant cell wall.
• Sporozoite. A motile, infective, asexual stage
of certain sporozoans, e.g., Cryptosporidium.
There are four sporozoites in each Cryptospo-
ridium oocyst, and they are generally banana-
shaped.
• Nucleus. A prominent internal structure seen in
Giardia cysts and Cryptosporidium oocysts.
Sometimes 2 to 4 nuclei can be seen in Giardia
cysts. In Cryptosporidium oocysts, one nucleus
per sporozoite sometimes can be seen.
Key Words
Antibody, Cryptosporidium parvum, cysts, fluo-
rescence, Giardia, immunoassay, oocysts, proto-
zoa.
End Notes
1 Januschka, M.M., et al. 1988. A comparison of
Giardia microti and Spironucleus muris cysts in
the vole: an immunocytochemical, light, and elec-
tron microscopic study. Journal of Parasitology
74(3):452-458.
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Chapter 2
Equipment
Sample Processing Apparatus
• Centrifuge, with swinging bucket rotors having
a capacity of 15 to 250 mL or larger per conical
tube or bottle.
• Mixer, vortexer.
• Vacuum source.
• Membrane filter holder, Hoefer manifold, model
FH 225V, 10 place holder for 25 mm diameter
filters.
• Slide warming tray, or incubator, 37°C.
• pH meter.
• Rubber policeman.
• Stomacher Lab Blender, model 3500 (BA 7022)2
(optional).
Sample Examination Apparatus
• Microscope, capable of epifluorescence and dif-
ferential interference contrast (D.I.C.) or
Hoffman modulation® optics, with stage and
ocular micrometers and 20X (N.A. = 0.6) to
100X (N.A. = 1.3) objectives. Equip the micro-
scope with appropriate excitation and band pass
filters for examining fluorescein isothiocyanate-
labeled specimens (exciter filter: 450-490 nm;
dichroic beam-splitting mirror: 510 nm; barrier
or suppression filter: 515-520 nm).
Reagents and Materials
Purity of Reagents
Reagent grade chemicals shall be used in all tests.
Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the
committee on Analytical Reagents of the Ameri-
can Chemical Society where such specifications
are available3.
Preparation of Reagents
Prepare reagents as specified by the formulations.
Purity of Water
Use distilled deionized, double distilled, or reagent
grade water.
Sample Collection Reagents
• Sodium Thiosulfate Solution (2.0%)- Dissolve
2.0 g of sodium thiosulfate (Na2S2O3 5H2O) in
50 mL water and then adjust to a final volume
of 100 mL.
Sample Processing Reagents
• Neutral Buffered Formalin Solution (10%) -
Dissolve 0.762 g disodium hydrogen phosphate
(Na2HPO4), 0.019 g sodium dihydrogen phos-
phate (NaH2PO4), and 100 mL formalin in wa-
ter to a final volume of 1 L.
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CHAPTER 2
EQUIPMENT
Phosphate Buffered Saline (PBS) - Prepare a
10X stock solution by dissolving 80 g sodium
chloride (NaCl), 2 g potassium dihydrogen phos-
phate (KH2PO4), 29 g hydrated disodium hydro-
gen phosphate (Na2HPO4 12 H2O) and 2 g po-
tassium chloride (KC1) in water to a final vol-
ume of 1 L. The 10X solution is used to prepare
IX PBS by diluting one volume of the 10X so-
lution with 9 volumes of water and adjust the
pH with a pH meter to 7.4 with 0.1 N HC1 or 0.1
N NaOH before use.
Sodium Dodecyl Sulfate Stock Solution (1%) -
Prepare solution by dissolving 1.0 g of sodium
dodecyl sulfate (SDS) in water to a final volume
of 100 mL.
Tween 80 Stock Solution (1%) - Mix 1.0 mL of
polyoxyethylenesorbitan monooleate 80 (Tween
80) stock solution with 99 mL of water.
Eluting Solution (Buffered Detergent Solution)
- Prepare solution by mixing 100 mL 1% SDS,
100 mL 1% Tween 80, 100 mL 10X PBS, and
0.1 mL Sigma Antifoam A (Cat. # A 5758) with
500 mL water. Adjust the pH to 7.4 using a pH
meter. Adjust the final volume to 1 L with addi-
tional water. Use within one week of prepara-
tion.
Sucrose Solution (2.5 M) - Dissolve 85.58 g of
sucrose in 40 mL prewarmed water then adjust
the final volume to 100 mL with water.
Percoll-Sucrose Flotation Solution, Sp. Gr. 1.10
- Mix 45 mL Percoll (sp. gr. 1.13; Sigma Cat.
# P 1644), 45 mL water and 10 mL 2.5 M su-
crose solution. Check the specific gravity with a
hydrometer. The specific gravity should be be-
tween 1.09 and 1.10 (do not use if less than 1.09).
Store at 4°C and use within a week. Allow to
reach room temperature before use.
Sample Examination Reagents
• Ensys Hydrofluor-Combo kit4 for detecting
Giardia cysts and Cryptosporidium oocysts in
water samples. The expiration date for the re-
agents is printed on the Hydroflour-Combo kit
label. Discard the kit once the expiration date is
reached. Store the kit at 2-8°C and return it
promptly to this temperature range after each use.
The labeling reagent should be protected from
exposure to light. Do not freeze any of the re-
agents in this kit. Diluted, unused working re-
agents should be discarded after 48 hours.
• Ethanol, (95%).
• Glycerol.
• Ethanol/Glycerol Series - Prepare a series of
solutions according to the following table:
95%
Ethanol
10ml_
20 ml
40 mL
80 mL
95 mL
Glycerol
5 mL
5 mL
5mL
5 mL
5mL
Reagent
Water
80 mL
70 mL
50 mL
10 mL
OmL
Final
Volume
95 mL
95 mL
95 mL
95 mL
100 mL
Final %
Ethanol
10
20
40
80
90.2
DABCO-Glycerol Mounting Medium (2%) -
Prewarm 95 mL glycerol using a magnetic stir
bar on a heating stir plate. Add 2 g 1,4
diazabicyclo [2.2.2] octane (DABCO, Sigma
#D-2522) to the warm glycerol with continuous
stirring until it dissolves. (CAUTION: hygro-
scopic; causes burns; avoid inhalation, as well
as skin and eye contact.) Adjust the final vol-
ume to 100 mL with additional glycerol. Store
at room temperature and discard after 6 months.
Bovine Serum Albumin (1%) - Sprinkle 1.0 g
bovine serum albumin (BSA) crystals over 85
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CHAPTER 2
EQUIPMENT
mL IX PBS, pH 7.4. Allow crystals to fall be-
fore stirring into solution with a magnetic stir
bar. After the BSA is dissolved, adjust the vol-
ume to 100 mL with PBS. For prolonged stor-
age, sterilize by filtering through a 0.22 Jim mem-
brane filter into a sterile tube or bottle. Store at
4°C and discard after 6 months.
Sample Collection Materials
• Filters and filter holder, either a 25.4 cm (10 in.)
long 1 fim nominal porosity, yarn-wound
polypropylene cartridge Commercial honey-
comb filter tube (M39R10A; Commercial Fil-
ters Parker Hannifin Corp., P.O. Box 1300, Leba-
non, IN) with a Commercial LT-10 filter holder
or a 25.4 cm (10 in.) long 1 \im nominal poros-
ity Filterite polypropylene cartridge (U1A10U;
Filterite Corporation, Timmonium, MD), with a
Filterite LMO10U-3/4 filter holder must be used.
• Garden hose or PVC tubing and connectors.
• Pressure regulator.
• Pressure gauge(s).
• Proportioner.
• Plastic sample bags, double-track, zipper-lock
or equivalent, approximately 15 in. (38 cm) x
15 in (38 cm).
• Cold packs or wet ice.
Sample Processing Materials
• Pans or trays, stainless steel or glass trays,
approx. 16.5 in. (41.91 cm) x 10 in. (25.4 cm) x
2 in. (5.08 cm) deep.
• Disposable knife/cutting tool, for cutting the
polypropylene filter fibers off filter core.
• Hydrometer, for liquids heavier than water
(range: 1.000-1.225), for adjusting specific grav-
ity of flotation solutions.
Sample Examination Materials
• Slides, glass microscope, 1 in. (2.54 cm.) x 3 in.
(7.62 cm) or 2 in. (5.08 cm.) x 3 in. (7.62 cm.).
• Cover slips, 25 mm2, No. 1 '/2.
• Filters, Sartorius brand5 cellulose acetate, 0.22
[im pore size, 25 mm diameter.
• Support Filters, ethanol-compatible membrane,
any pore size, 25 mm.
• Fingernail polish, clear, or clear fixative (Cat.
# 60-4890; PGC Scientifics6).
• Splinter forceps, fine tip.
• Blunt-end filter forceps.
End Notes
1 Hoefer Scientific Instruments, 654 Minnesota
Street, Box 77387, San Francisco, CA 94107
2Tekmar Company, P.O. Box 371856, Cincinnati,
OH 45222-1856
3 "Reagent Chemicals, American Chemical Soci-
ety Specifications", American Chemical Society,
Washington, DC. For suggestions on the testing of
reagents not listed by the American Chemical So-
ciety, see "Annular Standards for Laboratory
Chemicals," BDH, Poole, Dorset, U.K. and the
"United States Pharmacopeia."
4Ensys Environmental Products, Inc., P.O. Box
14063, Research Triangle Park, NC 27709
5 Sartorius Corp., Filter Div., 30940 San Clemente,
Hayward, CA 94544
6 PGC Scientifics, P.O. Box 7277, Gaithersburg,
MD 20898-7277
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Chapter 3
Filter Elution
B
efore beginning the assay procedure, check the valid-
ity and completeness of the data provided with the
sample. Essential information includes the:
• Name of the source water sampled.
• Number of gallons filtered.
• Turbidity of the sampled source water.
• Time and date when the sample collection began.
This information is required to properly report the results of
the analysis, and to ensure that the filter is eluted within 96
hours of the start of sampling.
Check the filter to ensure that the sample was collected
properly. If the fibers closest to the filter core appear
to be the dirtiest, the sampled water may have flowed
through the filter in the wrong direction. If improper
sampling is suspected, investigate the sampling procedures.
An improperly collected sample must not be analyzed.
The initiation of sample collection and elution from the col-
lection filter must be performed within 96 hours. Two ap-
proaches to eluting the particulates from the filter may be used:
either washing by hand or using a stomacher.
The materials used from this point on are potentially
of a biohazardous nature, and must be treated and dis-
carded appropriately. Before starting the analysis, make
sure you are wearing safety glasses and latex gloves.
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CHAPTER 3
FILTER ELUTION
Filter Washing
Pour the residual solution in the bag into a beaker,
rinse the bag with eluting solution, add the rinse solu-
tion to the beaker and discard the bag.
Handwashing
Using a razor knife or other appropriate disposable
cutting instrument, cut the filter fibers lengthwise down
to the core. Discard the blade after the fibers have been
cut.
Rinse the filter core with eluating solution into the beaker con-
taining residual solution from the filter bag.
Divide the filter fibers into a minimum of six equal portions
with one-sixth consisting of those cleanest fibers nearest the
core; the second one-sixth being the second, slightly dirtier,
layer of fibers, and so on to the final one-sixth consisting of
the outer-most filter fibers (the dirtiest fibers).
Beginning with the cleanest fibers (the one-sixth near-
est the core), hand wash the fibers in three consecutive
1.0 L volumes of eluting solution.
Wash the fibers by kneading them in the eluting solution con-
tained either in a beaker or a plastic bag.
Wring the fibers to express as much of the liquid as possible
before discarding.
Maintain the three 1.0 L volumes of eluate separate through-
out the washing procedure.
10
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CHAPTER 3
FILTER ELUTION
Using the same three 1.0 L volumes of eluate used in
Step 2, repeat the washing procedure on the second
one-sixth layer of fibers and then on sequentially to
the final outer one-sixth layer of fibers. The minimum
total wash time of fibers should be 30 minutes.
For extremely dirty filters, additional beakers of elut-
ing solution may be required.
After all the fibers have been washed, combine the
three 1.0 L volumes of eluate with the residual filter
water collected when the filter was unwrapped. Dis-
card the filter fibers before preparing the eluate and
residual water for concentration in a centrifuge.
Stomacher Washing
Using a razor knife or other appropriate disposable
cutting instrument, cut the filter fibers lengthwise down
to the core. Discard the blade after cutting the fibers.
Rinse the filter core with eluating solution into the beaker con-
taining residual solution from the filter bag.
After loosening the fibers, place all the filter fibers in a
stomacher bag that has a 3,500 mL capacity. To en-
sure against bag breakage and sample loss, place the
stomacher bag that contains the filter fibers into a sec-
ond stomacher bag.
11
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CHAPTER 3
FILTER ELUTION
Add 1.75 L of eluting solution to the fibers, and ho-
mogenize for two 5-minute intervals. Between each
homogenization period, knead the filter material by
hand to redistribute the fibers in the bag.
At the end of the second homogenization period, pour
the eluted particulate suspension into a 4-liter pooling
beaker. Wring the fibers out to express as much of the
liquid as possible before.
Return the fibers to the stomacher bag and add 1.0 L
of eluting solution. Homogenize, as in Step 3, for two
5-minute periods. Between each homogenization pe-
riod, knead the fiber materials by hand to redistribute
them
in the bag.
At the end of the second homogenization period, add
the eluted particulate suspension to the 4-liter pooling
beaker. Wring the fibers out to express as much of the
liquid as possible into the beaker. Discard the fibers,
and rinse the stomacher bag with eluting solution into the pool-
ing beaker.
12
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Concentration of Particulates
CHAPTER 3
FILTER ELUTION
After the filter fibers have been washed, and the eluate and
residual water has been pooled, the particulates in suspension
must be recovered using a centrifuge.
Pour the liquid into conical centrifuge bottles, then
balance the bottles in their shields.
Make sure to use the appropriate cushion for each bottle
in the shield. Otherwise, the conical bottle will col-
lapse during centrifugation.
Concentrate the combined eluate and residual water
into a single pellet by centrifugation at 1,050 x g for
10 minutes using a swinging bucket rotor and plastic
conical centrifuge bottles.
You must use a swinging bucket rotor for this opera-
tion.
At the end of 10 minutes, carefully aspirate and dis-
card the supernatant fluid. Resuspend the pellet in suf-
ficient elution solution by vortexing.
Vortexing is done between centrifugations to prevent
excessive packing of the particulates that form the pel-
let.
13
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CHAPTER 3
FILTER ELUTION
Continue centrifugations of the pooled eluate and re-
sidual water at 1,050 x g for 10 minutes until all the
particulates are concentrated in one conical bottle.
At the end of this period, remove the bottle from the
centrifuge and record the packed pellet volume.
Carefully aspirate and discard the supernatant fluid.
Resuspend the pellet by vortexing in an equal volume
of 10% neutral buffered formalin solution. If the packed
pellet volume is less than 0.5 mL, bring the pellet and
solution volume to 0.5 mL with eluting solution before adding
enough 10% neutral buffered formalin solution to bring the
resuspended pellet volume to 1.0 mL.
At this point, a break of up to 72 hours may be inserted
if the procedure is not going to progress immediately
to the Flotation Purification procedure described in
chapter 4. If a break is inserted at this point, be sure to
store the formalin-treated sample at 4°C for not more than 72
hours.
14
-------
Chapter 4
Flotation Purification
Eotation Purification is used to separate any Giardia cysts
md Cryptosporidium oocysts present in the sample from
:ertain other particulates and other debris which have a
greater specific gravity.
In one or more clear plastic 50 mL conical centrifuge
tubes, as necessary, vortex a volume of resuspended
pellet equivalent to not more than 0.5 mL of packed
pellet volume with enough eluting solution to make a
final volume of 20 mL.
Using a 50 mL syringe and 14 gauge cannula, under-
lay the 20 mL vortexed suspension of particulates with
30 mL Percoll-sucrose flotation solution (sp. gr. 1.1).
Some water samples will have heavier particulates that
penetrate the gradient before centrifugation begins in
Step 3. This is normal. However, if for any reason the
interface is disturbed and mixed, then another sample
aliquot must be used.
15
-------
CHAPTER 4
FLOTATION PURIFICATION
Without disturbing the pellet suspension/Percoll-su-
crose interface, centrifuge the preparation at 1,050 x g
for 10 minutes using a swinging bucket rotor.
To avoid disrupting the interface, slowly accelerate the
centrifuge over a 30-second interval up to the speed
where the tubes are horizontal. Similarly, at the end of
centrifugation, decelerate slowly. Do not use the
brake!
Using a polystyrene 25 mL pipet rinsed with eluting
solution, draw off the top 20 mL particulate suspen-
sion layer, the interface, and 5 mL of the Percoll-su-
crose below the interface.
Place all these volumes in a plastic 50 mL conical centrifuge
tube.
Add additional eluting solution to the plastic conical
centrifuge tube (Step 4) to bring the final volume to 50
mL. Centrifuge at 1,050 x g for 10 minutes.
Aspirate and discard the supernatant fluid down to 5
mL (plus pellet).
Resuspend the pellet by vortexing, and save this suspension
for further processing with fluorescent antibody reagents.
16
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Chapter 5
Indirect Fluorescent
Antibody Procedure
T
he Indirect Fluorescent Antibody, or IFA, Procedure
stains Giardia cysts and Cryptosporidium oocysts so
they can be seen with epifluorescence microscopy.
Determining Sample Volume per Filter
Determine the volume of sample concentrate from the
Flotation Purification procedure just completed that
may be applied to each 25-mm diameter membrane
filter used in the IFA assay.
Vortex the sample concentrate and apply 40 (iL to one
5-mm diameter well of a 12-well red heavy teflon-
coated slide1.
17
-------
CHAPTER 5
INDIRECT FLUORESCENT ANTIBODY PROCEDURE
Allow the sample to sit approximately 2 minutes at
room temperature.
Examine the flooded well microscopically at 200X total
magnification. If the particulates are distributed evenly
over the well surface area, and are not crowded or
touching, apply 1 mL of the undiluted sample to a 25-
mm diameter membrane filter in Step 6 of Sample Application
below.
When examining the flooded well, you must use an
objective lens with a very high working distance so
the meniscus does not touch or wet it.
If the particulates are too dense or too widely spread,
adjust the volume of the sample accordingly. Retest
on another well.
Always adjust the sample concentrate volume so that
the density of the particulates is just a little sparse. If
the layer of sample particulates on the membrane fil-
ters is too dense, any cysts or oocysts present in the
sample may be obscured during microscopic examination.
Make sure the dilution factor, if any, from this step is recorded
so that the number of parasite categories per 100 liters of
sampled water can be determined.
18
-------
CHAPTER 5
INDIRECT FLUORESCENT ANTIBODY PROCEDURE
Preparing the Filtration Manifold
Connect the filtration manifold to the vacuum supply
using a vacuum tube that has a "T"-shaped tubing con-
nector at one end.
ofHg.
Attach a Hoffman screw clamp to 4-6 cm of latex tub-
ing and then attach the latex tubing to the stem of the
"T" connector. The screw clamp is used as a bleeder
valve to regulate the vacuum to 2-4 inches (5-10 cm)
Close all the manifold valves and open the vacuum all
the way.
Using the bleeder valve on the vacuum tubing, adjust the ap-
plied vacuum to 2-4 inches (5-10 cm) of Hg.
Once adjusted, do not readjust the bleeder valve dur-
ing filtration. If necessary, turn the vacuum on and off
during filtration at the vacuum source.
Membrane Filter Preparation
One Sartorius 25 mm diameter cellulose acetate filter, 0.22
u.m pore size, and one 25-mm diameter ethanol-compatible
membrane support filter2, any porosity, are required for each 1
mL of adjusted suspension obtained previously. (See Deter-
mining Sample Volume per Filter, above.)
19
-------
CHAPTER 5
INDIRECT FLUORESCENT ANTIBODY PROCEDURE
Soak the required number of each type of filter sepa-
rately in Petri dishes filled with IX PBS. Using blunt-
end filter forceps to handle the filters, drop them, one
by one, flat on the surface of the buffer.
Be sure that the colored filter dividers provided in the
packaging have been removed.
Once the filters are wet, push the filters under the fluid surface
with the forceps. Allow filters to soak for a minimum of 1
minute before use.
Turn on the filtration manifold vacuum source. Leav- ._•
ing all the manifold well support valves closed, place p*
one support filter on each manifold support screen.
This filter ensures even distribution of sample.
Place one Sartorius 25-mm diameter cellulose acetate
filter on top of each support filter. Use a rubber po-
liceman to adjust the cellulose acetate filter, if neces-
sary. Open the manifold well support valves to flatten
the filter membranes. Make sure that no bubbles are trapped
and that there are no creases or wrinkles on any of the filtei
membranes.
20
-------
CHAPTER 5
INDIRECT FLUORESCENT ANTIBODY PROCEDURE
Use as many filter positions as there are sample vol-
umes to be assayed. Record the number of sample 25-
mm membrane filters prepared and the volume of
floated pellet (See Determining Sample Volume per
Filter, above) represented by these membranes.
In addition, include at least one positive control for Giardia
cysts and Cryptosporidium oocysts and one negative control
each time the manifold is used.
Position a 1 Ib (454 g) stainless steel well firmly over
each filter.
To ensure a good seal, use only wells with smooth
rims that are free of nicks or scratches.
Label each sample and control well appropriately with
little pieces of tape on the top of the stainless steel
wells and/or use manifold membrane labeling diagram
(see Appendix D, Sample Forms) to keep track of each
sample and control.
21
-------
CHAPTER 5
INDIRECT FLUORESCENT ANTIBODY PROCEDURE
Sample Application
Open the manifold support valve for each well contain-
ing filters.
Rinse the inside of each stainless steel well and mem-
brane filter with 2 mL 1% BSA. Drain the BSA solu-
tion completely from the membrane.
Close the manifold valve under each membrane filter.
22
-------
CHAPTER 5
INDIRECT FLUORESCENT ANTIBODY PROCEDURE
For the positive control, add to the appropriately la-
belled well 500-1,000 Giardia lamblia cysts and 500-
1,000 Cryptosporidium parvum oocysts, or use the
Ensys positive control antigen as specified in the kit.
For a negative control, add to the appropriately labelled
well 1.0 mL IX PBS.
Add 1.0 mL of the vortexed, adjusted water sample
(See Determining Sample Volume per Filter, above)
to an appropriately labelled well.
Open the manifold valve under each membrane filter
to drain the wells Rinse each stainless steel well with
2 mL 1% BSA, then close the manifold valve under
each membrane filter.
Do not touch the pipet to the membrane filter or to the
well.
23
-------
CHAPTER 5
INDIRECT FLUORESCENT ANTIBODY PROCEDURE
Indirect Fluorescent Antibody Staining
Dilute the primary antibody mixture and labeling re-
agent according to the manufacturer's instructions us-
ing IX PBS.
Pipet 0.5 mL of the diluted primary antibody mixture IT
onto each membrane and allow to remain in contact
with the filter for 25 minutes at room temperature.
At the end of the contact period, open the manifold
valve to drain the antisera.
24
-------
CHAPTER 5
INDIRECT FLUORESCENT ANTIBODY PROCEDURE
Rinse each well and filter 5 times with 2 mL IX PBS.
Close all manifold valves after the last wash is com-
pleted.
Do not touch the tip of the pipet to the membrane filter
or to the stainless steel wells.
Pipet 0.5 mL of the labeling reagent onto each mem-
brane and allow it to remain in contact with the filter
for 25 minutes at room temperature.
Cover all wells with aluminum foil to shield the re-
agents from light and to prevent dehydration and crys-
tallization of the fluorescein isothiocyanate dye dur-
ing the contact period.
At this point, start the Filter Mounting procedure pre-
sented in the following section.
At the end of the contact period, open the manifold
valves to drain the labeling reagent.
25
-------
CHAPTER 5
INDIRECT FLUORESCENT ANTIBODY PROCEDURE
Rinse each well and filter 5 times with 2 mL IX PBS.
Close all manifold valves after the last wash is com-
pleted.
Do not touch the tip of the pipet to the membrane filter
or to the stainless steel wells.
Dehydrate the membrane filters in each well by sequen-
tially applying 1.0 mL of 10, 20, 40, 80 and 90.2%
ethanol solutions containing 5% glycerol. Allow each
solution to drain thoroughly before applying the next
in the series.
Filter Mounting
Label glass slides for each filter and place them on a
slide warmer or in an incubator calibrated to 37°C.
Add 75 |iL 2% DABCO-glycerol mounting medium
to each slide on the slide warmer or in the incubator
and allow to warm for 20-30 minutes.
26
-------
CHAPTER 5
INDIRECT FLUORESCENT ANTIBODY PROCEDURE
Remove the top cellulose acetate filter with fine-tip
forceps and layer it over the correspondingly labeled
slide prepared with DABCO-glycerol mounting me-
dium. Make sure the sample application side is up.
If the entire filter is not wetted by the DABCO-glyc-
erol mounting medium, pick up the membrane filter
with the same forceps and add a little more DABCO-
glycerol mounting medium under the slide filter.
Repeat Step 3 for each filter. Use a clean pair of for-
ceps to handle each membrane filter. Soak the used
forceps in diluted detergent cleaning solution.
Allow the slides to remain on the slide warmer or in
the incubator for 20 minutes in order to clear.
If the membrane starts to turn white after clearing,
apply a small amount of DABCO-glycerol mounting
medium under the filter.
After the 20-minute clearing period, apply 20 |iL
DABCO-glycerol mounting medium to the center of
each membrane filter and cover with a 25 mm x 25
mm cover glass. Tap out air bubbles with the handle
end of a pair of forceps. Wipe off excess DABCO-glycerol
mounting medium from the edge of each cover glass with a
slightly moistened Kimwipe.
27
-------
CHAPTER 5
INDIRECT FLUORESCENT ANTIBODY PROCEDURE
Seal the edge of each cover glass to the slide with clear
fingernail polish.
Store the slides in a covered "dry box" at 4°C.
A dry box can be constructed from a covered
Tupperware®-type container to which a thick layer of
anhydrous calcium sulfate has been added. Cover the
desiccant with paper towels, and lay the slides flat on
the top of the paper towels.
Examine the slides microscopically as soon as pos-
sible but within 5 days of preparation. They may be-
come opaque if stored longer, and D.I.C. or Hoffman
modulation® optical examination would then no longer
be possible.
28
-------
Chapter 6
Microscopic Examination
The microscopic portion of this procedure depends on
very sophisticated optics. Without proper alignment and
adjustment, the microscope will not function at peak
efficiency. Therefore, it is imperative that all portions of the
microscope, from the light sources to the oculars, are adjusted
properly. These adjustments should be practiced until they be-
come second nature. The procedures for adjusting the
epifluorescent mercury bulb and the transmitted light filament
are presented in Appendix B.
Adjustment of Interpupillary Distance and
Oculars for Each Eye
These adjustments are necessary so that eye strain is reduced
to a minimum. These adjustments must be made for each indi-
vidual using the microscope. This section assumes the use of a
binocular microscope.
Interpupillary Distance
The spacing between the eyes varies from person to person
and must be adjusted for each individual using the microscope.
Place a prepared slide on the microscope stage, turn
on the transmitted light, and focus the specimen im-
age using the coarse and fine adjustment knobs.
29
-------
CHAPTER 6
MICROSCOPIC EXAMINATION
Using both hands, adjust the oculars in and out until a
single circle of light is observed while looking through
the two oculars with both eyes.
Ocular Adjustment for Each Eye.
This section assumes a focusing ocular(s). This adjustment can
be made two ways, depending upon whether or not the micro-
scope is capable of photomicrography and whether it is
equipped with a photographic frame which can be seen through
the binoculars.
Persons with astigmatic eyes should always wear their
contact lenses or glasses when using the microscope.
For microscopes not capable of photomicrography
This section assumes only the right ocular is capable of adjust-
ment.
Place a prepared slide on the microscope stage, turn
on the transmitted light, and focus the specimen im-
age using the coarse and fine adjustment knobs.
30
-------
CHAPTER 6
MICROSCOPIC EXAMINATION
Place a card between the right ocular and eye, keeping
both eyes open. Using the fine adjustment, focus the
image for the left eye to its sharpest point.
Now transfer the card to between the left eye and ocu-
lar. Without touching the coarse or fine adjustment,
and with keeping both eyes open, bring the image for
the right eye into sharp focus by adjusting the ocular
collar at the top of the ocular.
For microscopes capable of viewing a photographic frame
through the viewing binoculars
This section assumes both oculars are adjustable.
Place a prepared slide on the microscope stage, turn
on the transmitted light, and focus the specimen im-
age using the coarse and fine adjustment knobs.
31
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CHAPTER 6
MICROSCOPIC EXAMINATION
After activating the photographic frame, place a card
between the right ocular and eye, keeping both eyes
open. Using the correction (focusing) collar on the left
ocular, focus the left ocular until the double lines in
the center of the frame are as sharply focused as possible.
Now transfer the card to between the left eye and ocu-
lar. Again keeping both eyes open, bring the image of
the double lines in the center of the photographic frame
into as sharp a focus for the right eye as possible by
adjusting the ocular correction (focusing) collar at the top of
the right ocular.
Calibration of an Ocular Micrometer1
This section assumes that an ocular reticle has been installed
in one of the oculars by a microscopy specialist and that a
stage micrometer is available for calibrating the ocular mi-
crometer (reticle). Once installed the ocular reticle should be
left in place. The more an ocular is manipulated the greater the
probability is for it to become contaminated with dust particles.
This calibration should be done for each objective in use on
the microscope. If there is an optivar2 on the microscope, then
the calibration procedure must be done for the respective ob-
jective at each optivar setting.
Place the stage micrometer on the microscope stage,
turn on the transmitted light, and focus the microme-
ter image using the coarse and fine adjustment knobs
for the objective to be calibrated. Continue adjusting
the focus on the stage micrometer so you can distinguish be-
tween the large (0.1 mm) and the small (0.01 mm) divisions.
32
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CHAPTER 6
MICROSCOPIC EXAMINATION
Adjust the alignment of the stage and ocular microme-
ters so the 0 line on the ocular micrometer is exactly
superimposed on the 0 line on the stage micrometer.
Without changing the stage adjustment, find a point as
distant as possible from the two 0 lines where two other
lines are exactly superimposed.
Determine the number of ocular micrometer spaces as
well as the number of millimeters on the stage mi-
crometer between the two points of superimposition.
For example: Suppose 48 ocular micrometer spaces
equal 0.6 mm.
Calculate the number of mm/ocular micrometer space.
mm.
Since most measurements of microorganisms are given
in |im rather than mm, the value calculated above must
be converted to Jim by multiplying it by 1,000 |0,m/
Calculating Ocular Micrometer Space
0.6 mm
48 o.m. spaces
- = 0.0125 mm/o.m. space
Calculating Ocular Micrometer Space
0.6 mm
48 o.m. spaces
0.0125 mm/o.m. space
0.0125 mm/o.m. space x 1,000 urn/mm
= 12.5 nm/o.m. space
33
-------
CHAPTER 6
MICROSCOPIC EXAMINATION
Follow steps 1 through 6
for each objective. It is
helpful to record this in-
formation in a table, like
the example at right, which can be
kept near the microscope.
Item*
1
2
3
4
Objective
Power
10X
20X
40X
100X
Description
N.A.C =
N.A. =
N.A. =
N.A. =
No. of Ocular
Micrometer
Spaces
No. of Stage
Micrometer
millimeters'
(jm/Ocular
Micrometer
Space"
1 1,000 |jm/mm
" (Stage Micrometer length in mm x (1,000 pm/mml) 4- No. Ocular Micrometer Spaces
c N.A. stands for numerical aperture. The numerical aperture value is engraved on the barrel of
the objective.
Kohler Illumination
This section assumes that Kohler illumination will be estab-
lished for only the 100X oil D.I.C. or Hoffman modulation®
objective, which will be used to identify internal morphologi-
cal characteristics in Giardia cysts and Cryptosporidium oo-
cysts. If by chance more than one objective is to be used for
either D.I.C. or Hoffman modulation® optics, then each time
the objective is changed, Kohler illumination must be re-es-
tablished for the new objective lens. Previous sections have
adjusted oculars and light sources. This section aligns and fo-
cuses the light going through the condenser underneath the
stage at the specimen to be observed. If Kohler illumination is
not properly established, then D.I.C. or Hoffman modulation®
optics will not work to their maximal potential. These steps
need to become second nature and must be practiced regularly
until they are a matter of reflex rather than a chore.
Place a prepared slide on the microscope stage, place
oil on the slide, move the 100X oil objective into place,
turn on the transmitted light, and focus the specimen
image using the coarse and fine adjustment knobs.
34
-------
CHAPTER 6
MICROSCOPIC EXAMINATION
At this point both the radiant field diaphragm in the
microscope base and the aperture diaphragm in the con-
denser should be wide open. Now close down the ra-
diant field diaphragm in the microscope base until the
lighted field is reduced to a small opening.
Using the condenser centering screws on the front right
and left of the condenser, move the small lighted por-
tion of the field to the center of the visual field.
Now look to see whether the leaves of the iris field
diaphragm are sharply defined (focused) or not.
If they are not sharply defined, they can be focused distinctly
by changing the height of the condenser up and down with the
condenser focusing knob while you are looking through the
binoculars. Once you have accomplished the precise focusing
of the radiant field diaphragm leaves, open the radiant field
diaphragm until the leaves just disappear from view.
35
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CHAPTER 6
MICROSCOPIC EXAMINATION
The aperture diaphragm of the condenser should be
adjusted now to make it compatible with the total nu-
merical aperture of the optical system. This is done by
removing an ocular, looking into the tube at the rear
focal plane of the objective, and stopping down the aperture
diaphragm iris leaves until they are visible just inside the rear
plane of the objective.
After completing the adjustment of the aperture dia-
phragm in the condenser, return the ocular to its tube
and proceed with the adjustments required to estab-
lish either D.I.C. or Hoffman modulation® optics.
Microscopic Examination
Microscopic work performed by a single analyst should not
exceed 4 hours/day nor more than 5 consecutive days/week.
Intermittent rest periods during the 4 hours/day are encour-
aged.
Remove the dry box from 4°C storage and allow it to
warm to room temperature before opening.
36
-------
CHAPTER 6
MICROSCOPIC EXAMINATION
Adjust the microscope to ensure that the epifluores-
cence and Hoffman modulation® or D.I.C. optics are
in optimal working order.
Make sure that the fluorescein isothiocyanate cube is in place
in the epifluorescent portion of the microscope (see the sec-
tion on Sample Examination below.) Detailed procedures re-
quired for adjusting and aligning the microscope are found in
Appendix B.
Assay Controls
The purpose of these controls is to ensure that the assay re-
agents are functioning, that the assay procedures have been
properly performed, and that the microscope has been adjusted
and aligned properly.
Assay Giardia/Cryptosporidium Control
Using epifluorescence, scan the positive control slide
at no less than 200X total magnification for apple-green
fluorescence of Giardia cyst and Cryptosporidium
oocyst shapes. Background fluorescence of the mem-
brane should be either very dim or nonexistent.
Cryptosporidium oocysts may or may not show evidence of
oocyst wall folding, which is characterized under
epifluorescence by greater concentrations of FITC along sur-
face fold lines. Whether the oocysts show this characteristic
depends on the manner in which the oocysts have been treated
and the amount of turgidity they have been able to maintain3.
If no apple-green fluorescing Giardia cyst or
Cryptosporidium oocyst shapes are observed, then the
fluorescent staining did not work or the positive con-
trol cyst preparation was faulty. Do not examine the
water sample slides for Giardia cysts and Cryptosporidium
oocysts. Recheck reagents and procedures to determine the
problem.
37
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CHAPTER 6
MICROSCOPIC EXAMINATION
If apple-green fluorescing cyst and oocyst shapes are
observed, change the microscope from epifluorescence
to the 100X oil immersion Hoffman modulation® or
differential interference contrast objective.
At no less than 1,OOOX total oil immersion magnification, ex-
amine Giardia cyst shapes and Cryptosporidium oocyst shapes
for internal morphology.
The Giardia cyst internal morphological characteristics include
1-4 nuclei, axonemes, and median bodies. Giardia cysts should
be measured to the nearest 0.5 ^im with a calibrated ocular
micrometer. Record the length and width of cysts. Also record
the morphological characteristics observed. Continue until at
least 3 Giardia cysts have been detected and measured in this
manner.
The Cryptosporidium oocyst internal morphological charac-
teristics include 1-4 sporozoites. Examine the Cryptosporidium
oocyst shapes for sporozoites and measure the oocyst diam-
eter to the nearest 0.5 |0,m with a calibrated ocular micrometer.
Record the size of the oocysts. Also record the number, if any,
of sporozoites observed. Sometimes a single nucleus is ob-
served per sporozoite. Continue until at least 3 oocysts have
been detected and measured in this manner.
Assay Negative Control
Using epifluorescence, scan the negative control mem-
brane at no less than 200X total magnification for
apple-green fluorescence of Giardia cyst and Crypto-
sporidium oocyst shapes.
38
-------
CHAPTER 6
MICROSCOPIC EXAMINATION
If no apple-green fluorescing cyst or oocyst shapes are
found, and if background fluorescence of the mem-
brane is very dim or nonexistent, continue with ex-
amination of the water sample slides.
If apple-green fluorescing cyst or oocyst shapes are
found, discontinue examination since possible con-
tamination of the other slides is indicated. Clean the
equipment (see Appendix A), recheck the reagents and
procedure, and repeat the assay using additional aliquots of
the sample.
Sample Examination
Scan each slide in a systematic fashion, beginning with one
edge of the mount and covering the entire coverslip. An up-
and-down or a side-to-side scanning pattern may be used. The
diagram at the right illustrates two alternative patterns for sys-
tematic slide scanning.
Patterns ol Microscopic
Examination
Empty Counts, Counts with Amorphous Structure, Counts with
Internal Structure, and Total IFA Count
When appropriate responses have been obtained for
the positive and negative controls, use epifluorescence
to scan the entire coverslip from each sample at not
less than 200X total magnification for apple-green fluo-
rescence of cyst and oocyst shapes.
39
-------
CHAPTER 6
MICROSCOPIC EXAMINATION
optics.
When brilliant, apple-green fluorescing round-to-oval
objects (8 to 18 (im long by 5 to 15 |j,m wide) with
brightly highlighted edges are observed, switch the
microscope to either Hoffman modulation® or D.I.C.
Look for external or internal morphological characteristics
atypical of Giardia cysts (e.g., spikes, stalks, appendages,
pores, one or two large nuclei filling the cell, red fluorescing
chloroplasts, crystals, spores, etc.). If these atypical structures
are not observed, then categorize such apple-green fluorescing
objects of the aforementioned size and shape as either empty
Giardia cysts, Giardia cysts with amorphous structure, or Giar-
dia cysts with internal structures (nuclei, axonemes, and me-
dian bodies).
Record the shape and measurements (to the nearest 0.5 (0,m at
1,OOOX total magnification) for each such object. Record the
internal structures observed.
Sum the counts of empty Giardia cysts, Giardia cysts with
amorphous structure, and Giardia cysts with internal structures.
Report this sum as the total Giardia IFA count (see Report
Forms in Appendix zebbo).
Giardia cysts with internal structures must be con-
firmed by a senior analyst.
When brilliant, apple-green fluorescing ovoid or spheri-
cal objects (3 to 7 \\,m in diameter) with brightly high-
lighted edges are observed, switch the microscope to
either Hoffman modulation® or D.I.C. optics. Look
for external or internal morphological characteristics atypical
of Cryptosporidium oocysts (e.g., spikes, stalks, appendages,
pores, one or two large nuclei filling the cell, red fluorescing
chloroplasts, crystals, spores, etc.). If these atypical structures
are not observed, then categorize such apple-green fluorescing
40
-------
objects of the aforementioned size and shape as either empty
Cryptosporidium oocysts, Cryptosporidium oocysts with amor-
phous structure, or Cryptosporidium oocysts with internal struc-
ture (1 to 4 sporozoites/oocyst).
Although not a defining characteristic, surface oocyst
folds may be observed in some specimens.
Record the shape and measurements (to the nearest 0.5 (im at
1,OOOX total magnification) for each such object. Record the
number of sporozoites observed. Sum the counts of empty
Cryptosporidium oocysts, Cryptosporidium oocysts with amor-
phous structure, and Cryptosporidium oocysts with internal
structure. Report this sum as the total Cryptosporidium IFA
count (see Appendix D, Sample Forms).
Cryptosporidium oocysts with sporozoites must be con-
firmed by a senior analyst.
End Notes
1 Melvin, D.M. and M.M. Brooke. 1982. Laboratory Proce-
dures for the Diagnosis of Intestinal Parasites. U.S. Depart-
ment of Health and Human Services, HHS Publication No.
(CDC) 82-8282.
2 A device between the objectives and the oculars that is ca-
pable of adjusting the total magnification.
CHAPTER 6
MICROSCOPIC EXAMINATION
41
-------
Chapter 7
Calculations and Reporting
Calculations
It is necessary to calculate the percentage of floated sediment
examined microscopically. This percentage is calculated from:
• The total volume of the floated pellet obtained through filter
elution;
• The number of 25-mm membrane filters prepared together
with the volume of floated pellet represented by these mem-
brane filters; and
• The number of membrane filters examined.
Although the calculations can be performed with a hand cal-
culator, EPA strongly recommends that they be done with the
computer spreadsheet provided with the ICR Guidance Manual.
Calculate and record the percentage of floated sedi-
ment examined microscopically.
Calculate this value from the total volume of floated pellet ob-
tained (see Chapter 3, Filter Elution), the number of 25-mm
membrane filters prepared together with the volume of floated
pellet represented by these membrane filters (see the section
on Determining Sample Volume per Filter in Chapter 5), and
the number of membrane filters examined.
The following values are used in calculations:
V = Volume (liters) of original water sample.
P = Eluate packed pellet volume.
43
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CHAPTER 7
CALCULATIONS AND REPORTING
F = Fraction of eluate packed pellet volume (P) subjected to
flotation. It is determined by this equation:
ml P subjected to flotation
R = Percentage (expressed as a decimal) of floated sediment
examined.
TG = Total Giardia IFA cyst count (Empty Count, Count with
Amorphous Structure, and Count with Internal Structure).
EG = Count of Giardia cysts which are empty.
AG = Count of Giardia cysts with amorphous internal struc-
ture.
GW1S = Count of Giardia cysts with 1 internal structure.
GW2S = Count of Giardia cysts with more than 1 internal
structure.
TC = Total Cryptosporidium IFA oocyst count (Empty Count,
Count with Amorphous Structure, and Count with Internal
Structure).
EC = Count of Cryptosporidium oocysts which are empty.
AC = Count of Cryptosporidium oocysts with amorphous in-
ternal structure.
CWS = Count of Cryptosporidium oocysts with internal struc-
ture.
For positive samples, calculate the number of cysts or
oocysts per 100 liters of sample as follows:
X (TG, EG, AG, GW1S, GW2S, TC, EC, AC, orCWS)(100)
1OOL FVR
A sample calculation is shown in Appendix C.
44
-------
CHAPTER 7
CALCULATIONS AND REPORTING
For samples in which no cysts or oocysts are detected
(for example, TG or TC or GWS or CWS <1), calcu-
late the detection limit as follows:
-------
Chapter 8
Water Sample Controls
Water Sample Negative Control
This control is a check on equipment, materials, reagents and
technique. It involves processing a 1 u,m nominal porosity car-
tridge filter as if it were an unknown. All samples analyzed
over the course of a week are considered to be a batch. For
each batch, there must be a negative control.
Cut the fibers from an unused 1 \un nominal porosity
cartridge filter and process the fibers using the Filter
Elution, Concentration, Flotation Purification, and In-
direct Fluorescent Antibody procedures presented in
this guide.
Examine the entire concentrate from this sample, us-
ing the Microscopic Examination procedures presented
in Chapter 6.
If any cysts or oocysts are detected, do not process any
unknown samples until the source of the contamina-
tion is located and corrected. The results from samples
in the batch analyzed prior to the finding of a positive
in a negative control will be excluded from the ICR Data Base.
47
-------
CHAPTER 8
WATER SAMPLE CONTROLS
Water Sample Positive Control
The purpose of this control is to ensure that the laboratory can
recover cysts and oocysts when they are spiked into a sample
at a known level. All samples analyzed over the course of a
week are considered a batch.
Using the sample collection procedures (see Appen-
dix E) and the assay and microscopic examination pro-
cedures presented in Chapters 3 through 6, collect, pro-
cess, and examine a 40 L (10 gal) or larger sample of
reagent water or tap water that has been spiked with 1,000 Giar-
dia cysts and 2,000 Cryptosporidium oocysts.
Process the filtered water using the Filter Elution, Con-
centration, Flotation Purification, and Indirect Fluo-
rescent Antibody Procedures presented in this guide.
Examine the entire concentrate from this sample, us-
ing the Microscopic Examination procedures presented
in Chapter 6. It is not necessary to identify internal
morphological characteristics using differential in-
terference contrast microscopy.
If cysts and oocysts are not detected, do not process
any more unknown samples until the reason for not
recovering cysts and oocysts is determined and cor-
rected. The results from samples in the batch analyzed
prior to not finding cysts and oocysts in a positive control will
be excluded from the ICR Data Base.
48
-------
Appendix A
Cleaning the Manifold and
Stainless Steel Wells
Manifold
Stepl
After all the membrane filters have been mounted
on glass slides, discard the support filters.
Step 2
Open all the manifold valves and increase the
vacuum pressure to the manifold by closing the
bleeder valve associated with the vacuum tubing.
Step 3
Rinse each manifold filter support screen with 10-
20 mL of 0.01% Tween 80 solution.
Step 4
Rinse each manifold filter support screen with 10-
20 mL reagent water.
StepS
Disconnect the manifold from the vacuum and
wash the cover and fluid collection box in warm
detergent solution. Rinse with tap water and re-
agent water.
Stainless Steel Wells
Stepl
Place a cloth on the bottom of an autoclavable con-
tainer which is large enough to accommodate all
10 stainless steel wells in a single layer.
Step 2
Put the stainless steel wells top side down on the
cloth. The rim on the underside of the well is frag-
ile. Care must be taken to avoid scratching and
denting the rim.
Step 3
Add enough reagent water containing detergent to
cover the stainless steel wells by an inch or more.
Step 4
Autoclave the stainless steel container with the
stainless steel wells for 15 minutes at 15 lbs/in2
and 121°C. Use the slow exhaust mode at the
completion of the autoclave cycle.
StepS
Transfer the wells to a pan of hot detergent clean-
ing solution.
Step 6
Individually scrub the inside and bottom of stain-
less steel wells with a sponge.
Step?
Rinse each well with tap water followed by reagent
water. Drain and air dry the wells.
StepS
Always check the bottom ridge of each stainless
steel well for dents and scratches.
Step 9
If dents or scratches are found on the bottom of a
stainless steel well, do not use it until it is properly
reground.
A- 1
-------
Appendix B
Microscope Adjustments1
The microscopic portion of this procedure
depends upon very sophisticated optics.
Without proper alignment and adjustment
of the microscope the instrument will not function
at maximal efficiency and the probability of ob-
taining the desired image (information) will not
be possible. Consequently, it is imperative that all
portions of the microscope from the light sources
to the oculars are properly adjusted.
While microscopes from various vendors are con-
figured somewhat differently, they all operate on
the same general physical principles. Therefore,
slight deviations or adjustments may be required
to make these guidelines work for the particular
instrument at hand.
Adjustment of the Epifluorescent
Mercury Bulb and Transmitted
Light Bulb Filament.
The sole purpose of these procedures is to ensure
even field illumination.
Mercury Bulb Adjustment.
This section assumes that you have successfully
replaced the mercury bulb in your particular lamp
socket and reconnected the lamp socket to the lamp
house. These instructions also assume the con-
denser has been adjusted to produce Kohler illu-
mination. Make sure that you have not touched
any glass portion of the mercury bulb with your
bare fingers while installing it.
WARNING: Never look at the ultravio-
let light coming out of the mercury lamp
house or the ultraviolet light image with-
J out a barrier filter in place.
Stepl
Usually there is a diffuser lens between the lamp
and the microscope which either must be removed
or swung out of the light path.
Step 2
Using a prepared microscope slide, adjust the fo-
cus so the image in the oculars is sharply defined.
Step 3
Replace the slide with a business card or a piece of
lens paper.
Step 4
Close the field diaphragm (iris diaphragm in the
microscope base) so only a small point of light is
visible on the card. This dot of light tells you where
the center of the field of view is.
StepS
Mount the mercury lamp house on the microscope
without the diffuser lens in place and turn on the
mercury bulb.
Step 6
Remove the objective in the light path from the
nosepiece. You should see a primary (brighter) and
secondary image (dimmer) of the mercury bulb arc
on the card after focusing the image with the ap-
propriate adjustment.
B- 1
-------
APPENDIX B
MICROSCOPE ADJUSTMENTS
Step?
Using the other lamp house adjustments, adjust
the primary and secondary mercury bulb images
so they are side by side (parallel to each other)
with the transmitted light dot in between them.
StepS
Reattach the objective to the nosepiece.
Step 9
Insert the diffuser lens into the light path be-
tween the mercury lamp house and the micro-
scope.
Step 10
Turn off the transmitted light, remove the card
from the stage, and replace it with a slide of fluo-
rescent material. Check the field for even fluo-
rescent illumination. Adjustment of the diffuser
lens will most likely be required. Additional
slight adjustments as in step 6 above may be
required.
Step 11
Maintain a log of the number of hours the U.V.
bulb has been used. Never use the bulb for longer
than it has been rated. Fifty watt bulbs should
not be used longer than 100 hours; 100 watt
bulbs should not be used longer than 200 hours.
Transmitted Bulb Adjustment
This section assumes that you have successfully
replaced the transmitted bulb in your particular
lamp socket and reconnect the lamp socket to
the lamp house. Make sure that you have not
touched any glass portion of the transmitted
light bulb with your bare fingers while install-
ing it. These instructions also assume the con-
denser has been adjusted to produce Kohler il-
lumination.
Stepl
Usually there is a diffuser lens between the lamp
and the microscope which either must be removed
or swung out of the light path. Reattach the lamp
house to the microscope.
Step 2
Using a prepared microscope slide and a 40X (or
similar) objective, adjust the focus so the image in
the oculars is sharply defined.
Step 3
Without the ocular or Bertrand optics in place the
pupil and filament image inside can be seen at the
bottom of the tube.
Step 4
Focus the lamp filament image with the appropri-
ate adjustment on your lamp house.
StepS
Similarly, center the lamp filament image within
the pupil with the appropriate adjustment(s) on
your lamp house.
Step 6
Insert the diffuser lens into the light path between
the transmitted lamp house and the microscope.
End Notes
1 Smith, R.F. 1982. Microscopy and Photomicrog-
raphy: A Practical Guide. Appleton-Century-
Crofts, New York.
B-2
-------
Appendix C
Sample Calculations
Positive Samples
Assume that a 100 gallon (380 L) water sample
was collected. The sample was eluted, resulting in
5 mL of sediment. Fifty percent (2.5 L) of the sedi-
ment was purified by Percoll-surcrose flotation.
Forty percent of the floated material was exam-
ined microscopically. Eight empty Giardia cysts
and 3 Giardia cysts with internal structure were
found. No Cryptosporidium oocysts were ob-
served.
Using the formula in Chapter 7:
V = 380 L
P = 5mL
F = 2.5/5 = 0.5
R = 40% = 0.4
TG=11
GW1S = 3
Giardia cysts with structures
100 L
(GW1SK100)
FVR
(3)(100)
(0.05)(380)(0.4)
4;
and
Total IFA Giardia cysts _ (TG)(100)
100L FVR
(11X100)
" (0.5)(380)(0.4)
14
Negative Samples
Using the description given in the previous ex-
ample, no Cryptosporidium oocysts were observed.
The calculated detection limit per 100 liters would
be:
Total IFA Cryptosporidium oocysts _ (TC)(100)
100L ~ FVR
(0.5)(380)(0.4)
C- 1
-------
Appendix D
Sample Forms
Ten-Place Hoefer Manifold Membrane Labeling Diagram
1.
2.
3.
4.
5.
(topwtew)
6.
7.
8.
9.
10.
pressure gauge
o o o o o
Q O O O (D
D- 1
-------
Report Form
Slide prepared by:
Analyst:
Date prepared:
Date Analyzed:
-n*
|0
en
Object
Located by
I FA
No.
1
2
3
4
5
6
7
8
9
10
Shape (oval
or round)
Size
LxW
(Jim)
Total
Empty
Giardia
Cysts
(•)
(A)
Giardia Cysts with Internal Structure
(C)
Morphological Characteristics
Nucleus
(*)
Median Body
(•)
# with 1 morph. char.
# with >1 morph. char.
Axonemes
(•)
Total IFA
Count
(•)
(D = A+B+C)
A. Calculated number of Empty Giardia Cysts/100 liters
B. Calculated number of Giardia Cysts with Amorphous Structure/100 liters
C. Calculated number of Giardia Cysts with 1 Internal Structure/100 liters
C. Calculated number of Giardia Cysts with more than 1 Internal Structure/100 liters
D. Calculated Total IFA Giardia Count/100 liters
-------
CrvptosDoridium Report Form
Slide prepared by:
Analyst:
Date prepared:
Date Analyzed:
Object
Located by
IFA
No.
1
2
3
4
5
6
7
8
9
10
Shape (oval
or round)
Size
LxW
((im)
Total
Empty
CrvDtosDoridimn
Oo cysts
(•)
(A)
Crvptosporidium
Oocysts with Amorphous
Structure
(•)
(B)
CrvDtosDor^dimn Oocysts
with Internal Structure
(C)
Morphological
Characteristics
Sporozoite (#)
Total IFA
Crvotosooridium Count
(•)
(D = A+B+C)
A. Calculated number of Empty Crvptosporidiurn Oocysts/100 liters
B. Calculated number of Crvotosooridium Oocysts with Amorphous Structure/100 liters
C. Calculated number of Crvptosooridiurn Oocysts with Internal Structure/100 liters
D. Calculated Total IFA Crvptosooridium Count/100 liters
C/)
=
en
-------
Appendix E
Sampling
Sampling Apparatus Preparation
and Assembly
Sampling Apparatus
The sampling apparatus (see Figure 1) used for raw
water consists of a female hose connector, an inlet
hose, pressure regulator, pressure gauge, filter
holder, a 1 \im nominal porosity filter, an outlet
hose, a water meter, and a 1 gallon per minute flow
control valve or device (4 L/min). A pump will be
needed for unpressurized sources and a fluid
proportioner or proportioning injector and pressure
gauge will be needed before the filter for chlori-
nated or other disinfectant treated waters (see Fig-
ure 2 on the next page).
Label
Attach a water-resistant label containing the fol-
lowing information to the filter holder:
Start Time:
Stop Time:
Operator's
Date:
Meter Readina: Turbidity;
Meter Reading: Turbidity:
Name: Total Volume Filtered:
Sampling Location:
Hoses
Inlet and outlet hoses for the filter holder consist
of standard garden hoses and fittings. If desired
Filter Holder
Thoroughly wash the filter
holder with a stiff brush in hot
water containing detergent,
when sampling is completed.
Rinse the filter holder with tap
water until the soap residue is
gone. Follow with a thorough
rinse in reagent water and air
dry.
Figure 1: Raw-Water Sampling Apparatus
•_ Htrror fWKI
E- 1
-------
APPENDIX E
SAMPLING
Figure 2: Treated-Water Sampling Apparatus
• — HMFBTHMIKE
pressure, PVC tubing ('/2-inch I.D., 3/4-inch O.D.,
'/8-inch wall) and/or quick connects may be sub-
stituted for the standard garden hose and/or hose
clamps.
Hoses may be used repeatedly provided they are
rinsed with at least 20 gallons (76 liters) of the
water to be sampled prior to starting the sampling.
Pump
A pump is needed, when an unpressurized source
is being sampled.
Fluid Proportioner or Proportioning
Injector
If the water to be sampled is chlorinated or disin-
fected by any other chemicals, the disinfectant must
be neutralized during sample collection. While the
assay system allows detection of disinfected cysts
and oocysts, exposure to disinfectant may inter-
fere with the visualization of internal morpholo-
gies of these organisms.
Use sodium thiosulfate solution
to neutralize the disinfectant in
water samples. Add the sodium
thiosulfate solution to the wa-
ter during sample collection
with a mechanical fluid propor-
tioner pump or an in-line injec-
tor.'
Raw Water Sample
Collection
Stepl
Put on a pair of the latex gloves.
Step 2
Before connecting the sampling
apparatus (see Figure 1) to the tap or source to be
sampled, turn on the tap and allow the water to
purge residual debris from the line for 2 to 3 min-
utes, or until the turbidity of the water becomes
uniform.
Step 3
Connect the apparatus minus the filter to the tap
and allow 20 gallons (76 liters) to flush the sys-
tem. If a pressurized source is not available, use a
pump, following the manufacturer's instructions,
to get water through the sampling apparatus. While
the flushing of the apparatus is being done, adjust
the pressure regulator so the adjacent pressure
gauge reads no more than 30 pounds per square
inch (PSI).
Step 4
Turn off the water flow, when the flushing of the
apparatus is complete. Open the filter housing and
pour all the water out. Put the filter in, close, and
tighten the filter housing.
StepS
Use a water-resistant marking pen to record the
start time, meter reading, name of person collect-
E-2
-------
APPENDIX E
SAMPLING
ing the sample, turbidity, date and sampling loca-
tion on the filter holder label.
Step 6
Start water flow through the filter. Check the pres-
sure gauge after the pressure regulator to make sure
the reading is no more than 30 PSI. Readjust the
regulator, if necessary.
Step?
After the 100 L (27 gallons) of raw water has passed
through the filter, shut off the water flow, record
the stop time, final meter reading and turbidity of
the water at the end of filtration on the filter holder
label.
StepS
Disconnect the sampling apparatus while maintain-
ing the inlet hose level above the level of the open-
ing on the outlet hose in order to prevent
backwashing and the loss of particulate matter from
the filter.
Step 9
After allowing the apparatus to drain, open the fil-
ter housing and pour the residual water remaining
in the filter holder into a plastic sample bag.
Step 10
Aseptically remove the filter from the holder and
transfer the filter to the plastic sample bag con-
taining the residual water.
Step 11
Seal the bag and place it inside a second plastic
sample bag. Transfer the label or label informa-
tion from the filter holder to the outside of this sec-
ond (outer) bag.
Step 12
Transport the sample to the laboratory on wet ice
or with but not on cold packs. When the filter(s)
arrive at the laboratory, they should be immedi-
ately stored at 2-5°C. Do not freeze the filter dur-
ing transport or storage.
Finished Water Sample Collection
If the water must be neutralized, add sodium thio-
sulfate solution via the proportioner system. For
each 100 L of finished water sampled, 250 ml of
2.0% sodium thiosulfate solution will be needed.
Stepl
Put on a pair of the latex gloves.
Step 2
Before connecting the sampling apparatus (see
Figure 2) to the tap or source to be sampled, turn
on the tap and allow the water to purge residual
debris from the line for 2 to 3 minutes, or until the
turbidity of the water becomes uniform.
Step 3
Connect the apparatus minus the filter to the tap
and allow 20 gallons (76 liters) to flush the sys-
tem. If a pressurized source is not available, use a
pump, following the manufacturer's instructions,
to get water through the sampling apparatus.
While the flushing is being done, adjust the pres-
sure regulator, so the adjacent pressure gauge reads
no more than 30 PSI. Pour the 2% sodium thiosul-
fate solution into a graduated cylinder. Place the
injector tube into the solution and adjust the larger
top (vacuum) screw on the injector, so the pres-
sure on the pressure gauge following the injector
reads no more than 19 PSI.
Now adjust the smaller bottom (flow) screw on the
injector, so the flow rate of the thiosulfate solution
is 10 ml per minute. A hose cock clamp on the
injector tube may be required to achieve the cor-
rect thiosulfate flow rate. After this adjustment is
complete, transfer the injector tube to a graduated
carboy of thiosulfate solution.
E-3
-------
APPENDIX E
SAMPLING
Step 4
Turn off the water flow, when the flushing of the
apparatus is complete. Open the filter housing and
pour all the water out. Put the filter in, close, and
tighten the filter housing.
StepS
Use a water-resistant marking pen to record the
start time, meter reading, name of person collect-
ing the sample, turbidity, date and sampling loca-
tion on the filter holder label.
Step 6
Start water flow through the filter. Check the pres-
sure gauge after the pressure regulator to make sure
the reading is no more than 30 PSI. Also check to
make sure the thiosulfate solution is being drawn
into the sampling apparatus. Readjust the regula-
tor and injector, if necessary.
Step?
After the 1,000 L (264 gallons) of finished water
has passed through the filter, shut off the water flow,
record the stop time, final meter reading and tur-
bidity of the water at the end of filtration on the
filter holder label.
StepS
Disconnect the sampling apparatus while maintain-
ing the inlet hose level above the level of the open-
ing on the outlet hose in order to prevent
backwashing and the loss of particulate matter from
the filter.
Step 9
After allowing the apparatus to drain, open the fil-
ter housing and pour the residual water remaining
in the filter holder into a plastic sample bag.
Step 10
Aseptically remove the filter from the holder and
transfer the filter to the plastic sample bag con-
taining the residual water.
Step 11
Seal the bag and place it inside a second plastic
sample bag. Transfer the label or label informa-
tion from the filter holder to the outside of this sec-
ond (outer) bag.
Step 12
Transport the sample to the laboratory on wet ice
or with but not on cold packs and refrigerate at 2-
5°C. Do not freeze the filter during transport or
storage.
End Notes
1 Details on the operation and use of proportioner
pumps and injectors can be found in Standard
Methods for the Examination of Water and Waste-
water, Section 95 IOC, "Virus Concentration from
Large Sample Volumes by Adsorption to and EIu-
tion from Microporous Filters (PROPOSED)," 18th
ed., 1989, pp. 9-105 to 9-109.
E-4
------- |