MLS 108 Lab- Collection and Preservation of Stool

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L2: Specimen Collection and Processing
Laboratory Diagnosis
➔ Confirm a clinical impression that the condition has a
parasitic nature
➔ Rule-out a diagnosis
➔ Aid a clinician in the choice of proper medication
➔ Help in monitoring the effect of a treatment regimen. A. Collection
Ability of the Laboratory to Generate Reliable
Results depends on:
❖ Proper specimen Collection
❖ Handling
❖ Processing of specimens prior to examination
❖ Skill of the examiner
❖ Quality of the equipment
Diagnosis on the Identification of Parasitic Infection
➔ Demonstration of Parasites
◆ adults, eggs, larvae, cysts, oocysts, and
trophozoites
◆ Possible only in the patent stage of the
infection
➔ Detection of Host Immune Response to the
Parasites
◆ antibodies and Antigens
◆ Only a presumptive evidence of infection
Stool or Fecal Sample
➔ Most common specimen in the diagnosis of
gastrointestinal parasites
➔ Collected in a clean, wide – mouthed containers
made of waxed cardboard or plastic with tight
fitting lid to ensure retention of moisture and to
prevent accidental spillage.
➔ Must be submitted preferably in the fresh state or as
preserved samples.
➔ Data or Information
◆ Name
◆ Age and Sex
◆ Date and Time of Collection
◆ Requesting Physician / Requested Procedure
◆ Presumptive Diagnosis
◆ Prior Infections / Travel History
Factors to Consider:
➢ Intake of drugs / medicinal substances
○ Anti-diarrheal
○ Antacid
○ Bismuth
○ Barium and Laxatives
➢ Amount of stool to be collected (thumb-sized
specimen or 5 – 6 table spoon for watery. The
acceptable amount of stool required for parasite
study is 2 to 5 g, often referred to as the size of a
walnut.
Ova or Parasite Exam
1. The typical stool collection protocol consists of three
specimens, one specimen collected every other day
or a total of three collected in 10 days. One
exception is in the diagnosis of amebiasis, in which
up to six specimens in 14 days is acceptable. Stool
swabs are unacceptable for O&P exams.
2. Interfering substances- Certain substances and
medications interfere with parasite detection. These
are listed below:
a. Specimens should not contain water or
urine. Urine has been known to destroy
some parasites.
b. Stool should not be retrieved from toilet bowl
water because free-living protozoa and
nematodes may be confused with human
parasites.
c. Water may destroy select parasites, such as
schistosome eggs and amebic trophozoites.
d. Stool samples from patients whose therapy
includes barium, bismuth, or mineral oil
should be collected prior to therapy or not
until 5 to 7 days after the completion of
therapy. If the samples are taken during the
course of therapy, these interfering
substances may mask possible parasites
during examination.
e. Collection of specimens from patients who
have taken antibiotics or antimalarial
medications should be delayed for 2 weeks
following therapy.
f. Purging or bowel prep agents must be
cleared before the specimen can be
submitted for O&P exam, as they are
crystalline in nature and obscure any
parasitic elements that might be present.
g. Toilet paper in the stool specimen may
mask parasites or make examination of the
sample difficult.
3. All specimens should be placed in fixative ASAP
after collection.
❖ A two-vial system is currently in use for ova and
parasite fixation and preservation. Both vials
must be submitted for the complete OP exam.
MLS 108 Lab- Collection and Preservation of Stool
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➢ The pink cap vial contains formalin and
is used in an iodine concentration
procedure.
➢ The blue or gray cap vial contains PVA
and is used to prepare a permanent
stained smear.
❖ Pour over or use the "spork" provided in the cap
lid to sample the specimen, particularly areas of
blood or mucous.
❖ Add specimen to each vial until fluid level
reaches the fill-to-here line. Do not overfill the
vials.
❖ Tighten caps and shake vigorously to mix.
4. Note specimen consistency on the outside of the
vial and/or on the computer order.
a. Liquid = of pourable consistency
b. Soft = having no shape
c. Formed = having a definite shape
5. Include recent travel history of the patient, if
known.
6. Unpreserved liquid specimens may be submitted
to the lab in a clean, dry container, immediately
after passage, if amoebiasis is suspected. A direct
wet mount procedure can be performed to look for
motile protozoans.
B. Transport
1. Request should include all pertinent clinical
information, including suspected diagnosis and
travel history. Note any specific parasite the
physician mentions by name.
2. Transport to the laboratory ASAP. Do not
refrigerate.
3. Fresh liquid stool must be transported immediately
and rapidly to the lab and hand delivered to the
technologist on duty with verbal instructions to look
for amoebae.
C. Special Request
1. Giardia Antigen
a. Collect specimen as for O & P
b. Transfer stool to only the pink cap vial (formalin)
of the O &P collection set
2. Cryptosporidium
a. Collect specimen as for O & P.
b. Transfer stool to only the pink cap vial
(formalin) of the O & P collection set.
3. Pinworm
a. Contact the laboratory for a commercial
collection kit or clear Scotch tape and a glass
slide. Opaque tape is unacceptable.
b. As the patient sleeps at night, the pinworm
emerges from the rectum to deposit eggs on and
around the anus. Collect the specimen first thing
after patient awakens in the morning, before
arising from bed. Timing is critical for detection
of eggs.
c. Loop a strip of clear tape lengthwise down a
wooden tongue depresser, around the end and
back up the other side of the stick, sticky side
out. Hold both ends of the tape securely against
the tongue depressor.
d. Have the patient lie face down and spread the
buttocks.
e. Press the sticky side of the tape against several
areas of the perianal region.
f. Place the tape, sticky side down on a glass slide
and smooth with cotton or gauze.
g. Transport to laboratory ASAP
Occult Blood
A. Collection
1. There are no restrictions on the number of times an
occult blood test may be ordered.
a. Logically, if the 1st specimen is positive, the
reason for testing should be evaluated before
ordering additional tests.
b. with an initial negative test, additional specimens
may be required. Since gastrointestinal lesions
may bleed intermittently, the recommendation is
a specimen from 3 consecutive bowel
movements.
2. If patient preparation guidelines cannot be met,
be advised that results may be falsely positive or
falsely negative, and evaluate your patient
accordingly.
a. For females, do not submit specimens during, or
until three days after a menstrual period.
b. Do not submit specimens while the patient has
bleeding hemorrhoids or blood in the urine.
c. For 7 days prior to and during the collection
period, avoid aspirin or other nonsteroidal
anti-inflammatory drugs, anticoagulants, or any
MLS 108 Lab- Collection and Preservation of Stool
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substance which could irritate the ❖ SAF (Sodium Acetate-Acetic Acid Formalin)
gastrointestinal tract, including alcohol. ➢ It does not contain mercuric chloride and has a
d. For 72 hours prior to and during the collection long shelf-life
period, avoid: ➢ Images of the organism however are not as
- Vitamin C, or iron supplements sharp as those in PVA or Shaudinn’s.
containing Vitamin C, Red meat,
Artichokes, mushrooms, bean sprouts,
apples, oranges, bananas, grapes.

Stool Preservatives

❖ Formalin
➢ All purpose Fixative
➢ 5% concentration to preserve protozoan cysts,
10% helminth eggs and larvae
➢ May be buffered with NaPO4 (preserves the
morphological characteristics of the organism.
➢ Can be concentrated by FECT
❖ Schaudinn’s Solution
➢ To preserve fresh fecal specimens for staining of
smears
➢ Contains mercuric chloride that is too toxic for
humans
❖ PVA (Polyvinyl Alcohol)
➢ Plastic resin that adheres the stool to the slide
➢ Normally incorporated with Schaudinn’s Solution
➢ The main advantage is for the preservation of
protozoan cysts and trophozoites for permanent
staining.
➢ Can be concentrated by FECT and can be sent
to other laboratories for referral.
➢ Contains mercuric chloride but can be replaced
by Cupric Sulfate
❖ MIF (Merthiolate-Iodine-Formalin)
➢ Contains methiolate (thimerosal) and iodine
which act as a staining components, while
formalin acts as preservative.
➢ Useful for fixation of intestinal protozoan,
helminth eggs and larvae
➢ Preserved stools can be examined directly by
wet mount but difficulty in the specific
identification of protozoans can be encountered
(Logul’s iodine must be freshly prepared since it
is unstable, making unsatisfactory results.
MLS 108 Lab- Collection and Preservation of Stool
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Preservative Advantages Disadvantages

10% Formalin ● All purpose fixative ➢ Not suitable for some permanent smears
● Easy to prepare stained with trichrome
● Long shelf life ➢ Inadequate preservation of morphology of
● Good preservation of morphology of helminth protozoan trophozoites
eggs, larvae, protozoan cysts, and coccidia ➢ Can interfere with PCR, especially after
● Suitable for concentration procedures and UV extended fixation time
fluorescence microscopy
● Suitable for acid-fast, safranin, and chromotrope
stains
● Compatible with immunoassay kits and UV
fluorescence microscopy

MIF (merthiolate- ● Components both fix and stain organisms ➢ Not suitable for some permanent smears
iodine-formaldehyde) ● Easy to prepare stained with trichrome
● Long shelf life ➢ Inadequate preservation of morphology of
● Useful for field surveys protozoan trophozoites
● Suitable for concentration procedures ➢ Iodine interferes with other stains and
fluorescence
➢ Iodine may cause distortion of protozoa

LV-PVA (low viscosity ● Good preservation of morphology of protozoan
polyvinylalcohol) trophozoites and cysts
● Easy preparation of permanent smears stained
with such as trichrome (solution both preserves
organisms and makes them adhere to slides)
● Preserved samples remain stable for several
months
➢ Inadequate preservation of morphology of
helminth eggs and larvae, coccidia, and
microsporidia
➢ Contains mercuric chloride
➢ Difficult and expensive to dispose of
➢ Difficult to prepare in the laboratory
➢ Not suitable for concentration procedures
➢ Cannot be used with immunoassay kits
➢ Not suitable for acid-fast, safranin and
chromotrope stains

SAF (sodium ● Suitable for both concentration procedures and ➢ Requires additive (e.g., albuminglycerin) for
acetateacetic preparation of permanent stained smears adhesion of specimens to slides
acidformalin) ● Easy to prepare ➢ Permanent stains not as good as with PVA or
● Long shelf life Schaudinn's fixative
● Suitable for acid-fast, safranin, and chromotrope
stains
● Compatible with immunoassay kits

Schaudinn’s Fixative ● Good preservation of morphology of protozoan
trophozoites and cysts
● Easy preparation of permanent stained smears
➢ Less suitable for concentration procedures
➢ Contains mercuric chloride
➢ Inadequate preservation of morphology of
helminth eggs and larvae, coccidia, and
microsporidia
➢ Poor adhesion of liquid or mucoid specimens
to slides

Modified PVA copper ● Permanent smears can be made and stained ➢ Staining not consistent
or zinc with trichrome ➢ Organism morphology may be poor
● Zinc is preferred over copper ➢ Copper-morphology of cysts and
● No mercuric chloride trophozoites is poor
➢ Zinc-better morphology but not comparable
to LVPVA

One-Vial Fixatives ● Concentrate and permanent smear can be made ➢ Certain one-vial fixatives must use certain
(such as Ecofix, out of one vial stains
Parasafe, Unifix, ● Immunoassays can be done on most ➢ Color difference of stain
Proto-fix, STF, and ● No mercuric chloride ➢ Staining not always consistent
others that may be ➢ Sometimes more expensive than formalin
available) and LVPVA
Because 10% formalin and PVA have complementary advantages, it is recommended that the specimen be divided and
preserved in both types of preservatives (add one volume of stool to three volumes of the preservative)