Laboratory
5. Contact transmitted – readily infective., do not
Specimen Transport, Collection,
Ex. T. vaginalis
& Preservation
Parasitism: relationship between the host and parasite;
may or may not be harmful
Relationships Between a Host and Parasite
1. Obligatory – parasite cannot survive without a
host.
Ex. Ascaris lumbricoides
2. Facultative – parasite can survive without a host;
a parasite which have the power of changing from
one host to another of different species.
Ex. Strongyloides stercoralis (free living parasite)
3. Intermittent – parasites find a host during feeding
time.
Ex. Mosquito's carrying malaria
4. Commensalism – association between 2
organisms of different species in which one is
benefited while the other is neither benefited nor
injured; “eating at the same table”
Ex. Entamoeba coli and Man
5. Mutualism – both members benefit from each
other but life without the other is still possible.
6. Symbiosis – association between 2 organisms of
different species in which both members are so
dependent on each other that life apart is
impossible.
Effects of Parasite to Host
1. Pathogenic parasite – able to cause disease to its
host
2. Unpathogenic parasite – not able to cause
disease to its host (commensal)
Factors in Parasitism
1. source of infection
2. Mode of transmission
3. presence of a susceptible host
Example: Amoebiasis (E. histolytica)
#1 Source: water
#2 Mode: oral transmission
#3 mature cyst
Transmission
1. Soil transmitted – organisms develop further in
soil before they become infective to man.
Ex. Ascaris, Trichuris, Hookworm
2. Snail transmitted – requires development in
snails
Ex. Trematodes(flukes)
3. Arthropod transmitted – Ex. Malaria – mosquito
4. Food-animal transmitted –development in some
animals that serve as food to man. Ex. Taenia
solium (pork)
require further development
Types of Specimen for Parasite Exam
1. Fecal (stool) Specimens – most commonly
examined when infection with intestinal parasites
is suspected. Helminths, amoebae and other
intestinal protozoa can be identified during
microscopic examination performed on unstained
and concentrated fecal specimens particularly
their eggs, larvae adults, trophozoites, cysts and
oocysts form.
- also used for immunological tests that detect
Giardia, Cryptosporidium and Entamoeba antigens
in the specimens.
2. Purged and Enema Specimen – this type of
material is examined primarily to obtain
diagnostic evidence of infection with Entamoeba
histolytica, usually in cecal area when routine
examination of stool has been consistently
negative yet there is a strong clinical suspicion of
intestinal amoebiasis.
Purgative solution: a.) Magnesium sulfate b.) Sodium
sulfate
3. Urine Specimen/ Genital Specimen – urine is the
specimen of choice for the recovery of Schistosoma
haematobium eggs, microfilariae of Wuchereria,
Loa and Brugia and on rare occasions,
Strongyloides larvae. Trichomonas vaginalis are
often recovered from both male and female
patients.
a. Urine and vaginal discharge specimens and
prostatic secretions are examined by direct wet
mount for the presence of T. vaginalis. It
demonstrates a jerky motility under LPO.
4. Sputum Specimen - sputum specimens should be
collected in the early morning and transported in
a tight sealing, sterile container. A proper
specimen should be from lower respiratory
passages, not saliva.
- sample can be examined in direct wet mount with
saline or iodine or can be concentrated using N-
acetyl-L-cysteine and centrifugation.
5. Blood Specimen – second specimen commonly
examined in the laboratory. This is useful in the
recovery of blood parasites such as malarial
parasite, filarial worms, trypanosomes,
leishmanial and toxoplasma.
6. Aspirates
a. Proctoscopic aspirates and scrapings are used in
confirming suspicion of amebic ulcers in lower
sigmoid colon and rectum.
b. Duodenal aspirates are employed for the
demonstration of infections with Giardia lamblia,
Strongyloides stercoralis, Fasciolopsis buski or
Cryptosporidium.
- this specimen is collected by the use of tubing
(duodenal intubation) or by Entero-test method.
- Liver and lung aspirates are employed for the
demonstration of
Entamoeba histolytica. This sample should be examined in
direct wet mount and on permanent stained slides.
7. Biopsy Material – biopsy of skin, superficial
lymph nodes, visceral organs like the liver and
others can be processed by routine
histotechniques for demonstrating parasitic
stages.
8. Corneal Scrapings – diagnosis of Acanthamoeba
keratitis is best achieved by examination of this
specimen. Scrapings can be directly inoculated
into non-nutrient agar plates (Culbertson’s
medium) seeded with a suspension of live E. coli.
9. Cerebrospinal Fluid – CSF may be infected with
parasitic organisms such as Naegleria,
Acanthamoeba, Toxoplasma and Trypanosoma.
Patients with these infections generally exhibit
symptoms of meningitis and a lumbar puncture is
performed. CSF is collected in a sterile, tight sealed
container. Sample is examined in wet mount for
the presence of motile trophozoites.
10. Mouth and Nasal Discharge – Entamoeba
gingivalis and Trichomonas tenax cause parasitic
infections of the oral mucosa and gingival. They
are associated with poor oral hygiene and can be
recovered from mouth scrapings, particularly in
material from around the gum line or pyorrheal
pockets. Nasal discharge is collected and
examined for the presence of Naegleria fowleri.
Specimen is examined by direct wet mount for the
presence of parasites.
Stool Examination
Before you Collect your Sample
Do not take any of the following within 1 week of collecting
your sample:
- Medicines to treat heartburn, indigestion or to
prevent stomach ulcers (antacids)
- Barium or bismuth
- Medicines to treat diarrhea
- Oily laxatives such as castor oil
Your health care provider will give you instructions for
picking up your specimen container(s) for collecting your
sample.
You will also need a cleancollection decive such as a
shallow pan, plastics bag or clear plastics wrap (to place
over the toilet seat) in which to collect your sample before
transferring part of it to the specimen container(s).
Do not use specimen container if:
- The solution appearns cloudy of yellow
- It is past the expiration date
Keep you specimen container(s) away from heat or
flames. The liquid is flammable.
Keep your specimen container(s) out of the reach of
children and pets.
How to Collect your Sample
1. Unscrew the lid from the specimen container. Set
aside
2. Prepare the collection container (clean shallow
pan, plastic bag or clear plastic wrap) in which you
will collect your sample.
3. Collect the sample. Do not collect stool that has
been mixed with water or urine.
4. Using the plastic spoon attatched to the lid, scoop
out samples from bloody, slimy or watery areas of
the stool (if present). If the stool is hard, select
areas from each end and the middle of the stool.
5. Transfer enough of the selected stool to the orange-
and-green-cap specimen containers to raise the
level of liquid to the “fill to here” line. Do not
overfill.
If you have a screw-cap container without liquid,
transfer the liquid stool (about the size of a walnut)
to this container. There is not a “fill to here” line on
this container.
6. Screw the lid back on the container. Make sure it
is closed tightly. Shake to mix.
7. Write the following info on the container with a pen
or marker that will not run if the ink gets wet.
- Full name
Criteria for Specimen Rejection
Specimen contaminated with urine, residual soap, or
disinfectant.
Specimens received in grossly leaking transport
containers; diapers; dry specimens
Specimens submitted in fixative or additives
General Considerations in Stool Examination
Sample
• Stool after enemas are suitable for examination of
worms, eggs and protozoan parasites.
• Stool obtained after taking in oil laxatives, barium
or bismuth salts are not suitable for examination.
• Urine kills protozoan trophozoites rapidly, so
never accept stool mixed with urine.
• Stool should not be contaminated with water for it
will speed up growth of nonpathogenic organisms
making examination difficult.
• Trophozoites and cysts appear in stool at intervals.
A series of several specimens should be examined
to rule out a negative fecalysis result.
• Formed stool may contain protozoan cysts while
watery or diarrheic stool contain trophozoites.
• Prompt examination of specimen must be
observed to prevent disintegration of protozoan
trophozoites.
• Never leave stool specimen exposed to the air
without lids.
• Never freeze stool samples nor keep them in
incubators.
• Various structures may be mistaken for protozoan
cysts or worms by beginners.
• Epithelial cells and macrophages can be confused
with amoebic trophozoites, especially
macrophages that show slight amoeboid
movement and may contain red blood cells. The
nuclei which can be seen in BMB (Buffered
Methylene Blue) stained mounts, appear much
 larger than nuclei of amoeba and usually contain
several granules or particles of chromatin.
• Pus cells can be confused with amoebic cysts. The
nuclei appear as 3 or 4 rings and usually stain
heavily. The cytoplasm is ragged and the cell
membrane is often not seen. Amoebic cysts have a
distinct cell wall.
• Hair and fibers maybe confused with larvae.
However, hairs and fibers do not have the same
internal structure as larvae.
• Plant cells (e.g. molds or yeast) can be confused
with cyst or eggs. Plant cells usually have a thick
wall; cyst has a thin wall. Yeast and molds are
usually smaller than amoebic cysts and do not
have nuclei such as seen in amoebic cysts.
• Eggs of arthropods and plant nematodes may be
mistaken as parasites.
Amount and Timing of Collection
Formed: approximately 5 -7 grams of feces or about the size
of a marble or thumb size
Watery or diarrheic stool – 10ml or 10cc is required
A series of 3 specimens should be submitted
If amoebiasis suspected, a total of 6 specimens may be
ordered
Collection should be completed within no more than 14
days
Preservation of Stool Specimen
Refrigeration
- 4-8 C preserves protozoan trophozoites for several
days
- trophozoite can regain motility when we use warm
saline
- Stools should not be incubated
Formalin
- 5% preserve protozoans while 10% preserves
helminths and trematodes
- Advantages: simple to prepare,stable and does not
require last minute mixing
- Disadvantage: not suitable for permanent smears
Schaudinn’s Solution
- Fixation of fresh fecal material
- Used in preparing permanent-stained smears to
demonstrate protozoa
Polyvinyl Alcohol (PVA)
- for preservation of morphologic features of
intestinal protozoa, in particular trophozoite
stages, as well as helminth eggs and larvae
- Disadvantage: contains mercury which is
poisonous when indigested hard to dispose
Merthiolate-Iodine-Formaldehyde (MIF)
- a combination of preservative and stain for fecal
specimens
Composition: MF stock solution (9.4ml)
- tincture of merthiolate (eosin) 1:1000
- formaldehyde, glycerol,distilled water
- Disadvantage: needs last minute mixing
Sodium-Acetate Formaldehyde (SAF)
- Advantages: can be used as direct mount, in
permanent smears and concentrations procedures
does not contain mercury
- Disadvantages: (compared with PVA) quality of
staining in permanent stains is not as good as
Schaudinn’s or PVA fixatives
Dispatching/Mailing of Stool Specimens
10% Formalin (for wet mount) – prepare a mixture
containing 1 part of stool to 3 parts of formalin solution.
Place in a vial; crush the stool thoroughly. It preserves
specimen indefinitely if the bottle is tightly closed.
MIF (for wet mount) – just before dispatching, mix in a
tube or vial 4.7 ml of MIF solution and 0.3 ml of Lugol’s
iodine solution. Add a portion of stool, approximately 2 ml.
Crush with a glass rod or stick. It preserves specimen
indefinitely.
PVA (for permanent staining) – in a bottle or vial, pour
about 30 ml of PVA fixative or 3 quarters full; add enough
fresh stool to fill the container. Break the stool with a stick
and cover. It preserves all forms of parasites indefinitely.
Gross Exam of Stool
(Macroscopic Examination)
Look for
Color
Consistency
a. Hard – cannot be punctured by applicator stick
b. Formed – maintains shape can punctured by
applicator stick
c. Semi-formed – bottom side flattens in the
container
d. Soft – can be cut by applicator stick
e. Mushy – can be reshaped by applicator stick
f. Loose – stool shapes to the container
g. Watery – stool pours out of container
Gross or Macroscopic Examination of unpreserved stool
specimen is used to:
1. Determine the color
2. Determine the consistency
3. Screen for the presence of: blood, mucus, visible
parasites, foreign bodies
4. Help in the presumptive diagnosis of several
intestinal disorders
Careful observation of the color of the stool is AN
IMPORTANT PART OF THE PRELIMINARY DIAGNOSIS
BROWN: the normal color of stool
BLOOD: if present is considered to be an abnormal finding
and comes in different forms like:
1. 1.Bloody stool with bright red color indicates
bleeding at the lower GIT
2. Dark stool indicates bleeding the upper GIT
3. Loose or watery stool with bloody mucus
indicates amebic ulcerations
NOTE: OCCULT BLOOD in stool must be reported, there
might be any other causes aside of parasitic infection which
should not be ignored
The following are different consistency of stool that will
indicate as to what organisms are possibly present:
1. Soft to formed – normal
2. Loose, diarrheic, and liquid – contain protozoan
trophozoites
3. Formed – contain protozoan cysts
4. Any type of fecal specimen – may contain
Helminth eggs and larvae (which are difficult to
find in watery specimens, if present only a few)
Gross examination of the stool specimen may also
provide:
1. The opportunity to recover tapeworm proglottids
D’Antoni’s Method
- Detect protozoan cyst
(can be found on or within the specimen)
- Morphological appearance in increased with the
2. Recover adult worms like Ascaris and Enterobius
addition of iodine solution which act as stain
(can be found from the surface of the specimen or
in the container)
Materials
Materials Needed
1. Microscope
1. Fresh human stool sample
2. Stool sample
2. Applicator stick
3. Clean glass slides
4. Applicator stick
Procedure 5. Cover slip
1. Poke all areas of specimen and take note of color 6. Mark pen
and consistency 7. D’Antoni’s reagent
2. Record results a. Potassium iodide 1.0g
a. Name of px b. Iodine crystals 1.5g
b. Date of exam c. Distilled water 100ml
c. Age
d. Gender Procedure
e. Micro exam
1. On clean glass slide, place D’Antoni’s reagent
- Color
2. Using applicator stick, pick 2mg feces (enough to
- Consistency form low cone at the end of applicator stick)
- Result 3. Mix with reagent
- Name of examiner 4. Feces should be thoroughly comminuted in a drop
Wet Mount so that that a uniform suspension will spread
evenly to all edges of the cover slip
5. Cover with cover slip and exclude bubbles
- detection of motile protozoan trophozoites 6. Examine entire preparation systematically and
and liquid specimens are received thoroughly under the microscope under LPO
- determine cellular morphology 7. Confirmation of parasite by switching to HPO
a. For helminths a positive result is determined
as number of eggs with the name of the
Materials helminth per cover slip
1. stool sample microscope b. No ova seen for negative result
2. clean class sides c. For protozoa is named positive for the name of
3. applicator stick protozoa and the stage
4. cover slips d. Non-seen for negative result
5. marking pens
6. NSS (normal saline slxn) 0.85% NaCl slxn Note: a properly prepared DFS (direct fecal smear) will
a. 0.85% NaCl powder allow ordinary newspaper print to be barely legible through
b. 100ml distilled H2O the preparation
Thick smear will make observation difficult
Procedure Thin smear make observation unsatisfactory
1. On a clean slide, place a drop of saline solution
2. Using app stick pick about 2 mg of feces enough to
form a low cone at the end of app stick and mix Wet Mount
with NSS A simple and efficient procedure
3. Feces should be evenly comminuted in a drop so - Should be examined immediately after
that a uniform suspension will be evenly spread preparation to avoid evaporation of liquid
evenly to all edges of the cover slip - Preservation for several hours may need mixture
4. Cover with cover slip and exclude bubbles of equal parts of the following:
5. Examine systematically and thoroughly under the 1. petroleum jelly
microscope using LPO 2. paraffin
6. Confirmation of parasite by switching to HPO 3. nail polish (colorless)
a. For helminths confirmation is made by the Recommended for motile trophozoites detection,
number of eggs with name of helminths per if specimen is watery and diarrheic
cover slip
b. No ova seen for negative Principle
c. For protozoa positive result is reported as Direct Wet Mount is performed to detect motile protozoan
positive for the name of protozoa and the stage trophozoites and to determine cellular morphology
d. And non-seen for negative result
Note: a properly prepared DFS (direct fecal smear) will Entameoba coli Balantidium coli
allow ordinary newspaper print to be barely legible through
the preparation
Thick smear will make observation difficult
Thin smear make observation unsatisfactory
 Results
Helminthes
- if positive, report number of eggs
- name of the helminthes per coverslip or in plusses
- if negative, no ova seen
Protozoa
- if positive, name the protozoa the stage
- if negative, none seen

Remember
• A properly prepared DFS will allow ordinary
newspaper print to be barely legible through
preparation.
• Too thick smear – difficult microscopic observation
• Too thin smear – unsatisfactory examination
• Addition of stain – temporarily to aid the location
and identification of protozoa

Common Temporary Stains

Dilute Iodine
MIF solution (Merthiolate Iodine Formaldehyde)
Nair’s Buffered Methylene Blue solution

Dilute Iodine Solutions

• Most commonly used
• Useful in cystic stage identification, since iodine
kills and distorts the trophozoites
• Visibility and number of nuclei is enhanced
• Morphologic features are more clearly seen
• Freshly prepared
• Amoebic cysts stained with iodine results:
1. glycogen – takes a dark red-brown color
2. cytoplasm – yellowish color
3. nuclei – better define
4. karyosome – position is easier to observe

Dilute Iodine Solutions are employed in the following

methods:
1. D’Antoni’s
2. O’Connor’s
3. Lugol’s

Other Stains
MIF solution
- easy to prepare and use
Nair’s Buffered Methylene
- requires several steps to prepare
- pH must be adjusted for proper staining results