Laboratory

5. Contact transmitted – readily infective., do not

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