PHASES OF LABORATORY IDENTIFICATION OF • watery stool: 5 to 6 tablespoons

PARASITES: • Contamination with toilet water, urine, or soil must be

Pre-analytic phase: specimens received in the laboratory that prevented.
are compromised because of improper collection, labeling, or • Age of the stool sample
transport should be rejected and a new specimen should be • Delay in examination of specimens may require preservation
requested. to ensure that parasites are present in the identifiable stage.
Analytic phase: laboratory techniques performed in the testing • Temporary storage of fecal samples
of the samples should be completed with care to ensure that
accurate results are obtained. SPECIMEN TYPE, STABILITY AND PRESERVATION
Post-analytic phase: Interpretation and reporting of results 1. Fresh specimen is mandatory for the recovery of motile
obtained should be accurately reported in a timely manner trophozoites.
2. Liquid stool should be examined or preserved within 30
SPECIMENS AVAILABLE FOR PARASITIC minutes of passage. Trophozoite form are found in this stool.
EXAMINATIONS: 3. Soft stool should be examined or preserved within 1 hour
• stool of passage
• urine 4. Formed stool should be examined or preserved within 24
• blood hours of passage. Cyst form are found in this stool.
• sputum
• cerebrospinal fluid STOOL PRESERVATIVES:
• tissue aspirate 1. Formalin
• tissue biopsies • all purpose fixative
• 5% formalin for protozoan cyst
• orifice swabs
• 10% formalin for helminth eggs and larvae
• solution may be buffered with sodium phosphate to preserve
FACTORS TO CONSIDER IN SPECIMEN COLLECTION:
the morphological characteristics of the organisms
- Life cycle (parasite species and stage of development)
- Frequency and timing of collection

FACTORS THAT SHOULD PRECIPITATE TESTING:

1. Travel history
2. Immune status of patients
3. Clinical symptoms
4. Documented previous infection
5. Contact with infected individuals
6. Screening outbreak situation
7. Occupational screening for food handlers
8. Therapeutic failure (retested after therapy)

SPECIMEN FOR FECAL SAMPLE

The most common method of diagnosis of intestinal parasites is
through the demonstration of eggs, larvae, adults, trophozoites,
cysts, or oocysts in the stool.
CONTAINER: clean, wide-mouthed containers made of waxed
cardboard or plastic with tight fitting lid (screw-capped tops)
LABEL: patient name
age
sex
date/time of collection
requesting physician
requested procedure
presumptive diagnosis
prior infections
travel history
Advantages:
FACTORS TO CONSIDER FOR STOOL MATERIALS
• it is easy to prepare
TO BE USEFUL IN PARASITIC DIAGNOSIS:
• it preserves specimens for up to several years
• Intake of drugs/medicinal substances (antacids, anti
• it has a long shelf life
diarrheals, barium, bismuth, mineral oil and laxatives)
Disadvantages:
• Intake of antibiotics and antimalarial drugs
• it does not preserve parasite morphology adequately for
• Amount of stool to be collected is dictated by the techniques
permanent smears
that will be used.
• trophozoites usually cannot be recovered and morphologic
• routine stool examination: thumb-sized specimen of
details of cysts and eggs may fade with time
formed stool
• considered a potential health hazard or soft specimen and 5-6 spoonful of watery specimen. In
formed stool, try to collect a small amount of stool from each
2. Schaudinn’s solution – most often combined with PVA, end and the middle part. Do not overfill the cup.
preserve fresh stool for staining purposes, contains HgCl2 • For specimen with preservatives, use spoon to build into the
(highly toxic) lid or sampling to raise the sample into “Fill to Here” line. Do
not overfill the vial. Mash the formed stool against the side of
3. Polyvinyl Alcohol (PVA) – plastic resin, which serves to the vial with a spoon. Tighten the lid and shake firmly until the
adhere a stool sample onto a slide. specimen is well mixed.
Advantage:
• Main advantage of using PVA is related to the preservation of
protozoan cysts and trophozoites for permanent staining. • The stool must not contain any contaminant of soil, urine or
• PVA-preserved specimens have a long shelf life when stored toilet bowl water. Sample areas of stool which appears bloody,
at room temperature. slimy or watery should have represented the sample.
Disadvantage: • The specimen should be transported to the laboratory within
• contains mercuric chloride which can cause potential health half an hour after the passage for unpreserved specimen.
problems Stability of sample with preservatives depends on the type of
preservative is present to them.
4. Merthiolate-Iodine Formalin – useful for fixation of • Each specimen should be labeled with:
intestinal protozoans, helminth eggs and larvae. • Patient name
• Age
5. Sodium Acetate-Acetic Acid Formalin (SAF) – viable • Sex
alternative to the use of PVA and Schaudinn fixative, can be • Date/time of collection
used for performing concentration techniques and permanent
stained smears
Advantages: Laboratory Safety in Parasitology
• mercury-free
• easy to prepare
• long shelf life
• can be used for preparing smears for staining with the
modified acid-fast stain to detect coccidian oocysts.
Disadvantage:
• not as sharped as PVA and Schaudinn’s fluid
• adhesive properties of SAF are not good, the addition of
albumin to the microscope slide may be necessary to ensure
adhesion of the specimen to the slide

6. Modified Polyvinyl Alcohol – alternatives to mercury-based

PVA are the use of substitute compounds containing copper
sulfate or zinc sulfate
Advantage:
• can be used for concentration methods and permanent stained
smears
Disadvantage:
• do not provide the same quality of preservation for adequate
protozoan morphology on a permanent stained slide as the
mercury-based fixatives
7. Alternative Single-Vial Systems – non-toxic fixatives that
are free of formalin and mercury and can be used for
concentration techniques and permanent stained smears. Some
of these products can also be used for performing fecal
immunoassays.

STOOL COLLECTION PROCEDURE

• Give the patient a clean, dry glass jar or plastic cup with
widemouthed and tight-fitting lid specimen container.
• Instruct the patient to directly pass the stool specimen on the
specimen container or pass the stool in a clean pan and transfer
the stool to the specimen container with a spatula.
• For specimen with no preservatives, tighten the lid to prevent
leakage and refrigerate the sample if will not be transported
immediately. The stool should be about thumb size of forming
 10. Specimen autoclaving before discarding is suggested.
11. Applicator sticks, tongue depressors and pipettes may be
discarded in the same container as slides.
12. Spillage must be soaked with disinfectant. Residue should
be cleaned and discarded as contaminated trash.
13. Chemicals must be considered poisonous, flammable and
corrosive. Distinct labels must be affixed.
14. Mercuric compounds cannot be disposed as regular trash.
A closed steel container is suitable.
15. Sandbox is required for strong acids and base. Avoid
storing strong acids and bases together.

16. Flammables are considered to be explosives. Laboratory

should have adequate ventilation and no open flames.
17. Poisons can intoxicate to the extent of death when
ingested or inhaled. Pipetting by mouth should be avoided.
18. Prolonged contact with xylene and formaldehyde can be
carcinogenic.

Routine Stool Examination

PHASES OF ROUTINE STOOL EXAMINATION
A. Physical Analysis
B. Chemical Analysis
C. Microscopic Analysis
• Routine stool examination is useful in determining patients
clinical status based on the stool sample collected.
• Those three results also correlate with one another in
identifying parasitic infection in an individual

ROUTINE STOOL EXAMINATION

A. Physical Examination
• Stool specimens submitted for parasitic study should first be
examined macroscopically to determine the consistency and
color of the samples and should be screened and examined for
the presence of gross abnormalities.
Consistency
• degree of moisture in a stool specimen may serve as an
indication of the types of potential parasites present
• If the stools are fresh, the laboratory can classify the
1. Handle all fresh specimens carefully. Use the universal consistency as formed, semi-formed, soft, loose, or watery.
precaution. Fecal specimen may contain parasites and many
potential pathogenic bacteria and contagious viral
agents.
2. Handling specimens and disposal of waste material are of the
utmost importance to maintain good health for the laboratory
personnel and the community.
3. No food and drink allowed in the laboratory. They can be
contaminated.
4. Smoking is not allowed in the laboratory.
5. Frequent hand washing can help to prevent the spread of
parasites.
6. Laboratory gown and uniform must be washed at the
working site. Special attention should be given to avoid
contamination of other items.
7. Obtain immunizations to prevent pathogenic organisms and
treat such. Color
• Color of a stool is important because it may indicate the
8. Dispose contaminated materials on properly labelled waste condition of the patient.
containers with instructions for the waste collection. • The normal color of the stool is brown
9. Container for used slides must be filled with one third to
one half disinfectant. The container should be autoclaved and
emptied.
 • Vitamin C >250 mg/d
• Iron supplements containing vitamin C
• Failure to wait specified time after sample is applied to add the
developer reagent

C. Microscopic Examination
• To detect the presence of parasites in a stool specimen,
microscopic examinations are performed.
• It can also reveal many elements present in the intestinal tract
aside from parasites and normal fecal constituents (artifacts).
• It should be performed on a fresh specimen.
• Involves three distinct procedures:
• direct wet preparations
• concentrated technique
• permanently stained smear
Reporting:
• NOPS- No ova and/or parasite seen
• NIPS- No intestinal parasite seen

Gross abnormalities
• Adult worms, proglottids and parasitic indications (pus and
mucus)
ü The surface of the stool should be examined for parasites,
such as pinworms, tapeworm proglottids, and adult worms.
ü The sample should then be broken up using a wooden
applicator stick and examined once more for macroscopic
parasites, especially adult helminths.
• Samples containing adult worms may be carefully washed
through a wire screen. This process allows for the retrieval and
examination of the parasites for identification purposes.

B. Chemical Examination
• By far the most frequently performed fecal analysis is the
detection of occult blood (hidden blood).
• Principle: detection of the pseudoperoxidase activity of
hemoglobin.

Hema-screen Guiac Slide Test Kit

1. Hema-Screen Slide
2. Hema-Screen developer
(<6% H2O2)
Results: ARTIFACTS, PSEUDOPARASITES, AND CONFUSERS
(+) any trace of blue coloration 1. White blood cells
(-) no detectable blue coloration • Polymorphonuclears (PMNs), which may indicate
inflammation.
Interference of FOBT • Eosinophils, which may indicate an immune response to a
False-Positive parasitic infection.
• Aspirin and anti-inflammatory medications 2. Red blood cells
• Red meat • may indicate ulcerations or bleeding
• Horseradish 3. Macrophages
• Raw broccoli, cauliflower, radishes, turnips • usually present in both bacterial and parasitic infections
• Melons • one can mistake the active macrophages for amebic
• Menstrual and hemorrhoid contamination trophozoites
False-Negative
 • pollen grains
• starch granules
• vegetable spirals

4. Charcot-Leyden crystals
• released with the disintegration of eosinophils
• may indicate presence of hypersensitivity or parasitic
infections, especially amebiasis 9. Plant and animal hairs
5. Epithelial cells • may look like helminth larvae.
• from the intestinal tract 10. Muscle fibers
6. Eggs of arthropods, plant nematodes, and other spurious • undigested striated muscle fibers are associated with
parasites pancreatic insufficiency
• may be mistaken for human parasites 11. Fat globules
• found in greasy stools

7. Fungal spores DIRECT FECAL SMEAR

• Candida spp., yeast, and yeast-like fungi • About 2 mg of stool (amount forming a low cone at the tip of
8. Elements of plant origin: an applicator stick) is comminuted thoroughly with a drop
• plant cells/fibers
of 0.85% sodium chloride solution (NSS) and then covered • Very good in detecting eggs with thick shells (Ascaris and
with cover slip. Trichuris) but not with thin shells (hookworm eggs, become
• Primary useful in the detection of motile protozoan distorted and transparent).
trophozoites (pale and transparent). • Usefulness is limited if stools are diarrheic and watery, not
• Weak iodine solution (Lugol’s and D’ Antoni) are temporary able to detect protozoan cysts and trophozoites.
stain to demonstrate nuclei. Helminth eggs and larvae can be
detected using this preparation.
• Cytoplasm: Golden yellow
• Nucleus: Pale and refractile
• Glycogen: Deep brown

Kato-Thick Smear

ROUTINE STOOL EXAMINATION PROCEDURES

I. Direct Fecal Smear
Direct Fecal Smear 1. Label slide and place one drop of NSS and one drop of
Lugol’s iodine.
2. Place a match head size portion of the stool sample and
make a smooth uniform emulsion by rotary motion of the
applicator stick.
3. Place the coverslip.
4. Examine under the microscope.
II. Kato-thick
1. Place 50-60 mg stool (size of 2 munggo bean) at the
center of the slide and place a piece of cellophane soaked
in pretreated glycerin-malachite green solution.
2. Using a test tube press cellophane gently in order to
spread the stool sample.
3. Leave at room temperature (10-20 minutes) or 1 hour.

KATO THICK SMEAR

• About 50-60 mg of stool (approximately the size of two
munggo bean) is placed over a glass slide and covered with
cut cellophane paper soaked in a mixture of glycerine and
malachite green solution.
• Glycerine: clearing solution
• Malachite green: to give color to the cellophane in order to
give a pale green background to the eggs and to minimize the
brightness of the microscope
• Preparation is best examined within 10 to 20 minutes.
• Simple, economical and useful for mass stool examination