Clinical Parasitology

LABORATORY: DFS, Iodine Staining, KOH Wet Mount (Part 1)

Lecturer: Mr. Fritz A. Bucao, RMT
DFS, Iodine Staining, KOH Wet Mount (Part 1)

day or every other day) so a 10-day period will ensure a

DFS = Direct Fecal Smear higher chance/likelihood in recovering the parasite,
especially the intestinal protozoa.
Specimens in Parasitology
● Stool When a patient is suspected of having intestinal
○ Most commonly submitted sample for the amoebiasis, 6 specimens may be recommended.
examination of parasites because the
largest number of parasites affecting 6 specimens are recommended to ensure recovery of
man affects the intestines. parasite especially if the infection is still starting (light
○ Very important in detecting protozoa. infection stage).
○ In stool protozoa, we can find here cysts
(stage ?)/trophozoid stage, helminth Take note: A patient that has received treatment for
eggs, larva, adult worm/proglottids in protozoan infection should be checked 3-4 weeks after
cestodes. therapy and those treated with Taenia infection should be
● Tissue aspirate checked 5-6 weeks after therapy.
○ Not a common specimen.
○ For tissue aspirate, we can use liver Purpose of checking is to check whether the treatment is
aspirate, duodenal aspirate or working properly because if protozoa can still be seen
bronchoalveolar lavage. after therapy (even after the given time period), then that
● Blood means that the therapy did not work, and the
○ For blood specimens, we can find doctor/physician should give an alternative and effective
plasmodium species, Leishmania intervention.
species, or Trypanosoma species.
● Urine All stool samples should be collected in a suitable, clean,
○ We can use urine samples in detecting wide mouthed container like a plastic container with a
Trichomonas vaginalis and tight-fitting lid, waxed cardboard box (½ print) or
Schistosoma haematobium. matchbox.
● Sputum
○ Detects specifically, Paragonimus Sample of stool container:
westermani.
● Muscle Biopsy
○ Detects Trichinella spiralis.
● Cerebrospinal Fluid
○ This is the site where the
Acanthamoeba goes (in the nervous
system). Acanthamoeba is contracted
when swimming in contaminated pools
when you are immunocompromised.
● Orifice Swab
○ Orifice - openings in the body e.g. vagina
The second container in the left box has a spoon used to
(where we can detect Trichomonas
collect the sample.
vaginalis), and perianal areas where we
could get Enterobius vermicularis and
Tight fitting lid = to avoid spillage and to contain the
Taenia species.
moisture within the container.

Ova and Parasites (O&P)

Wide mouth = facilitate in the collection (makes the
- Most common procedure performed in the
collection easier).
examination of stool specimen, also called as the
Routine Stool Examination. In some books, they
Make sure labels are present in the sample. Labels
call it Routine Ova and Parasites (O&P).
should be placed on the body of the container, not on
- O&P translates to making a fecal smear on the
the cover to avoid mismatching samples which could lead
sample.
to erroneous results.

Patient Preparation
DID YOU KNOW?
Certain substances and medications also impede
The typical stool collection protocol consists of 3
detection of intestina protozoa: mineral oil, bismuth,
specimens: one specimen collected every other day or
antibiotics antimalarial agents and non-absorbable
a total of 3 collected in 10 days.
antidirrheal preparations. After administration of any of
there compounds, parasitic organisms may not be
Why 3 specimens and why in 10 days?
recovered for a week or several weeks. This is actually
So that we can recover all the parasites that can
stipulated in the history of the patient; the doctor himself
be found in the stool sample or all the parasites that are
should note what medications or substances a patient is
affecting the patient. A period of 10 days in needed
taking.
because protozoa species (the intestinal protozoa) are
not released/shed in the stool samples daily (it can be per

YCONG, Yasmin Belle – BSMT 2E

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Clinical Parasitology
LABORATORY: DFS, Iodine Staining, KOH Wet Mount (Part 1)
Lecturer: Mr. Fritz A. Bucao, RMT
DFS, Iodine Staining, KOH Wet Mount (Part 1)

Specimen collection should be delayed after parasites will not be preserved well, or chances are that
barium (for 5-10 days) or antibiotics (for at least 2 weeks) their morphology will be distorted.
are administered.
Specimen should be mixed well with the preservatives to
Routine stool examination requires a thumb-sized or achieve thorough fixation and the specimen must be fixed
pea-sized formed stool or about 5-6 tablespoons (10 mL) in the preservative for at least 30 minutes before
of watery stool. It is not necessary to measure the stool processing again.
sample, just do estimation.
In the US (and in other countries), O&P examination is
*Instruct your patients properly regarding the proper stool composed of 3 stages: (1) DFS, (2) concentration
collection. techniques, (3) permanent stained smears.

Liquid specimens are examined within 30 minutes of In other countries, their preferred sample is the
passage; semi-formed specimens within 1 hour. This is preserved stool, not fresh stool because according to
because in liquid specimens, we are looking for them, fresh stool may be submitted at a later time,
trophozoites and these protozoan trophozoites are very meaning there’s a delay in the submission. If there’s a
sensitive. Once they are outside the body, they delay, there is a possibility that the parasites have already
disintegrate rapidly so liquid specimens or semi-formed deteriorated, deeming the procedure useless.
specimens need to be processed immediately/ASAP.
However, the Philippines accepts any samples, even
Trophozoites (the _____ form of protozoa) like liquid/fluid fresh ones.
environments so they like to swim. Once the stool sample is mixed with preservatives, DFS
cannot be performed anymore (will be explained later on).
1. Formed stool specimens do not likely contain
trophozoites hence they can be held for 24 hours
following the collection.
2. Never freeze stool samples nor keep them in
incubators. Do not freeze to avoid killing the Stool Preservatives:
trophozoites and do not incubate to avoid
proliferating (multiply) the bacteria in the stool
sample. Incubating the sample will cause the
bacteria to increase in number, therefore making
it difficult to look for a protozoa or parasite.

Every specimen should be identified with the following

information:
● Patient’s name
● Age/sex
● ID number First picture:
● Physician’s name Ecofix (PVA) - eco-friendly.
● Date & time specimen was collected Second picture:
● Requested procedure Total Fix - universal fixative but still
● Presumptive diagnosis Eco-friendly.
● Prior infections Third picture:
● Travel history 2-vial system - most common
collection: one vial is 5%-10%
*This information is book based but now, each laboratory formalin or buffered formalin (pink one; used
has their own standard operating procedures (SOP) for concentration and fecal immunoassays) and
which will include protocols on what information to the other vial is fixative containing PVA (blue
include. Always refer to the SOPs of the one; used for permanent stained smears).
hospital/laboratory you are working in.

Stool Preservatives *remember this in exams: preservative:stool ratio is 3:1

and stool:preservative ratio is 1:3 (don’t get tricked!)
It is inevitable that some samples will experience delays,
so in such situations, the samples should be subjected DIFFERENT STOOL PRESERVATIVES
first to preservatives or fixatives.
Formalin
When selecting a preservative, we should consider the ● All-purpose fixative, buffered with sodium
possibility of preparing stained slides so whatever fixative phosphate to preserve morphological
is used, the recommended fixative ratio is 3:1 (3 characteristics.
fixative: 1 specimen). ● 5% concentration: recommended for protozoan
cysts.
If fixative is lesser than specimen, it could lead to ● 10% concentration: recommended for helminth
improper or inadequate fixation which means that the eggs and larvae.

YCONG, Yasmin Belle – BSMT 2E

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Clinical Parasitology
LABORATORY: DFS, Iodine Staining, KOH Wet Mount (Part 1)
Lecturer: Mr. Fritz A. Bucao, RMT
DFS, Iodine Staining, KOH Wet Mount (Part 1)

Preserved stool can be concentrated using FECT 2 stages of Stool Examination:

(Formalin Ether Concentration Technique) also known 1. Macroscopic or Gross Examination
as FEACT (Formalin Ethyl Acetate Concentration 2. Microscopic Examination
Technique).
Macroscopic or Gross Examination
Schaudinn’s Solution ● Importance: gives us the primary assessment on
● Used to preserve fresh stool/fresh fecal specimen the condition of the patient.
in preparation for staining the stool smears. ● Includes finding parasites, undigested food,
● Provides excellent preservation of protozoan mucus present upon examination.
trophozoites and cysts. ● This includes color, consistency, gross blood,
● Contains mercuric chloride which is highly toxic to mucus, presence of undigested foods, and
humans. proglottids.
● For many years, Schaudinn’s solution has been
considered the “gold standard”. Consistency of the stool can be reported as the following:
● Formed
Polyvinyl Alcohol (PVA) ● Soft
● PVA is a plastic resin that serves to adhere to a ● Loose
stool sample onto a slide. ● Watery
● Disadvantage: the use of mercury chloride, hence
the modification below. Classification of the different stool consistency: Bristol
● Main advantage: preservation of protozoan cyst Stool Chart
and trophozoites for permanent staining.
● Normally incorporated into the Schaudinn’s
solution.
● Modified PVA: using non-mercuric compounds
such as copper sulfate (used with trichrome
stain).

Merthiolate Iodine-Formalin (MIF)

● Components both fix and provide stain color.
● Contains merthiolate (thimerosal) and iodine
that act as staining components.
● Formalin acts as a preservative.
● Useful for fixation of intestinal protozoa, helmith
eggs and larvae.
● Disadvantages: contains mercury compounds MEMORIZE THIS:
(thimerosal).

Sodium Acetate Acetic Acid Formalin (ASF)

● Advantage: does not contain mercuric chloride;
long shelf-life.
● Disadvantage: images are not as sharp after
staining as compared with those fixed with PVA
or Schaudinn’s solution.

Single-Vial Collection Systems

● Both the concentration and permanent stained
smears can be prepared. Liquid consistency of stools has a greater frequency of
● It is also possible to perform fecal immunoassay recovering protozoan trophozoites.
procedures from some of these vials.
If the consistency is formed, there’s a greater frequency
Examination Of Stool Samples (Routine Fecalysis) of detecting cysts.

Importance: Soft (semi-formed) consistency can recover both cysts

● To detect the presence of intestinal parasite. and trophozoites.
● For the detection of GIT (gastrointestinal tract)
bleeding. Any consistency is for Helminth eggs and larvae.
● Used as a clue in medical and surgical diagnosis.
● For the detection of excessive fats in stool As seen in the diagram, cysts can be recovered the
(steatorrhea). greatest in formed samples and least in any consistency
● For the detection of evidence of malfunction of vehicle trophozoites can be seen the greatest in watery
some parts of the GIT, liver, and pancreas. samples and becomes lesser as it goes up.

YCONG, Yasmin Belle – BSMT 2E

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Clinical Parasitology
LABORATORY: DFS, Iodine Staining, KOH Wet Mount (Part 1)
Lecturer: Mr. Fritz A. Bucao, RMT
DFS, Iodine Staining, KOH Wet Mount (Part 1)

Stool Color
● Indicative for the presence of parasite. Charcot-Leyden Crystals
● Presence of blood should always be reported. ● Disintegration of eosinophils, indicate presence
● Dark-colored stool: bleeding in the UGIT (upper of hypersensitivity, parasitic infection.
gastrointestinal tract).
● Blood and mucus in soft/watery: presence of Epithelial Cells
trophozoite. ● Usually normal (normally seen in stool samples)
● Blood and mucus in soft or watery stools may
possibly yield the presence of trophozoites. Yeast
● By gross examination, tapeworm proglottids and ● Fungal infections
adult nematodes may be found on the stool
sample (this can provide presumptive diagnosis). Plant elements
● Temporary storage of fecal samples in a ● Serve as confusers; there’s no clinical
refrigerator (3-5 degrees Celsius) may be significance for plant elements.
acceptable if there is a delay.
● Prolonged refrigeration can bring about Microscopic appearance of the elements:
desiccation (dryness). Trophozoites are killed by
refrigeration.

*If lab is busy, process the liquid samples first because of

the trophozoites especially if there is a presence of blood
and mucus.

Study and memorize this table:

Microscopic Examination

After macroscopic examination, proceed now to

microscopic examination however, a fecal smear or
direct fecal smear (DFS) must be made first before
proceeding.

Microscopic examination reveals elements in the

gastrointestinal tract (GIT) such as:

WBC/PMNs
● Presence of this indicates inflammation,
eosinophils (increase during parasitic infections
and allergies), immune response to parasitic
infection.
● Neutrophils increase during bacterial infection.

RBC
● Presence of RBCs is indicative of bleeding or
uleratives (caused by parasite cells).

Macrophages
● Indicative of bacterial and parasitic infections.

YCONG, Yasmin Belle – BSMT 2E

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