Laboratory 1. Parasitic protozoa
For this lab, we have chosen to focus on a few parasites representing four protozoan orders that are
areas of concern to humans around the world.
Organisms to study: Giardia lamblia
Trichomonas vaginalis
Entamoeba histolytica
Entamoeba coli
Balantidium coli
The organisms you will study in this lab are unicellular protozoan parasites. Protozoans represent
some of the oldest known eukaryotic groups that are highly adapted for each of their habitats.
Unicellular protozoans are single celled organisms containing a nucleus (or at some stages nuclei),
cytoplasm, plasma membrane, and highly specialized organelles. Protozoans are strictly aquatic
organisms. They must have an aqueous environment for feeding, reproduction, and locomotion.
Many protozoans form cysts that provide protection for stages of reproduction that do not occur
entirely in a fluid. Members of this group are highly successful, having invaded every ecological
niche imaginable. Nearly every species of metazoan has a complement of protozoans living in it.
Many human and animal diseases are caused by unicellular organisms, making any knowledge of
parasitic protozoans important to both doctors and parasitologists! This first lab will be one of your
most challenging simply because these organisms are small and because the preparations are fecal
smears and tissue smears. To make your job easier, we have included pictures within the lab
manual to help you form a search image. The best way for you to get through this material is to
become familiar with these pictures…..and to be patient. Have fun!
1. Order Diplomonadida
The order Diplomonadida contains parasites that are bilaterally
symmetrical. Giardia lamblia is considered one of the deepest
branching or most primitive eukaryotes in existence. Some
scientists have called Giardia the “missing link” in the evolution of
eukaryotic cells from prokaryotic cells. Giardia lamblia was the
first eukaryotic cell to be seen using one of the first good quality
microscopes developed by Antoine Van Leeuwenhoek back in the
1600’s.
Giardia lamblia is an intestinal parasite of humans and a variety of other mammalian hosts.
Giardia is highly contagious and is the cause of a number symptoms that can include severe
diarrhoea. A human or animal host becomes infected when it ingests food or water that is
contaminated with the feces from another infected host. Transmission depends on the swallowing
of mature cysts. Once swallowed, the cysts pass though the stomach, excyst in the duodenum and
develop into trophozoites. Trophozoites develop into cysts as they pass through the intestine and
into the colon. The feeding stage, or trophozoite, is 12-15 µm long, rounded at the anterior end and
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pointed at the posterior end. The ventral surface bears
a concave, bi-lobed adhesive disc. The adhesive disc
is a rigid structure that allows this organism to attach
to the host intestinal cells. Trophozoites feed on the
intestinal mucosal surface. In severe infections the
free surface of nearly every cell is covered by a
parasite and one person can pass millions of cysts each
day.
Giardiosis is diagnosed by finding cysts or
trophozoites in the feces. Both life cycle stages have a
characteristic appearance making it the easiest human
intestinal protozoan to diagnose.
Trophozoite
Slide: Giardia lamblia trophozoite: this parasite
is generally tear-drop shaped with two visible nuclei.
If you use the fine focus knob to focus in and out on
the parasite you will be able to see the bi-lobed adhesive
disc.
Note: Tear-drop shape may or may not be visible
depending on your plane of vision.
Cyst
Slide: Giardia lamblia cyst: Giardia cysts are slightly
smaller than trophozoites and have 4 visibly distinct nuclei.
A median rod, known as an axostyles, is often visible down
the centre of the organism.
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2. Order Trichomonadida
Members of the Order Trichomonadida are nearly all parasitic. They
are small flagellates that exist only as trophozoites. Members of this
family are easy to recognize because they have an anterior tuft of
flagella, a median rod (axostyle), and an undulating membrane that
extends the length of the body. The characteristic number of flagella
is
five,
though there is some deviation from
this number. In addition,
Trichomonads have a single nucleus
and a parabasal body (golgi
apparatus). Trichomonads lack
mitochondria but do have granules
that serve roughly the same purpose
(these granules contain many of the
Kreb’s cycle enzymes).
Trichomonads are found widely as
parasites of both vertebrates and
invertebrates. Most are found in the
intestinal tract but can be found in
the reproductive system, urinary
tract, and the respiratory tract.
Transmission is direct.
Trichomonads have no cyst stage.
Reproduction is asexual (binary
fission)
Trichomonads in humans
Slide: Trichomonas vaginalis: found in the
genital and urinary tract of humans. T. vaginalis is
transmitted from one person to the next through
sexual intercourse. Although both men and women
are equally infected with T. vaginalis, it causes
disease almost exclusively in women. Diagnosis
involves finding living organisms in material taken
from an infected person. Swabs are most
commonly examined under a microscope.
Pentatrichamonis hominis: There is no slide for P. hominis but know that it occurs in the large
intestine of humans and is non-pathogenic.
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Trichomonads in other animals
Tritrichomonas foetus: causes trichomonad abortion, or bovine trichomonas. The principle host
of T. foetus is the domestic ox however closely related organisms are also known to infect the horse
and the pig. T. foetus infects the reproductive tract of both cows and bulls.
Trichomonas gallinae: causes avian trichomonas. T. gallinae infects the digestive tract, liver,
and lungs. Infections to the liver and lungs will often result in death.
3. Order Amoebida:
Family Entamoebidae
The term “amoeba” refers to an organism
that moves and feeds with the aid of
pseudopodia.
The Entamoebidae lack
flagella at all stages in their development.
Species in the Entamoebidae are parasites
or commensals of the digestive system.
Several species occur in humans; most are
harmless commensals but Entamoeba
histolytica is a highly pathogenic species.
The life cycles of all species in the
Entamoebidae are similar: A host ingests
food or water contaminated with
Entamoeba cysts. After excysting in the
small intestine, the cytoplasm and nuclei
divide to form trophozoites. Trophozoites
live in the intestinal tract where they feed
on bacteria. The trophozoites are then
passed with the faeces into the colon. As
fecal matter becomes dehydrated the
amoeba encysts. Transmission occurs when
a host swallows these specialized multinucleate cysts.
Species 1: Entamoeba histolytica: This species is the third most common
cause of parasitic death in the world and is estimated to be responsible for
between 50,000 and 100,000 deaths each year. It is the causative agent of
amoebic dysentery. Transmission is direct, involving ingestion of
contaminated food and water. It normally feeds on bacteria in the intestine but
will also erode the mucosa coating of the epithelium and enter the mucosa of
the gut causing a typically flask-shaped ulcer. Trophozoites may even be carried by the lymphatics
to other organs, most often the liver, where they cause further problems.
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Slide: Entamoeba histolytica
trophozoite: Trophozoites are
round with large lobular
pseudopodia and a clock-face
nucleus. The nuclear endosome is
typically (but not always)
centrally located. You may see
erythrocytesin the
cytoplasm, indicating digestion of erythrocytes.
Trophozoite
Cysts
Slide: Entamoeba histolytica cyst: Cysts are smaller
than trophozoites. They contain 4 nuclei and
chromatoid bars (tightly
packed messenger RNA with
rounded ends).
Slide: Entamoeba histolytica in colon tissue. Trophozoites
in tissue sections are often larger than those found in luminal
samples. You will find the trophozoites within ulcers in the
tissues. Search the slides for ulcerations and look for the
inflammatory response.
Species 2: Entamoeba coli: This is another species that occurs in the intestine of humans. It is a
harmless commensal of the human large intestine, but looks similar to Entamoeba histolytica. It
feeds on bacteria, other protozoa, and yeasts. E. coli is more common than E. histolytica, however
multiple infections are common therefore it is important to be able to distinguish between these two
forms.
Slide: Entamoeba coli trophozoite: distinguished
Trophozoite
Cyst
from E. histolytica by its coarser chromatin,
eccentrically located endosome and the absence of
erythrocytes in the cytoplasm.
Slide: Entamoeba coli cyst: E. coli cysts contain 8
nuclei (compared to 4 nuclei seen in E. histolytica) and
smaller chromatid bodies that are often filamentous.
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4:Phyllum Ciliophora:
Family Balantidiidae
The family Balantidiidae contains one major genus,
Balantidium, which can be found in the intestines of
crustaceans, insects, fish, amphibians, and
mammals.
The species you will examine,
Balantidium coli, is the largest protozoan parasite of
humans. B. coli is the cause of balantidiosis or
balantidial dysentery. Transmission is direct and
usually occurs when the cyst stage is accidentally
ingested via contaminated food or water. It is
generally non-pathogenic but can cause ulceration
of the intestinal epithelium followed by secondary
bacterial infections.
Slide: Balantidium coli trophozoite: trophozoites are oblong
Trophozoite
with a prominent kidney shaped
macronucleus
and
a
smaller
micronucleus. B. coli is uniformly
ciliated over its body but this is rarely
visible on these slides.
Slide: Balantidium coli cyst: cysts
are round with a rounder and
thicker macronucleus
Cyst
Slide: Balantidium coli intestinal tissue: search the tissue crosssection for ulcerations caused by the trophozoites.
Trophozoites are easily distinguishable by their large size.
The trophozoites attack intestinal epithelial tissue, causing
flask-shaped ulcers (similar to amoebic ulcers). The liver,
lungs and urogenital tract may also become infected.
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Learning Objectives
1. Know general features of protozoans
2. Order Diplomonadida
- Visual ID Giardia lamblia – cyst + troph
- Life cycles and roles of cyst + troph
- Anatomy of troph + cyst
- Transmission: from who, how, what stage, to who
- Tissue it is found
- Pathology = symptoms and disease
3. Order Trichomonadida
- What makes them special? (2 things)
- Visual ID Trichomonas vaginalis
- Life cycle, anatomy and transmission
4. Order Amoebida, Family Entamoebidae
- How do they move
- Visual ID Entamoeba histolytica and Entamoeba coli (trophs + cyst)
- Be able to visually differentiate between the two species
- How do they differ in other ways
- Life cycle, Transmission, Epidemiology, Pathology
5. Phyllum Ciliophora, Family Balantidiidae
- Visual ID Balantidium coli troph + cyst
- What makes B. coli special
- Life cycle, Pathology
- Host, Tissue, Transmission
Vocabulary
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Protozoan
Trophozoite
Cyst
Giardiosis
Flagella
Axostyle
Amoeba
Pseudopodia
Pathology
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Transmission
Epidemiology
Erythrocyte
Erythrocytosine
Cilia
Commensal
Pathogenic
Endosome
Ulcer
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