PROTOZOA
OBJECTIVES
1. To learn the common characteristics of organisms of Protozoa.
2. To identify free-living and parasitic forms of Protozoa.
MATERIALS
Samples
live cultures
Amoeba proteus
Euglena
prepared slides of
(trophozoite form
only):
Giardia lamblia
Trichomonas vaginalis
Entamoeba histolytica
Trypansoma gambiense
Balantidium coli
Plasmodium malariae
Paramecium caudatum
Other materials:
microscope, slides, coverslips, droppers
BACKGROUND
This handout begins your introduction to Domain Eukarya. All living organism are put into a taxon (group) according
to relatedness (Figure 1-1). The domain is separated into three well-defined kingdoms—Fungi, Animalia and Plantae
— along with Protists that lack the characteristics of the those three kingdoms. Despite many structural similarities
among Protists, the organisms arise out of diverse origins. Protists include a number of unicellular and multicellular
eukaryotes — Algae, Protozoa, and Fungi-like slime molds — that live in moist habitats. Some organisms in this
kingdom provide the evolutionary connection between unicellular and multicellular life forms. Currently, Protists are
divided into hierarchal 6 supergroups — Excavata, Archaeplastida, Chromalveolata, Amoebozoa, Rhizaria, and
Ophisthokonta.
Protozoans (proto=first, zoan=animals) are unicellular, animal-like heterotrophs. Protozoa is no longer a taxonomic
group, but is rather a descriptive term to include supergroups— Excavata, Chromalveolata, Amoebozoa, Rhizaria, and
Ophisthokonta. There are several distinguishing characteristics of these simple animals. For one, Protozoans lack cell
walls. Instead, many have a flexible layer, a pellicle, or extracellular membrane comprised of inorganic substances,
such as silica. Most protozoans have food vacuoles for enzymatic digestion, and contractile vacuoles to maintain water
balance by accumulating and expelling excess water. Their single cells structures use a variety of locomotive organelles
or gliding mechanisms to move through a range of ecological habitats from pond water to host cells.
The free-living trophozoite is found in fresh water and feed on small organisms (i.e., bacteria, yeast, and algae). Most
reproduce asexually by binary fission, budding or schizogony although few members can reproduce sexually. Parasitic
protozoans feed on nutrient derived from the host and may cause disease. Some parasites may form resistant cyst
1
structures for survival when outside of the host or in unfavorable environments. Diagnoses of Protozoan infections are
usually made by examining the cyst of individual species in formed stool or diarrhea. Both trophozoite and cyst forms
are released from the feces in soil, water, or vegetation.
Supergroups of Protozoa
In this exercise, you will examine living and prepared slides of Protozoans. Currently, all are separated into
supergroups. Review some of Protozoan supergroups below and in Tables 1-1 and 1-2.
1. SUPERGROUP AMOEBOZOA. Cytoplasmic streaming produces outwardly lobe-like projections called
pseudopods (“false feet”) to power its movement. Amoeba proteus, and its parasitic relative Entamoeba
histolytica are in Phylum Amoebozoa.
2. SUPERGROUP EXCAVATA. Long whip-like proteinaceous appendage or flagella propel the organism.
Autotrophic Euglena and heterotrophic Trypanosoma are Phylum Euglenozoa. Since Euglena contain
chloroplast, many scientists consider it an algae. Other parasitic members in this supergroup—include Giardia
lamblia in Phylum Diplomonads and Trichomonas vaginalis in Phylum Parabasalids. Both Trichomonas and
Giardia lack functional mitochondria.
3. SUPERGROUP CHOMALEVEOLATA. Some have numerous rhythmic waving, shorter hair-like structures
called cilia for motility. Paramecium caudatum and parasitic Balantidium coli are in Phylum Ciliates. These
ciliates contain 2 nuclei, macronucleus and micronucleus. Some nonmotile members of this supergroup include
genus Plasmodium in Phylum Apicomplexa.
Domain
Kingdom
Supergroup*
Phylum
Class
Order
Family
Genus
Species
Figure 1-1. Taxonomic hierarchy. A supergroup is a category high in the taxonomic hierarchy. It is placed between Kingdom and
Phylum to properly cluster organisms that derived from a common ancestor.
2
Table 1-1 . Classification of Protozoans used in this lab
Supergroup
Phylum
Species
Amoebozoa
(pseudopods)
Amoebozoa
Amoeba proteus
Entamoeba histolytica
Excavata
(flagellates)
Euglenozoa
Euglena
Trypansoma cruzi
Trypansoma
gambiense
Diplomonads Giardia lamblia
Chromalveolata
(ciliated except
(Apicomplexans)
Parabasalids
Trichomonas vaginalis
Ciliates
Paramecium
caudatum
Balantidium coli
Apicomplexa
Plasmodium sp.
Distinguishing
Characteristics
Found in fresh water
Human parasite
Disease
Chloroplast; an algae
found in fresh water
Undulating
membrane for
movement
None
None
Chagas disease
Triatoma bite
(kissing bug)
Tsetse fly bite
no mitochondria
No cyst stage;
nonfunctional
mitochondria;
undulating membrane
Found in fresh water
None
Amebic
dysentery
African sleeping
sickness
Enteritis;
possible
dysentry
urethritis;
vaginitis
Source of Human
infection
None
Fecal
contamination of
water
Fecal
contamination of
water
Contact with
vaginal or urethral
discharge
None
None
Only human parasitic
ciliate
Balantidium
dysentery
Intracellular
parasites; Complex
life cycle with multiple
host; apical end
containing invading
organelles
Malaria
Fecal
contamination of
water
Anopheles
mosquito bite
3
Table 1-2. Characteristics of free-living Protozoa
Supergroup
AMOEBOZOA
Image
Pseudopod
Food vacuole
Nucleus
Contractile vacuole
Structures
1. Pseudopods: cytoplasmic projections that allow
movement
2. Nucleus: only one present
3. Food vacuoles: structure for digestion of food
4. Contractile vacuole: structure that regulates
osmotic pressure
Cytoplasm
Amoeba
EXCAVATA
1. Flagella: one or more whip-like structures that
allow locomotion
2. Pellicle: clear elastic layer on outside of cell
3. Nucleus: only one present
4. Cytostome (mouth): site where food enters in to
the oral groove
5. Chloroplast: organelles that containing pigments
or photosynthesis (only in some members of
Excavata, i.e., Euglena)
6. Eye spot: structures that senses light (only in
some members of Excavata; i.e., Euglena)
Chloroplast
Nucleus
Pellicle
Eye spot
Cytostome
Flagellum
Euglena
CHOMALEVEOLATA
1. Cilia: several hair-like structures that allow
locomotion
2. Pellicle: clear elastic layer on outside of cell
3. Macronucleus: one or more large nuclei that
controls cellular activities
4. Micronucleus: small nucleus that functions in
conjugation, a form of sexual reproduction
5. Cytostome (mouth): site where food enters in to
the oral groove
6. Food vacuoles: structure for digestion of food
Cilia
Food vacuole
Micronucleus
Macronucleus
Cytostome
Contractile vacuole
Paramecium
4
Parasitic Protozoa
Many parasitic protozoa multiply in humans for their survival. Review the parasitic Protozoans below and in Table
1-3.
Parasitic Protozoa with no cyst
Trichomonas vaginalis. The pear-shape trophozoite has 1 flagellum within an undulating membrane for movement
and four anterior flagella. There is not any cyst stage. The parasitic flagellate is transmitted via direct contact
causing the Trichomonasis, a sexually transmitted disease (STD). T. vaginalis infects the genitourinary tract,
inducing inflammation. Men usually are asymptomatic, whereas women often present a green discharge and foul
odor.
Parasitic Protozoa with no intermediate host
1. Entamoeba histolytica. The quadri-nucleated, thick walled cysts are ingested, and travels through the acidic
contents of the stomach to the colon. The division of the nuclei produces 8 trophozoites that grow in the colon
causing Amoebiasis. These growing trophozoites may cause amebic dysentery (bloody diarrhea).
2. Balantidium coli. The only protozoan ciliate that infects humans. The free-living form contains a macro- and
micro- nuclei. Upon ingestion, the multi-layered wall cyst, allows its passage through the stomach to the large
intestine, where it grows and causes intestinal ulcers. Infected individual symptoms may also include and vary
between constipation and diarrhea (possible dysentery).
3. Giardia lamblia. The ingestion multinucleated cyst, which allows movement through the gastric acid. The nuclei
produce 2 trophozoite that attach to the small intestines. Symptoms include abdominal discomfort, weight loss,
or severe diarrhea (possible dysentery).
Parasitic Protozoa with intermediate host
1. Trypansoma species. These hemoflagellates have an undulating membrane. Trypansoma cruzi causes Chagas
disease transmitted by the Triatoma Kissing Bug. The other species Trypansoma brucei is carried by
invertebrate vectors, such as Tsetse fly (Glossina sp.) or Reduviid bug, and include three subspecies include —
(1) Trypansoma b. brucei (does not infect humans); (2) Trypansoma b. rhodesiense found in cases of East
African sleeping sicknesses and (3) Trypansoma b. gambiense found in cases of West Africa African sleeping
sicknesses. The latter of the subspecies is the cause of the most deadly infections.
African sleeping sickness is characterized by fever, muscle aches, abnormal neurological responses, and
disturbed sleeping pattern. The parasite has two hosts, (1) the intermediate invertebrate vector — secondary
host, and (2) a definitive vertebrate host (i.e., human) — primary host. Blood sucking Tsetse fly injects
infectious metacyclic trypomastigotes (shorter; only found in vertebrates) into the lymph or bloodstream of its
host. Inside the host, metacyclic trypomastigotes transform into life-threatening bloodstream trypomastigotes,
where the parasites reach other areas of the body and continue to divide by binary fission.
An uninfected Tsetse fly becomes infected when taking a blood meal from its host. In the fly’s digestive tract,
parasites transform into procyclic trypomastigotes, multiply by binary fission, leave the mid-gut, and transform
into epimastigotes. The epimastigotes (longer; only found in invertebrates) continue to multiply by binary
fission in the salivary glands of the fly. It takes approximately 3 weeks for the epimastigotes to reach its salivary
gland.
5
2. Plasmodium species. Four malaria parasites including P. vivax, P. ovale, P. falciparum, P. malariae (highest
mortality). The parasite has two hosts, (1) the intermediate vertebrate human — secondary host and (2) a
definitive invertebrate host, female mosquito Anopheles — primary host. Review the life cycle of Plasmodium in
Figure 1-4.
The infected ravenous pregnant, female mosquito bites human injecting parasitic sporozoites into blood. During
the pre-erythrocytic stage, the sporozoites infect the liver and after reproduction by schizogony (multiple nuclear
division followed by cell division). Merozoites released from the liver infect red blood cells. Within the
erythrocytic stage, merozoites continue to divide by schizogony forming schizonts, tropohooites, and a
“ring” (useful diagnostic stage). Eventually, and numbers of merozoites are released from ruptured red blood
cells. Some merozoites turn into male and female gametocytes into the blood.
An uninfected mosquito becomes infected with gametocytes when taking a blood meal from its host. In the gut
of the mosquito, mating or sporogamy produces an elongated zygote called ookinete. The ookinetes transform
oocyst, which eventually release sporozoites into the mosquito’s salivary gland. After development, it takes
approximately 10 to 18 days for each parasite to reach its salivary gland.
6
Table 1-3. Characteristics of parasitic Protozoa
Supergroup
Structures
AMOEBOZOA
Trophozoite: Pear shape with
1 nucleus present
Entamoeba histolytica
Infection: Amoebic dysentery
Locomotive
organelles
Pseudopods
Site of
infection
Ingestion of
cyst;
trophozoite
form in large
intestines
Isolation of Parasitic
Form
Stool
Entry into
bloodstream
by way of
Tsetse fly
Blood
Nucleus
15 – 20 µM
(in diameter)
Nuclei
Cyst: 1 – 4 nuclei present in
mature cyst
None
Trophozoite: Crescent shape
with 1 nucleus
Flagella
10 – 15 µM
(in diameter)
EXCAVATA
Trypansoma gambiense
Infection: African sleeping sickness
Flagellum
Cyst: None
Undulating membrane
Nucleus
10 – 35 µM
(in length)
7
Phylum
Structures
Locomotive
organelles
Site of
infection
Isolation of Parasitic
Form
EXCAVATA
Trophozoite: Pear shaped
with 2 nuclei
4 pairs of flagella
Ingestion of
cyst;
trophozoite
form in small
intestines
Stool
Cyst: Oval shaped with 2 – 4
nuclei present
4 pairs of flagella
inside of cyst
Trophozoite: Oval shaped
with 2 nuclei (macronucleus
and micronucleus)
Cilia
Ingestion of
cyst;
trophozoite
form in large
intestines
Stool
Giardia lamblia
Infection: dysentery
Nucleus
Flagellum
12 – 15 µM
(in length)
Nuclei
7 – 10 µM
(in length)
CHOMALEVEOLATA
Balantidium coli
Infection: dysentery
Micronucleus
Macronucleus
Cilia inside of cyst
50 – 130 µM
(in length)
Macronucleus
Micronucleus
50 – 70 µM
(in length)
8
ANOPHELES MOSQUITO
(Secondary host)
HUMAN – DEFINITIVE HOST
(Primary host)
sporozoites
Infected
mosquito
Sporozoites
moving to salivary
gland
Pre-erythrocytic
stage
merozoites
schizonts
oocyst in
gut wall
Erythrocytic stage
trophozoite
“ring"
Uninfected
mosquito
zygote
gametocytes
gametes
Figure 1-4. Life Cycle of Plasmodium
9
OBSERVE LIVING PROTOZOA
Procedure
1. Review microscope use and procedures.
2. Locate individuals on bottom of the culture.
3. Prepare a wet mount of living Euglena and Amoeba by using an eye dropper to remove a few drops form the
bottom of the culture.
4. Put 1-2 drops on a standard slide.
5. Cover the preparation with coverslip and examine It under low power (10 X).
6. Draw your organisms.
10
Name ________________________________
TITLE
Date _________________________________
PROTOZOA LAB
PURPOSE _________________________________________________________________________________
________________________________________________________________________________________________
RESULTS
Image of Amoeba proteus (live cells)
Organelle of locomotion: ____________________________
Total mag. of _______ X
Phylum: ________________________________
Total mag. of _______ X
Description _________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
11
Image of Euglena (live cells)
Organelle of locomotion: ____________________________
Total mag. of _______ X
Phylum: ________________________________
Total mag. of _______ X
Description _________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
Image of Entamoeba histolytica
Organelle of locomotion: ____________________________
Total mag. of _______ X
Phylum: ________________________________
Total mag. of _______ X
Description _________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
12
Image of Trichomonas vaginalis
Organelle of locomotion: ____________________________
Total mag. of _______ X
Phylum: ________________________________
Total mag. of _______ X
Description _________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
Image of Giardia lamblia
Organelle of locomotion: ____________________________
Total mag. of _______ X
Phylum: ________________________________
Total mag. of _______ X
Description _________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
13
Image of Trypanosoma gambiense
Organelle of locomotion: ____________________________
Total mag. of _______ X
Phylum: ________________________________
Total mag. of _______ X
Description _________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
Image of Plasmodium vivax
Organelle of locomotion: ____________________________
Total mag. of _______ X
Phylum: ________________________________
Total mag. of _______ X
Description _________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
___________________________________________________________________________________________
14
Questions
I. Multiple choice. Circle the correct answer choice
1. Which of the following statements regarding protozoa is FALSE?
A. Protozoa are common in water and soil.
B. Some protozoan pathogens are transmitted by arthropod vectors.
C. Nearly all protozoa cause disease.
D. Most protozoa reproduce asexually.
E. Protozoa are unicellular eukaryotes.
2. Giardia and Trichomonas are unusual eukaryotes because they
A. do produce cysts.
B. lack nuclei.
C. are motile.
D. lack functional mitochondria.
E. do not produce cysts
3. Which of the following is the most effective control for malaria?
A. treating patients
B. eliminate Anopheles mosquitoes
C. vaccination
D. eliminate the intermediate host
E. None of these is an effective control.
4. Which of the following organisms is photoautotrophic protozoan?
A. Balantidium coli
B. Euglena
C. Trichomonas vaginalis
D. Entamoeba histolytica
E. Giardia lamblia
5. Which phylum of protozoa contains organisms that are non-motile, obligate intracellular parasites?
A. Apicomplexa
B. Amoebozoa
C. Ciliates
D. Euglenozoa
E. None
6. Which of the following statements about protozoa is true?
A. All protozoa are photosynthetic.
B. Some protozoa reproduce by schizogony, a process that is virtually identical to the budding process that happens
in some yeast.
C. All protozoa can undergo sexual reproduction.
D. When conditions become harsh, some protozoa can form a protective capsule, which is called a cyst.
E. None
7. Trichomonas vaginalis can be distinguished from other parasitic protozoa by which of the characteristics listed below?
A. It is usually found in drinking water and is associated with fecal contamination.
B. It has an undulating membrane, infects the vagina, and is frequently transmitted by sexual contact.
C. It is a photosynthetic organism that lives in fresh water.
D. It infects Anopheles mosquitoes and can be transmitted by a bite.
E. None
15
8. The resistant stage of protozoa is called the ________.
A. merozoite
B. endospore
C. cyst
D. amoeba
E. trophozoite
9. Which of the following protozoa has pseudopods for motility?
A. Balantidium coli
B. Euglena
C. Trichomonas vaginalis
D. Entamoeba histolytica
E. Giardia lamblia
10. Which Phylum of protozoa contains flagellated microorganisms?
A. Mastigophora
B. Ciliophora
C. Sarcodina
D. Sporozoa
E. Cestode
11. In disease malaria, the ______ merozoites infect the ____ of the human host cells.
A. Plasmodium; liver
B. Anopheles; red blood cells
C. Plasmodium; red blood cells
D. Anopheles; liver
E. Tsetse; red blood cells
12. Which of the following statements regarding protozoa is FALSE?
A. Protozoa have a cell wall.
B. Some protozoa reproduce asexually.
C. Some protozoa cause disease.
D. Protozoa are unicellular eukaryotes.
E. Arthropod vectors transmit some protozoan pathogens.
II. Short Answer.
13. Indicate the functions of:
A. Food vacuole ……………………………………………………………………………………………….…………………………………………
B. Contractile vacuole ……………………………………………………………………………………………….…………………………………………
C. Cytostome……………………………………………………………………………………………….………………………………………………………………
D. Undulating membrane……………………………………………………………………………………………….…………………………………………
E. Macronucleus ……………………………………………………………………………………………….…………………………………………
F. Micronucleus ……………………………………………………………………………………………….…………………………………………
G. Apical ends ……………………………………………………………………………………………….…………………………………………
16
14. List 5 characteristics of protozoa.
A. ……………………………………………………………………………………………….
B. ……………………………………………………………………………………………….
C. ……………………………………………………………………………………………….
D. ……………………………………………………………………………………………….
E. ……………………………………………………………………………………………….
15. How are do protozoa different from bacteria? ……………………………………………………………………………………………….
……………………………………………………………………………………………….………………………………………………………………………
16. What Kingdom does protozoa belong to? ………………………….………..………………………
17. Why are Euglena not considered Protozoa? ……………………………..………………………
18. Name one protozoa that causes an STD. ……………………………………..……………………
19. What importance it the cyst stage to the protozoa? ……………………………………………………………………………
……………………………………………………………………………………………….……………………………………………………………
20. What is the genus name of the vector involved in African Sleeping Sickness? ……………………………………………………
21. Both Plasmodium and Trypanosoma are found in the blood stream. Where are these Protozoans located in respect to the
red blood cells?
……………………………………….……………………………………………………………………………………………………….…………………………………………
22. What is the name of the female mosquito involved in Malaria? …………………………………………………………………
23. Why are humans the definitive host of the disease Malaria? …………….………………………………………………………………
……………………………….……………………………………………………………………………………………….…………………………………
17