Experiment no.

9
Protozoa of Medical Importance

2019
 I. Introduction

Protozoa are eukaryotic organisms that has a membrane bound nucleus which exists as a

structurally and functionally independent individual cells. Most protozoa are microscopic

organisms and only a few grow to a size that is large enough to be visible to the naked eye. They

have developed a relatively complex sub-cellular features such as organelles and membranes which

enable them to survive their environment. Moreover, As a unicellular eukaryotes, protozoa display

all the same essential life activities as higher metazoan eukaryotes wherein they move about to

survive, feed and breed.

Protozoa has a four groups: the Amoeboid, Flagellated, Ciliates Protozoans and

Sporozoans. Amoeboid use pseudopodia to creep or crawl over solid substrates. Flagellates use

elongate flagella which undulate to propel the cell through liquid environment while Ciliates uses

numerous small cilia which undulate in waves allowing cells to swim in fluids. And in Sporozoans,

it has been shown that the pre-spore stages move using tiny undulating ridges waves in the cell

membrane imparting a forward gliding motion. Protozoan biodiversity includes counts of some

32,000 living species and another 34,000 extinct (fossil) species, especially foraminifera. Of those

alive today, some 21,000 species occur as free-living organisms in the aquatic or terrestrial

environments, whereas the remaining 11,000 species are parasitic in vertebrate and invertebrate

hosts. There are approximately 6,900 flagellate species (1,800 parasitic, 5,100 free-living), 11,550

amoebae species (250 parasitic, 11,300 free-living), 7,200 ciliate species (2,500 parasitic, 4,700

free-living) and 5,600 sporozoan species, all parasitic.

II. Objectives

There are two objectives that is needed to be done in this experiment. The first one is to examine

the prepared parasitic protozoans through the use of a microscope while the other one is to differentiate
 the parasites based on their classification, organs or structures that are present and the type of the

developmental stages that are recovered from the specimens.

III. Materials and Methodology

A. Materials

This experiment only requires a microscope with an illuminator and prepared slides of

some parasitic protozoans.

B. Methodology

During this experiment, the permanent mounted slides of the prepared slides of representative

protozoans were observed by the group members under the microscope in order to find them in their trophic,

cystic and other developmental stages. Photos were, then, taken in order for the group members to easily

draw them and label their parts with colored pencils. After having distinguished their parts, the protozoans

observed were classified based on their distinguishing features, pathogenicity, mode of transmission, stages

and their hosts in the form of a table.

IV. Results

Organism Classific Distinguishin Diseases it Mode of Infective Intermed Definitive

ation g Features may cause transmissi Stage iate Host Host
on

Chilomasti Protozoa: -Pear or Considered Ingestion Cystic None Humans

x mesnili, Retortam lemon shaped Non of the stage
trophozoite on adea with a pathogenic contaminat
flagella - ed food
located at the and water
anterior end

Chilomasti Protozoa: -has a large Considered Ingestion Cystic None Humans

x mesnili, Retortam single nucleus Non of cyst via Stage
cyst on adea -thick nuclear pathogenic contaminat
membrane ed food
with small and water
central
 karyosome

Trichomon Protozoa: -pyriform, Vaginitis in Sexual Trophic None Humans

as vaginalis Parabasal jerky (on- women and intercourse stage
id spot) urethritis in but also by
-non- men communal
directional bathing,
motility in sharing of
fresh wash
specimen clothes,
toilet
equipment
seats and
mother to
daughter
during
birth.

Gardia Protozoa: -pyriform Giardiasis; Ingested of Cystic None Humans

lambia, Intestinal (pear- Chronic the stage
trophozoite Flagellate shaped), diarrheal contaminat
Scientific s -rounded disease ed food or
name: anteriorly and water or
Giardia pointed by fecal-
intestinalis posteriorly oral route
and
G.duodenal -progressive,
e rapid,
tumbling and
spinning
often linked
to a “falling
leaf” type of
motility in
fresh liquid
stools

-Bilaterally
symmetrical

Gardia Protozoa: - oval shaped Duodenitis; Infection Cystic None Humans

lambia, Intestinal with thick steatorrhea; occurs by stage
cyst Flagellate cyst wall Chronic ingestion
s diarrheal of mature
-finely disease tetra
granular nucleated
cytoplasm cyst with
clearly contaminat
separated ed food,
from cyst drink,
 wall finger, etc.

Table 1.
Mastigophora

Organism Classific Distinguishin Diseases it Mode of Infective Intermed Definitive

ation g Features may cause transmissi Stage iate Host Host
on

Lodamoeb Tubuline nucleus is Diarrhea -Direct Cystic None Humans

a butchili, arca fairly large contact stage
trophozoite and a large orally
endosome (kissing)
-
contaminat
ed water or
drinks
(fecal oral
route)

Endolimax Archaem - oval-shaped Intermittent or - fecal oral Cystic None Humans

nana, cyst oeba -irregular chronic routine stage
karyosome Diarrhea -ingestine
-has 4 nuclei of cysts
from
contaminat
ed water

Entamoeba Tubuline -has large Amoebiasis; -exposure Cystic None Humans

hystolica a central amoebicliver to stage
trophozoite karuosome abscess contaminat
s -active but ed (with
less vigrous cyst) water
indirectional and food
mtotilitu

Entamoeba Tubuline -spherical- Amoebiasis; -exposure Cystic None Humans

hystolica, a shaped amoebic liver to stage
cyst -minute rice- abscess contaminat
shaped ed food
chromatoidal and drink
bars -trhough
-has glycogen direct
mass in contact(by
immature hands)
stages

Table 2.
 Sarcodina

Organism Classific Distinguishin Diseases it Mode of Infective Intermed Definitive

ation g Features may cause transmissi Stage iate Host Host
on

Balantidiu Protozoa: - only 2 GI tract; Through Cystic Cows, Humans

m coli, Litostom nuclei are balantidial the fecal- Stage horses
Trophozoit atea visible. dysentery oral route, and pigs
e -its which is
macronucleus most
is long and common in
kidneyshaped contaminat
and the ed water.
spherical
micronucleus
is nestled
next to it.

Balantidiu Protozoa: - smaller than GI tract; Through Cystic Cows, Humans

m coli, Litostom trophozoites balantidial the fecal- Stage horses
Cysts atea - round and dysentery oral route, and pigs
have a tough, which is
heavy cyst most
common in
contaminat
ed water.

Opalina Protozoa: - has many They are Encyst in Cystic Frogs No

Opalinea rows of symbionts and the Stage Definite
flagella do not harm intestine of host
- has their hosts frogs and
numerous be
nuclei excreted in
the feces

Table 3.
Cilliates

V. Discussion

According to cK-12 Foundation (2019), Sporozoans , are able to form spore-like cells, from which

they get their name. They are not motile as they do not have a flagella, cilia, or a pseudopodia. All

sporozoans are parasites of animals and may cause diseases. They possess a certain organelle that comprises

a apicoplast, which allows them to enter a host. This group is morphologically diverse. Different organisms
within Sporozoans, as well as different life stages for a given apicomplexan, can vary substantially in size,

shape, and subcellular structure (Wikipedia, 2019). Within this phylum are three groups—coccidians,

gregarines, and haemosporidians. Most of the sporozoans observed in the microscope during this

experiments were Haemosporida, namely the Plasmodium malariae, Plasmodium falciparum, and the

Plasmodium vivax, which can all cause Malaria and are transmitted by certain insects. The Monocystis, on

the other hand, belongs to the group gregarines. These parasites inhabit the intestines of a large number of

invertebrates, because majority of earthworms are infected by this parasite Coccidia are intracellular

parasites of the phylum Apicomplexa that cause a range of pathologies collectively termed coccidiosis.

(Science Direct, 2019)

For the mastigophora, 5 specimens were observed. Chilomastix mesnili is considered

nonpathogenic. The presence of cysts and/or trophozoites in stool specimens can however be an indicator

of fecal contamination of a food or water source, and thus does not rule-out other parasitic infections.The

characteristic lemon shaped cysts can be seen in a formol-ether concentrate. Motile organisms can be seen

in a wet preparation of a fresh stool however the characteristic morphology is evident in a permanently

stained preparation.

Trichomonas vaginalis is an anaerobic, flagellated protozoan parasite and the causative agent of

trichomoniasis. It is the most common pathogenic protozoan infection of humans in industrialized countries.

Cysts are resistant forms and are responsible for transmission of giardiasis. Both cysts and trophozoites can

be found in the feces (diagnostic stages) . The cysts are hardy and can survive several months in cold water.

Infection occurs by the ingestion of cysts in contaminated water, food, or by the fecal-oral route (hands or

fomites) . In the small intestine, excystation releases trophozoites (each cyst produces two trophozoites) .

Trophozoites multiply by longitudinal binary fission, remaining in the lumen of the proximal small bowel

where they can be free or attached to the mucosa by a ventral sucking disk . Encystation occurs as the

parasites transit toward the colon. The cyst is the stage found most commonly in nondiarrheal feces.

Because the cysts are infectious when passed in the stool or shortly afterward, person-to-person

transmission is possible. While animals are infected with Giardia, their importance as a reservoir is unclear.
 Among the protozoans observe, there were four Sarcodina that were examined in the experiment

namely Iodameba butschili troph, Endolimax nana cyst, Entamoeba histolytica trophozoite and Entamoeba

histolytica cyst. Iodameba butschili troph is a leaf-like organism that has a nucleus with no chromatin

granules. It also has a large karyosome that is surrounded by achromatic granules. During the process of

binary fission, the trophozoites reproduce in the large intestine, having the mature cyst as its infective stage.

Exposure to this organism (contaminated food and drinks) can lead into infection. Various laboratory

diagnosis are utilized to detect this specie, however the examination of dirct fecal smear examination is

widely used. The second organism that was observed is the Endolimax nana cyst which is also found in the

large intestine. It has an oval, deep-yellow colored cytoplasm that is seen when smeared with iodine. The

presence of 1-4 large nuclei and irregular karyosome (a mass of chromatin) in the nucleus makes it more

unique as compare to other protozoans. The students also examine Entamoeba histolytica trophozoite and

Entamoeba histolytica cyst. The difference between the two is there environment they survive in and their

shapes. The trophozoite is observed in the large intestine, abscesses, etc. While, the cyst survives in stools

of chronic dysenteric patients and carriers. In line with this, the trophozoites exhibit an elongated structure

when actively motile and rounded at rest. In contrast, Entamoeba hystolyca cyst is seen spherical even if

motile or at rest.

VI. Conclusion

Protozoans are a group of eukaryotic single-celled organisms. Several species of protozoans infect humans

and inhabit the body as commensals or parasites . The parasitic protozoans of major medical importance

include certain species of amoebae, flagellates, and sporozoans.

Some of the intestinal protozoa are nonpathogenic and produce no disease; however, microscopists must

be able to distinguish pathogenic from nonpathogenic species. The presence of nonpathogenic species

indicates that the person has been exposed to fecal contamination.

Several species can cause mild to severe gastrointestinal symptoms, and E. histolytica may produce

extraintestinal lesions. However, pathogenic or potentially pathogenic protozoa do not always produce

symptoms or they may remain after symptoms have resolved. Asymptomatic individuals may serve as

reservoirs for the infection. Detection of a potentially pathogenic protozoan does not necessarily prove that

the organism is causing the illness. Patients may have diarrhea caused by other organisms, such as

Salmonella spp., Shigella spp., Escherichia coli or rotavirus. Current intestinal protozoan pathogens

included in this chapter are: Entamoeba histolytica, Blastocystis spp., Giardia lamblia, Dientamoeba fragilis

and Balantidium coli. Trichomonas vaginalis, a urogenital flagellate, is also considered pathogenic, and

may cause mild to severe vaginitis and other urogenital problems.

Questions to Answer

1. What stage of Entamoeba histolytica can be observed in a. diarrheic stool b. formed stool?

The stage of Entamoeba Histolytica that is present in diarrheic stool is primarily trophozoites while

the mature cysts are observed in the formed stool.

2. What are the different specimens that can be collected in the diagnosis of Giardiasis?
 There are various specimens that can be used for the diagnosis of Giardiasis such as the collection

and examination of fluid from the duodenum or biopsy of the small intestine. However, the most effective

way for diagnosing this disease is through the antigen testing of the stool.

3. What is the distinguishing characteristic of Balantidium coli cyst and trophozoite?

There are various distinguishing characteristics that can identify Balantidium coli cyst from

trophozite. The first one is the presence of two nuclei during the stage of trophozoites as well as the opening

on its end known as the peristome. In comparison, the cysts are smaller than trophozoites measuring 40-60

mm across. In cases with macronucleus, the only parts that are visible in the cysts are cilia and contractile

vacuoles.

4. What is the organ of locomotion of B. coli

Balantidium coli is considered to be a ciliate, therefore it uses its cilia to move from one place to

another. It is a harilike projections that is responsible for locomotion and transporting fluid materials.

5. What is the characteristic movement of the B. coli trophic stage?

B.coli trophozites exhibit a distinctive errastic twisting motion, that is comparable to a falling leaf.

They are commonly found attached to epithelial cells of the small intestine and rarely in stools. It absorbs

nutrients from intestinal lumen through pinocytosis. And, utilizes an asexual replicaion, wherein both nuclei

divide on the same time. The process of cytokinesis restores the binucleated state of each daughter cell.

Both cells receive one pair of nuclei that possess gene expression and other properties.

6. What are the characteristic features of the different stages of Plasmodium species?

Below is the picture that depicts the various characteristic feature of the different stages of

plasmodium species.
 All four species exhibit a very similar ringforms. However, P.falciparum rings are a little smaller

and is more numerous than others. It has a large number of rings to cope for the abaence of more mature

stages and multiply-infected erythrocytes. In contrary, erythrocytes that are infected with P.vivax and

P.ovale are evidently larger thus exhibiting Schüffner’s dots in the maturation of trophozoites. The

trophozites of P.vivax are considered amoeboid while P.ovale are compact. In connection, p.malariae is

compact yet its erythrocyte host is not enlarged.

The P.falciparum exhibit a crescent-shaped gametocytes as compared to the rest of the species in

exception to P.malariae that does not modify its erythrocyte host. The differences in sizes can ba

adistinguishable factor between trophozoites and a nucleus. In line with this, mature crogametocytes stain

lighter and have a more diffuse nucleus than macrogametocytes .

7. What are the specimens and methods of laboratory diagnosis of malaria?

 The most common specimen to be used for diagnosing Malaria is a blood smear. The use of the

stains such as Giemsa and Wright is utilized to detect the presence of the parasite. In connection, there are

various methods to do diagnose this disease, some of which are thick and thin blood smears which is

considered the “gold standard” for malaria detection. Another is the rapid diagnostic tests (antigen testing)

which utilize a testing strip to identify if a person is positive. Molecular test (polymerase chain reaction,

PCR) is also used for diagnosis. This amplifies the parasite’s DNA which allows the detection and

identification of such Plasmodium species. Antibody Testing (serology) in contrary, is used to detect people

who are previuosly exposed to the parasite. The last test, is the susceptibility test which is testing the parasite

in the presence of incresing amounts of a drug to observe its effect and the parasite’s resistance.

Todar, K. (2012). Mechanisms of Bacterial Pathogenicity. Retrieved from

http://textbookofbacteriology.net/pathogenesis.html
Vorland, L. (2001). What makes bacteria pathogenic? Retrieved from
https://www.ncbi.nlm.nih.gov/m/pubmed/11757445/
Laboratory Info. (2019). Various shapes and arrangements of bacterial cells. Retrieved from
https://laboratoryinfo.com/various-shapes-and-arrangements-of-bacterial-cells/