Protozoa Parasitology Labs.
Second Grade
University Of Baghdad College of science
Department of Biology
Protozoa Parasitology Lab Second Course – Feb. 2019 – May. 2019
Course Title: Parasitology
Lab Hours: 2 Hours
Instructor: Dr. Hayder Zuhair, Dr. Entsar Saheb, Dr. Harith Saeed, Dr. Amjed Qays, Dr. Rasha Hussein Israa Salim, Batool Kathim, Rula Raad
Grading:
Final grade for the Lab work will be based on the following:
Dates Total Points % of Final Grade
Lab Quizzes Each week 60 6%
First exam 40 7%
Second exam 40 7%
Total 20%
Student participation:
Attendance is expected in all classes. Students are expected to come prepared for class, and to contribute to and participate in all class discussion. Any absences must be justified and explained to the instructor in advance in order to make up any of the graded material.
Parasitic Protozoa
1-Sarcodina (Amoebae): Entameba histolytica, Entameba coli,
Endolimax nana, Iodameba butchlii, Entamoeba gingivalis
2- Ciliates: Balantidium coli
3-Mastigophora (Flagellates): Dientameba fragilis, Giardia lamblia,
Trichomonas vaginalis, Trypanosoma brucci, T. cruzi, Leishmania
donovani, L. tropica
4- Sporozoa: Plasmodium falciparum, Toxoplasma gondi,
Cryptosporidum parvum
Lab 1
Lab 1
General objectives of our Lab.
1- Scientific name and common name
• He is known as the father of modern taxonomy,
Many of his writings were in Latin
Carl Linnaeus
2- Identification: Shape – Nucleus – Cyst and/or Trophozoite – locomotion- Vacuoles.
3- Pathogenicity: - Location of parasite
- Name of Disease
- Symptoms
General objectives of our Lab.
Protozoa general characteristics:
1- Unicellular eukaryotes.
2- Have organelles that often function similarity to
organs and systems of multicellular organisms
3- locomotion: pseudopodia, flagella or cilia
4- Reproduction: Asexual (binary fission or multiple
fission) Sexual (conjugation or syngamy).
Phylum: Protozoa
• Super class: Sarcodina
• Super class: Flagellata
• Super class: Ciliata
• Super class: Sporozoa
Taxonomy
Protozoa Super Class: Sardcodina
Types of nuclei in Protozoa Karyosome or endosome
Chromatin granules
Class Sarcodina- Entamoeba histolytica • Trophozoite 10-60 µM Cyst 10-30 µM diameter.
• Found in the large intestine, cecum and terminal ileum.
• Endoplasm / ectoplasm.
• Endosome of nucleus is small and centric, peripheral
chromatin is identical.
• Pseudopodia are thin.
• Food vacuoles usually contain RBC.
• Cysts is the infective stage and has 4 nuclei, chromatoid
bodies are thick.
• Pathogenic, causes amebic ulcer/ amoebiasis/ amoebic
dysentery -blood in stool, complications- liver abscess.
Entamoeba histolytica
Trophozoite of E. histolytica Cyst of E. histolytica A:Nuclei B:Chromatoid bar
Entamoeba histolytica
Amoebic ulcer caused by E. histolytica
Cup or Flask shape
Class Sarcodina- Entamoeba coli
• Trophozoite (15-50 µM diameter) and Cyst.
• Found in the large intestine, colon.
• Endoplasm = ectoplasm.
• Endosome of nucleus is acentric, peripheral chromatin is non-
identical.
• Pseudopodia usually thick.
• Food vacuoles do not contain RBC.
• Cysts has 8 nuclei, chromatoid bodies are thin.
• Non-pathogenic, commensal.
Class Sarcodina- Entamoeba coli
Endosome of nucleus is acentric, peripheral
chromatin is non-identical
nuclei
Chromatoid bar
Lab 2
Lab 2
Class Sarcodina- Entamoeba gingivalis
• Only trophozoite, 10-20 µM diameter.
• Found in mouth, in teeth roots and gum, it is found
with 95% of gum diseases.
• Endosome is large, centric or acentric, peripheral
chromatin is non-identical.
• Highly vacuolated.
• Non-pathogenic.
Class Sarcodina- Entamoeba gingivalis Endosome is large, centric or acentric,
peripheral chromatin is non-identical
Entamoeba gingivalis
Class Sarcodina- Iodamoeba butschlii
• Trophozoite 8-20 µM, cyst 5-20 µM diameter.
• Found in large intestine.
• Endoplasm = ectoplasm.
• Large endosome surrounded by chromatic
globules.
• Non-pathogenic (commensal)
• Cyst has only one nucleus.
Class Sarcodina- Iodamoeba butschlii
Large endosome surrounded by chromatic globules, Cyst has only one nucleus
Class Sarcodina- Endolemax nana
• Very small – Trophozoite 6-12 µM
• Cyst 5-8 µM
• Live in large intestine, non-pathogenic
• Ectoplasm / Endoplasm
• Large endosome with no peripheral
chromatin.
Class Sarcodina- Endolemax nana
Large endosome with no peripheral chromatin
Trophozoite
Cyst – 4 nuclie غير الكروماتين )
(موجود
2- SUPERCLASS: Ciliata
(CILIOPHORA)
Balantidium coli
• Location: Ileum, colon and rectum
• Pathogenic: Balantidiasis
• Two forms: Trophozoit + Cyst
Cytostome
Cilia
macronecleus
Contractile vacuole
Trophozoite
• Oval shape
• Course cilia
• Contractile vacuoles
• Kidney shape macronucleous
Life cycle of Balantidium coli
Lab 3
Flagellata
INTESTINAL FLAGELATES
& Genital tract flagellates
HAEMOFLAGELATES
BLOOD& TISSUE FLAGELLATES
Lab 3
3- SUPERCLASS: (Flagellata)
MASTIGOPHORA
Include all protozoa that have one flagellum or more in
trophozoite stage
Dientamoeba fragilis
• Trophozoite 5-12 µM
• Found in large intestine
• Ectoplasm / endoplasm.
• Endosome consist of aggregated chromatin granules.
• 60% has 2 nuclei.
INTESTINAL FLAGELATES
Dientamoeba fragilis
Endosome consist of aggregated chromatin granules.
60% has 2 nuclei.
Giardia lamblia
• Location: Small intestine (Duodenum)
• Pathogenic: Giardiasis or lambiasis
• Two forms: Trophozoite + Cyst
INTESTINAL FLAGELATES
Trophozoit of Giardia lamblia Sucking disc Axostyle
Parapasal body
Trophozoite
• Bilaterally symmetrical
• Pear shape
• Broad round anterior and tapering
posterior. Convex dorsal surface and
concave sucking disc. Flat ventral
surface
• Two nuclei with large central
karyosomes
• Two axostyles, two parabasal bodies
• Four pairs of flagella
Cyst of Giardia lamblia
Cyst
• Oval in shape
• They have smooth well defined wall
• Contains four nuclei
• Contain parabasal bodies and flagellates
Trophozoit of Trichomonas
4 flagella 4 flagella
1 flagellum 1 flagellum
quiescent formفي دور السكون الدور المغتذي يتم انتقال العدوى بواسطة *
الذي يتكون بتكور الدور المغتذي الذي يصبح عديم الحزكة
Location: Vagina, Urethra
Pathogenic: Vaginitis, Urithritis
One form: Trophozoite
Genital tract flagellates
Lab 4
Lab 4
Blood and tissue flagellates or
Haemoflagellates
Haemoflagellates Forms
Leishmania tropica
Location:- Cutaneous
Pathogenic:- Baghdad boil or oriental sore
Form:- Amastigote + Promastigote
Vector:- Phlebotomus (Sand fly)
Leishmania donovani
Location:- visceral
Pathogenic:- Dum – Dum fever or kalazar
Form:- Amastigote + Promastigote
Vector:- Phlebotomus
Leishmania braziliensis
Location:-mucocutaneous
Pathogenic:- Uta, Chiclero ulcer, Espundia
Forms:- Amastigote + Promastigote
Vector:- Lutzomyia
In monocyte or phagocytic cells
macrophage
Nucleus of
macrophage
Amastigote of Leishmania
In the invertebrate vector or
the culture
Simple binary
fission
Leishmania promastigote
Life cycle of Leishmania spp.
Amastigotes phagocytized by macrophage
Macrophage
nucleus
Amastigotes
Nucleus
In vitro infection of macrophage with Leishmania amastigotes, DAPI
stain (Hayder Z. Ali et al. 2012)
Oriental sore or Baghdad boil
Case infected with kalazar
Chiclero ulcer or Uta
Lab 5
Lab 5
Trypanosoma gambiense
Location:- In plasma of blood
Pathogenic:- Mid and west African sleeping sickness
Forms:- Epimastigote and trypomastigote
Vector:- Tse-tse or Glossina palpalis
Anterior station development
Trypanosoma gambiense elongated spindle shape
trypomastigote
Trypanosoma cruzi
Location:- Muscles of heart and nervous system muscles
Pathogenic:-South America trypanosomiasis (Chaga’s
disease)
Forms:- Amastigote+ promastigote +
epimastigote + trypomastigote
Vector:- Triatomidae (Kissing bug)
Posterior station development
Trypanosoma cruzi
U or C shape Trypomastigote
Kinetoblast (big)
Lab 5
Lab 6
4- SUPERCLASS: Sporozoa
Lab 6
Sporozoa
o All of the species belong to this group are parasitic
o Produce spores, oocyst
o No clear organs for movement or locomotion
o Reproduction:
Asexual: Binary fission, Multiple fission, Endodyogeny
Sexual: Anisogametes, Isogametes
♀gametes ♂gametes
Zygote
Ookinate
Oocyst
sporocyst
spores
Sexual Reproduction
of humans caused by infectious diseaseborne -mosquitois a Malaria
Plasmodiumof the genus protists eukaryotic
and subtropical regions, including much of tropicalIt is widespread in
Americasand the Asia, Africa
red The disease results from multiplication of malaria parasites within
, headacheand fever, causing symptoms typically include blood cells
, and deathcomain severe cases progressing to
Four species of Plasmodium can infect humans:
Plasmodium falciparum
vivaxPlasmodium
ovalePlasmodium
malariaePlasmodium
Malaria
Plasmodium
• Include the sporozoa that have two replication cycle:
Schizogony (asexual)
• In the vertebrate host
Sporogony (sexual)
• In the invertebrate host
P.falciparum P.ovale P.malariae P.vivax
Malignant malaria Ovale tertian
malaria
Quatrain malaria Tertian malaria
36-48 h 48 h 72 h 48 h
Life cycle of Plasmodium in mammals (asexual)
P. falciparum P. ovale P. malariae P. vivax
Scanty cytoplasm and
small vacuoles,
multiple rings
common
Resembles that of P.
vivax, ring larger in
size and thicker.
smaller in size than
P. vivax, occupied
1/6 of the RBC,
heavy chromatin
dot, vacuole may
appeared filled in
pigment
Delicate cytoplasmic
ring measuring 1/3
RBC diameter, single
chromatin dot, ring
surround a vacuole
Ring form (early trophozoites)
Amoeboid stage (developing trophozoite)
P. falciparum P. ovale P. malariae P. vivax
Fine pigment
granules are
common, only
detected in sever
infection
Amoeboid
tendencies not as
evident as in P.
vivax
Solid cytoplasm,
band form
Irregular amoeboid
appearance. ring
remnant is common,
Mature Schizont P. falciparum P. ovale P. malariae P. vivax
Smaller merozoites
single pigment
mass , an average
of 8-26 merozoites
Meriozoite larger
than P.malariae
,irregular rosset ,
usually 8
Schizont smaller
but merozoites
larger an average
of 6-12
merozoites
Parasites occupy ¾
of RBCs, rossete of
an average of 12-24
merozoites
Microgametocyte P.falciparum P.ovale P.malariae P.vivax
Usually sausage
shape, chromatin
diffused
Similar to P.vivax
but smaller, never
abundant
Similar to P. vivax
but smaller, less
numerous
Spherical, compact,
no vacuole, large
nucleus, cytoplasm
stains light blue
Macrogametocyte P. falciparum P. ovale P. malariae P. vivax
Crescent often
longer and more
slender , chromatin
central , cytoplasm
stain darker blue
Similar to P. vivax
but smaller
Similar to P. vivax
but smaller and
less numerous
spherical , compact
,acentric chromatin
mass
Exflagellation: formation of flagelliform
microgametes from a microgametocyte
in some sporozoa
Zygote: the diploid cell that results from
the fusion of two gametes
Ookinete: The motile stage of the zygote
preceding the oocyst stage
Life cycle of Plasmodium in Mosquito (Sexual)
Oocyst : The resistant stage of the life
cycle of some sporozoal parasites. It
contains a zygote and under
appropriate conditions sporulates to
become a mature infective oocyst.
Sporozoite: Spore formed after
fertilization; a sickle shaped nucleated
germ formed by division of the
protoplasm of a spore of
an organism during sporogony.
Life cycle of Plasmodium in Mosquito
Lab 7
Lab 7
Toxoplasma gondii Final host:- members of family Felidae (domestic
cats and their relatives).
Intermediate host:- many warm-blooded animals, including humans.
Forms:- Oocyst (contains two sporocysts, each one contain four sporozoites
Tackyzoite, Bradizoite
Diagnosis:- is based on serology(IgM and IgG ElISA Test) and on
histologic examination of tissue.
Oocyst Tackyzoite
Toxoplasma gondii Life Cycle
T. gondii primarily exists in three forms: oocysts, tachyzoites, and bradyzoites.
Oocysts are only produced in the definitive host, members of the family Felidae.
When passed in feces and then ingested, the oocysts can infect humans and other
intermediate hosts. They develop into tachyzoites, which are the rapidly
multiplying trophozoite form of T. gondii. They divide rapidly in cells, causing
tissue destruction and spreading the infection. Tachyzoites in pregnant women are
capable of infecting the fetus. Eventually tachyzoites localize to muscle tissues and
the CNS where they convert to tissue cysts, or bradyzoites. This is thought to be a
response to the host immune reaction. Ingestion of cysts in contaminated meat is
also a source of infection, as bradyzoites transform back into tachyzoites upon
entering a new host
Toxoplasma life cycle
Eimeria
Host:- Small and large intestine of herbivores and carnivores
cattle, poultry, and smaller ruminants including sheep and goats.
Oocyst:- The sporulated oocyst is said to be tetrasporic meaning
it contains four sporocysts, each one contain two sporozoites
Disease:- Coccidiosis (diarrheal disease).
Diagnosis:- Oocyst in faeces.
Eimeria Oocyst
Eimeria life cycle
Eimeria species are
considered to be
monoxenous because
the life cycle is
completed within a
single host.
Cryptosporedium
Host:- Humans and a wide range of animals including
mammals, birds ,reptiles ,amphibians and fish.
Oocyst:- Sporulated oocysts, containing 4 sporozoites
Disease:- Cryptosporidiosis
Diagnosis:- Oocyst in faeces.
Cryptosporedium Oocyst
Cryptosporedium life cycle
Laboratory diagnosis and protozoan parasitic infection
The laboratory diagnosis is important in order to know
which parasite causes the disease, so it helps and provides
the clinical diagnosis. The responsibility for accurate
diagnosis requires special training, skill, and ability to
diagnose the parasite and to distinguish it from impurities,
and foreign matter, as well as the availability of essential
equipment and materials.
Lab 7 Laboratory diagnosis and protozoan parasitic infection
Specimen: it depends on the parasite habitat and its
distribution:
Stool
Blood
Urine
Sputum
Other body fluid
Fecal specimen: Immediately have to examine.
Liquid specimen should examining after 30 min. of passage
Soft (semi-formed) specimen after 1 hr.
Formed specimen after 24 hr. from passage.
Collection of specimen
Collecting Stool Samples for Testing
Preparation and Procedure
• Stool should be collected into a clean, dry container.
• Specimens must not be contaminated with urine or
water, so collection from toilet bowls is unacceptable.
• Print the patient name, date and time on the container
with the sample.
• Deliver the specimen to the lab immediately.
Direct examination:
This test is used to find parasites that cause diarrhea like Giardia
lamblia, loose or watery stools, cramping, flatulence (gas) and other
abdominal illness. CDC recommends that three or more stool samples,
collected on separate days, be examined. This test looks for ova (eggs)
or the parasite. You must put your stool specimens into special
containers. Specimens should be refrigerated, but not frozen, until
delivered to the lab. In the lab special stains or those special tests are
used to look for parasites. The stool examination consists of:
Macroscopy: Color, Consistency, Composition
Microscopic: Permanent, Temporary
Direct saline smear (1 drop of saline on slide + small amount of stool
take it by wooden stick).
Iodine smear (1 drop of Iodine solution on slide + small amount of
stool take it by wooden stick).
Concentration technique: separate parasites from fecal debris and
increase the chances of detecting parasitic organisms when these are in
small numbers. They are divided into flotation techniques and
sedimentation techniques.
1- Floatation: (most frequently used: zinc sulfate or Sheather’s sugar)
use solutions which have higher specific gravity than the organisms to
be floated so that the organisms rise to the top and the debris sinks to
the bottom. The main advantage of this technique is to produce a
cleaner material than the sedimentation technique. The disadvantages of
most flotation techniques are that the walls of eggs and cysts will often
collapse, thus hindering identification. Also, some parasite eggs do not
float.
• Liquefy small amount (1 gram) fecal with 5 ml of normal saline 9%.
• Apply the suspension with two layers of wet gauze.
• Collect the filtered suspension in centrifuge tubes
• Put the tubes in the centrifuge (1500 rpm for 1 min)
• Discard supernatant add 5 ml of N.S. and re-centrifuge suspension.
• Discard supernatant and add ZnSo4 to deposit (5 ml) and centrifuge.
2- Sedimentation: • Mix the specimen well.
• Strain 5ml of the fecal suspension (more or less depending on its consistency)
through wetted cheesecloth-type gauze placed over a disposable paper funnel into a
15 ml conical centrifuge tube. (Conical paper cups with the tips cut off are
sufficient).
• Add 0.85% saline or 10% formalin through the debris on the gauze to bring the
volume in the centrifuge tube to 15 ml. Distilled water may be used;
• Centrifuge at 500 × g for 10 minutes.
• Decant supernatant. Add 10 ml of 10% formalin to the sediment and mix
thoroughly with wooden applicator sticks.
• Add 4 ml of ethyl acetate, stopper the tube, and shake vigorously in an inverted
position for 30 seconds. Carefully remove the stopper.
• Centrifuge at 500 × g for 10 minutes.
• Free the plug of debris from the top of the tube by ringing the sides with an
applicator stick. Decant the top layers of supernatant.
• Use a cotton-tipped applicator to remove debris from sides of the centrifuge tube.
• Add several drops of 10% formalin to re-suspend the concentrated specimen.
Proceed with applicable testing.
Blood tests
Some, but not all, parasitic infections can be detected by
testing blood. Blood tests look for a specific parasite
infection; there is no blood test that will look for all parasitic
infections. There are two general kinds of blood tests that
the doctor may order:
1- Serology This test is used to look for antibodies or for
parasite antigens produced when the body is infected with a
parasite like: (Toxoplasma gondii) and the immune system
is trying to fight off the invader. This test is done in the lab
by different methods:
Latex test
ELISA test
Immunofloresent test
2- Blood smear This test is used to look for parasites that are found
in the blood. By looking at a blood smear under a microscope,
parasitic diseases can be diagnosed.
A- Thick and Thin Blood Smears for Malaria
Thick and thin blood smears are used to determine whether you have
malaria (Plasmodium spp.). If one test is negative and no parasites
are found, you will have repeated blood smears every 8 hours for a
couple of days to confirm that there is no malaria infection.
Blood smears are taken most often from a finger prick. Thick and thin
blood smears help to know the percentage of red blood cells that are
infected (parasite density) and what type of parasites are present.
A thick blood smear is a drop of blood on a glass slide.
Thick blood smears are most useful for detecting the
presence of parasites, because they examine a larger
sample of blood. (Often there are few parasites in the
blood at the time the test is done.)
Thick blood smear
A thin blood smear is a drop of blood that is spread
across a large area of the slide. Thin blood smears help
doctors discover what species of malaria is causing the
infection.
Thin blood smear
Results
Results of thick and thin blood smears may show:
Normal
No parasites are present in red blood cells. Your doctor will repeat the
test every 8 hours for 1 or 2 days if he or she still suspects that you
have malaria.
Abnormal
Parasites are present in red blood cells. The infecting species of
Plasmodium is identified. Also, the percentage of red blood cells
infected by the Plasmodium parasite (density) is determined.