Lab 9 – Eukaryotic Microbes and Serology
In this lab you will be examining several eukaryotic organisms of medical
and/or commercial importance. Recall that eukaryotic organisms are divided into
four kingdoms: animalia, plantae, fungi, and protista. You will view slides from all
kingdoms, with the exception of the plant kingdom. We will also be looking at
human blood smears in this lab and doing blood typing.
Exercise 1: Kingdom Fungi
Fungi are non-motile organism that secrete exoenzymes into the
environment, and then absorb the digested materials. They can either obtain
their nutrients by decomposing dead organic matter (saprophytes) or living
plants, animals, or humans (parasites).
The life cycle of a fungus usually involves both a sexual and an asexual
form. Gametes (sexual reproduction) are produced by gametangia, while
spores (asexual reproduction) are produced by sporangia.
In lab 1, you viewed unicellular yeast. This week, you will focus on
several multicellular, filamentous molds including Rhizopus, Aspergillus, and
Penicillium. The individual filaments in these fungi are called hyphae, and are
collectively termed mycelium. Rhizoids anchor the hyphae and the sporangia
supporting sporangiophores.
Rhizopus
Rhizopus is a fast-growing species of fungus that darkens with age, giving
it a “salt and pepper” appearance. Under the microscope, Rhizopus can be
identified by its large, circular sporangia. The sporangium is supported by a
hemispherical columella.
Rhizopus stolonifer is a common bread mold. Other species of Rhizopus,
including, R. arrhizus, are responsible for zygomycosis, a potentially fatal
infection. Infection occurs when spores are inhaled and delivered to the tissues
by the blood. This may result in necrosis in diabetic and immunocompromised
patients.
Aspergillus
Aspergillus is a common environmental fungus. Species of Aspergillus
can be found in a variety of colors, ranging from yellow to green to brown, and
sometimes black. Aspergillus forms distinctive chains of spores at the end of
hyphae, called conidia.
Some species of Aspergillus cause an opportunistic infection called
aspergillosis. Symptoms of aspergillosis vary based on conditions of exposure.
Pulmonary aspergillosis colonizes bronchial tissue that has been damaged by
conditions such as tuberculosis. Allergic aspergillosis causes asthma-like
symptoms in individuals sensitized to the spores. Invasive aspergillosis results in
necrotizing pneumonia and may spread to other organs.
Penicillium
Penicillium is also a common environmental fungus. Green, powdery
colonies that radiate to a white apron characterize Penicillium when viewed on a
plate or other surface. The colonies are a lighter color on the opposite surface.
When viewing microscopically, brush-shaped conidiophores are observed.
While Penicillium may cause an infection known as penicilliosis, it is better
known for the production of the antibiotic penicillin. Penicillium is also used as
the fermenting agent in the production of cheese.
Objective: To identify each of these fungi based on their appearance and be
able to associate each with its medical/commercial importance.
Results:
1. Draw a sample of each organism
2. What is the medical/commercial importance of Rhizopus?
3. What is the medical/commercial importance of Aspergillus?
4. What is the medical/commercial importance of Penicillium?
Exercise 2: Kingdom Protista
The organisms we will examine from this Kingdom are the protozoans.
Protozoans are unicellular, heterotrophic microorganisms. The life cycles of
protozoans are generally important in their pathogenicity. The basic life cycle
consists of a vegetative trophozoite stage and a resting cyst (egg) stage. The
differences between life cycles of each protozoan included in this lab can be
found in the diagrams below.
Protozoans are classified based on their method of locomotion:
 Members of the subphylum Mastigophora move using flagella. In this lab
you will view the flagellate Giardia lamblia and Trypanosoma.
 Members of the subphylum Sarcodine move using cytoplasmic extensions
called pseudopods. In this lab you will view the amoeboid Entamoeba
histolytica.
 Members of the phylum Ciliophora move via cilia covering the cell. In this
lab you will view the ciliate Balantidium coli.
 And members of the phylum Apicomplexa are generally nonmotile. In this
lab you will view the Apicomplexa Plasmodium malaria.
Giardia lamblia
Giardia causes an intestinal illness called giardiasis. Giardiasis is one of
the most common waterborne diseases in the United States. It is contracted
by ingesting fecally contaminated food or water or by putting objects in the
mouth that have come in contact with feces contaminated with Giardia.
Symptoms of giardiasis including diarrhea, gas, greasy stools, stomach
cramps, and nausea may appear 1 to 2 weeks following infection and may
persist for 2 to 6 weeks. Giardia infection is diagnosed by observing
trophozoites or cysts in stool samples.
In this lab, you will observe Giardia lamblia trophozoites in a fecal smear.
Note that because you are viewing a fecal smear, there will be many
organisms other than Giardia, which may make it difficult to find the Giardia.
Objectives:
 Identify Giardia trophozoites in a fecal smear.
 Know the method of transmission for Giardia.
 Know the symptoms of giardiasis.
 Know how Giardia infections are diagnosed.
Results:
1. How are Giardia infections diagnosed?
2. How is Giardia transmitted to humans?
3. What diseases are caused by Giardia infections and what are the
symptoms?
Trypanosoma
Trypanosoma causes the potentially fatal trypanosomiasis, or sleeping
sickness. While there are approximately 25,000 cases reported to the World
Health Organization each year, many go unreported because they occur in
parts of Africa that lack the infrastructure to get accurate reporting of cases.
The only cases seen in the U.S. have been among individuals who had
traveled to Africa. Symptoms of sleeping sickness generally begin 1 to 4
weeks after being bit by an infected tsetse fly. The bite may be painful and
develop into a chancre. The infected individual may experience fever, severe
headaches, irritability, fatigue, swollen lymph nodes, and aching muscles and
joints. Untreated, the infected individual will develop confusion, slurred
speech, seizures, and difficulty walking and talking due to invasion of the
central nervous system. Death will occur in several weeks to months if
treatment is not received. Trypanosoma infection is diagnosed by
observation of blood samples or spinal fluid.
In this lab you will observe Trypanosoma in blood smears.
Objectives:
 Identify Trypanosoma blood smear.
 Know the method of transmission for Trypanosoma.
 Know the symptoms of sleeping sickness.
 Know how Trypanosoma infections are diagnosed.
Results:
1. How are Trypanosoma infections diagnosed?
2. How is Trypanosoma transmitted to humans?
3. What diseases are caused by Trypanosoma and what are the
symptoms?
Entamoeba histolytica
Entamoeba causes the intestinal disease amebiasis, which is
characterized by loose stool, stomach pain and cramping in most instances.
In severe cases, amebic dysentery (bloody stool and fever) can occur.
Entamoeba is transmitted by ingesting cysts from fecally contaminated food,
water, or other objects. Symptoms of the disease generally set in 1 to 4
weeks following ingestion. Entamoeba infection is diagnosed by observation
of trophozoites or cysts in a fecal sample. It should be noted that the
pathogenic Entamoeba histolytica looks identical to a more common, and
nonpathogenic species of Entamoeba (E. dispar) under the microscope. This
leads to false positives in laboratory tests.
Objectives:
 Identify Entamoeba in a fecal smear.
 Know the method of transmission for Entamoeba.
 Know the symptoms of amebiasis.
 Know how Entamoeba infections are diagnosed.
Results:
1. How are Entamoeba infections diagnosed?
2. How is Entamoeba transmitted to humans?
3. What diseases are caused by Entamoeba and what are the symptoms?
Plasmodium malaria
Plasmodium, the causative agent of malaria, is spread to human by the
female Anopheles mosquito. As seen in the figure below, Plasmodium has a
more complex lifecycle than the other eukaryotic microbes we are examining
in this lab. After a mosquito introduces the sporozoite form of the protozoan
into a human host, it goes through development stages in both the liver and
the blood. The liver stage involves multiplication of the parasite. Once
released from the liver, the Plasmodium enters red blood cells, where they
multiply and are released to continue invading other red blood cells. It is the
blood stage that causes the symptom of malaria, including fever, chills,
sweats, fatigue, headaches, muscle pains, nausea, and vomiting. Severe
malaria may result in neurological complications, anemia, pulmonary edema,
cardiovascular collapse, kidney failure, and death.
Plasmodium infections are diagnosed by observing the red blood cells
infected with the parasite in a blood smear. While this is a simple procedure,
malaria often goes undiagnosed for a number of reasons. In places, such as
the United States, where malaria is not endemic, clinicians often do not
consider malaria as a possible diagnosis. In places where malaria is
endemic, many people are carrying the parasite, but have developed a
resistance to it. In this case, illness cannot always be attributed to the
malaria. In these same countries, resources may be lacking to make a
reliable and timely diagnosis.
Objectives:
 Identify Plasmodium in a blood smear.
 Know the method of transmission for Plasmodium.
 Know the symptoms of malaria.
 Know how Plasmodium infections are diagnosed.
Results:
1. How are Plasmodium infections diagnosed?
2. How is Plasmodium transmitted to humans?
3. What diseases are caused by Plasmodium and what are the symptoms?
Exercise 3: Kingdom Animalia
While worms themselves may not be microscopic, there is often
microscopic evidence found in clinical specimens that is used to diagnose an
infection, therefore the helminthes are often included in the discussion of
eukaryotic microbes. The life cycle of the parasitic worms is often complex,
involving several hosts. We will not go into the life cycle of these organisms,
but rather focus on their clinical importance and diagnosis.
The three major categories of clinically important parasitic worms are the
trematodes (flukes), cestodes (tapeworms), and the nematodes (round
worms). In this lab we will look at one example of each.
Schistosoma mansoni
Schistosoma is a parasitic worm that has a complicated life cycle involving
both humans and aquatic snails. It causes schistosomiasis when contracted
via contact with fecally contaminated water containing juvenile worms. These
worms penetrate the skin and enter the body’s circulation, where they
continue to develop. Eggs laid by the adults penetrate the intestinal wall and
are excreted in the feces. While many infections are asymptomatic, fever,
cough, abdominal pain, diarrhea, hepatospenomegaly, and eosinophilia may
be experienced.
There are over 200 million people infected worldwide. This disease is
seen in many parts of the world, including Africa, South America, The Middle
East, and Southeast Asia. Diagnosis is made by identification of eggs in stool
or urine samples, depending on the species suspected.
Objectives:
 Identify Schistosoma eggs.
 Know the method of transmission for Schistosoma.
 Know the symptoms of schistosomiasis.
 Know how Schistosoma infections are diagnosed.
Results:
1. How are Schistosoma infections diagnosed?
2. How is Schistosoma transmitted to humans?
3. What diseases are caused by Schistosoma and what are the symptoms?
Taenia
Members of the genus Taenia are commonly known as tapeworms.
Different species of Taenia infect different animals, such as T. saginata, the
beef tapeworm, and T. solium, the pork tapeworm. Like other animal
parasites, Taenia has a complex lifecycle involving multiple hosts. Humans
may become infected with the tapeworms by ingesting raw or undercooked
meat of an infected animal. Inside the human host, the tapeworm develops
into an adult. The adult tapeworms head region consists of a structure called
a scolex to attach to the small intestine. The length of the tapeworm varies by
species, but is typically between 2 and 7 meters. The adult produces
proglottids (segments containing both male and female reproductive organs).
Near the head of the tapeworm, the proglottids are immature. As you move
away from the head, the proglottids become mature and develop sex organs.
At the distal end of the tapeworm the proglottids are gravid, meaning they
contain fertilized eggs. The gravid proglottids break off from the tapeworm
and may exit in the feces.
Infection by the tapeworm may cause mild abdominal symptoms, or the
infected person may be asymptomatic. The main symptom is often the
passage of proglottids in the feces. Diagnosis is made by identification of
eggs and proglottids in the feces. This cannot be done in the first 3 months
following infection, as time is needed for the tapeworm to mature and produce
gravid proglottids.
Objectives:
 Identify the different regions of Taenia.
 Know the method of transmission for Taenia.
 Know the symptoms of a tapeworm infection.
 Know how Taenia infections are diagnosed.
Results:
1. How are Taenia infections diagnosed?
2. How is Taenia transmitted to humans?
3. What diseases are caused by Taenia and what are the symptoms?
Ascaris lumbricoides
Ascaris infections are the most common type of worm infection, occurring
worldwide. Infections are found most frequently in areas of poor hygiene and
sanitation. People with infections are frequently asymptomatic, but may
experience slow growth and weight gain. Infections occur when fecally
contaminated soil is ingested. Eggs hatch in the stomach. Larvae migrate to
the lungs and up the throat where they are once again swallowed. The larvae
develop into in the intestines into adult worms. Eggs laid by the female are
passed in the feces. Diagnosis of Ascaris infection is made by identifying
eggs in the stool.
Objectives:
 Identify Ascaris eggs.
 Know the method of transmission for Ascaris.
 Know the symptoms of an Ascaris infection.
 Know how Ascaris infections are diagnosed.
Results:
1. How are Ascaris infections diagnosed?
2. How is Ascaris transmitted to humans?
3. What diseases are caused by Ascaris and what are the symptoms?
Information in this lab modified from:
Centers for Disease Control and Prevention. “CDC – Diseases and Conditions.”
[Online] 4 April 2008. <http://www.cdc.gov/DiseasesConditions/ >.
Exercise 4: Human Blood Smears
A human blood smear will consist mainly of erythrocytes or red blood cells
(RBC’s), but also contains many leukocytes or white blood cells (WBC’s).
Leukocytes, which play very important roles in the immune system, come in a
variety of forms. We can divide leukocytes in two main groups, granulocytes
(have cytoplasmic granules) and agranulocytes (do not have granules). Each of
these groups can be further subdivided. Neutrophils, eosinophils, and basophils
are all granulocytes; while lymphocytes and monocytes are agranulocytes. With
each playing a different role in the immune system, the relative abundance of
each type of leukocytes can often be used as an indicator of a specific type of
infection or illness. The abundance of leukocytes is determined by performing a
differential blood cell count.
Neutrophils, the most abundant type of leukocyte, are phagocytes that
respond to bacterial infections. As you may deduce, increased concentrations of
neutrophils are indicative of systemic bacterial infections. Anything causing a
decrease in neutrophil numbers (congenital disorder, leukemia, chemotherapy,
etc) will leave a person more susceptible to bacterial infections. Neutrophils are
variable in appearance.
Eosinophils, which account for 1-4% of leukocytes, play a role in the
immune response in parasitic infections. An increase in eosinophil numbers is
indicative of a parasitic infection, while a decrease in eosinophils is seen in
conditions that result in increased glucocorticoid production, such as stress.
Eosinophils appear as red granular cells with a two-lobed nucleus.
Basophils, the least abundant leukocytes, account for 0.1-1% of
leukocytes. They participate in the inflammatory response by secreting
histamine. Low basophil counts, in conjunction with low neutrophil counts,
indicate a high likelihood of leukemia. Basophils appear as purple granular cells
with either a two-lobed or unlobed nucleus.
Lymphocytes, a relatively abundant agranuloctye, account for 25-33% of
leukocytes. There are several varieties of lymphocytes including natural killer
cells, B cells, and T cells. Natural killer cells destroy virally infected or cancerous
cells, while B and T cells participate in specific acquired immunity. An increase
in lymphocyte numbers indicates a viral infection. Decreases in lymphocyte
numbers are seen in immune deficiency disorders, such as HIV.
Monocytes, like neutrophils, are phagocytic, but also act as antigen
presenting cells for the immune system. When found in the bloodstream, these
leukocytes are called monocytes, but are referred to as macrophages when they
migrate into tissues. These cells are identifiable by their horseshoe-shaped
nucleus.
Objective:
 Identify each type of leukocyte.
 Understand the main functions of each type of leukocyte.
 Identify disorders/diseases associated with abnormal levels of each
leukocyte.
Materials:
 Commercially prepared human blood smears
 Leukocyte models
 Blood poster
Procedure:
1. View the commercially prepared slide set up at the back of the lab.
a. You may wish to refer to the poster available on the wall for “ideal”
leukocytes.
2. View the leukocyte models.
3. Draw an example of each type of leukocyte.
Results:
1. What are the two main groups of WBCs and which leukocytes fall into
each category?
2. What is the major function of each WBC?
3. What is generally the most abundant type of WBC?
4. Which WBCs are phagocytic?
5. Which WBCs are involved in specific acquired immunity?
6. What is indicated by an increased eosinophil number?
7. What is indicated by an increased neutrophil concentration?
8. Which WBCs act as antigen presenting cells in the immune system?
9. What is the difference between a monocyte and a macrophage?
Exercise 5: Blood Typing
ABO and Rh blood typing are both types of hemagglutination (test in
which clumping of red blood cells indicates a positive reaction). ABO blood
typing looks for the presence of A and/or B antigens on the surface of red blood
cells. Individuals with one type of antigen (antibody generating molecule) will
produce antibodies against the other type. Refer to the chart below for the blood
type, antigen found on the blood cell, and antibodies produced:
Blood Type
A
B
AB
O
Antigen(s) on RBC’s
A
B
A and B
none
Antibodies Produced
Anti-B
Anti-A
None
Anti-A and Anti-B
Notice that people with type AB blood do not produce antibodies against
either antigen. This makes them “universal recipients” of blood. People with
type O blood produce antibodies against both antigens, meaning they can only
receive blood from another type O individual. However, the lack of antigens on
their RBC’s make them “universal donors” of blood.
Antibodies against a specific antigen cause the blood to agglutinate
(clump) when it comes in contact with that antigen. We can determine blood type
by adding our blood to both anti-A and anti-B antisera (blood serum that contains
antibodies). Refer to the chart below for all possible reactions:
Anti-A Antiserum
Agglutination
Anti-B Antiserum
No Agglutination
No Agglutination
Agglutination
Agglutination
Agglutination
No Agglutination
No Agglutination
Interpretation
A antigen present
B antigen absent
A antigen absent
B antigen present
A antigen present
B antigen present
A antigen absent
B antigen absent
Blood Type
A
B
AB
O
The Rhesus factor, or Rh factor, is determined in the same manner.
Individuals that are Rh positive have the antigen on their RBC surface, while
individuals that are Rh negative do not have the antigen on their RBC surface.
Addition of anti-Rh antiserum to an Rh positive sample will cause agglutination.
Objectives:
 Understand the principles behind hemagglutination reactions.
 Determine an individual’s blood type based on these reactions.
 Identify which blood types are most abundant in a population and which
are least abundant.
Materials:
 Blood typing kits
o Lancets
o Alcohol wipes
o Well plates
o Toothpicks
o Bandages
o Gloves
o Sharps container
o Anti-A antiserum
o Anti-B antiserum
o Anti-Rh antiserum
o Artificial blood
Procedure:
1. This can either be done either on your own blood or using the artificial blood
supplied.
2. Extreme caution must be used in this lab. Universal precautions must be taken
anytime bodily fluids, such as blood, are present.
3. Place a drop of anti-A antiserum in the well labeled “A”.
4. Place a drop of anti-B antiserum in the well labeled “B”.
5. Place a drop of anti-Rh antiserum in the well labeled “Rh”.
6. If using your own blood, clean the tip of the index finger with an alcohol pad. Let
the alcohol dry.
7. Shake the hand you are going to prick and massage the index finger from the
base to the tip.
8. Remove the blue tip of the lancet, place the narrow white tip against the side of
the clean index finger and depress the button. If you are performing this step on
a classmate, you must wear gloves.
9. Squeeze the finger over each of the wells to dispense a drop of blood into each
drop of antiserum. Do not touch your finger to the antiserum.
a. If you are using the artificial blood, squeeze a drop into each of the wells.
10. Discard the lancet into the sharps container.
11. Put a bandage over the wound.
12. Use a toothpick to mix the blood and antisera. Use a different toothpick for each
well.
13. Gently rock the slide back and forth for a few minutes.
14. Look for agglutination in each of the wells.
15. You may use the chart above or the poster on the wall to help in determining
your blood type.
16. Collect the class data to determine the percentage of students with each blood
type
Blood Type
A
B
AB
O
# of students with
blood type
Total # of students
in class
% of students with
blood type
Results:
1. Which blood type is the universal donor? Why?
2. Which blood type is the universal recipient? Why?
3. What is the difference between an antigen and an antibody?
4. What antigens will present on RBCs of a B+ individual?
5. What type of antibodies will a B+ individual make?
6. What is hemagglutination?
7. If the blood clumps in the well is it positive or negative for that blood type?