REMOTE LAB ACTIVITY
SUBJECT SEMESTER: ________________
TITLE OF LAB: Parasitology
Lab format: This lab is a remote lab activity.
Relationship to theory (if appropriate): In this lab you will learn to identify the several life stages
and differences in appearance of several reprehensive parasites.
Instructions for Instructors: This protocol is written under an open source CC BY license. You may use
the procedure as is or modify as necessary for your class. Be sure to let your students know if they
should complete optional exercises in this lab procedure as lab technicians will not know if you want
your students to complete optional exercises.
Instructions for Students: Read the complete laboratory procedure before coming to lab. Under the
experimental sections, complete all pre-lab materials before logging on to the remote lab. Complete
data collection sections during your online period, and answer questions in analysis sections after your
online period. Your instructor will let you know if you are required to complete any optional exercises in
this lab.
Remote Resources: Primary – Microscope, Secondary – Introduction to Parasitology slide set.
CONTENTS FOR THIS NANSLO LAB ACTIVITY:
Learning Objectives....................................................................................................................
Background Information ...........................................................................................................
Equipment .................................................................................................................................
Pre-lab Exercise 1: Microscopic Examination of Giardia lambia
cysts and trophozoites .......................................................................................................
Pre-lab 1 Question ...................................................................................................................
Pre-lab Exercise 2: Microscopic Examination of Entamoeba Granulosus Cysts .......................
Pre-lab 2 Question ...................................................................................................................
Pre-lab Exercise 3: Microscopic Examination of Taenia Saginata Eggs ....................................
Pre-lab 3 Question ...................................................................................................................
Pre-lab Exercise 4: Microscopic Examination of Plamodium Falciparum Rings
and Plasmodium Vivax All Stages .......................................................................................
Pre-lab 4 Question ...................................................................................................................
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Pre-lab Exercise 5: Microscopic Examination of Ascaris Lumbricoides Eggs ...........................
Pre-lab 5 Question ...................................................................................................................
Experimental Procedure ...........................................................................................................
Exercise 1: Microscopic Examination of Giardia Lamblia Cysts and Trophozoites ..................
Exercise 2: Microscopic Examination of Entamoeba Granulosus Cysts ...................................
Exercise 3: Microscopic Examination of Taenia Saginata Eggs ................................................
Exercise 4: Microscopic Examination of Plamodium Falciparum Rings and
Plasmodium Vivax All Stages ...............................................................................................
Exercise 5: Microscopic Examination of Ascaris Lumbricoides Eggs ........................................
Summary Questions ..................................................................................................................
Preparing for this NANSLO Lab Activity ....................................................................................
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LEARNING OBJECTIVES:
After completing this laboratory experiment, you should be able to do the following things:
1. Identify and distinguish between the different kinds of intestinal parasites: Giardia lamblia,
Entamoeba granulosus, Taenia saginata, Plasmodium falciparum, Plasmodium vivax, and Ascaris
lumbricoides.
2. Identify and distinguish between the different larval stages of certain parasites.
3. Identify and distinguish between cysts, eggs, and trophozoites.
4. Measure the relative size of cysts and eggs and provide a comparison between them.
BACKGROUND INFORMATION:
Worldwide, parasitic diseases constitute a major health problem in several developed and developing
countries. Each year, the World Health Organization (WHO) publishes reports relevant to the prevalence
of parasitic diseases across the World. The lack of sanitation, poor education, and poverty has led to the
increase of their prevalence in developing countries. The severity of human parasitic diseases ranges
from minimal to life-threatening such as the case of malaria and schistosomiasis. Certain infections lead
to nutritional loss such as the case of the Ascaris infection common among children in developing
countries.
Parasitology is the study of animal and plant parasitism as a biological phenomenon. Parasites occur in
virtually all major animal groups and in many plant groups with hosts as varied as the parasites
themselves.
Parasitism is a type of association between two species where one benefits and the other is harmed.
A parasite is an organism that lives on or in a host and gets its food from or at the expense of its host.
Parasites can cause disease in humans. Some parasitic diseases are easily treated and some are not. The
burden of these diseases often rests on communities in the tropics and subtropics, but parasitic
infections also affect people in developed countries. Throughout evolution, parasites have developed
the ability to grow either inside or on their host cells and require more than one host as part of their life
cycle.
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Certain parasites are transmitted via direct contact from a carrier. Others require a biological vector
where the parasite completes part of its life cycle before being transmitted to another host. Several
parasites have the ability to produce either cysts or eggs. Cysts are forms that are resistant to the
environment allowing the parasites to survive during unfavorable conditions. Eggs are also resistant to
environmental changes but they get their protection from a thick protective coat. Studying life cycles of
several human parasites has led to an advance in control and prevention of the infections, but we need
to also recognize that some parasites along with their vectors have developed resistance to certain
drugs and chemicals making it difficult to completely eradicate certain parasitic infections.
Numerous parasites can be transmitted by food including many protozoa and helminthes. In the United
States, the most common food borne parasites are protozoa such as Cryptosporidium spp., Giardia
intestinalis, Cyclospora cayetanensis, and Toxoplasma gondii; roundworms such as Trichinella spp. and
Anisakis spp.; and tapeworms such as Diphyllobothrium spp. and Taenia spp. (Click on the italicized
words to get more information about each type of parasite). Many of these organisms can also be
transmitted by water, soil, or person-to-person contact. Occasionally in the U.S., but often in developing
countries, a wide variety of helminthic roundworms, tapeworms, and flukes are transmitted in foods
such as:
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undercooked fish, crabs, and mollusks;
undercooked meat; raw aquatic plants such as watercress; and
raw vegetables that have been contaminated by human or animal feces.
Some foods are contaminated by food service workers who practice poor hygiene or who work in
unsanitary facilities.
Symptoms of food borne parasitic infections vary greatly depending on the type of parasite. Protozoa
such as Cryptosporidium spp., Giardia intestinalis, and Cyclospora cayetanensis most commonly cause
diarrhea and other gastrointestinal symptoms. Helminthic infections can cause abdominal pain,
diarrhea, muscle pain, cough, skin lesions, malnutrition, weight loss, neurological and many other
symptoms depending on the particular organism and burden of infection. Treatment is available for
most of the food borne parasitic organisms.
The human body can be infected by several prokaryotic and eukaryotic parasites. In this lab you will
learn to identify the different phases of the life cycle of several represented parasites that infect
humans.
References:
http://www.britannica.com/EBchecked/topic/443269/parasitology
http://www.cdc.gov/parasites/food.html
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EQUIPMENT:
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Paper
Pencil/pen
Slides
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Giardia lamblia Cysts
Giardia lamblia Trophozoites
Entamoeba histolytica cysts
Taenia saginata eggs
Plasmodium falciparum rings
Plasmodium vivax all stages
Ascaris lumbricoides eggs
Computer with Internet access for the remote laboratory and for data analysis
PRE-LAB EXERCISE 1: Microscopic Examination of Giardia Lamblia
Cysts and Trophozoites
Giardiasis is a diarrheal illness caused by a microscopic parasite Giardia intestinalis (also known as
Giardia lamblia, or Giardia duodenalis) found on surfaces or in soil, food, or water that has been
contaminated with feces from infected humans or animals. Giardia intestinalis is a protozoan flagellate
(Diplomonadida) which is protected by an outer shell that allows it to survive outside the body for long
periods of time and makes it tolerant to chlorine disinfection. While the parasite can be spread in
different ways, water (drinking water and recreational water) is the most common method of
transmission. The Giardia life cycle has several forms. The two we are most interested in are cysts and
trophozoites.
Left: G. intestinalis trophozoites in Kohn stain.
Center: G. intestinalis cyst stained with trichrome.
Right: G. intestinalis in in vitro culture, from a quality control slide.
http://www.cdc.gov/parasites/giardia/
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Cysts are a resistant form 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.
In this lab exercise you will observe and measure the relative size of Giardia lamblia cysts.
References:
http://www.cdc.gov/parasites/giardia/biology.html
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PRE-LAB 1 QUESTION:
1. What kind of shape do you think the Giardia lamblia cysts will have?
PRE-LAB EXERCISE 2: Microscopic Examination of Entamoeba Granulosus Cysts
Amebiasis is a disease caused by the parasite Entamoeba histolytica. It can affect anyone, although it is
more common in people who live in tropical areas with poor sanitary conditions. Entamoeba histolytica
is well recognized as a pathogenic ameba associated with intestinal and extraintestinal infections.
Just like Giardia lamblia the cysts and trophozoites of Entamoeba histolytica are passed in feces .
Cysts are typically found in formed stool, whereas trophozoites are typically found in diarrheal stool.
Infection by Entamoeba histolytica occurs by ingestion of mature cysts in fecally contaminated food,
water, or hands. Excystation occurs in the small intestine and trophozoites are released, which
migrate to the large intestine. The trophozoites multiply by binary fission and produce cysts , and both
stages are passed in the feces . Because of the protection conferred by their walls, the cysts can
survive days to weeks in the external environment and are responsible for transmission. Trophozoites
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passed in the stool are rapidly destroyed once outside the body, and if ingested would not survive
exposure to the gastric environment. In many cases, the trophozoites remain confined to the intestinal
lumen ( : noninvasive infection) of individuals who are asymptomatic carriers, passing cysts in their
stool. In some patients, the trophozoites invade the intestinal mucosa ( : intestinal disease), or,
through the bloodstream, extraintestinal sites such as the liver, brain, and lungs ( : extraintestinal
disease) with resultant pathologic manifestations. It has been established that the invasive and
noninvasive forms represent two separate species, respectively E. histolytica and E. dispar. These two
species are morphologically indistinguishable unless E. histolytica is observed with ingested red blood
cells (erythrophagocystosis). Transmission can also occur through exposure to fecal matter during sexual
contact (in which case not only cysts, but also trophozoites could prove infective).
References:
http://www.cdc.gov/parasites/amebiasis/biology.html
In this lab exercise you will measure the relative size of Entamoeba histolytica cysts and compare them
with the ones produced by Giardia lamblia.
PRE-LAB 2 QUESTION:
1. Do you predict the size of Entamoeba histolytica cysts to be smaller, bigger or the same as the
Giardia lamblia ones?
2. Rewrite your answer to question one in the form of an If … Then … hypothesis.
PRE-LAB EXERCISE 3: Microscopic Examination of Taenia Saginata Eggs
Taeniasis in humans is a parasitic infection caused by the tapeworm species Taenia saginata (beef
tapeworm), Taenia solium (pork tapeworm), and Taenia asiatica (Asian tapeworm). Humans can
become infected with these tapeworms by eating raw or undercooked beef (T. saginata) or pork (T.
solium and T. asiatica). People with taeniasis may not know they have a tapeworm infection because
symptoms are usually mild or nonexistent.
Humans pass the tapeworm segments and/or eggs in feces and contaminate the soil in areas where
sanitation is poor. Taenia eggs can survive in a moist environment and remain infective for days to
months. Cows and pigs become infected after feeding in areas that are contaminated with Taenia eggs
from human feces. Once inside the cow or pig, the Taenia eggs hatch in the animal’s intestine and
migrate to striated muscle to develop into cysticerci, causing a disease known as cysticercosis. Cysticerci
can survive for several years in animal muscle. Humans become infected with tapeworms when they eat
raw or undercooked beef or pork containing infective cysticerci. Once inside humans, Taenia cysticerci
migrate to the small intestine and mature to adult tapeworms which produce segments and eggs that
are passed in feces. Infection with T. solium tapeworms can result in human cysticercosis which can be a
very serious disease that can cause seizures and muscle or eye damage.
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Life Cycle:
Taeniasis is the infection of humans with the adult tapeworm of Taenia saginata or Taenia solium.
Humans are the only definitive hosts for T. saginata and T. solium. Eggs or gravid proglottids are passed
with feces ; the eggs can survive for days to months in the environment. Cattle (T. saginata) and pigs
(T. solium) become infected by ingesting vegetation contaminated with eggs or gravid proglottids . In
the animal's intestine, the oncospheres hatch , invade the intestinal wall, and migrate to the striated
muscles, where they develop into cysticerci. A cysticercus can survive for several years in the animal.
Humans become infected by ingesting raw or undercooked infected meat . In the human intestine, the
cysticercus develops over two months into an adult tapeworm which can survive for years. The adult
tapeworms attach to the small intestine by their scolex and reside in the small intestine . Length of
adult worms is usually 5 m or less for T. saginata (however it may reach up to 25 m) and 2 to 7 m for T.
solium. The adults produce proglottids which mature become gravid, detach from the tapeworm, and
migrate to the anus or are passed in the stool (approximately 6 per day). T. saginata adults usually have
1,000 to 2,000 proglottids, while T. solium adults have an average of 1,000 proglottids. The eggs
contained in the gravid proglottids are released after the proglottids are passed with the feces. T.
saginata may produce up to 100,000 and T. solium may produce 50,000 eggs per proglottid respectively.
In this lab exercise you will observe and measure the relative size of Taenia saginata eggs and compare
it to the size of Entamoeba histolytica cysts measured in the previous exercise.
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References:
http://www.cdc.gov/parasites/taeniasis/biology.html
PRE-LAB 3 QUESTIONS:
1. Do you predict the size of normal Taenia saginata eggs to be smaller, bigger or the same as
Entamoeba granulosus cysts?
2. Rewrite your answer to question one in the form of an If … Then … hypothesis.
PRE-LAB EXERCISE 4: Microscopic Examination of Plamodium Falciparum Rings
and Plasmodium Vivax All Stages
Malaria is a mosquito-borne disease caused by a Plasmodium parasite that can be spread to humans.
Diseases that are spread between animals and humans are said to be zoonotic. Malaria is a serious and
sometimes fatal disease that commonly infects a certain type of mosquito which feeds on humans.
People who get malaria are typically very sick with high fevers, shaking chills, and flu-like illness.
Although malaria can be a deadly disease, illness and death from malaria can usually be prevented. Left
untreated, they may develop severe complications and die. In 2010 an estimated 219 million cases of
malaria occurred worldwide and 660,000 people died, most (91%) in the African Region. About 1,500
cases of malaria are diagnosed in the United States each year. The vast majority of cases in the United
States are in travelers and immigrants returning from countries where malaria transmission occurs,
many from sub-Saharan Africa and South Asia.
Malaria parasites are micro-organisms that belong to the genus Plasmodium. There are more than 100
species of Plasmodium, which can infect many animal species such as reptiles, birds, and various
mammals. Four species of Plasmodium have long been recognized to infect humans in nature. In
addition there is one species that naturally infects macaques which has recently been recognized to be a
cause of zoonotic malaria in humans. (There are some additional species which can, exceptionally or
under experimental conditions, infect humans).
The human naturally infecting species are:
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P. falciparum is found worldwide in tropical and subtropical areas. It is estimated that every
year approximately 1 million people are killed by P. falciparum, especially in Africa where this
species predominates. P. falciparum can cause severe malaria because it multiples rapidly in the
blood and can thus cause severe blood loss (anemia). In addition, the parasites can clog small
blood vessels. When this occurs in the brain, cerebral malaria results, a complication that can be
fatal.
P. vivax is found mostly in Asia, Latin America, and in some parts of Africa. Because of the
population densities, especially in Asia, it is probably the most prevalent human malaria
parasite. P. vivax (as well as P. ovale) has dormant liver stages ("hypnozoites") that can activate
and invade the blood ("relapse") several months or years after the infecting mosquito bite.
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P. ovale is found mostly in Africa (especially West Africa) and the islands of the western Pacific.
It is biologically and morphologically very similar to P. vivax. However, differently from P. vivax,
it can infect individuals who are negative for the Duffy blood group, which is the case for many
residents of sub-Saharan Africa. This explains the greater prevalence of P. ovale (rather than P.
vivax) in most of Africa.
P. malariae, found worldwide is the only human malaria parasite species that has a quartan
cycle (three-day cycle). (The three other species have a tertian, two-day cycle.) If untreated, P.
malariae causes a long-lasting, chronic infection that in some cases can last a lifetime. In some
chronically infected patients P. malariae can cause serious complications such as the nephritic
syndrome.
P. knowlesi is found throughout Southeast Asia as a natural pathogen of long-tailed and pigtailed macaques. It has recently been shown to be a significant cause of zoonotic malaria in that
region, particularly in Malaysia. P. knowlesi has a 24-hour replication cycle and so can rapidly
progress from an uncomplicated to a severe infection; fatal cases have been reported.
The natural life cycle of malaria involves the malaria parasites infecting successively two types of hosts:
humans and female Anopheles mosquitoes. In humans, the parasites grow and multiply first in the liver
cells and then in the red cells of the blood. In the blood, successive broods of parasites grow inside the
red cells and destroy them, releasing daughter parasites ("merozoites") that continue the cycle by
invading other red cells. The blood stage parasites are those that cause the symptoms of malaria. When
certain forms of blood stage parasites ("gametocytes") are picked up by a female Anopheles mosquito
(see Figure below) during a blood meal, they start another, different cycle of growth and multiplication
in the mosquito.
After 10-18 days, the parasites are found (as "sporozoites") in the mosquito's salivary glands. When the
Anopheles mosquito takes a blood meal on another human, the sporozoites are injected with the
mosquito's saliva and start another human infection when they parasitize the liver cells. Thus the
mosquito carries the disease from one human to another (acting as a "vector"). Differently from the
human host, the mosquito vector does not suffer from the presence of the parasites.
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The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female
Anopheles mosquito inoculates sporozoites into the human host . Sporozoites infect liver cells and
mature into schizonts , which rupture and release merozoites . (Of note, in P. vivax and P. ovale a
dormant stage [hypnozoites] can persist in the liver and cause relapses by invading the bloodstream
weeks, or even years later.) After this initial replication in the liver (exo-erythrocytic schizogony ), the
parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony ). Merozoites
infect red blood cells . The ring stage trophozoites mature into schizonts, which rupture releasing
merozoites . Some parasites differentiate into sexual erythrocytic stages (gametocytes) . Blood stage
parasites are responsible for the clinical manifestations of the disease. The gametocytes, male
(microgametocytes) and female (macrogametocytes) are ingested by an Anopheles mosquito during a
blood meal . The parasites’ multiplication in the mosquito is known as the sporogonic cycle . While in
the mosquito's stomach, the microgametes penetrate the macrogametes generating zygotes . The
zygotes in turn become motile and elongated (ookinetes) which invade the midgut wall of the
mosquito where they develop into oocysts . The oocysts grow, rupture, and release sporozoites ,
which make their way to the mosquito's salivary glands. Inoculation of the sporozoites into a new
human host perpetuates the malaria life cycle.
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Anopheles Freeborni Mosquito Pumping Blood
http://www.cdc.gov/malaria/about/biology/mosquitoes/freeborni_large.html
References:
http://www.cdc.gov/malaria/diagnosis_treatment/diagnosis.html
http://www.cdc.gov/malaria/about/biology/
http://www.cdc.gov/malaria/about/biology/parasites.html
In this lab exercise you will observe and measure the diameter of Plasmodium falciparum rings and
compare it with the one of Plasmodium vivax rings.
PRE-LAB 4 QUESTION:
1. In this lab you will examine the ring stage of two different species of the Plasmodium parasite.
Do you expect to see a difference in the size of the rings? Explain your reasoning.
PRE-LAB EXERCISE 5: Microscopic Examination of Ascaris Lumbricoides Eggs
Ascariasis: An estimated 807-1,221 million people in the world are infected with Ascaris lumbricoides
(sometimes called just "Ascaris"). Ascaris, hookworm, and whipworm are known as soil-transmitted
helminths (parasitic worms). Together, they account for a major burden of disease worldwide. Ascariasis
is now uncommon in the United States.
Ascaris lives in the intestine and Ascaris eggs are passed in the feces of infected persons. If the infected
person defecates outside (near bushes, in a garden, or field) or if the feces of an infected person are
used as fertilizer, eggs are deposited on soil. They can then mature into a form that is infective.
Ascariasis is caused by ingesting eggs. This can happen when hands or fingers that have contaminated
dirt on them are put in the mouth or by consuming vegetables or fruits that have not been carefully
cooked, washed or peeled.
People infected with Ascaris often show no symptoms. If symptoms do occur they can be light and
include abdominal discomfort. Heavy infections can cause intestinal blockage and impair growth in
children. Other symptoms such as cough are due to migration of the worms through the body. Ascariasis
is treatable with medication prescribed by your health care provider.
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Left/Right: Fertilized eggs of A. lumbricoides in unstained wet mounts of stool.
Center: Adult female A. lumbricoides
http://www.cdc.gov/parasites/ascariasis/
Life Cycle:
Adult worms live in the lumen of the small intestine. A female may produce approximately 200,000
eggs per day which are passed with the feces . Unfertilized eggs may be ingested but are not infective.
Fertile eggs embryonate and become infective after 18 days to several weeks , depending on the
environmental conditions (optimum: moist, warm, shaded soil). After infective eggs are swallowed ,
the larvae hatch , invade the intestinal mucosa, and are carried via the portal, then systemic
circulation to the lungs . The larvae mature further in the lungs (10 to 14 days), penetrate the alveolar
walls, ascend the bronchial tree to the throat, and are swallowed . Upon reaching the small intestine,
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they develop into adult worms . Between 2 and 3 months are required from ingestion of the infective
eggs to oviposition by the adult female. Adult worms can live 1 to 2 years.
References:
http://www.cdc.gov/parasites/ascariasis/
PRE-LAB 5 QUESTION:
1. Several of the parasites you are examining in this lab use cysts to protect themselves from the
environment. The round worm Ascaris lumbricoides uses eggs to accomplish the same functions
what differences and similarities do you expect to see between eggs and cysts.
In this lab exercise you will observe Ascaris lumbricoides eggs.
EXPERIMENTAL PROCEDURE
Once you have logged on to the remote lab system, you will perform the following laboratory
procedures. See Preparing for the Microscope NANSLO Lab Activity below.
EXERCISE 1: Microscopic Examination of Giardia Lamblia Cysts and Trophozoites
Data Collection:
1. Select the Giardia lamblia cysts slide (Slide Cassette 3: #1) from the microscope interface. Using
the 10X objective, identify the cysts and bring them into focus.
2. Carefully work your way through all the objectives focusing with each one until you reach the
60X objective and capture an image of Giardia lamblia cysts. Insert the image below.
3. Select the Giardia lamblia trophozoites slide (Slide Cassette 3: #2) from the microscope
interface. Using the 10X objective, identify the trophozoites and bring them in to focus.
4. Carefully work your way through all the objectives focusing with each one until you reach the
60X objective and capture an image of Giardia lamblia trophozoites. Insert your images below.
Analysis:
5. Using your image from step 2, label the cysts. Insert your image below.
6. Describe the shape of the cysts.
7. Next we are going to measure the size of the cysts. To determine the size of the cysts, we are
going to use the ratio method. In order to do this, you will need one piece of information which
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is the width of your field of view. On our microscopes, the field of view is 305µm at 40X
magnification and 205µm at 60X magnification.
8. Using the image in Figure 1 as an example, we can see that the total width of the field of view is
13.6 cm or 136 mm (Image A). The cell (Gray) is 3.7 cm or 37mm (Image B).
Figure 1: Measurements
9. Dividing 37mm/136mm = 0.272 which we multiply by the total length of the field of view so
(60x (0.272 * 205µm = 55.77 µm) rounded for significant figures gives us a cell size of 56µm.
10. Using your image from step 4, label the trophozoites, the nuclei and the flagella. Insert your
image below.
11. Describe the shape of the throphozoites.
EXERCISE 2: Microscopic Examination of Entamoeba Granulosus Cysts
Data Collection:
1. Select the Entamoeba histolytica cysts slide (slide Cassette 3: #10) from the slide loader. Using
the 10X objective identify the cysts and bring them into focus.
2. Carefully work your way through all the objectives focusing with each one until you reach the
60X objective and capture an image. Insert your image of Entamoeba histolytica cysts below.
Analysis:
3. Using your picture from step 2, label the cysts. Inset your labeled image below.
4. Utilizing the method from Exercise 1, determine the length of the Entamoeba histolytica cysts.
5. Based on your observation and measurement, describe the difference between shape of Giardia
lamblia cysts and Entamoeba histolytica cysts
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6. Are your results in correlation with what you have predicted earlier?
7. Rewrite your hypothesis to take into account the new information you have learned in this
exercise.
8. What is the impact of drinking water contaminated with cysts on the digestive system and the
overall function of the human body?
EXERCISE 3: Microscopic Examination of Taenia Saginata Eggs
Data Collection:
1. Select the Taenia saginata eggs slide (Slide Cassette 3: #5) from the slide loader. Using the 10X
objective identify Taenia saginata eggs and bring them into focus.
2. Carefully work your way through all the objectives focusing with each one until you reach the
60X objective and capture an image. Insert your image of Taenia saginata eggs below.
Analysis:
3. Utilizing the image from step 2, label the eggs. Insert the labeled image below.
4. Utilizing the method from Exercise 1, determine the diameter of Taenia saginata eggs
5. Based on your observation and measurement, describe the difference between the size of
Taenia saginata eggs and Entamoeba granulosus cysts?
6. Are your results in correlation with what you have predicted earlier?
7. Rewrite your hypothesis to take into account the new information you have learned in this
exercise.
8. What structure is more resistant the cyst or the egg? Explain your answer.
EXERCISE 4: Microscopic Examination of Plamodium Falciparum Rings and
Plasmodium Vivax All Stages
Data Collection:
1. Select the Plasmodium falciparum rings slide (Slide Cassette 3: #16) from the slide loader. Using
the 10X objective identify the Plasmodium falciparum rings and bring them into focus.
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2. Carefully work your way through all the objectives focusing with each one until you reach the
60X objective and capture an image. Insert your image Plasmodium falciparum rings below.
3. Select the Plasmodium vivax all stages slide (Slide Cassette 3: #17) from the slide loader. Using
the 10X objective identify the different stages and bring them into focus.
4. Carefully work your way through all the objectives focusing with each one until you reach the
60X objective and capture an image. Insert your image Plasmodium vivax below.
Analysis:
5. Using the image from step 2, label the Plasmodium falciparum rings. Insert your image below.
6. Using the image from step 4, label the different larval stages of Plasmodium vivax. Insert your
image below.
7. Utilizing the method from Exercise 1, determine the diameter of Plasmodium falciparum rings
and Plasmodium vivax rings.
8. Were there any difference between the rings produced by the 2 species. Does this observation
match your prediction form the pre-lab? Why or why not.
EXERCISE 5: Microscopic Examination of Ascaris Lumbricoides Eggs
Data Collection:
1. Select the Ascaris lumbricoides eggs slide (Slide Cassette 3: #3) from the slide loader. Using the
10X objective, identify the cysts and bring them into focus.
2. Carefully work your way through all the objectives focusing with each one until you reach the
60X objective and capture an image. Insert your images of Ascaris lumbricoides eggs.
Analysis:
3. Using the image from step 2, label the eggs. Insert your images of Ascaris lumbricoides eggs.
4. Were your predictions about the differences between an egg and cysts from the pre-lab
correct? Why or why not.
SUMMARY QUESTIONS:
1. What is the difference between a cyst and a trophozoite? Define each one.
Which one is less infective when found in a feces sample? Explain your answer.
2. Compare and contrast the life cycle of Taenia saginata and Plasmodium falciparum.
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3. What is the impact of an infection by Plasmodium falciparum on red blood cells, blood fluidity
and blood circulation overall?
4. You have been assigned to work on a Worldwide project with the World Health Organization
(WHO), the National Health Institute (NIH) and the Center for Disease Control (CDC) aiming at
reducing the prevalence of Schistomiasis (infection par the parasite: Schistosomia mansoni) in
the World.
Write a proposal describing:
a) The different strategies that might be developed and implemented to act at different
levels of the parasite cycle.
b) Provide the rationale behind each one of your strategies, their outcomes, benefits and
their limitations.
5. Research a different eukaryotic human parasite than the ones you studied in this lab. Describe
the mode of transmission, the host, the different stages of its cycle and ways to prevent
transmission to human.
6. Write a paragraph describing the latest initiatives and actions taken Worldwide to eradicate
Malaria.
What obstacles have been encountered so far by governmental and non-governmental
agencies?
Discuss pharmaceutical companies’ future plans.
7. One of your friends returned from a trip to Kenya and has been complaining in the last few
weeks from fatigue, dizziness and blood in feces.
Based on your knowledge of different parasites, what parasitology test will you recommend him
to do? And why?
8. Research more details on the following parasitic infections: leishmaniasis and trypanosomiasis
and contrast them in term of causative agents, signs, symptoms and transmission vectors.
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PREPARING FOR THIS NANSLO LAB ACTIVITY:
Read and understand the information below before you proceed with the lab!
Scheduling an Appointment Using the NANSLO Scheduling System
Your instructor has reserved a block of time through the NANSLO Scheduling System for you to complete
this activity. For more information on how to set up a time to access this NANSLO lab activity, see
www.wiche.edu/nanslo/scheduling-software.
Students Accessing a NANSLO Lab Activity for the First Time
You must install software on your computer before accessing a NANSLO lab activity for the first time.
Use this link to access instructions on how to install this software based on the NANSLO lab listed below
that you will use to access your lab activity – www.wiche.edu/nanslo/lab-tutorials
1. NANSLO Colorado Node -- all Colorado colleges.
2. NANSLO Montana Node -- Great Falls College Montana State University, Flathead Valley
Community College, Lake Area Technical Institute, and Laramie County Community College.
3. NANSLO British Columbia Node -- Kodiak College.
Using the Microscope for a NANSLO Remote Web-based Science Lab Activity
We've provided you with three ways to learn how to use the microscope for this NANSLO lab activity:
1. Read these instructions.
2. Watch this short video https://www.youtube.com/watch?feature=player_embedded&v=m7w9ssIgVdw.
3. Print off these instructions to read (PDF version of the instructions.)
NOTE: The conference number in this video tutorial is an example. See “Communicating with
Your Lab Partners” below to determine the toll free number and pin to use for your NANSLO lab
activity.
MICROSCOPE RWSL LAB INTERFACE INSTRUCTIONS
The Remote Web-based Science Lab (RWSL) microscope is a high quality digital microscope located at
the NANSLO Node. Using a web interface as shown below, you can control every function of the
microscope just as if you were sitting in front of it.
The equipment control software shown below is written using the LabVIEW software from National
Instruments. The user interface is presented as a LabVIEW control panel which will be referred to as the
lab interface for the remainder of the document.
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Figure 1: Remote Web-based Science Lab (RWSL) Microscope Lab Interface
COMMUNICATING WITH YOUR LAB PARTNERS
As soon as you have accessed this lab interface, call into the toll free conference number shown on the
control panel to communicate with your lab partners and with the Lab Technicians. Use the PIN code
noted to join your lab partners. Only one person can be in control of the equipment at any one time so
talking together on a conference line helps to coordinate control of the equipment and creates a more
collaborative environment for you and your lab partners.
GAINING CONTROL OF THE MICROSCOPE
Right click anywhere in the grey area of the lab interface and choose “Request Control of VI” from the
dialogue box that appears when multiple students are using the microscope at the same time,. After
you request control, you may have to wait a short time before you actually receive control and are able
to use the features on this lab interface.
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Figure 2: Selecting "Request Control of VI"
RELEASING CONTROL OF THE MICROSCOPE
To release control of the microscope so that another student can use it, right click anywhere in the grey
area of the lab interface and choose "Release Control of VI" from the dialogue box that appears.
Figure 3: Selecting "Release Control of VI"
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MICROSCOPE CONTROLS
The Stage Controls allow you to adjust the visual of the specimen that has been placed on the stage of
the microscope, select lenses with various magnifications, and select whether or not the condenser lens
is in the light beam. Below are more specific instructions on using these controls. When using the
arrows on this lab interface, click and hold the arrow until the desired effect is achieved or click and wait
to view the result before clicking again. Quick clicks on the arrows may cause the system to lock up.
Figure 4: Microscope Controls - Stage, Objective & Condenser
Stage Controls: Using the left and right and up and down arrows found to the right of the microscope
image in the Stage Control area, moves the microscope stage which holds the specimen. These arrows
allow you to precisely control the position of the specimen on the stage.
1. Use the "Right" and "Left" arrows to move the Stage so that you can view the specimen from
left to right.
2. Use the "Backward" and "Forward" arrows to move the Stage so that you can view the top,
middle or bottom of the specimen.
3. Use the "Up" and "Down" arrows to move the stage closer or farther away from the objective
lens to bring a specimen into focus. BE CAREFUL! Don't move the stage too close to the lens.
When selecting the button between the "Up" and "Down" arrows, you can toggle between “Coarse” and
“Fine” focus. When the button is dark green and “Coarse/Fine” is displayed to the right of the button,
the microscope is in “Coarse” focus. When the button is bright green and “Fine” is displayed, the
microscope is in “Fine” focus. Typically, you will start with coarse focus which moves the stage in large
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increments and then use fine focus to complete your final focusing as it moves the stage in smaller
increments. There is no difference between the course and fine focus when using the 60X objective
NOTE: When you click on these arrows, the specimen appears to move in the opposite direction. Since
the objective stays fixed, the image moves in the opposite direction of the stage. This is how these
controls work on most microscopes so the "feel" of the microscope is preserved over the web.
Figure 5: Right/Left & Backward/Forward Stage
Controls
Figure 6: Up/Down Stage Controls & Coarse/Fine
Focus Control
Objective: A microscope mounts an objective lens very close to the object to be viewed. Depending on
need, different lenses with different power will be used on the microscope. This microscope feature
multiple objectives, each with different power, mounted on a rotating turret. The larger the
magnification numbers the greater the magnification. For example, if a specimen is viewed through a
40X objective lens, the magnifier in that lens displays the specimen 40 times larger than an equivalent
view as seen by the unaided eye. Remember that the ocular or other lenses also add to the
magnification.
This microscope has five lenses – 4X, 10X, 20X, 40X, and 60X. Use the arrows below the objective lens
box that indicates the magnification of the current objective lens to move to a higher or lower
magnification lens. If you have activated the “Picture-in-Picture” Preset 2 (see below) you will be able to
see the objective lens move when you select a new magnification.
Condenser: The condenser controls whether or not the condenser lens is in the light beam. You want to
have the condenser OUT for the 4x objective but IN for all the others.
SELECTING A CASSETTE AND LOADING SLIDES ONTO THE STAGE
There are two tabs on the lab interface. When you first access the lab interface, the "Microscope" tab is
displayed by default. Click on the Slide Loader tab at the top of the screen to access the controls for the
Slide Loader robot. There can be up to four cassettes available on the Slide Loader. These cassettes are
used to store slides, and each can hold up to 50 slides. The cassettes available to you are dependent on
the lab activity to be completed. Once a cassette has been selected, you will use the drop-down list to
select your slides.
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Figure 7: Select the Slide Loader Tab to select a cassette and slides.
EXAMPLE OF HOW TO LOAD SLIDES
In this example, we have selected Cassette #1. Using the drop-down menu, we have selected "1:
Colored Threads Whole Mount." Then, we selected the "Load" button. A message indicates that the
slide is loading. Using the picture-in-picture camera, you can watch this happening. The robotics selects
the slide and places it on the microscope stage.
Figure 8: Selecting the slide
"1: Colored Threads Whole Mount" from Cassette #1
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Notice that when a slide is actually on the microscope (or when it is being loaded or unloaded), the
cassette controls are greyed out so you cannot load a second slide until the first is removed. Once the
slide is on the microscope stage, it will be listed in the "Current Slide on Stage" box. The only thing that
the Slide Loader robot can do is return it to the cassette when the "Return Slide to Cassette" button is
selected.
Figure 9: "LOADING SLIDE ... PLEASE WAIT" is displayed
in the "Current Slide on Stage" window
Select the "Microscope" tab to perform the NANSLO lab activity. Once you are finished with the slide,
select the "Slide Loader" tab and select "Return Slide to Cassette" button. Once the slide is returned to
the cassette, the Slide Loader controls are again available to select another slide from the cassette.
ENHANCING THE MICROSCOPE IMAGE
The digital camera mounted on the microscope has a camera control unit that is equipped with a series
of image processing functions that enable you to quickly and easily correct imaging problems that arise
from low or high contrast, poor focus, insufficient or uneven illumination, sample shading or
discoloration and noise. The most common reason for uneven elimination is a light source that does not
completely fill the field of view on lower magnifications. The White Balance should be used only if the
image appears to be brown or gray, and you think you might need to adjust it (although it won't hurt
anything to click this button).
A choice of color modes can be selected in the Microscope Image area and are used to display the image
in different color palettes in order to highlight certain features. The default setting is "Normal."
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Figure 10: Microscope Image Special Effects and Other Image Controls for Camera
Here is a description of each option:
1. In the “Normal” mode, the sample is displayed in its true colors.
2. In the “Negative” mode, the sample is displayed in a color-inverted form, where red, green, and
blue values are converted into their complementary colors. The technique is useful in situations
when color inversion can be of benefit in exposing subtle details or in quantitative analysis of
samples.
3. In the “Blue Black” mode, the black portions of a grayscale negative sample are displayed in
blue. This mode is often useful to reveal details in samples having a high degree of contrast. The
“Blue Black” filter can aid you in examining a wide spectrum of difficult samples.
4. In the “Black & White” mode, a grayscale image of the sample is displayed.
5. In the “Sepia” mode, a brown scale (black and white) image of the sample is displayed. Although
typically this filter is of little utility, it can be employed to alter image color characteristics to
improve the visualization of sample detail.
6. At times, the sample may have an unacceptable color quality. Use “White Balance” calibration
to remove the color cast. This process is often referred to as white balancing.
7. Auto Exposure is on automatically. You do not need to do anything with Auto Exposure unless
you are adjusting the luminance. If you are doing so, you should turn off Auto Exposure by
clicking on the button. The green light is now off. Now adjust the luminance. See explanation
below.
Reference: http://www.microscopyu.com/articles/digitalimaging/dn100/correctingimages.html
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Auto Exposure is normally turned on, but you can turn it off if you want to play around with the
brightness of the light source and not have the microscope camera automatically adjust it. It is usually
best, though, to leave it turned on.
When you turn off the Auto Exposure, the button turns dark green. Some new controls appear that let
you turn the LED off or on, and also adjust the intensity of the light source. The intensity of the light
source can be increased or decreased manually with the dial that now appears next to the Objective
control when Auto Exposure is turned off.
Figure 11: Additional controls available when Auto Exposure is turned off
CAPTURING AND SAVING A MICROSCOPE IMAGE
When the “Capture Image” button is pressed, a high-resolution image of what is currently in the field of
view of the objective is captured. While the image is being captured, the button will be illuminated
bright green. The capture is complete when the light turns off. Be patient as this may take several
seconds to complete.
After the Capture Image light turns off, select the “View Captured Image” tab on the bottom of this
control panel to view the image.
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Figure 12: Click the capture image button (#1), wait till the green light goes off,
and then select the View Captured Image tab (#2)
After opening this image through the View Captured Image tab, you will need to take a snapshot of it
and save it to your computer. There are several ways to do this, depending on your operating system.
WINDOWS:
1.
Pressing the two keys ALT and Print Screen simultaneously will copy the active window into
your computer clipboard. Then you can past it into a document.
2. Windows 7 and above has a Snipping Tool program under Programs/Accessories which can
capture selected areas of the screen.
3. Right click on it and select "Copy" from the menu presented. After right clicking and selecting
Copy, just open a document and right click and select Paste. You can either paste it directly into
your lab report document or into another one for safe keeping until you use it later. You can
use drawing tools in your word processing editor to annotate this image so you can show your
instructor that you know what you were suppose to be looking for!
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Figure 13: Right click and select Copy to paste the image into a document.
MAC:
1. Press these three keys simultaneously –
. This will change your cursor icon into a
little cross.
2. Now press the spacebar, and the icon becomes a camera. Click in the image window you want
to take a snapshot of, and it will save the image to a file on your desktop.
There are lots of free screenshot utilities you can also use to capture this image.
If you are familiar with saving a document to your computer, you also can select “Save Image As” from
the pop-up menu, give the image a name and then select a location on your computer where you want
this image to be saved for future use.
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MICROSCOPE IMAGE VIEW WINDOW
The Image View Window displays the real-time video feed from the digital camera “looking through” the
microscope.
Figure 14: Image View Window
PICTURE-IN-PICTURE CONTROLS - CAMERA PRESET POSITIONS AND PAN-TILT-ZOOM CONTROLS
When you click on the "Picture-in-Picture" button, it turns bright green. A second real-time video feed
from another digital camera appears in the Image View Window. The controls shown in Figure 15 are all
operational when the Picture-in-Picture feature is selected.
Figure 15: Picture-in-Picture Image Controls
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CAMERA PRESETS
There are six camera preset positions.
Figure 16: Picture-in-picture Camera Preset 1 and 6
- Displays the microscope, microscope camera, and a
camera control unit projecting the sample on the
Stage.
Figure 17: Picture-in-picture Camera Preset 2:
Displays a closeup of the objective lens.
Figure 18: Picture-in-picture Camera Preset 3 Displays a closeup of the camera control unit
projecting the sample on the Stage.
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Figure 19: Picture-in-picture Camera Preset 4 Displays the microscope eye piece and
the camera mounted to the microscope.
Figure 20: Picture-in-picture Camera Preset 5 Displays the Condenser Lens underneath the Stage
that focuses the light on the sample. The
Condenser Lens controls the width of the beam. In
some instances you will want a tighter beam while
in other cases you will want a broader beam to
control the image quality. This setting has been
optimized for you.
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PAN, TILT, ZOOM CONTROLS FOR PICTURE-IN-PICTURE
For each camera preset view, additional camera options are available.
1. Use the up and down arrows to tilt the camera up or down.
2. Use the right and left arrows to pan right or left.
3. Use the left "Zoom OUT" arrow and right "Zoom IN" arrow to zoom out and in.
Figure 21: Picture-in-picture Camera - Example of "Zoom In" capability
For more information about NANSLO, visit www.wiche.edu/nanslo.
All material produced subject to:
Creative Commons Attribution 3.0 United States License 3
This product was funded by a grant awarded by the U.S.
Department of Labor’s Employment and Training
Administration. The product was created by the grantee and
does not necessarily reflect the official position of the U.S.
Department of Labor. The Department of Labor makes no
guarantees, warranties, or assurances of any kind, express or
implied, with respect to such information, including any
information on linked sites and including, but not lim ited to,
accuracy of the information or its completeness, timeliness,
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PRE-LAB EXERCISE 3: Microscopic Examination of Taenia