NANSLO REMOTE LAB ACTIVITY

 

SUBJECT SEMESTER: XXXXXXXXXXXXXXXXXXXXX

TYPE OF LAB:  XXXXXXXXXXXXXXXXXXXX

 

 

 

 

 

 

 

 

TITLE OF LAB:  Parasitology

Parasitology NANSLO Lab Activity in Word Format last updated May 15, 2014.

 

Lab format: This lab is a remote lab activity.

 

Relationship to theory (if appropriate):  In this lab you will learn cell types and the identifying characteristics between the kingdoms.

 

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 exercise.

 

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 on-line period, and answer questions in analysis sections after your on-line period.  Your instructor will let you know if you are required to complete any optional exercises in this lab.

 

Remote Resources:  Primary - Microscope; Secondary - 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

Pre-lab 4 Question

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 saginate 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

 

LEARNING OBJECTIVES:

 

After completing this lab, 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.

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:

• 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

 

EQUIPMENT:

 

• Paper
• Pencil/pen
• Slides
  
  

o    Giardia Lamblia Cysts
o    Giardia Lamblia Trophozoites
o    Entamoeba Histolytica Cysts
o    Taenia Saginata Eggs
o    Plasmodium Falciparum Rings
o    Plasmodium Vivax All Stages
o    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/

 

 

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

 

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 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 QUESTIONS:

 

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.

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.

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:

• 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.
• 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 pig-tailed 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.

 

 

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.

 

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.

 

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, 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 shapes 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 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.
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.
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.
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:

 

• The different strategies that might be developed and implemented to act at different levels of the parasite cycle.
• 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.

 

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. 
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.

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.

 

 

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" 

 

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 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.

 

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

 

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."

 

 

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

 

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.

 

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!

 

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. 

 

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

 

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.

 

 

 

 

 

 

 

 

 

 

 

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.

 

 

 

 

 

 

 

 

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.

 

 

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Creative Commons Attribution 3.0 United States License 3  

 

 

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