Part 1 - Get a Lab Appointment and Install Software:

Set up an Account on the Scheduler (FIRST TIME USING NANSLO):Find the email from your instructor with the URL (link) to sign up at the scheduler.Set up your scheduling system account and schedule your lab appointment. NOTE: You cannot make an appointment until two weeks prior to the start date of this lab assignment. You can get your username and password from your email to schedule within this time frame.

Install the Citrix software: – go to http://receiver.citrix.com and click download > accept > run > install (FIRST TIME USING NANSLO).You only have to do this ONCE. Do NOT open it after installing. It will work automatically when you go to your lab. (more info at http://www.wiche.edu/info/nanslo/creative_science/Installing_Citrix_Receiver_Program.pdf)

Scheduling Additional Lab Appointments: Get your scheduler account username and password from your email.Go to the URL (link) given to you by your instructor and set up your appointment.(more info at http://www.wiche.edu/nanslo/creative-science-solutions/students-scheduling-labs)

Changing Your Scheduled Lab Appointment: Get your scheduler account username and password from your email. Go to http://scheduler.nanslo.org and select the “I am a student” button. Log in to go to the student dashboard and modify your appointment time. (more info at http://www.wiche.edu/nanslo/creative-science-solutions/students-scheduling-labs)

Part 2 – Before Lab Day:

Read your lab experiment background and procedure below, pages 1-22.

Submit your completed Pre-Lab 1-5 Exercises (pages 4-15) per your faculty’s instructions.

Watch the Microscope Control Panel Video Tutorial http://www.wiche.edu/nanslo/lab-tutorials#microscope

Part 3 – Lab Day

Log in to your lab session – 2 options:1)Retrieve your email from the scheduler with your appointment info or 2) Log in to the student dashboard and join your session by going to http://scheduler.nanslo.org

NOTE: You cannot log in to your session before the date and start time of your appointment. Use Internet Explorer or Firefox.

Click on the yellow button on the bottom of the screen and follow the instructions to talk to your lab partners and the lab tech.

Remote Lab ActivitySUBJECT 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...................................................................................................... 2Background Information .............................................................................................. 2-4Pre-lab Exercise 1: Microscopic Examination of Giardia Lamblia Cysts and Trophozoites ......................................................................................... 4–6Pre-lab 1 Question ...................................................................................................... 6Pre-lab Exercise 2: Microscopic Examination of Entamoeba Granulosus Cysts........... 6-7Pre-lab 2 Question ...................................................................................................... 7Pre-lab Exercise 3: Microscopic Examination of Taenia Saginata Eggs ........................ 7-9Pre-lab 3 Question ...................................................................................................... 9Pre-lab Exercise 4: Microscopic Examination of Plamodium Falciparum Rings and Plasmodium Vivax All Stages .......................................................................... 9-12 Pre-lab 4 Question ...................................................................................................... 12Pre-lab Exercise 5: Microscopic Examination of Ascaris Lumbricoides Eggs ............... 12-14

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Pre-lab 5 Question ...................................................................................................... 15Equipment ................................................................................................................... 15

CONTENTS FOR THIS NANSLO LAB ACTIVITY – CONT’D

Preparing for this NANSLO Lab Activity ........................................................................ 15-16Experimental Procedure .............................................................................................. 16Exercise 1: Microscopic Examination of Giardia Lamblia Cysts and Trophozoites ...... 16–17Exercise 2: Microscopic Examination of Entamoeba Granulosus Cysts ....................... 17-18Exercise 3: Microscopic Examination of Taenia Saginata Eggs .................................... 18 Exercise 4: Microscopic Examination of Plamodium Falciparum Rings and Plasmodium Vivax All Stages .................................................................................. 18-19Exercise 5: Microscopic Examination of Ascaris Lumbricoides Eggs ............................ 19Summary Questions ..................................................................................................... 19-20Creative Commons Licensing ....................................................................................... 21U.S. Department of Labor Information ........................................................................ 21

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.

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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

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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

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.

Figure 1:  Shows three microscopic slides. The one on the left is a G. intestinalis trophozoites in Kohn stain. The center one is a G. intestinalis cyst stained with trichrome. The one on the right is a G. intestinalis in vitro culture, from a quality control slide. http://www.cdc.gov/parasites/giardia/

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Life Cycle:

Figure 2: Life cycle of Giardiasis in a human.

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.

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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.

Life Cycle:

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Figure 3: Life cycle of Entamoeba Granulosus Cysts in a human.

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

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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.

Life Cycle:

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Figure 4: Life cyle of Taeniasis in a human.

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:

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.

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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.

Life Cycle:

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Figure 5: Life cycle of Malaria Parasite.

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,

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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.

Figure 6: Anopheles Freeborni Mosquito Pumping Bloodhttp://www.cdc.gov/malaria/about/biology/mosquitoes/freeborni_large.html

References:

http://www.cdc.gov/dpdx/malaria/ http://www.cdc.gov/malaria/diagnosis_treatment/diagnosis.htmlhttp://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

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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.

Figure 6: Left/Right: Fertilized eggs of A. lumbricoides in unstained wet mounts of stool. Center: Adult female A. lumbricoides

http://www.cdc.gov/parasites/ascariasis/

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Life Cycle:

Figure 7: Life cycle of the Ascaris lumbricoides.

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:

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http://www.cdc.gov/parasites/ascariasis/biology.html 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.

EQUIPMENT:

Paper Pencil/pen Slides

o Giardia lamblia Cystso Giardia lamblia Trophozoites o Entamoeba histolytica cystso Taenia saginata eggso Plasmodium falciparum ringso Plasmodium vivax all stages o Ascaris lumbricoides eggs

Computer with Internet access for the remote laboratory and for data analysis

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

For those accessing a NANSLO laboratory for the first time, you may need to install software on your computer to access the NANSLO lab activity. Use this link for detailed instructions on

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steps to complete prior to accessing your assigned NANSLO lab activity – www.wiche.edu/nanslo/lab-tutorials.

Video Tutorial for RWSL: A short video demonstrating how to use the Remote Web-based Science Lab (RWSL) control panel for the air track can be viewed at http://www.wiche.edu/nanslo/lab-tutorials#microscope.

NOTE: Disregard the conference number in this video tutorial.

AS SOON AS YOU CONNECT TO THE RWSL CONTROL PANEL: Click on the yellow button at the bottom of the screen (you may need to scroll down to see it). Follow the directions on the pop up window to join the voice conference and talk to your group and the Lab Technician.

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

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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 8: 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.

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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.

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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.

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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?

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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.

8. Based on your knowledge of different parasites, what parasitology test will you recommend him to do? And why?

9. 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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For more information about NANSLO, visit www.wiche.edu/nanslo.

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