Biology 220 - Laboratory - Investigating Symbiotic Relationships
(Sillman, 2009)
Please refer to your textbook and lecture notes for background information about symbiosis. Be able to
define and give examples of commensalism, mutualism and parasitism which are different from the examples
we investigate in this laboratory exercise. Questions based on the material in this lab will be included on
lecture exam 3. The Study Guide on the last page will assist you in focusing on important concepts.
Mutualism
(Study Guide questions 1 and 2)
Examine the bacteria (genus Rhizobium) contained within the nodules growing on the roots of the clover (a
legume) provided. Carefully following this procedure:
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Crush one nodule in a small drop of water on a slide.
Remove the root debris and spread the water into a thin film. Allow this film to air dry.
Do not blow on it!
Heat fix the bacteria onto the slide by passing the bottom of the slide over a flame. The slide will get
hot, so use a clothespin to hold it.
Stain the bacterial cells with 2 drops of crystal violet for 30 seconds and rinse with tap water.
Examine the bacteria using the oil immersion lens of the compound microscope (See below).
Do not add a cover slip.
Use of the oil immersion lens:
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Focus at low (100X) power, then at high (430X) power.
Position the nosepiece halfway between the high dry and the oil immersion objectives.
Place one drop of oil on the slide
Click the oil immersion objective in place, making sure the oil "connects" the slide and the objective
lens. Use only fine focus to clearly see image.
When finished, dispose of the slide properly and thoroughly clean the oil immersion objective with a
chemwipe and alcohol. CAUTION: Do not get oil on any of the other objective lenses when removing
the slide.
Bacteria residing inside root nodules change in both appearance and physiology when compared to freeliving individuals of the same species. The altered form is called a ‘bacteroid’. Draw a single Rhizobium
bacteroid you observe from the clover root nodule and estimate the length and width of this cell below:
Parasitism (Study Guide question 3)
Study the life cycle and morphological adaptations of the dog tapeworm Dipylidium caninum, using the slides
and information provided below.
Basic Life Cycle of Dipylidium caninum ( also refer to Figure 1)
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tapeworm eggs (on grass or in soil) are ingested by larval fleas
tapeworms hatch inside fleas, which are the intermediate host
dogs, cats or humans (especially children) ingest fleas. Children are often infected when licked on
mouth by a dog which has been biting its own fleas.
tapeworm attaches to intestinal wall of dog or cat, the final host, using a structure called the scolex,
which usually has sucker plates and hooks.
tapeworms grow and develop inside final host, absorbing nutrients directly through their special skin the tegument. In the environment of the intestines, the tapeworm is bathed in 'nutrient soup', its food
predigested by the host.
tapeworm 'segments' are called proglottids. When mature, proglottids contain both male and female
reproductive structures and produce both sperm and eggs.
eggs are fertilized and develop within the proglottids – a gravid proglottid contains hundreds of
fertilized eggs.
These gravid proglottids break off and are passed out with the feces, releasing the fertilized eggs,
which become 'flea food' to continue the cycle.
What to look for on Slides
Each slide contains 3 'sections' of the tapeworm which you can see with the naked eye. Examining the slides
at 100X magnification you should be able to see the following:
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the scolex, neck and immature proglottids visible on the top, thin section. The hooks on the scolex
may be difficult to see, but sucker plates are clearly visible.
mature proglottids are visible on the middle section. Note the 2 sets of male and 2 sets of female
reproductive organs in each proglottid.
gravid proglottids are visible on the bottom section. Note the eggs encased in small clusters.
Reproductive organs are no longer functional or visible.
Mature Proglottid
Gravid Proglottid
Commensalism
(Read about Apicomplexans) (Study Guide questions 4 - 6)
According to the Centers for Disease Control, malaria kills more than 1 million people each year, most of them
African children. Plasmodium, the organism that causes malaria, belongs to the protist phylum Apicomplexa
and has a complex life cycle. The organism we will study as an example of commensalism is also a member of
the Apicomplexa - Monocystis lumbrici - a symbiont of the earthworm Lumbricus terrestris. Keep in mind that,
unlike Monocystis, most apicomplexans are parasitic.
The following definitions, along with Figure 2, will help you learn the complex life cycle.
sporocyst - small oval sac with a hardened outer shell. This structure is
resistant to temperature and moisture extremes and contains 8
sporozoites, which are too small to be seen with a light microscope.
Sporocysts are released into the soil when a worm dies and decomposes.
sporozoite - the infectious life stage of Monocystis. The sporozoite stage (remember - too small to be seen
with a light microscope) infects a new host when a sporocyst containing sporozoites is ingested by a worm.
The sporozoites are released, penetrate the gut wall and travel via the bloodstream to the seminal vesicles.
seminal vesicles - structures within which sperm form and mature in earthworms. You will be making a wetmount slide of the seminal vesicles to look for Monocystis. You will also see seminal vesicle cells and sperm
cells (with hair-like tails) on your slide.
trophozoite - a large, ameboid life stage of Monocystis which develops from the sporozoite within the seminal
vesicles and feeds on developing sperm cells.
oocyst - Two trophozoites pair and form a large, round cyst within which each
trophozoite forms gametes. Gametes (from different trophozoites) fuse to form
diploid zygotes (the zygote is the only diploid stage in the life cycle). Each
zygote then forms a hard wall around it (we're back to the sporocyst!) and
undergoes meiosis, followed by mitosis. These events result in 8 haploid cells,
the sporozoites, contained within the sporocyst.
Procedure to Examine Monocystis lumbrici
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Obtain a small (1 square mm) piece of seminal vesicle from the earthworm specimen and place on a
slide. Spread the tissue out so it is a relatively thin layer.
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Add a drop of saline and a cover slip. Push down gently to spread the tissue.
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Observe at 100X and answer questions 6 - 12 on your study guide.
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Earthworms are host to many other internal symbionts, including nematodes and other protists.
Locate the nephridia and make a slide of this tissue and examine for symbionts. You may also do a
'coelomic wash' to look for symbionts of the coelomic cavity.
Figure 1 Some Structures of Dipylidium caninum, the Dog Tapeworm
Figure 2 Life Cycle of Monocystis lumbrici
Study Guide Questions
1. Describe the benefits each partner receives in the mutualistic relationship between leguminous plants
and bacteria of the genus Rhizobium.
2. Explain why yearly crop rotation of leguminous and non-leguminous plants is a productive farming
strategy.
3. List at least 3 adaptations of the dog tapeworm to its life style.
4. Which life stage of the apicomplexan Monocystis initiates infection of a new host?
5. "Encystment" is a common strategy of many internal symbionts. It enables them to withstand adverse
conditions and to "wait" for proper conditions to infect a new host. Does this strategy occur in the life
cycle of Monocystis? If so, when?
6. "Magnification" (a great increase in population size) is also a common strategy of internal symbionts.
This increases the chances of successful infection of new hosts. Describe when magnification greatly
increases the number of infectious organisms in the life cycle of Monocystis.
7. Describe the symbiotic relationship exhibited by lichens. Is this relationship best characterized as
mutualism, parasitism or commensalism? Describe the contributions made by each member of this
symbiotic pair.
8. Name the organism that causes malaria and the vector that enables the spread of this disease. Know
the life cycle of the organism that causes malaria. (There is a nice figure outlining this in your
textbook).
9. In 1348, bubonic plague (the Black Death) killed nearly 80% of the world's population, according to
some authorities. This disease is caused by a bacterium, Pasteurella pestis, which is transmitted to man
mainly by the rat flea (the vector). For each of the following human diseases, name the pathogen (the
organism which causes the disease) and the vector (the organism responsible for transmitting the
pathogen from one host to another.
Lyme disease
Rabies
Yellow fever