Zoology Name: Block: Exercise #6: Protozoan Groups Lab Guide

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1 Zoology Name: Block: Exercise #6: Protozoan Groups Lab Guide The protozoa are a diverse assemblage of unicellular eukaryotic organisms having at least two animal like properties: 1) absence of a cell wall, 2) presence of at least one motile stage in the life cycle. All functions of life are performed within the limits of a single plasma membrane. There is a division of labor within the cytoplasm where complex organelles carry out specific tasks. Protozoans are widespread ecologically. Some are free living while others live in a symbiotic relationship. The following is a condensed classification of the protozoan groups as presently recognized. Amebas: Locomotion by pseudopodia; body naked or with external or internal test or skeleton; asexual reproduction by fission; sexuality, if present, is associated with flagellated gametes; most are free living, some are parasitic. Phylum Euglenozoa: move by means of a flagella. (ex. Euglena) Phylum Chlorophyta: unicellular and multicellular algae; photosynthetic chlorophyll pigments, flagella of equal length and smooth, mostly free living photoautotrophs. (ex. Volvox) Phylum Ciliophora: Cilia or ciliary organelles present in at least one stage of the life cycle; usually two types of nuclei; binary fission; budding and multiple fission also occur; sexuality involving conjugation, autogamy, and cytogamy; heterotrophic nutrition; mostly free living; contractile vacuole typically present. (ex. Paramecium) Exercise 6A: Amebas The amebas may be naked or enclosed in a shell. Live in both fresh and salt water and in soil. Feed on algae, bacteria, protozoa, rotifers, and other microscopic organisms. General features The outer cell membrane is the plasmalemma. It has tiny, hair like extensions which are thought to be involved in adhesion of the cell surface to the substrate or to nutrient particles. The cytoplasm is enclosed by the plasmalemma is differentiated into a thin, perifpheral rim of stiff ectoplasm and inner, more fluid, endoplasm. The amoeba moves and changes shape by thrusting out pseudopodia (false feet), which are extensions of the cell body. The ameba creeps forward when the pseudopodium grasps a surface and a motile force pulls the rest of the cell forward. The advancing end is the hyline cap. As endoplasm flows into the hyline cap, it is converted into stiff ectoplasm, thus extending the sides of the pseudopodium, much like a tube or sleeve. As the pseudopodium lengthens, ectoplasm at the temporary tail end converts again into streaming endoplasm to replenish the forward flow. The food vacuoles that are formed contain food particles surrounded by water and are enclosed in a membrane. Cells and unicellular animals that engulf foreign particles are called phagocytes, and this type of ingestion is known as phagocytosis. It involves encircling the prey by pseudopodia to form a food cup. Along with water, the prey becomes completely enclosed by cytoplasm to for the food vacuole. The contents are digested by enzymes that are secreted by the membrane bound organelles called lysosomes. Undigested elements are eliminated at any point along the plasmalemma. They can also take in tiny fluid droplets and minute food particles using a process that forms channels, called pinocytosis. Osmorgulation (fluid regulation) is controlled by a clear, bubble containing no particles. Called a contractile vacuole. This bubble will gradually increase in size by the accumulation of fluid and then rupture and disappear. It rids the ameba of excess water that has been taken in with food particles or osmosis. The nucleus is disc shaped and finely granulated. You can distinguish it from the contractile vacuole because the latter is perfectly spherical, increases in size, and finally disappears. The nucleus is often found towards the center of the animal. Amebas reproduce asexually by a type of mitotic cell division known as binary fission.

2 Procedure: Live specimens 1. Place a drop of culture water drawn from the bottom of the culture bottle. Add a cover slip and adjust the lighting to provide low light. With low power, explore the contents of the slide. 2. There may be some very tiny cilites swimming about, but the ameba will be gray, irregularly shaped, and finely granular in appearance. If it does not appear to be moving, watch it for a moment to see if the granules are in motion or if the shape is slowly changing. 3. After locating a specimen, please SKETCH below and label the following parts: (Plasmalemma, Nucleus, Food Vacuole, Hyline cap, Endoplasm, Ectoplasm, Psuedopodia, Food Vacuole) 4. Now, make a series of 5-6 outline SKETCHES to show changes in shape that occur in an ameba during a period of about 10 minutes. Use arrows to show the direction of flow of the cytoplasm. 5. Now, use the stained, prepared slides provided and locate any cellular parts you were not able to identify in step 3. Questions 1. What is the average size (µm) of specimens on your slide? (µm) 2. Do you think amebas react to simuli? How could you test this and what have you observed? 3. What is the main function of the contractile vacuole.

3 4. How do you think the ameba will respire (take in oxygen and release carbon dioxide)? 5. Describe how an ameba will reproduce. How would you describe the genetic diversity of the amebas in the culture, assuming they all originated from one specimen? 6. Explain how these protozoa will take in food. Use abundant details. Make sure to mention phagocytosis and pinocytosis. 7. Describe ameboid movement. 8. Drinking untreated water when on vacation in a foreign country can sometimes result in infection by Entamoeba histolytica. What disease does this organism cause and what are the symptoms? Exercise 6B: Euglenozoa Euglena Members of this phyla are flagellated protozoa having one or more flagella undulating, whiplike organelles that move these protozoa efficiently through their fluid environment. There are two major groups: The plantlike flagellates are a pigmented species which possess chlorophyll and consequently can synthesize their own food from carbon dioxide and water like any green plant. They are autotrophic, but some are also heterotrophic. The second group are animal like flagellates and are colorless, lack photosynthetic pigments, and obtain their nutrients by absorbing them through the plasma membrane or engulfing their prey. They are heterotrophic. Euglenoids are found in still pools and ponds. They are spindle shaped and greenish in color. This is caused by the chloroplasts which contain chlorophyll. A nucleus is centrally located but difficult to see. The blunt anterior end bears a little whiplike flagellum which may be seen with reduced light. It emerges from a reservoir which is a clear, flask shaped space in the anterior end. Movement involves generation of waves originating at the base of the flagellum and transmitted along its length to the tip. Often when they stop swimming they change shape. These movements are called euglenoid movement. The body is covered with a protective, but flexible pellicle that is secreted by the ectoplasm which covers the endoplasm. A stigma, or eyespot, is reddish near the base of the flagellum is thought to be light sensitive. Osmoregulation is accomplished by a large contractile vacuole. Most take in no solid food and are photosynthetic, but during times of extreme hardship, no light, they can become saprozoic and taken in small food molecules. Euglena reproduce by longitudinal fission.

4 Procedure 1. Place a drop of protoslo on a clean slide and then add a drop of rich, Euglena culture. Then add a coverslip. Please SKETCH below and label the following parts: (Flagellum, stigma, reservoir, pellicle, nucleus, chloroplast) 2. Now, use the stained, prepared slides provided and locate any cellular parts you were not able to identify in step 1. Questions 1. What is the average length (µm) of Euglena specimens in your culture? (µm) 2. Describe the movement you noticed in the Euglena. 3. How does the Euglena satisfy most of its nutritional requirements? 4. What is the function of the stigma?

5 5. Following the survival strategy of making a cyst to protect itself, are Euglena that emerge from a cyst genetically similar to or different from organisms of previous generations? 6. Given that cyst formation is probably adaptive to conditions in a Euglena s habitat, in what type of habitat do you think a Euglena would be found in? Volvox These are large, beautiful, spherical colonial protists. They are found in stagnant pools and ponds. They may reach a diameter of 2mm are easily visible to the naked eye. They represent a transition between single celled protozoa and the many celled metazoans (like us). They have the beginnings of cell differentiation, which can result in a division of labor among the cells. This group also illustrates an important stage in the development of sex. Each colony is composed of various one celled individuals called zooids. They are connected to each other by protoplasmic strands. The individual cells are separated into somatic (body) cells and a smaller number of reproductive cells. These somatic cells handle nutrition, locomotion, and response to simuli. Most contain chloroplasts and a stigma for light sensitivity. They also contain a pair of flagella for locomotion. Volvox reproduce both sexually and asexually. During spring and summer they reproduce asexually by repeated division of the cells which form miniature daughter colonies. These eventually rupture from the mother colony. In the fall, these asexual colonies will develop sex cells. Some form macrogametes (eggs), while others form packets of microgametes (sperm). The sperm escape and fertilize the eggs, which become zygotes. Each zygote forms a cyst wall around itself to protect it from the harsh conditions of winter. They break out of the cyst in the spring and give rise, by cell division, to new asexual colonies. Procedure 1. Use a pipette and transfer a colony (visible to the naked eye) to a depression slide and cover with a plastic cover slip. 2. Study with low power. If you use high power, be careful so you don t break the slide or objective. 3. Please SKETCH below and label the following parts: (daughter colonies, flagellum) 3. Now, use the stained, prepared slides provided and locate any cellular parts you were not able to identify in step 3.

6 Questions 1. What is the average length (µm) of Volvox specimens in your culture? (µm) 2. In what ways is Volvox similar to Euglena? In what ways does it differ? 3. Were the colonies you observed reproducing asexually or sexually? How would you know? 4. When scientists say that Volvox shows beginnings of cell differentiation, what do they mean? 5. How do somatic and reproductive cells of Volvox differ in function? Which do you think would be more abundant in a colony? 6. Why might sexual reproduction in this group be more common in the fall, whereas asexual reproduction tends to dominate in the spring?

7 Exercise 6C: Ciliophora Paramecium is an active ciliate protozoan common in most fresh water that contains vegetation and decayed organic matter. The paramecium is slipper shaped, rather transparent and colorless. They are very active. It can swim at a rate of 1 to 3mm per second. They have an oral groove that extends from the anterior end to about the middle of the body. At the posterior end of the oral groove is the mouth (a.k.a the cytostome). The oral groove extends from the mouth into the body as a little canal (a.k.a the gullet or cytopharynx). The groove and gullet are lined with strong cilia that are used to draw in food. The cytoplasm is made up of two zones, ectoplasm and endoplasm. Outside the ectoplasm is the pellicle with a delicate cell membrane that lies just underneath the pellicle. Fluid regulation (osmoregulation) is accomplished using a contractile vacuole that is usually located at each end. Cilia perform like the oars of a boat. They have an effective stroke that propels the animal forward and a recovery stroke. The nuclei may be difficult to see in living specimens. Most will have two nuclei; a large macronucleus (which regulates metabolism) and a smaller micronucleus that is used in reproduction and it contains the animals DNA. Sometimes the micronucleus can be covered up by the macronucleus. Paramecium have trichocysts, which are very small and lie just under the pellicle. Under certain conditions, these will explode and release a very fine thread-like filament. They are believed to have a protective function. Some trichocysts can also be used to anchor the animal while feeding. Ciliates are holozoic (a particulate feeder), feeding on very small particles such as bacteria, which it will draw towards its cytostome by action of the cilia in the oral groove. A food vacuole will form which will allow digestion of the food. The anal pore (a.k.a the cytoproct) is formed between the mouth and the posterior end of the body. It is a temporary opening where indigestible food is discharged. Ciliates like paramecium will undergo binary fission to reproduce asexually with a constriction happening across the middle of the body. Sexually, they will exchange genetic material by undergoing conjugation. This does not produce new paramecium, but does allow genetic diversity to take place. In this type of reproduction, they will attach at the oral groove side. The exchange of micronuclei material will take place. Procedure 1. Using a few strands of absorbent cotton, spread these out evenly on a slide and then add a drop or two of Paramecium culture. Cover with a coverslip. 2. Use your scanning objective first, then switch to low power for study.

8 3. Find a paramecium that has been stopped by the cotton fiber, SKETCH and label the following cell parts: (oral groove, cilia, cytostome, macronucleus, micronucleus, contractile vacuole, endoplasm, ectoplasm, food vacuole) 4. Now, use the stained, prepared slides provided and locate any cellular parts you were not able to identify in step 3. Questions 1. What is the average length (µm) of Volvox specimens in your culture? (µm) 2. How is the paramecium similar yet different from Euglena? (Compare/Constrast) 3. Using your textbook and/or online resources, describe how conjugation occurs. \ 4. Cilia are often thought of as a structure which will assist an organism in locomotion, but in paramecium, it has a dueal function. What is the second way that cilia are important to paramecium?

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