Day 2: Introduction to protozoa and algae

by Charlotte Omoto

Protozoa and algae are simple organisms that are useful in biology lab classes. Many of them have cilia/flagella for movement. The two protozoa we will study today are Paramecium and Tetrahymena. I will also briefly introduce the single cell biflagellate algae, Chlamydomonas.

Paramecium and Tetrahymena are both ciliated protozoa that swim and feed by the use of their cilia. They are fun little critters to observe. In fact, I think it is a good exercise to first observe without doing any manipulations, and without telling the students exactly what to observe. With careful observation, students may observe ciliary motion, food vacuole formation, contractile vacuole filling and emptying, and cytoplasmic motion within the cell including the food vacuoles.

Chlamydomonas is a biflagellate algae. Like other algae, Chlamydomonas has chlorophyll and thus is green. They also have flagella which is very much like the cilia found on Paramecium and Tetrahymena and totally unlike the flagella found on bacteria. The only thing common between Chlamydomonas flagella and bacterial flagella is the unfortunate choice of the same word. Many things can be done with Chlamydomonas, but for this time, I will simply demonstrate a way to show positive and negative phototaxis. Soon there will be a web site for a NSF-funded project to show how to use Chlamydomonas in the classroom. I will put a link to that site here when it becomes available.

One very useful addition to a simple microscope to help observe these processes is a dark-field filter (see article on dark-field filter). Which I will demonstrate.

In this workshop, we can only touch on a couple of processes, but these organisms are great for showing many other interesting behavioral phenomena including sex. When I use these organisms in class, I like to make sure that students realize that this is not just an empty lab exercise but that it has real connections to processes that occurs in human cells.

For example, anyone observing live ciliated protozoa will soon see that they swim in particular directions, occassionally back up or go around objects. These movements are not random walks as we discussed for E. coli. These cells have some of the same cellular machinery as our nerve cells. For example, if the front end of a paramecium is bumped, paramecium will stop, and even back up a few cell length if the bump is strong enough and swim off in a different direction. On the other hand, if the cell is bumped at its rear end, it can swim faster. These behavior makes sense and seems to give the cells a ``brain'', but these behaviors are mediated through membrane ion channels found on the cell surface that detects these bumps. The channels allow certain ions to flow into or out of the cell that causes changes similar to that in our nerve cells when they are activated. Of course, we don't have the equipment to give these single celled critters bumps but we can get similar behavior from paramecium by introducing specific ions that flow through these membrane ion channels.

Paramecium and Tetrahymena ``eat'' by phagocytosis through their oral apparatus and by pinching in food vacuoles. We will observe this process in detail. Students might ask how this could possibly be related to any human cell processes. But phagocytosis is a class of processes known as endocytosis (meaning bringing into cells) which is involved in numerous human cell processes. For example, our cells bring in cholesterol and iron into the cells in this way. Macrophages, a type of white blood cells, protect us from foreign bacteria that invade our bodies by literally ``eating'' them by endocytosis. Babies acquire immunity early in life from their mother's milk which has antibodies in it. Transcytosis, a type of endocytic process where by molecules go across a cellular layer, brings the antibodies from the mother's blood to her milk and how the antibodies are transferred from babies tummies to its blood. Endocytosis also plays a role in pathological process of asbestosis. Asbestos fibers are endocytosed by cells in our lungs. But since its not organic material and can't be broken down by our cells, it remains in the cells and the fibers eventually kill the cells! Naturally, studying protozoa do these things are a lot easier than trying to study these other processes.

All three of the critters we mention today have sex and Carolina even sells ``Mating Kits'' to demonstrate it.

The experiment

Quickie Experiments

All these experiments can be modified to be a full experiment or student initiated inquiry.

Effect of ions on paramecium behavior

This experiment can be done either by introducing the test solution into a dish of paramecium, or conversely placing a drop of paramecium into a dish of test solution. The behavior of the paramecium is viewed in the petri dish under the dissecting microscope. Take a pasteur pipette with one of the test solutions and try it.

Positve phototaxis by Chlamydomonas

Place a dilute culture of Chlamydomonas in a test tube (just slightly green)

Make a light mask so that only a middle ~cm band of light shines on the tube and the remainder is dark.

Note the accumulation of cells to the lighted band over time.

This experiment can be added to and modified to include different light intensity (can be done by distance from the same light source or with filters), different wavelength, and measuring the speed of phototaxis by noting the rate in change of the accumulated band of cells and comparing it to a standard.

Negative phototaxis by Chlamydomonas

Place a drop of fairly dense culture on a microscope slide with the light covered with a red filter. This red filter should still allow you to observe the cells swimming. Now quickly withdraw the red filter which suddenly lets in bright white light.

This experiment can be expanded by changing the ionic composition of the solution. Chlamydomonas tolerate plain distilled water for quite a duration so one can first observe whether the same reaction is observed in distilled water as in their growth media and then add back ions that were in their media.

Endocytosis of India Ink by Tetrahymena

1. Add an equal volume (2 mls) of Tetrahymena to an equal volume (2 mls) of 1% India Ink.
2. Sample immediately after mixing, and over the next 30 minutes by putting two drops of cells + ink suspension into 1 drop of 0.3% glutaraldehyde in a test tube. Place on a microscope slide, cover with slide cover. The glutaraldehyde kills the cells so that the process of endocytosis is stopped.
3. Note the number of food vacuoles by counting the number of vacuoles per cell for at least 20 cells for each time point. Since the cells are killed by glutaraldehyde, as long as you mix the cells with glutaraldehyde at the correct times, you can do the counting at your leisure.

Endocytosis and change in pH observed in Paramecium

1. Add 1 drop of yeast congo red to a tube of Paramecium. Note the time.
2. Mix the cells with enough Protoslo on the microscope slide to slow down their movement for observation. Do not use formalin since it kills the cells and the pH you observe would not reflect the pH inside the vacuole. Note the changes with time in the number, size and color of the yeast in the food vacuoles. Continue observation until you have observed a change in the color of some of the food vacuoles.
3. Describe and quantify your observations. You may need to repeat this several times to get enough observations to be sure of your results.

Pre-Lab Preparation

Paramecium culture procedure

In a culture dish, half fill with tap water. Let stand several hours.Add a small amount of fresh horse manure (about the same volume as that of a hazelnut) filter out debris through cheesecloth and paper towel.
Innoculate with Paramecium culture.

Tetrahymena.

Get bacteria-free cultures, if buying from a company, ie Carolina.
Prepare the following Tetrahymena media plates in 500 ml flask:

• 4.0 g proteose peptone
• 1.0 g dextrose
• 0.4 g yeast extract
• 2.0 ml FeCl3 (45 mM)*
• 100 ml glass distilled water.

Mix well.
Dissolve:

• 3.0 g agar in
• 30 ml glass distilled water.

Add the agar to the other mixture. Bring the volume to 200 ml. Autoclave for 20 minutes. Pour 25 ml into 95 x 15 mm sterile plastic petri dishes. If prepared ahead of time, store wrapped and upside down in refrigerator.
Also prepare same media without agar.
To grow Tetrahymena add 5.0 ml sterile liquid media to each dish of solid medium.
Transfer a loop of cells from stationary liquid culture or another solid culture. Cells should be transferred every three days. (can start enough cultures for whole class from one plate). It seems that with 95 x 15 mm plates, you can keep a culture going by transferring once a week.

If the air is dry, the plates may be kept in a covered container kept moist with a towel kept moist with distilled water.

Chlamydomonas and methods for culturing them can be obtained from Carolina

yeast congo red.

Add 3 g dry yeast to 10 ml water and stir. Add 0.3 g Congo Red.
Boil gently for 10 minutes. This will keep in the refrigerator for a week.
This thick solution may be diluted by buffer prior to use.

India ink (Hunt-Speedball super black, or similar without detergent)

Make 1% in distilled water.

Paramecium behavior test solutions

All solutions contain 1mM Ca, 0.01mM EDTA, 1mM Hepes pH 7.2
Test solutions contain in addition either:

• 10 mM NaCl
• 6 mM BaCl₂
• 30 mM KCl

Continue to Day 3...

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