Tetrahymena Movement Experiment Manual

John Giannini & Michele Severson

St. Olaf College, 2015

1520 St. Olaf Ave, Northfield MN 55057

giannini@stolaf.edu

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Preface

This manual is intended to provide students with additional experiments to try

using Tetrahymena movement analysis. See our Tetrahymena movement manual for

the methods to measure movement.

Acknowledgements

We would like to thank Chris Stewart for his technical support during the

development of this manual. We would also like to thank Eric Cole for his assistance

and providing us with Tetrahymena stock culture.

Stewart_Giannini_Manual_on_Analyzing_Tetrahymena_Movement_2016

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Experiments with Tetrahymena Movement

Introduction As described in the introduction to this manual, Tetrahymena have a natural pattern of movement. This can be modified as they respond to changes in their environment. Experiments 1. You can simply observe and record the normal behavior of cells in a log

phase culture grown in modified NEFF media.

Grow a 24 hour culture of Tetrahymena in modified NEFF media (31° C with gentle mixing should produce Tetrahymena in the log phase of growth)

Use the chamber of your choice (instructions begin on page 4). o The slide chamber works best for long term viewing

Fill your chamber with culture and wait 10 minutes and observe Collect data, descriptions, photos, movies

2. You can also observe movement in a starved Tetrahymena culture Grow a 24 hour culture of Tetrahymena Centrifuge the cells in a clinical centrifuge for 1 minute at a setting

of 4

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Re-suspend cells in buffer 25mL of H20 TRIS-HCl pH 7.4 Centrifuge the cells once again and re-suspend Let the cells incubate in a culture flask for 4-24 hours at 31° C

with gentle shaking Observe their behavior Collect data

3. You can observe cell movement at different stages of growth (log-phase, stationary phase)

Grow a tetrahymena culture and monitor cell movement at 24, 48, 60, and 72 hours

4. You can observe the movement of cells in media at different pHs. Grow a 24 hour culture of Tetrahymena in modified NEFF media Set the pH of some fresh growth media using 0.1 M HCl or 0.1 M

KOH to various pHs (4.0, 5.0, 6.0, 7.0, 7.5, 8.0) In a table top centrifuge, spin 1.5 ml of Tetrahymena culture at

14,000 RPM for 20 seconds.

Re-suspend the pellet in 1.5 μl of the desired media that was pH modified.

5. You can observe the changes in movement of Tetrahymena in the presence of various environmental pollutants (organics, salts, heavy metals)

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Grow a 24 hour Tetrahymena culture in modified NEFF media Place 500 μl of culture in a 1.5 ml microfuge tube Add various amounts of the pollutant Make sure you control for the volume change due to the added

pollutant Wait 30 minutes and observe (includes chamber equilibration)

6. You can measure the effect of various ion channel blockers on movement of Tetrahymena

Grow a 24 hours Tetrahymena culture Place 500 μl of culture in a 1.5 ml microfuge tube Add various K+ or Ca2+ channel inhibitors Wait 30 minutes and observe (includes chamber equilibration)

Example:

NN C 55-0396 1μl final concentration (T Type Ca2+ channels)

1-Octanol 10μl final concentration (T Type Ca2+ channels)

Flunarizine 1-10 μM final concentration (general Ca2+ blocker)

B. Experiments on Chemotaxis in Tetrahymena

Introduction Tetrahymena have the ability to sense their environment and respond to the presence of various repellents and attractants. As described earlier, they demonstrate different movement patterns when placed into media with attractants and repellents.

Experiments

Grow a 24 hour culture of Tetrahymena in modified NEFF media One way to do the experiment is to add attractants or repellents to

500 μl of Tetrahymena culture, wait 30 minutes, and then observe (includes chamber equilibration)

o Note: the culture media is itself an attractant Another way to perform this study is to centrifuge the cells in a

clinical centrifuge for 1 minute at a speed setting of 4, then: o Re-suspend in 10 μM TRIS-HCl pH 7.4 o Repeat this once more o Add repellents and attractants in 500μl of culture free of media

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C. Experiments with Galvanotaxis in Tetrahymena Introduction

When Tetrahymena are placed in an electric field, they go from a random pattern of movement and align their movement with the electric field. They will move specifically toward the cathode. The reason for this response is believed to be due to a depolarization of the membrane potential on the side of the cell facing the cathode and a hyperpolarization of the membrane potential on the side of the cell facing the anode. The depolarization cause Ca2+ channels to open, which causes the cilia to reverse direction on the end of the cell facing the cathode. The hyperpolarization on the anode end forces K+ channels to open, causing the cilia to beat faster. The net result is movement toward the cathode.

List of Attractants & Repellents Attractants: Leucine Methionine Proteose Peptone (media component) Yeast Extract (media component) Repellents: Lysozyme (1μM final concentration) GTP (10 μM final concentration)

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Experiments

1. Grow a 24 hour culture of Tetrahymena in modified NEFF media

Construct a special chamber with electrodes following directions below:

Take a microscope slide and place a thin snake of Fun-Tak on each end.

Make two electrodes using 26-gauge wire.

Place the wires in the putty.

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Place another slide over the top and squeeze tightly.

Next, fill the chamber with Tetrahymena and wait 10 minutes. Observe and document the movement patterns of Tetrahymena.

Now hook up a 9-volt battery to the electrodes using alligator clips.

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Observe and document movement (the cells will die when they contact the electrode, so only keep the power on for a short time)

You can reverse the electrode connection and observe the movement

2. You can do the above experiment with starved cells (see previous experiments for how to starve cells)

3. Observe galvanotaxis in the presence of a Ca2+ channel inhibitor 4. By using different combinations of batteries, you can vary the voltage 5. Observe the phenomenon in the presence of a calcium ionophore, A23187

(100 μM final concentration) 6. Observe galvanotaxis in the presence of various concentrations of CaCl2

Grow a 24 hour Tetrahymena culture in NEFF media Spin the cells in a clinical centrifuge for 1 minute at a setting of 4 Pour off the supernatant and suspend cells in H20 (24μl) Repeat the above step Place 100 μl of the washed cells in a 1.5μl microcentrifuge tube Add various amounts (μl) of a 0.1 M CaCl2 stock solution Wait 30 minutes (includes chamber equilibration) Observe and document Tetrahymena behavior with and without

the 9 volt battery D. Experiments with Aerotaxis in Tetrahymena

Introduction Aerotaxis in Tetrahymena is when the organism moves toward an O2-rich environment. It is a dramatic occurrence in Tetrahymena, but not much is known about its underlying mechanism. Experiments 1. You can observe the movement patterns of cells close to the O2 source

and further away from the O2 source Grow a late log phase Tetrahymena culture in modified NEFF

media (24-34 hrs at 31 degrees with gentle swirling) Load a slide chamber or capillary tube with Tetrahymena culture

(For the capillary tube, be sure to leave one end open) Wait and observe your chamber every 5 minutes; cells should

accumulate at the end that is exposed to the air Be sure to compare cell movement in the middle of the

chamber or in the middle of the capillary tube to the O2 exposed region

Collect data (pictures, observations, movies, etc.)

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2. You can investigate aerotaxis with starved cells or cells at the stationary phase of growth

Grow a Tetrahymena culture in modified NEFF media for 24 hours To starve cells, wash them 2 times in 10μM TRIS-HCl pH

7.4 by spinning them in a clinical centrifuge at a setting of 4 for 1 minute

Suspend in 10μM TRIS-HCl pH 7.4 and wait 4-24 hours For stationary cells, simply let your culture grow for 50-

70 hours 3. You can test the effect of various pollutants on aerotaxis. 4. Test the effect of Ca2+ and K+ channel blockers on aerotaxis.

E. Experiments with Geotaxis in Tetrahymena Introduction

Tetrahymena cells demonstrate negative geotaxis. When placed in a vertically oriented capillary tube, they move opposite the force of gravity toward the top of the tube. Experiments 1. Observe and document geotaxis behavior in Tetrahymena.

Grow a log phase culture of Tetrahymena in modified NEFF media for 24 hours

Fill a capillary tube with Tetrahymena culture, cap both ends with Fun-Tak, and place on a microscope slide

o Make sure all the air is removed!! Observe the cell movement after 10 minutes Now, orient the capillary so that it is vertical Wait 30 minutes and then observe Tetrahymena distribution in

the capillary tube (includes chamber equilibration) 2. Try and figure out how long it takes for geotaxis to take place

Repeat the above experiment, but rather than wait 30 minutes to observe geotaxis, make observations every 5 minutes until geotaxis is obvious

3. Do Ca2+ channel or K+ channel inhibitors affect geotaxis? 4. See previous experiments for aerotaxis and galvanotaxis for more ideas.

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References

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Aonuma, M., Kadono, T., & Kawano, T. (2006). Inhibition of anodic galvanotaxis of

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Ferracci, J., Ueno, H., Numayama-Tsuruta, K., Imai, Y., Yamaguchi, T., & Ishikawa, T.

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Hellung-Larsen, P., Leick, V., & Tommerup, N. (1986). Chemoattraction in

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Hennessey, T. (2005). Responses of the ciliates Tetrahymena and Paramecium to

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Kim, D., Kim, P., Lee, K., Kim, J., & Kim, M. (2013). Galvanotactic behavior of

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Lampert, T., Nugent, C., Weston, J., Braun, N., & Kuruvilla, H. (2013). Nociceptin

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Ogawa, N., Oku, H., Hashimoto, K., & Ishikawa, M. (2006). A physical model for

galvanotaxis of Paramecium cell. Journal of Theoretical Biology, 242, 314-328.

Schwab, A., Hanley, P., Anke, F., & Stock, C. (2008). Potassium channels keep mobile

cells on the go. Physiology, 23, 212-220.