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Project Summary

Difficulty	8
Time required	Long (a couple of weeks)
Prerequisites	An introductory course in chemistry would be useful.
Material Availability	You will need to order the "Chemotaxis in Physarum" kit online; see the Materials and Equipment list for details. This item may have to be ordered by your teacher.
Cost	Average ($50 - $100)
Safety	Be careful working with sharp blades. Adult supervision is recommended.

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Abstract

Objective

Explore chemotactic responses of P. polycephalum in culture exposed to various amounts of glucose.

Introduction

If you have walked through a wooded area that's still damp from a recent rain, you may well have seen a Physarum growing on a tree stump or among the fallen leaves. Members of the genus Physarum are also called slime molds. Slime molds are classified with protists. More than 700 different species of slime molds exist. They have a two-part life cycle. During warm, moist weather, a slime mold lives as a shapeless, growing blob called a plasmodium. The plasmodium may be gray, cream, colorless, bright yellow, or orange. A plasmodium slowly creeps across the ground, moving like an amoeba and consuming bacteria, fungi, and organic debris as it moves. When the environment dries out, the plasmodium transforms into many small, often stalk-like, fruiting bodies that are full of dust-like spores. The tiny spores can remain dormant in the soil for years, waiting for wet weather, at which time they release small, motile (capable of movement) cells. Two motile cells fuse together and grow to become a new plasmodium, starting a new cycle of life.

In order to survive, a Physarum plasmodium searching for food has to respond appropriately to environmental cues. It is constantly "deciding" which way to move: toward areas that will have food or away from areas that contain harmful substances. Part of the "input data" the plasmodium relies on to make this kind decision is based on chemical cues. If the organism senses chemicals that indicate the presence of food, it will move toward the area with more of that chemical. If it senses chemicals that are harmful, it will move to avoid further contact. Movement in response to a chemical signal is called chemotaxis.

Because chemotaxis is easy to observe and measure in Physarum, you can investigate various factors that affect it. In this biology science fair project, you will determine how the chemotactic responses of a Physarum polycephalum (P. polycephalum) plasmodium growing in culture depends on the concentration of glucose.

Terms, Concepts and Questions to Start Background Research

• Physarum
• Slime mold
• Protist
• Plasmodium
• Fruiting body
• Environmental cue
• Chemical cue
• Chemotaxis
• Physarum polycephalum
• Millimolar concentration

Questions

• To what other environmental cues do you think a moving plasmodium might respond?
• How many species of Physarum are found in the state in which you live?
• Based on your research, what are the various stages in the Physarum's life cycle?
• If you go to the kitchen when you smell something cooking, is that an example of chemotaxis?

Bibliography

• Bozzone, D. M. (2005). Using Microbial Eukaryotes for Laboratory Instruction and Student Inquiry. Retrieved December 18, 2009, from http://www.ableweb.org/volumes/vol-26/05-Bozzone.pdf
• Note: Scroll down to see the section about Physarum.
• Lachish, U. (2002). Moles and Molarity. Retrieved March 18, 2010, from http://urila.tripod.com/mole.htm
• Gregory, M.J. (n.d.). Chemotaxis and Phototaxis in Physarum. Retrieved December 18, 2009, from http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/bio
• Chet, I., et al. (1977). Chemotaxis of Physarum polycephalum towards Carbohydrates, Amino Acids and Nucleotides. Journal of General Microbiology 102:145-148. Retrieved December 18, 2009, from http://mic.sgmjournals.org/cgi/reprint/102/1/145

Materials and Equipment

• Chemotaxis in Physarum polycephalum Kit; available from Carolina Biological at www.carolina.com, item #155825B. The kit contains the following:
• Physarum culture, as plasmodium on agar plate
• Petri dishes
• Non-nutrient agar
• Oatmeal flakes (Physarum "food")
• Filter paper
• Aluminum foil
• Microwave
• Oven mitt
• Disposable gloves
• Plastic knives (1 box)
• Ruler, metric
• Plastic cups (12)
• Permanent marker
• Distilled water (1 gallon)
• Glucose tablets, 4 g/tablet; available at most drug stores
• Beaker, 250 mL; available from Carolina Biological at www.carolina.com, item #721502
• Graduated cylinder, 10 mL; available from Carolina Biological at www.carolina.com, item #721600
• Graduated cylinder, 100 mL; available from Carolina Biological at www.carolina.com, item #721603
• Scissors
• Forceps (tweezers)
• Lab notebook
• Optional: Digital camera
• Graph paper

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Experimental Procedure

Important Notes Before You Begin:

1. Be sure you thoroughly read the procedure before beginning so you know how much time each step will take and can plan your schedule accordingly.
2. Follow the steps below to make culture plates of Physarum for your experiments. The cultures will be grown on agar, and then transferred to fresh agar plates when the Physarum covers the old plate. The goal is to establish a healthy stock of growing plasmodia for your experiments.

1. Loosen the top of a bottle of non-nutrient agar.
2. Heat the non-nutrient agar in a microwave until it is liquid. Use an oven mitt to remove it if the bottle is hot.
3. Pour the agar into 10 petri dishes so that the bottoms are covered.
4. Let the agar harden.
1. For long-term storage, wrap the petri dishes in their plastic sleeves and store them in a refrigerator until you are ready to use them.
5. Put on your disposable gloves. Count out 25 sterile oatmeal flakes for each agar surface and place them on top of the agar.
6. Cut out a piece of agar with plasmodium attached from the Physarum polycephalum culture that came with the kit. Read the following before you begin:
1. Use a plastic knife.
2. The piece should be about 1 cm by 1 cm.
3. Place the agar block, with the plasmodium facing down, on one of the agar plates (which are the petri dishes now containing agar) that you made in steps 3–5.
4. Place a lid on the plate.
7. Wrap the plate in aluminum foil.
8. Monitor the culture daily, until you see enough growth. Use your judgment.
1. To keep the culture healthy, transfer the culture to fresh plates (the additional ones you made in steps 3–5) every 3–4 days. To transfer to fresh plates, follow steps 6–8 of this section, except cut out a piece from your culture, not from the kit.
2. To dispose of older cultures, wrap them in aluminum foil and place them in your regular garbage.
3. Begin your experiments once you have established a stock of growing Physarum cultures.

Making Solutions with Varying Concentrations of Glucose

1. Label five plastic cups: 100, 10, 1.0, 0.10, and Blank.
2. Place a glucose tablet in the cup labeled 100 and add 222 mL of water from the 250-mL beaker.
3. Let the tablet of glucose dissolve completely.
1. This is a 100-mM solution.
• mM stands for millimolar. It is a way of measuring the concentration of an ingredient in solution. For more information about molarity, see the reference by Uri Lachish in the Bibliography, above.
2. The solution may be slightly hazy due to additives in the tablet. You can ignore this.
4. Make a 10-mM solution:
1. Use the cup labeled 10.
2. Mix 10 mL of the 100-mM solution with 90 mL of distilled water.
3. Use the 10-mL graduated cylinder and the 100-mL graduated cylinder to measure volumes.
4. Mix the solutions in a clean plastic cup.
5. Thoroughly wash the graduated cylinders between uses.
5. Make a 1-mM solution.
1. Use the cup labeled 1.
2. Mix 10 mL of the 10-mM solution with 90 mL of distilled water.
3. Use the 10-mL graduated cylinder and the 100-mL graduated cylinder to measure volumes.
4. Mix the solutions in a clean plastic cup.
5. Thoroughly wash the graduated cylinders.
6. Make a 0.1-mM solution.
1. Use the cup labeled 0.1.
• Mix 10 mL of the 1.0-mM solution with 90 mL of distilled water.
2. Use the 10-mL graduated cylinder and the 100-mL graduated cylinder to measure volumes.
3. Mix the solutions in a clean plastic cup.
7. Pour 200 mL of distilled water into the cup labeled Blank.

Preparing Plates with Paper Strips

1. Label five clean petri dishes (without agar), as follows: 100, 10, 1.0, 0.10, and Blank.
2. Cut filter paper into 10 strips, each approximately 8 cm x 2 cm.
3. Soak six filter paper strips in the distilled water, the Blank cup.
4. Using a permanent marker, label four dry strips 100, 10, 1.0, and 0.10.
5. Soak the labeled strips of filter paper in the appropriate cups containing glucose: 100 mM, 10 mM, 1.0 mM, or 0.10 mM.
6. Allow the filter paper strips to soak for at least 15 min.
7. Using forceps, take a filter paper strip from the distilled water, the cup labeled Blank. Allow excess fluid to drip off.
8. Place five of the wet strips from the Blank cup on the bottom of each of the five sterile petri dishes, near the middle. See Figure 1.

Figure 1. Experimental setup to test Physarum polycephalum chemotaxis using the filter paper strip method. Two strips of filter paper are placed below a growing Physarum culture. One strip was dipped in a test solution (marked with an "x") and the other strip was dipped in distilled water, the cup labeled Blank. In this example, all of the tests are "positive" for growth toward the strip with the test substance.

9. Place the last wet strip from the Blank solution next to the strip in the plate labeled Blank.
10. The two strips should abut against one another. See Figure 1.
11. Remove a filter paper strip from the 100-mM glucose beaker; allow excess fluid to drip, and place the strip right next to the distilled water strip on the bottom of the petri dish labeled 100. The two strips should abut against one another.
12. Repeat for the strips and plates marked 10, 1.0, and 0.10.

1. Use a clean plastic knife to cut a plasmodium culture into blocks sized 0.5 cm x 0.5 cm.
1. The plates you made with growing Physarum are plasmodium cultures. Now you can cut them up and use them for your experiments.
2. Cut as many blocks as you need.
2. Transfer four such blocks, plasmodium-side down, onto the junction of the two filter paper strips. See Figure 1 for an idea of spacing.
1. When you select the agar block with Physarum to transfer, be sure that you cut a thick vein or sheet of "tissue" located toward the edge of the plasmodium. Do not transfer pieces of oatmeal from the stock culture.
2. Repeat for all of the filter strip petri dishes.
3. Wrap the dishes in aluminum foil and incubate them right side up at room temperature.
4. Observe plasmodium movement every hour for several hours, and again the next day. You will need to unwrap the plates to observe them, but be sure you wrap them back up again. The yellow plasmodia will be easily seen if they migrate onto the white filter paper.

Observing the Plates

1. Record all observations in your lab notebook, as follows. As an option, you could take pictures with a digital camera for your display board.
1. For each plate, record the number of cultures that are growing farther into the glucose as Positive Toward Glucose in your lab notebook.
2. Record the number of cultures that are growing equally in the glucose or the water strips as Neutral.
3. Record the number of cultures that are growing farther toward the Blank strip as Negative, Away from Glucose.
4. Use your judgment about what to score as positive, neutral, or negative.

Repeating the Procedure

1. Perform the entire procedure two more times with fresh materials. This will show that your results are repeatable.

Graphing Your Results

1. Combine the data for each trial.
2. Graph the concentration of glucose on the x-axis and the number of Positive Toward Glucose on the y-axis.
3. On a separate graph, graph the concentration of glucose on the x-axis and the number of Neutral on the y-axis.
4. On a separate graph, graph the concentration of glucose on the x-axis and the number of Negative, Away from Glucose on the y-axis.

Variations

• Try other chemicals, such as sugars, salts, amino acids, etc.
• Try soaking the strips in cultures of Physarum's "natural" diet, such as bacteria or yeast.
• Try mixing an attractant chemical with a chemical you've found to be a repellent.
• Make a 1-cm-diameter round punch to cut out and transfer Physarum polycephalum cultures.

• For more science project ideas in this area of science, see Microbiology Project Ideas.

Credits

David B. Whyte, PhD, Science Buddies

The procedure is modified from Bozzone, D. M. 2005. Using microbial eukaryotes for laboratory instruction and student inquiry. Retrieved December 18, 2009 from http://www.ableweb.org/volumes/vol-26/05-Bozzone.pdf

Last edit date: 2010-03-03 12:00:00

Career Focus

If you like this project, you might enjoy exploring careers in Microbiology.

	Biological Technician What do the sequencing of the human genome, the annual production of millions of units of life-saving vaccines, and the creation of new drought-tolerant rice varieties have in common? They were all accomplished through the hard work of biological technicians. Scientists may come up with the overarching plans, but the day-to-day labor behind biotech advances is often the work of skilled biological technicians.			Microbiologist Microorganisms (bacteria, viruses, algae, and fungi) are the most common life-forms on Earth. They help us digest nutrients; make foods like yogurt, bread, and olives; and create antibiotics. Some microbes also cause diseases. Microbiologists study the growth, structure, development, and general characteristics of microorganisms to promote health, industry, and a basic understanding of cellular functions.
	Biologist Life is all around you in beauty, abundance, and complexity. Biologists are the scientists who study life in all its forms and try to understand fundamental life processes, and how life relates to its environment. They answer basic questions, like how do fireflies create light? Why do grunion fish lay their eggs based on the moon and tides? What genes control deafness? Why don't cancer cells die? How do plants respond to ultraviolet light? Beyond basic research, biologists might also apply their research and create new biotechnology. There are endless discoveries waiting to be found in the field of biology!

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