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Project Summary

Difficulty	7  –  9
Time required	Long (a couple of weeks)
Prerequisites	None
Material Availability	You will need a Physarum polycephalum culture kit. See the Materials and Equipment list for details.
Cost	Average ($50 - $100)
Safety	Use caution when working with sharp blades and when working with hot solutions.

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Abstract

Objective

Establish a culture of Physarum polycephalum and determine if it can find the shortest distance between two food sources in a maze.

Introduction

With a name like slime mold, it's understandable that you would expect these organisms to be ugly and disgusting. Despite their unappealing name, slime molds are fascinating and, for some observers, beautiful organisms. They form colorful gliding masses as they creep over decaying wood and leaves. A particularly interesting and well-studied slime mold is called Physarum polycephalum, the many-headed slime mold.

Figure 1. Physarum polycephalum growing in Olympic State Park. (Wikipedia, 2009.)

In its growing stage, Physarum polycephalum looks like a giant amoeba that spreads out like a fan, enveloping and eating everything in its path. It consumes bacteria and decaying organic matter. In this plasmodial stage, Physarum polycephalum is a bright yellow glistening mass that can grow to more than a foot across. If the conditions that the organism finds itself in become challenging—for example, the ground becomes dry or food becomes scarce—the plasmodium will begin to shrink and form sporangia. The sporangia produce spores that can be spread by the wind to new regions for the organism to grow. Germination of the spores leads to the formation of a new plasmodium.

A curious variation occurs when the plasmodium encounters conditions that are too severe for it to continue growing, but not severe enough to cause it to form sporangia. It goes into a kind of dry, dormant state, called sclerotium from which it can revive itself if it becomes moist. The organism is sometimes shipped as a sclerotium when purchased from scientific supply companies.

Physarum polycephalum is inexpensive to obtain and it can be easily cultured on a moist surface, such as a damp piece of filter paper or an agar plate (agar plates are easier to use because they stay moist). It can be fed rolled oats as it grows, and divided to a fresh plate when it has grown to cover the first surface.

Slime molds, like Physarum polycephalum are part of the kingdom Protoctista. This is probably the least understood of the five kingdoms of life; the others being animals, plants, fungi, and bacteria. One of the most interesting things about Physarum polycephalum is that it shows a quality that could be called primitive intelligence. Studies done by Dr. Nagakaki and colleagues in Japan have shown that Physarum polycephalum can find the shortest distance through a maze. When the Physarum polycephalum organism is chopped up and dropped into a labyrinth (a maze), they put themselves back together and start to move. If a food source is placed at the entrance and exit to the maze, they avoid dead ends in the maze and form a connection (as a single tube) between the food sources. In all cases, the Physarum polycephalum chose the path that was the shortest between the two food sources. In a way, it "solved" the puzzle of finding the shortest path through the maze!

According to the abstract of the paper, here is how the authors sum up the findings:

The procedure, as described in the Nakagaki paper, is quoted below:

Figure 2. Maze-solving by Physarum polycephalum. 2.a. Structure of the organism before finding the shortest path. Blue lines indicate the shortest paths between two agar blocks containing nutrients: α1 (4151 mm); α2 (3351 mm); β1 (4451 mm); and β2 (4551 mm). 2.b. Four hours after the setting of the agar blocks (AG), the dead ends of the plasmodium shrink and the pseudopodia explore all possible connections. 2.c. Four hours later, the shortest path has been selected. Plasmodium wet weight, 90 +/- 10 mg. Yellow, plasmodium; black, "walls" of the maze; scale bar is 1 cm. 2.d. Path selection. Numbers indicate the frequency with which each pathway was selected. "None:" no pseudopodia (tubes) were put out. See Supplementary Information at the Nature website noted in the Bibliography for an animated versions of 2.a-c. (Nakagaki, et al., 2000.)

Quoting again for the Nakagaki paper, here is a summary of their results:

In this advanced biology science fair project, you will attempt to repeat the research results of Nakagaki, et al. Independent confirmation of results is a common and important practice in science. The goal is to address the question "Does the organism Physarum polycephalum demonstrate qualities that can be termed primitive intelligence?" The procedure below provides an outline for approaching this research problem, but you will have to work out many of the details independently. There are plenty of ways to expand on Nakagaki, et al.'s findings, some of which are suggested in the Variations section at the end of the procedure, but you may be able to think of others.

Terms, Concepts and Questions to Start Background Research

• Slime mold
• Physarum polycephalum
• Plasmodium
• Sporangia
• Germination
• Sclerotium
• Agar plate
• Protoctista
• Primitive intelligence
• Amoeba-like cell
• Dendritic network
• Pseudopodia
• Minimum-length solution

Questions

• How many species have been described in the kingdom Protoctista?
• What is a pseudopod?
• What is meant by the term dendritic network?
• What are the various forms that Physarum polycephalum can assume in its life cycle?
• Do you agree with the authors of the paper by Nakagaki, et al. that slime molds are capable of demonstrating a form of "primitive intelligence"?

Bibliography

• University of California, Berkeley. (2000). Introduction to the "slime molds." Retrieved August 18, 2009, from http://www.ucmp.berkeley.edu/protista/slimemolds.html
• Thomas, A. (2000, September 28). Slime mold solves maze puzzle. Australian Broadcasting Corporation, Science News. Retrieved August 18, 2009, from http://www.abc.net.au/science/news/stories/s189608.htm
• Conover, A. (2001). Hunting Slime Molds. They're not animals and they're not plants, and biologists want to know a lot more about them. Smithsonian Magazine. Retrieved August 18, 2009, from http://www.smithsonianmag.com/science-nature/phenom_mar01.html
• Nakagaki, T, et al. (28 September, 2000). Maze-solving by an amoeboid organism. Nature 407, 470.
• The abstract to the paper is available here: http://www.nature.com/nature/journal/v407/n6803/abs/407470a0.html.
• The complete version of the original paper is available for a fee from www.nature.com, or you could find it at a local library that carries Nature.
• The author may supply PDF versions of the paper on request.

Materials and Equipment

Note: Keep in mind as you evaluate how many materials you'll need that you will be running at least six trials of this experiment, and they can be run concurrently.

• Physarum polycephalum culture kit; available from WARD'S Natural Science at wardsci.com, catalog # 85 W 4752
• The kit contains agar, petri plates, and Physarum polycephalum food (oatmeal).
• If you supply the food, use "old -fashioned" oatmeal. Physarum polycephalum does not like "instant" oatmeal.
• The culture is shipped separately because it is a living organism.
• You may need to have the kit shipped to your school if there are restrictions on delivering to a residential address.
• Transparency paper; available at office supply stores (1 box)
• Exacto cutting tools; available at hardware, office supply, and craft stores. Alternatively, you can use small scissors.
• Printer that can use transparency paper
• Heat-protective gloves
• Forceps or tweezers
• Distilled water (1 gallon)
• Optional: Digital camera
• Lab notebook

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Experimental Procedure

1. Prepare the agar plates following the directions supplied with the Physarum polycephalum culture kit.
   1. Microwave the agar to melt it. Loosen the caps prior to microwaving.
   2. Use heat-protective gloves to handle the hot agar.
   3. Uncover one plate at a time and pour just enough agar to cover the bottom of the plate.
   4. Allow the plates to cool for about 1 hour.
2. Start growing the Physarum polycephalum culture.
1. Follow the directions that came with the kit to establish actively growing plasmodia.
2. Avoid exposing the culture to direct light.
3. Use a swab applicator to transfer a portion of the plasmodia supplied to the center of an agar plate.
4. Using forceps or tweezers, transfer three or four pre-moistened oat grains around the plasmodial mass. Pre-moisten the oatmeal with a few drops of distilled water.
5. Keep feeding the Physarum polycephalum culture moistened oats to enhance its growth.
6. Transfer part of the culture to new agar plates, as needed, to maintain the culture. Note: The culture can also be maintained using moist filter paper as a substrate, and oatmeal you buy at a grocery store for food.
3. Make the Physarum polycephalum labyrinth.
1. Look at Figure 2 to see the shape of the labyrinth used in the original paper. It is about 4 cm x 4 cm in size. The design is reproduced as a template in Figure 3.
2. Print the design below on transparency paper. You may need to adjust the size of the printed figure so that it is about 4 cm x 4 cm.
3. Cut out the clear portion of the labyrinth using Exacto blades or a small pair of scissors.
4. Place the maze on the agar surface of a clean agar plate.
5. Note that the maze has three pieces.
6. Placing the maze on the agar while it is still hardening may create a better attachment between the maze and the agar surface.

Figure 3. Template of a Physarum polycephalum labyrinth. It should be about 4 cm x 4 cm when printed out. See the paper by Nakagaki, et al. in the Bibliography for more details.

4. Grow the Physarum polycephalum in the labyrinth, as follows.
1. Transfer several small pieces of the growing plasmodia culture in the maze. Note in your lab notebook the time and location at which you added the food.
2. Allow the Physarum polycephalum to grow until it fills the maze.
5. Add food to the labyrinth.
1. Place a pre-moistened piece of oatmeal at each spots labeled "F" in Figure 2.
2. You can also use agar blocks that already contain nutrients.
6. Observe the pattern of growth of the Physarum polycephalum. Record the Physarum polycephalum's pattern of growth using photographs or drawings.
7. Does the Physarum polycephalum establish a connection between the two pieces of food using the shortest route? Record all observations in your lab notebook.
8. Repeat the procedure at least five more times. These trials could be done in parallel, with replicates of the maze.
9. Record the results in a data table. How often did the Physarum polycephalum "choose" the shortest path to connect the two food sources?

Variations

• Try other maze designs. Determine the lengths between the food sources for all possible connecting routes. What is the minimum percentage difference in length between routes that produces repeatable differences in the Physarum's choice of routes?
• Devise other ways of making mazes. For example, you could try printing the maze on the filter paper directly, then coloring in the maze with crayons or other material the Physarum polycephalum will not grow over.
• View the Physarum polycephalum growing on an agar plate with a microscope. Place the culture plate on the microscope stage and view at 40x magnification. Make the diaphragm small and focus on the bright yellow veins. Can you see the rapid fluid motion (streaming) of the cytoplasm? Does the streaming change at various times during the course of the experiment?
• Place food (moistened oats) on an agar plate in a triangle and inoculate the plate with Physarum polycephalum. What is the final shape of the organism? Is it the minimal distance possible to connect the food sources? Try other patterns. What happens if one food source has more food than the others?

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

Credits

David B. Whyte, PhD, Science Buddies

Last edit date: 2010-01-07 12:00:00

Career Focus

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

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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