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Project Summary

Difficulty	7  –  8
Time required	Long (a couple of weeks)
Prerequisites	Note: Pilobolus is grown on rabbit dung embedded in plain agar. A strong stomach is a prerequisite for working with this culture.
Material Availability	You will need to order a Pilobolus culture kit online. See the Materials and Equipment list for details. You should work with your teacher to have the kit delivered to your teacher at school, since there are restrictions on sending it to a residential address.
Cost	Average ($50 - $100)
Safety	No issues

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Abstract

Objective

Introduction

The fungus Pilobolus is a common inhabitant of cow and horse manure, or dung. While you and I might consider that a less than ideal place to live, this is high-value real estate for a fungus. The conditions are warm and moist, and there are abundant nutrients. The fungus grows in the dung by forming a network of hyphae, which are threadlike structures composed of cells that are attached end to end. It is the main tissue of the growing fungus. After growing throughout the dung pile for two to three days, the fungus begins a process of forming structures, called fruiting bodies, that are required for asexual reproduction. Fruiting bodies contain spores. These spores will grow into new fungi under the right growing conditions.

The ideal place for a young spore to start out is on a blade of grass. From there, it can be eaten by a horse or a cow. After passing through the digestive tract unscathed, the spore will, if it is lucky, find itself in a fresh dung pile all its own. But how does the parental fungus solve the problem of getting the spores out of the dung pile and onto the grass? Pilobolus has solved this problem by developing two interesting features: a light-sensitive structure that allows the fruiting body to locate light sources, and a mechanism for firing the sac full of spores toward the light source (a spore sac is also called a sporangium). Why shoot toward the light? Well, imagine a dung pile that is surrounded by trees on three sides. If it shoots the spore sac randomly, three out of four sacs will hit a tree and be lost to the next generation. The side with light is a better bet—it has a good chance of having grass, thus putting the fungal spores in a place where they stand a chance to be eaten by a passing cow or horse.

Pilobolus has evolved mechanisms that allow it to aim and fire in order to place its spores in the best possible spot for survival and reproduction. How does it do it? The stalk below the sporangium becomes swollen with liquid (due to osmotic pressure), with a black mass of spores on the top (see Figure 1, below). Below the swollen tip is a light-sensitive area, which is critical in directing the growth of the Pilobolus so that it faces toward the light. As the fungus matures, pressure builds in the stalk until the tip explodes, launching the spores into the light.

Figure 1. Parts of the Pilobolus fruiting body. The sporangium contains spores and sits on top of a vesicle containing fluid at high pressure. The sporangium is shot toward a light source, having been aimed in the correct direction by the sporangial stalk. The spore sac reaches accelerations that are among the fastest in nature.

In this biology science fair project, you will grow a culture of Pilobolus and experiment with how its "spore cannon" responds to various light conditions. This science fair project will involve some creative problem solving on your part, since you are working with a live organism. It would be a good idea to give yourself enough time to become adept at culturing the Pilobolus before you begin experimenting. Also, the experiments will most likely need several rounds of troubleshooting and fine-tuning, so allow time for this as well.

Terms, Concepts and Questions to Start Background Research

• Fungus
• Pilobolus
• Hyphae
• Asexual reproduction
• Fruiting body
• Spore
• Spore sac
• Sporangium
• Osmotic pressure
• Sporangial stalk
• Mycelia
• Trophocyst
• Inoculate

Questions

• What are the various structures that make up the fruiting body of a Pilobolus fungus?
• Based on your research, how does the fungus generate the force required to fire the spore sac?
• What is the speed with which a spore sac is launched? How far does the spore sac travel?
• What makes the spore sac stick to the surface on which it lands?
• What is the definition of positive phototropism?

Bibliography

• Wikipedia Contributors. (2009, August 22). Pilobolus. Retrieved September 15, 2009, from http://en.wikipedia.org/w/index.php?title=Pilobolus&oldid=309388056
• Eddleman, H., PhD. (1999). Dung Jars. Retrieved September 16, 2009, from http://www.disknet.com/indiana_biolab/kids102.htm

This paper by Coble and Bland provides background and ideas for experimenting with Pilobolus.

• Coble, C.R. and Bland, C.E. (1974). Open-Ended Experimentation with the Fungus Pilobolus. Retrieved August 30, 2009, from http://www.eric.ed.gov/ERICWebPortal/custom/portlets/recordDetails/detailmini.jsp

This paper describes how the authors used super-high-speed photography (250,000 frames per second!) to capture the entire spore launch process in four species of fungi.

• Yafetto, L., et al. (2008). The Fastest Flights in Nature: High-Speed Spore Discharge Mechanisms among Fungi. Retrieved August 30, 2009, from http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003237

This website provides good background information about Pilobolus, along with a fun animation.

• Fogel, R. (1996). Pilobolus—Fungal Shotgun. Retrieved August 10, 2009, from http://herbarium.usu.edu/fungi/funfacts/pilobfct.htm

Materials and Equipment

• Pilobolus culture kit; available from WARD'S Natural Science at www.wardsci.com, Item # 853923.
  • The kit comes with six rabbit dung plates. Include in the order a request for a "special order" of 10 additional rabbit dung plates.
  • Consider ordering two kits if you plan to try variations on the basic project.
  • You should work with your teacher to have the kit delivered to your teacher at school, since there are restrictions on sending it to a residential address.
• Magnifying glass
• Lab notebook
• Lamp with 75-watt (W) bulb
• Optional: Camera
• Aluminum foil
• Permanent marker
• Pin
• Metric ruler

Disclaimer: Science Buddies occasionally provides information (such as part numbers, supplier names, and supplier weblinks) to assist our users in locating specialty items for individual projects. The information is provided solely as a convenience to our users. We do our best to make sure that part numbers and descriptions are accurate when first listed. However, since part numbers do change as items are obsoleted or improved, please send us an email if you run across any parts that are no longer available. We also do our best to make sure that any listed supplier provides prompt, courteous service. Science Buddies receives no consideration, financial or otherwise, from suppliers for these listings. (The sole exception is any Amazon.com or Barnes&Noble.com link.) If you have any comments (positive or negative) related to purchases you've made for science fair projects from recommendations on our site, please let us know. Write to us at scibuddy@sciencebuddies.org.

Experimental Procedure

For health and safety reasons, science fairs regulate what kinds of biological materials can be used in science fair projects. You should check with your science fair's Scientific Review Committee before starting this experiment to make sure your science fair project complies with all local rules. For more information, visit these Science Buddies pages: Projects Involving Potentially Hazardous Biological Agents and Scientific Review Committee.

Important Note Before You Begin: You will need to perform a total of three trials to demonstrate that your results are accurate and repeatable. The best way to do this is to start trial #2 one day after you've started trial #1, and trial #3 one day after you've started trial 2 (two days after you started trial #1). Keep careful notes in your lab notebook for all steps.

Day 1:

1. Using a sterile cotton swab (part of the kit), transfer a few sporangia from the tube culture to four dung plates. Inoculate directly on the exposed dung surface.
2. Invert the plates and incubate at room temperture overnight.

Day 2:

1. The next day, place the plates agar-side-down under a light source, such as a ceiling light fixture.
2. Incubate for two days.
3. Use a permanent marker to number the plates 1 to 4. Label the bottom and the lid.

Day 4:

1. Remove the covers from plates #2, #3, and #4 and wrap the plates in aluminum foil.
2. Make small holes in the aluminum foil with the pin, one hole per plate: 1-mm diameter for plate #2, 2-mm diameter for plate #3, and 4-mm diameter for plate #4. Put each hole in the middle of each foil disk.
3. Put the plastic lids back on plates #2, #3, and #4.
4. Leave the plastic lid on plate #1.
5. Place the plates under a light source; for example, a lamp with a 75-W bulb.

Day 5 and Beyond:

1. Observe plate #1 with the clear plastic lid. After you see black sporangia adhering to the underside of the cover of plate #1 (4–7 days), carefully remove the aluminum foil from plates 2–4.
2. Look for sporangia on the underside of the foil, near the holes. Observe the pattern around the light holes and record all observations in your lab notebook. Use a magnifying glass, if necessary.
3. How accurate is the aim of the Pilobolus toward the light source?

Analyzing Your Results

1. Draw concentric circles around the light source holes and count the number of sporangia. Graph the results.
2. Graph the size of the light hole on the x-axis and the size of the region with sporangia on the y-axis.
3. Does the accuracy depend on the size of the hole?

Variations

• What happens if there are two point sources?
• Use different colors of light to determine which color the Pilobolus responds to best. You can buy colored plastic filters (or gels) online. Or you could experiment with different colors of light emitting diodes (LEDs).
• Try varying the timing of light exposure, keeping the intensity the same. For example, expose separate cultures to 2 hours of light in the morning, mid-day or evening.
• Cover a culture with a 5-gallon bucket with a hole in it (with paper on the inside and tape over the hole). How accurate is the Pilobolus when the light source is at this increased distance?
• Devise a way to measure the maximum distance (vertical and horizontal) a spore sac can be fired.
• Repeat the procedure using Pilobolus that you cultured from nature. See the website in the Bibliography, authored by Dr. Eddleman, for information on growing Pilobolus.

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

Credits

David B. Whyte, PhD, Science Buddies

• Scotch® is a registered trademark of 3M.

Last edit date: 2010-01-25 00:00:00

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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