"Honey Dew from Bunny Too!" is an activity that may be incorporated into many different classroom situations.  The experiment will demonstrate the the mechanism for spore dispersal and show the different stages of the life cycle of the Pilobolus crystallinus.  This lab was adapted from the original inspiration of Donald Cronkite, Professor of Biology at Hope College, Holland Michigan.  He teaches Introductory Biology, Embryology, and Science and Human Values.  His research interests center around using Paramecium as a model organism for studies of cellular and developmental processes.
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• To observe the spore dispersal mechanism of the Pilobolus
• To cultivate Pilobolus populations from the wild using various herbivores (cow dung or horse dung) manure.
• To compare the Pilobolus in various samples(cow patties, horse apples, or bunny pellets).
• To understand the life cycle of the Pilobolus crystallinus.
• To observe and draw the stages in the life cycle of the Pilobolus.

• Biology, Ecology, or Environmental Science Students
• 6th - 12th

Notes to the Teacher:
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• "Wow Lab"- The students will make observations from a pre formatted lab protocol to observe the life cycle of Pilobolus.
• "Investigative"- The students will design a lab to test the mechanism that triggers spore dispersal.  They should have some clues after making observations of the life cycle.  Part of the fun here is to let the students devise a method for testing this mechanism and be able to repeat the protocol with similar results.

• Order equipment and cultures from a biological supply company.  Allow plenty of time for ordering and plan accordingly (2 weeks).
• If you would like to culture your own samples directly from the field, visit a horse farm, dairy, or cattle ranch (make sure the animals have grazed in a field not grain fed).
• Place a large pile of fresh manure in a old glass baking dish (9x12).
• Cover with plastic wrap to keep the moisture in the container.
• Store in a relatively dark area at room temperature for 24-48 hours or until a whitish-grey lawn with black dots appears on the surface of the manure.
• Optimal growth temperature is 24^o C but not higher than 30^o C.
• One day prior to the lab you need to prepare the (RDA) "rabbit dung agar"
• Autoclave or use a presser cooker to sterilize the rabbit dung collected in a jar.
• Make a 2% non-nutrient agar medium by dissolving 20 grams in 1 liter of water and autoclave (makes approximately 50  plates).
• Now place 6 - 8 rabbit pellets in a sterile petri dish and pour agar over the pellets.
• Allow the plates to solidify and place in the refrigerator.  They will keep for about a month.   DON'T FREEZE !
• Prepare at least 10 culture plates 6 - 7 days prior to the lab.  This will enable the students to make initial observations and to inoculate their plates.

Class time needed

• The initial class set up will be one class period.  The duration of the lab may run over two weeks depending on the growth conditions and types of activities the students will be performing with the fungus.  However, if you just use the "Wow" section, it could be completed in one class period.

Hazards/Precatuions

• The students should practice sterile technique.   Students should wash the work areas with appropriate disinfectant (10% Bleach Solution) and wash their hands with soap and water.
• Disposal of the cultures should not be a problem since theses organisms are not pathogenic.  However, if contamination occurs pour a bleach solution on to plates and allow them to set for 24 hours before disposing in a sealed trash bag.

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•           Ward's  page 49     Living Catalog #  - 85W4671                   Price     $7.30

•           Ward's  page 49     Demo Plates Catalog #  - 85W8671          Price     $8.90

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•           Carolina  page 278    Catalog #  K3-21-6721        500g        Price    $80.00

Suggestions to reduce cost:

• Groups of four use 1 petri dish (class of 30 -approximately 10 per class).
• 20 grams of non-nutrient agar mixed with 1 liter of water makes approximately 50 plates with 20 ml of agar in each plate (enough to do 5 classes of 30 students).
• Rabbit Dung Pellets figure no less than 6 - 8 per plate.
• Stock Cultures - culture your own FREE from the field.
• Pressure cooker to sterilize RD rabbit dung)  and agar at 15 psi for 15 minutes.
• You may want to experiment with a microwave to sterilize the RD.

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• Asexual Reproduction
• Life Cycle
• Spore
• Sporangium
• Sporangiophore
• Zygomycete
• Haploid number
• Diploid number
• Mitosis

• Life Cycles
• Asexual Reproduction
• Experimental Design
• Mechanisms for Spore Dispersal

Introduction:

• Do herbivores eat dung?
• How do the spores get off the dung and on to the plants?

Purpose:

• To observe the asexual life cycle of Pilobolus crystallinus
• To practice sterile technique
• To design a lab to observe the spore dispersal mechanism

Material/Equipment:

• Some type of inoculating instrument (loop, Q-Tip, toothpick)
• A culture of Pilobolus to observe and for inoculation
• 1 petri dish of RDA (rabbit dung agar) per group
• Dissection microscope (2X) or compound microscope (5X or 10X)
• Brown paper or shoe box (with lid) to cover the petri dishes from the light

• Obtain all the material from the list above before starting experiment.
• Clean work area with disinfectant.
• Make initial observation of the growing cultures before inoculating the RDA.  Use either a compound microscope (5X or 10X) or dissection scope (2X).
• Use a sterile inoculating loop, or sterile toothpick, swab, etc. to gather the black spores usually located on the lid of the petri dish.
• Gently, rub loop across the RDA plate in order to place the spores on the new medium.
• Place inoculated petri dish in a brown bag or shoe box with a lid in a cool dark place (24^o C).
• Make observations daily for the next 6 - 7 days. (Complete the table)
• Clean work area with disinfectant.
• Return equipment and dispose of all contaminated materials as directed by the teacher.
• WASH HANDS !
• When black specks start to appear in the culture ........
• prepare a wet mount slide of the spores
• observe the total structure before placing the cover slip
• gently place the cover slip on top of the structure and push (squashing the specks)
Can you find the spores? ............ Count them >>>>______________
 

Observations:
 
 

Date	Qualitative	Quantitative	Sketch

 

Questions:
 

1. What are the bulbous orange swelling?
2. What are the clear stalk structures with liquid drops?
3. What are the brown - black structures at the top of the clear stalks?
4. How did the black specks get on the lid of the petri dish?
5. Propose three ways the black specks appeared on the lid?
 

Conclusion:
 
 
 
 

Part 2

Directions:
 
• Design a lab to explore the possible mechanisms used to disperse the spores.
• Make sure to include 2 groups
• Follow the model protocol used in Part 1 include:
• Title
• Purpose
• Hypothesis
• Material List
• Procedure
• Observations
• Conclusion

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1. Evaluate the proposed hypothesis, observations, conclusion, and questions together.

• Is the hypothesis written in the If Then format as a statement?
• Do they have both types of observations (Quantitative & Qualitative)?
• Does their conclusion uphold or refute their hypothesis and have the students provided evidence to prove their argument?
• Answers to Questions:
 
1. What are the bulbous orange swelling? (beginnings of sporangiophore & sporangium)
2. What are the clear stalk structures with liquid drops? (sporangiophore)
3. What are the brown - black structures at the top of the clear stalks? (sporangium)
4. How did the black specks get on the lid of the petri dish? (answers will vary)
5. Propose three ways the black specks appeared on the lid? (answers will vary)
 
1. Use a Rubric to evaluate the Experimental Design Section.

 
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Student Directions:

    You will develop your own spore dispersal model.  The model must include the following:
 

• A mechanism for spore dispersal.
• The model must move to demonstrate the dispersal of spores.
• An explanation of how your method of dispersal will ensure the success of the next generations.
• A jingle, poem, song, rap, etc. that will accompany the model explaining the dispersal mechanism.

Benjamin, C.R. 1996. The Encyclopedia Americana, International Edition. Volume 12 under "Fungus"

Fun Facts About Fungi   .... Pilobolus -- Fungal Shotgun
http://www. herb.lsa.umich.edu/kidpage/pilobfct.htm

A Fungus That Shoots Like A Cannon
http://www.eduzone.com/Tips/Science/GAfungus.htm
http://www.eduzone.com/_vti_bin/shtml.dll/search.htm (type in Pilobolus)

Dung Jars
http://www.disknet.com/indiana_biolab/a2.htm

Fungi: The Hidden Kingdom
http://www.herb.lsa.umich.edu/kidpage/kingfact.htm

A Fungus That Blows Its Top
http://www.pages.prodigy.com/HRLW46A/creat006.html

Forest pathology "Ethnopathology"
http://www.esf.edu/course/jworrall/ethnog.htm
 

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