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<center> Bioluminescence in Fireflies<BR> The luciferase-luciferin reaction in Photinus pyralis.<BR? By Terry Lynch>

I was 18 years old living in Jacksonville, Alabama, when I first performed this experiment. It is a repeat of the classic experiment in bioluminescence first performed by Robert Dubois in 1887 using photogenic material of the West Indies elaterid Pyrophorus and the molluscs Pholas, a bivalve. The experiment is performed using crude extracts from firefly lanterns and demonstrates that the luminescence of fireflies is an enzyme-substrate reaction.

This experiment may be conducted with under ten firefly lanterns but it is more spectacular when a larger number of fireflies are collected and sacrificed for the demonstration. I recommend using Photinus pyralis or any other Photinid as these fireflies are easy to collect and have large lanterns.

One of the keys to getting a good result is to collect fireflies early in the evening, at twilight when they first begin their courtship flights. Immediately upon catching fireflies with an insect net, remove them from the net and place them in a bottle or jar that is set into an ice pack. This immobilizes the fireflies, preserving their energy and avoids depletion of the chemicals used in the biochemical reaction which is responsible for firefly luminescence.

Try to collect between 100-300 adult males, the more the merrier! Keep the fireflies on ice until they are ready to use. I recommend they be used within several hours as this experiment must be conducted in total darkness, and when better to do it then later in the evening on the same night that the fireflies are collected.

Total darkness is best achieved by going into a windowless room (or a room that has had the windows covered with black plastic, foil or other material so as to make them opaque) at night such that when room lights are turned off, absolutely no light can be seen, not even a pen hole of light leaking through a door crack. So be certain you have access to such a darkroom before even attempting this experiment.

You will be preparing two firefly lantern extracts, one of Luciferase, a heat sensitive enzyme, and another of Luciferin, an oxidizable substrate. Begin by counting your fireflies and dividing them into two equal groups of about 50-100 fireflies each, more or less depending upon how many fireflies you are able to collect. Then use a one edge razor blade to dissect and remove the firefly lanterns. This operation is best done by placing the immobilized fireflies upon a metal jar lid that is set upon an ice pack so as to maintain the fireflies in a cold state; in fact, the fireflies may even be placed in a freezer first, to kill them if exposure to the ice pack has not already done that.

After removing all the firefly lanterns, being careful to discard any wings and wing covers, place the lanterns in a chilled mortar and pestle, add a small amount of chilled distilled water and ground up the lanterns. Use an eyedropper or syringe to add only enough water to cover the lanterns and equal their bulk which will be about 1- 5 mls, more or less depending upon how many firefly lanterns you are mashing up.

After you grind up the first batch of lanterns, note that a luminescent liquid is produced. View this liquid in a totally darkened room. You will need to allow your eyes to adapt to the darkness 3-5 minutes. Note how bright the firefly extract is initially and that over time the brightness decreases. In fact you want to leave this extract setting at room temperature (68 to 78 degrees Fahrenheit) until no more or only slight luminescence is observed. This will result in an extract which contains luciferase.

Next prepare the second extract. Begin by boiling a beaker of distilled water for several minutes. Then let the boiled water cool slightly. Add an equal amount of this hot, boiled distilled water to the second batch of firefly lanterns, ie, the same amount of water you used to produce the first luciferase extract. Then grind these lanterns in the hot water inside a mortal and pestle that has been warmed slightly by running hot water over it. When the lanterns are completely ground, place the crude extract into a test tube and set it into the beaker of hot water. The idea is to use the heat from the water to destroy all the luciferase enzyme leaving only luciferin which is not destroyed by heat. No more than a minute or two of heat exposure should be required to destroy all the enzyme; the exact amount of time depends upon the amount of fireflies you are processing and the initial temperature of the water. If you turn out the room lights and watch what happens when hot water is added to the lanterns, you should be able to see all glow promptly disappear. When the lanterns are no longer glowing you know all the luciferase has been destroyed and you may return the luciferin extract to room temperature.

To observe the luciferase-luciferin reaction place equal amounts of each extract in separate test tubes and go into a room totally darkened. Let your eyes adjust to the absence of light 3-5 minutes. Be sure that there is no light, or only very dim light, coming from the luciferase extract. Then add the luciferin extract to the luciferase extract and shake the test tube vigorously! You should note an immediate increase in the brightness of the light emitted from the extract as the luciferase and luciferin react. (Smaller amounts of extract may be used by placing a few drops of each extract upon two separate microscope slides and then combining the two extracts simply by pressing the two microscope slides together. This allows the experiment to be performed repeatedly or by a greater number of students.)

The chemical reaction for luciferase-luciferin is shown below:

luciferace ATP + D-luciferin + O2 ---------> oxyluciferin + AMP + PPi + CO2 + light

Once you have performed this basic experiment you may improve upon it by trying to: (a) increase the purity of extracts; (b) increase the oxygen available for the reaction; or (c) use commercially available extracts to perform the same experiment. Variations of this experiment may be done using extracts made from different species of fireflies or different types of luminescent animals.

Conclusion

From this experiment students should learn: (a) how to collect and handle large numbers of fireflies; (b) how to make crude extracts of luciferase and luciferin; (c) that luciferase is destroyed by heat; and (d) that luciferin is not destroyed by heat (e) that bioluminescence is a chemical reaction that involves oxidation of luciferin in the presence of luciferase. By actually doing this experiment themselves using fireflies they have collected, students gain an appreciation for that self learning and discovery process which they may later apply to original nature studies. This is a much superior method of learning than is just reading about bioluminescence out of a textbook. In fact, it is a fun and exciting learning process which should inspire students to learn about life and the natural sciences.

Related Links

Blinks and Links

The Amateur Scientist by TAL

Copyright 1999 by Terry Lynch. All Rights Reserved.

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