Teacher setup information for this lesson is available here.

Fireflies, jack-'o-lantern mushrooms, and anglerfish are all examples of bioluminescence - light produced within a living organism by a chemical reaction. We'll explore part of the chemical reaction that results in the bioluminescence of a firefly and see how temperature can affect it.

To measure the light output of the chemical reaction we'll use a test tube color sensor with the light off. First connect the test tube color sensor to your Arduino. The test tube color sensor has an LED on one side and a color sensor on the other. Attach the LED to digital pin 2 and the color sensor to an I2C pin on your grove shield. Then, if you haven't already, connect your Arduino to your computer.

In many examples of bioluminescence, a type of chemical called a luciferin reacts with oxygen in the presence of a type of enzyme called a luciferase to produce light. In fireflys another chemical called adenosine-triphosphate (ATP), is also necessary. ATP is used to transport chemical energy within cells and is the main energy source for the majority of cellular functions.

First, read the procedure below before you start. Then, if you can, dim the lights. (You'll be able to see the reaction much better). Finally, follow the procedure below.

Procedure:

1. Your teacher will have buffer solution available. Follow your teachers instruction on how prepare an ATP solution and a firefly lantern solution using the buffer.
2. Transfer the firefly lantern solution to a test tube that will fit into the test tube color sensor.
3. Place the test tube in the test tube color sensor and cover it with the test tube cover
4. Prepare another test tube of the lantern solution and place this in an ice bath. We'll use it later.
5. Click Record Baseline below. This will zero the reading to account for any light that is leaking into the color sensor.
6. Remove the test tube from the color sensor and add 2-3 drops of the ATP solution. It should start glowing almost immediately.
7. Quickly place the test tube into the color sensor and place the cover over it.
8. Watch the brightness level from the color sensor. Notice how bright the reaction starts, and how quickly the brightness decreases.

Remove the test tube from the color sensor and touch the bottom. Do you notice anything? It should still be about room temperature. Because the reaction produces almost no heat, we say that bioluminescence produces cold light. On the other hand, the chemical reaction that takes place when something is on fire is a good example of a reaction that produces light and a lot of heat. A reaction that produces heat is also called exothermic. Now that you've seen the room temperature reaction, we're going to try again with a cooled and then a heated lantern solution.

Procedure:

1. Retrieve the test tube you prepared earlier from the ice bath.
2. Place the test tube in the test tube color sensor.
3. Click Record Baseline below. This will zero the reading to account for any light that is leaking into the color sensor.
4. Remove the test tube from the color sensor and add 2-3 drops of the ATP solution. It should start glowing almost immediately.
5. Quickly place the test tube into the color sensor and place the cover over it.
6. Watch the brightness level from the color sensor. Notice how bright the reaction starts, and how quickly the brightness decreases.

Procedure:

1. Prepare another test tube of firefly lantern solution.
2. Place this test tube in a hot water bath for about 1 minute. The water should be at least 65 degrees Celsius.
3. Add 2-3 drops of ATP solution to the test tube.

When the luciferase enzyme gets too hot it denatures. This means that the shape of the enzyme changes so that it no longer functions, which prevents the light emitting reaction from occurring. As with other chemical reactions, cooling the solution should've slowed down the reaction. This makes it last longer, but produce less light. If we had heated the solution, but kept the temperature lower (under 50 degrees Celsius), the reaction would not have lasted as long as the room temperature reaction, but it would've been brighter.

If your teacher has sodium chloride, they can demonstrate that adding this to a glowing vial of the lantern solution will quench the light almost immediately. We don't actually know how a firefly flashes its lantern, but one possibility is that chloride ions are involved.