Ocean Bioluminescence: Investigate When and How Dinoflagellates Glow

Setting Up Your Cultures

1. When the cultures arrive, their natural dark/light cycle will have been disturbed during shipment. You will need to adjust them to a new dark/light cycle before starting the experiment.
   1. Read the care instructions for the cultures carefully. Follow any directions that are sent with the cultures to get them started.
   2. If you buy cultures that do not come in test tubes, you will need to transfer your culture into test tubes.
      1. Rinse the test tubes with hot tap water before you use them and let them dry. Do not use any dish soap or other cleaning agents as the cultures are sensitive to these.
      2. Label nine test tubes with the culture name.
      3. Gently swirl the culture. With the transfer pipette, carefully transfer 10 mL culture from the vial into each test tube. Suck the culture liquid up slowly to reduce the stress on the organisms.
      4. You should have at least nine test tubes filled with 10 mL culture.
   3. Place all the culture test tubes in one or two plastic cups inside one of the cardboard boxes. Tape the cups to the box, so they do not fall over. Place the box with the cultures under a light with an LED or CFL light bulb. You can now close the lid of the box if you want to keep the cultures in the dark and open the lid of the box to expose the cultures to light.
   4. The type of light you use is important. The best lighting for optimum growth and health of the cells is a 25–40 watt LED bulb placed at least 1 meter from the flasks (Figure 5). You can place the lights on a timer for any length of time for experimentation purposes.
      
      
      Image Credit: Svenja Lohner, Science Buddies / Science Buddies
      Figure 5. Lamp setup for the dinoflagellates experiment.
   5. To adjust the cultures to a new dark/light cycle, keep the cultures in the dark during the daylight hours. This will allow you to study their bioluminescence (which typically happens during the dinoflagellate's dark cycle) during your normal waking hours. Our suggestion is to keep the cultures in the dark from 8:00 AM to 8:00 PM each day and in the light from 8:00 PM to 8:00 AM. If this timing does not fit with your family's schedule, you can vary it depending on your time availability. Make sure, however, that you keep a consistent 12 hours of light / 12 hours of dark schedule every day.
   6. Another important variable for the cultures is temperature. It should be between 68–78°F (20–26°C).
   7. Follow steps 1 and 2 of the "Making Observations" section below to check the cultures for bioluminescence every evening around 7:00 PM (or an hour before you switch to their light cycle). They should be most active at the end of their dark cycle. If they are glowing at this time for two days in a row, you are ready to start with your experiment. It can take up to a week for the cultures to get used to a new dark/light cycle. If they do not glow after more than 10 days at the new dark/light cycle, your culture might have died.

   When working with living cultures, there is always a risk that the microorganisms die due to improper handling or other circumstances. If your ordered dinoflagellate cultures fail to glow even when following all the steps in the provided care instructions, do not get discouraged. You might not have done anything wrong! When your culture is alive, you should be able to see tiny brown cells suspended in the sea water. Dead cells are whitish in color and settle on the bottom of the test tubes. If you are unsure wheter your culture is still alive or not and you do not see the dinoflagellates glowing, contact the seller and speak to their technical support team. Explain your observations and that you think your cultures might have been dead upon delivery. Most likely, you will be reimbursed or sent a new culture.

2. To start your experiments, place one clean plastic cup in each of the three boxes near the middle. The cups will be used to keep the cultures upright. Glue or tape the cups in the boxes so that they won't fall over. Label the boxes as follows:
   1. Dark
   2. Light
   3. Light/Dark
3. Label your nine culture test tubes as follows:
   1. A: Dark
   2. B: Dark
   3. C: Dark
   4. D: Light
   5. E: Light
   6. F: Light
   7. G: Light/Dark
   8. H: Light/Dark
   9. I: Light/Dark
4. Wrap tubes A, B, and C in aluminum foil.
   1. These will be kept in constant darkness.
   2. Place tubes A, B, and C in the plastic cup in the box marked Dark. It is ok if the test tubes are not completely upright—leaning against the side of the cup is fine.
   3. Place the top on the Dark box and set it aside.
5. Place tubes D, E, and F in the plastic cup in the box labeled Light. It is ok if the test tubes are not completely upright—leaning against the side of the cup is fine.
6. Put the lamp with a compact fluorescent (CFL) or LED bulb in an area where it can stay on 24 hours a day.
7. Place the Light box under the lamp.
8. Turn on the lamp.
   1. Do not put the top on the Light box.
   2. These tubes will be kept in constant light.
   3. The cell cultures will be taken out of the light for brief periods of time, described later in the procedure, to determine if they will produce bioluminescence in response to mechanical stress.
9. The Dark box should be placed next to the Light box, also under the lamp.
   1. Keep the boxes in the same area to minimize variations in temperature, etc.
   2. The boxes will be heated equally since they are both under the lamp.
   3. Keep the lid on the Dark box at all times so as not to expose these dinoflagellate cultures to any light.
10. Place tubes G, H and I in the cup in the box labeled Light/Dark.
    1. These cultures will be kept in darkness for 12 hours of the day and in light for 12 hours of the day, and in light for part of the day.
    2. Keep tubes G, H, and I in the dark during the daylight hours and in the light during night.
    3. Cover tubes G, H, and I with aluminum foil in the morning. Place the wrapped tubes in the Light/Dark box and cover the box with the lid.
    4. Place the Light/Dark box near the other two boxes, also under the lamp.
    5. Unwrap tubes G, H, and I in the evening. Unwrap them at the same times each day. Our suggestion is to continue with the same dark/light cycle as before. For example, keeping them in the dark from 8:00 AM to 8:00 PM each day and in the light from 8:00 PM to 8:00 AM or whatever schedule you settled on previously.
    6. Expose the Light/Dark tubes to the lamp light during the night.
    7. Re-wrap them in aluminum foil in the morning and place the lid back on the Light/Dark box.

Making Observations

Now that you have your dinoflagellate cultures set up, you can determine how the light and dark conditions affect their bioluminescence. The bioluminescence of the cultures can be viewed in a darkened room.

Create a scale for how bright the cultures glow. You will rank the brightness on a scale from 1 to 4, with 4 being the brightest.

1. Take the cultures into a dark room several times a day at the same times each day. You can have a dim light on, such as a nightlight or a flashlight, to help you work in the room. Keep the light several feet away from the cultures. Keep track of which tubes belong in which boxes.
2. To make observations, you will need to unwrap the tubes wrapped with aluminum foil. Make sure to wrap them again afterward.
3. To stimulate bioluminescence, make sure the caps are tight on the test tubes and, one at a time, turn the test tube upside down three times. You should see a clear blue light where the bubble in the tube is moving through the culture. This region has the highest mechanical stress. Do not shake the cultures too much, otherwise, they will die. Record your observations and brightness ranking in your lab notebook.
4. Test all of the cultures for bioluminescence. Record all observations and brightness rankings in your lab notebook. Try to treat each tube in the same way so that you can compare the relative amount of light produced.
5. Observe each of the cultures at least four times per day for five days. Space each observation several hours apart. Keep track of dates, times, and all data in your lab notebook.
6. Make sure you observe the light/dark culture during both its light phase and its dark phase. Aim for once before the light cycle starts, twice during the light cycle and again after the dark cycle starts.

Analyzing Your Data

1. Graph your data.
   1. Graph the time on the x-axis and the brightness score on the y-axis.
   2. Suggestion: Divide the x-axis into 24 hours, with noon in the middle. Mark the period of darkness for the light/dark cycle with a black rectangle under the x-axis. Add bars for the bioluminescent scores to the graph at the appropriate times. Use different colors for the all-light, all-dark, and light/dark cultures.
2. How does varying their light exposure affect the bioluminescence of the dinoflagellates?
3. Repeat the procedure at least two more times so that you have three sets of data. You can reuse the cultures you already have for the second and third trial.