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Bioluminescence
Objective: In this experiment students will isolate luminescent bacteria from different
fish species and/or seawater on solid culture medium.
Introduction
Bioluminescence is the ability of living things to emit light. It is found in
• many marine animals, both invertebrate (e.g., some cnidarians, crustaceans, squid)
and vertebrate (some fishes);
• some terrestrial animals (e.g., fireflies, some centipedes);
• some fungi and bacteria.
Bioluminescence in Marine Animals
Ninety percent of deep-sea marine life is estimated to produce bioluminescence. The
widespread occurrence of luminescence among deep-sea animals reflects the
perpetual darkness in which they live.
• At least one fish has its luminescent organ located at the tip of a protruding stalk
and uses it as bait to lure prey within reach of its jaws.
• When disturbed, one species of squid emits a cloud of luminescent water instead
of the ink that its shallow-water relatives use.
• Some marine animals that live near the surface have luminescent organs on their
underside. These probably make it more difficult for predators beneath them to
see them against the light background of the surface.
In the case of fishes, the light is emitted by luminescent bacteria that grow in
luminescent organs. The above photos show the flashlight fish, Photoblepharon
palpebratus, with the lid of its luminescent organ open (left) and closed (right). The
light is produced by continuously-emitting luminescent bacteria within the organs, but
its display is controlled by the fish. Most marine animals appear to use their
luminescent organs for such varied functions as
1.
2.
3.
4.
5.
Camouflage
Attraction prey for feeding e.g. Luminous lure.
Defense, Repulsion, expelling, or confusing a potential predator.
Communication (in the dark) for Mating or Schooling of fish.
Illumination.
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Biotechnological Applications
Bioluminescent organisms are a target for many areas of research. Luciferase systems
are widely used in the field of genetic engineering.
Some proposed applications of engineered bioluminescence include:
1. glowing trees to line highways to save government electricity bills
2. agricultural crops and domestic plants that luminesce when they need watering
3. novelty pets that bioluminesce (rabbits, mice, fish etc.)
How does bioluminescence work?
The group of chemicals responsible for light is known as luciferins. The molecular
details vary from organism to organism, but each involves a luciferase, the enzyme
behind bioluminescence that catalyzes luciferin is luciferase. When luciferin reacts
with oxygen, it is oxidized. Once a molecule of luciferin is oxidized, it cannot flash
any more until is reduced to form luciferin again.
In bacteria, luciferase catalyzes the oxidation of reduced flavin mononucleotide
(FMNH2) and a long-chain fatty aldehyde in the presence of molecular oxygen to
yield FMN, carboxylate and blue light of 490nm.
The reaction is as follows:
FMNH2 + RCHO + O2 ----------- FMN + RCOOH + H2O + light (490 nm),
where R represents a long-chain alkyl group.
Figure . The bioluminescence chemistries of bacterial, firefly and Renilla luciferases. The actions
of luciferase from three genera on the substrates aldehyde (bacterial), luciferin (firefly) and
coelenterazine (a coelenterate, Renilla reniformis) and the resulting end products, carboxylate,
oxyluciferin and coelenteramide, respectively.
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Materials
• L-shaped glass rods.
• Laminar flow biosafety
cabinet
• Petri dishes.
• Parafilm.
• Cotton.
• Aluminum foil
•
Bunsen burners
• 70% Ethanol.
• Inoculating loops.
• 1-L Flask.
• Four 150-200 ml flasks.
Culture Media Recipe "seawater complete (SWC) medium"
• NaCl
8.88 g
• MgCl2.6H2O
1.62 g
• MgSO4.7H2O
24.6438 g
• KCl
0.238
• Peptone
2g
• Glycerol
1.2ml
• Yeast extract
1.2g
• Agar
6g
• Distilled water
400 ml
Method
1. Measure out the materials in the above recipe to make 400 ml of agar medium and
place them in a 500 ml flask.
2. Mix the ingredients. To ensure complete dissolution.
3. Bring to a gentle boil on a hotplate until all ingredients are dissolved.
4. Dispense into four 150 ml flasks (100 ml each).
5. Stopper the flasks with nonabsorbent cotton and aluminum foil..
6. Autoclave the agar at 121oC, 15 lbs pressure for 15 min.
7. If you do not have an autoclave, you can pressure-cook it for 30 minutes.
8. While autoclaving, clean the working area inside the laminar airflow that you will
be using, by spraying a small amount of 70% ethanol on the surface and wipe it
around with a clean paper towel, while UV light turned off. Be sure to allow the
alcohol to completely evaporate before lighting any flames!
9. Put all the materials you need including the medium in the laminar airflow and
turn on UV lamp.
10. Allow the medium to cool to 55 oC.
11. Use sterile plates to prepare agar plates as you need them for culturing bacteria.
12. Work quickly, remove the cap of a flask, flame the mouth, and pour the liquid
agar into a sterile petri dish while the cover is partially raised.
13. Swirl the liquid so it covers the entire bottom surface.
14. Allow to solidify.
15. It is good to pour them a day in advance of inoculation so condensed water on the
inside does not cause problems. Alternatively, you could leave them partially
opened for about 15 minutes while in laminar flow and UV turned on.
16. Replace the cover.
17. Aseptically inoculate the center of the agar plates with a single loopfull taken
(transferred) from the fish surface and/or gills.
18. Don’t forget to sterilize the loop and set it down before and after doing anything
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19. Use sterilized bent glass rod to spread the inoculum over the entire surface of the
media.
20. Replace the lid of the dish.
21. Label bottom of plates (not the top) along the edge. Label in small letters name,
lab number, and fish name.
22. Wrap the Parafilm around the edge of the Petri dish to slow evaporation.
23. Incubate for 1 to 2 days in an inverted position; observe the growth of luminescent
microorganisms.
24. For pure cultures, students can pick out different luminescent colonies from their
primary culture plates, and streak them on secondary culture plates.
Bioluminescent Algae
In this experiment, you will examine bioluminescence in dinoflagellates.
Procedure
1. Prepare a wet-mount slide of dinoflagellates. To do so:
a. Transfer a drop of dinoflagellates to a slide.
b. Cover the drop with a cover slip.
2. Place the slide on a compound light microscope, turn on the microscope light,
and focus on low power. Observe organisms for a minute or two.
3. Without disturbing the slide, turn off the microscope light and the room lights.
Leave both off for about two minutes.
4. While the lights are off, look through the eyepiece of the microscope. Watch
the dark slide for a minute or two.
5. While observing through the eyepiece, gently tap the slide to disturb the
organisms.
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