1. Marine Biotechnology Lab
Bioluminescence

2. Bioluminescence
is the ability of living things to emit light.

3. 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.

4. 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.

5. 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.

6. The 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.

7. Most marine animals appear to use their luminescent organs for such varied functions as :
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.

8. 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:
Glowing trees to line highways to save government electricity bills
Agricultural crops and domestic plants that luminesce when they need watering
Novelty pets that bioluminesce (rabbits, mice, fish etc.)

9. How does bioluminescence work?
The group of chemicals responsible for light is known as luciferins.
The enzyme behind bioluminescence that catalyzes luciferin is luciferase.

10. The reaction is as follows:
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 + O FMN + RCOOH + H2O + light (490 nm).
where R represents a long-chain alkyl group.

12. Materials

13. Culture Media Recipe "seawater complete (SWC) medium"
NaCl 8.88 g MgCl2.6H2O 1.62 g MgSO4.7H2O g KCl Peptone 2 g Glycerol 1.2ml Yeast extract 1.2g Agar 6g Distilled water 400 ml

14. Method
Measure out the materials in the above recipe to make 400 ml of agar medium and place them in a 500 ml flask.
Mix the ingredients. To ensure complete dissolution.
Bring to a gentle boil on a hotplate until all ingredients are dissolved.
Dispense into four 150 ml flasks (100 ml each).
Stopper the flasks with nonabsorbent cotton and aluminum foil.

15. Autoclave the agar at 121oC, 15 lbs pressure for 15 min.
While autoclaving, clean the working area inside the laminar airflow that you will be using.
Put all the materials you need including the medium in the laminar airflow and turn on UV lamp.
Allow the medium to cool to 55 oC.

16. Use sterile plates to prepare agar plates as you need them for culturing bacteria.
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.
Swirl the liquid so it covers the entire bottom surface.
Allow to solidify.
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.
Use sterilized bent glass rod to spread the inoculum over the entire surface of the media.
Replace the lid of the dish.
Label bottom of plates (not the top) along the edge.
Wrap the Parafilm around the edge of the Petri dish to slow evaporation.
Incubate for 1 to 2 days in an inverted position; observe the growth of luminescent microorganisms.

18. Bioluminescent Algae
In this experiment, you will examine bioluminescence in dinoflagellates.

19. Procedure 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.
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.
Without disturbing the slide, turn off the microscope light and the room lights. Leave both off for about two minutes.
While the lights are off, look through the eyepiece of the microscope. Watch the dark slide for a minute or two.
While observing through the eyepiece, gently tap the slide to disturb the organisms.