Bioluminescent Bacteria
There are some bacteria found in seawater, marine sediments, and the guts of marine animals that emit light. Some fish such as flashlight fish, anglerfish and also bobtail squid have evolved and developed the ability to use these microbes for their own benefit and establish a symbiotic relationship. These animals have developed special organs that provide bioluminescent bacteria with a source of food and a place to live, and in return these animals can use the glowing bacteria as a means to camouflage and protect them- selves from their predators, hunt for food, or even attract their mates.
Objectives
Students will be able to isolate luminescent bacteria from seawater and grow them on seawater complete agar.
Students will observe the luminous colonies in a dark room, and learn about bioluminescence.
Recipe for SWC (seawater complete agar can also be ordered from media supply catalogs.)
|
·
|
750 ml artificial seawater sea.
|
|
·
|
250 ml distilled water
|
|
·
|
5 grams Bacto peptone
|
|
·
|
3 grams yeast extract
|
|
·
|
3 ml glycerol
|
|
·
|
15 grams agar
|
-
For storage vials, add 1.0 g of CaCO
3
/liter
Mix the media in a container large enough to hold 1 L of media, and boil the media for ~ 30 minutes making sure that it does not boil over. Allow it to cool down enough to be touched (50-60 degrees C). Then pour media into sterile petri dishes (~15 mL media per standard petri dish)-Agar will solidify once the temperature falls below 40 degrees C.
Materials needed
|
·
|
5-10 mL sample of seawater
|
|
·
|
4-20 plates of sea water complete medium
|
|
·
|
Glass spreading rod
|
|
·
|
Incubator
|
|
·
|
Sterile tooth picks
|
|
·
|
Pipettes
|
|
·
|
Gloves, safety goggles and aprons
|
Procedure
|
1.
|
Collect a sample of about 5 - 10 ml of seawater to use as inoculum.
|
|
2.
|
Take 2 plates of SWC agar medium and use a pipette to measure 0.1 ml of the seawater inoculum and pour it on the center of a plate. Repeat for the second plate.
|
|
3.
|
Pour 0.2 ml of seawater inoculum onto the third plate. Repeat for the fourth plate.
|
|
4.
|
Use the glass-spreading rod, spread the inoculum thoroughly over the surfaces of the plates. Allow the sample to be absorbed by the medium.
|
|
5.
|
Invert the plates and incubate at 20 degrees C.
|
|
6.
|
Examine the plates after 24 hours in a dark room. Watch for 'spreaders'-bacteria that can spread across the plates contaminating and engulfing other bacteria.
|
|
7.
|
In the dark room, (with red light) use sterile toothpicks to pick 2 or 3 isolated luminous colonies and transfer to fresh SWC plates, then use an inoculating loop to streak the fresh SWC plates to get isolated colonies of the luminous bacteria. Again, watch out for spreaders.
|
Analysis Questions
|
1.
|
Describe the chemical reaction of the luciferase enzyme that bioluminescent bacteria use to emit light. What is the role of oxygen in this reaction?
|
|
2.
|
Describe the colonies. What do they look like?
|
|
3.
|
Where can you find these bacteria?
|
|
4.
|
Define Quorum Sensing. Why is it important?
|
|
5.
|
Describe the symbiotic relationship between bioluminescent bacteria and other organisms.
|
|
6.
|
Do you think these bacteria can be beneficial in the medical field? Why?
|
