Bi 6 Biotechnology for Everyone, Laboratory

Isolation of Bioluminescent Bacteria

Quorum sensing is the language of bacteria in the form of chemical signals, which they release.
Bacteria communicate with each other in order to react to the rapidly changing conditions in their
environment like the availability of nutrients, evasion of toxic molecules, protection against other
bacteria and so forth. The chemical signals must be in sufficient concentration and that means there
should be enough number of cells releasing them to trigger the expression of specific genes as a
response to a specific environmental condition. Quorum sensing therefore can never be achieved by a
single bacterium but only through a collective effort hence the term ‘quorum’ which is defined as ‘a
group with members competent of accomplishing an objective’. This is an amazing phenomenon
wherein unicellular microorganisms are reacting to their environment in unison like a single
multicellular organism. It accomplishes a task and reaps benefits as a group that can not be attained as
a single cell.

The ability of living organisms to emit light is called bioluminescence. In 1970, Nealson, Platt
and Hastings reported that the glow from the nocturnal Hawaiian bobtail squid is emitted by millions
of marine bacterial cells called Vibrio fischeri, which are living underneath the skin of the squid. The
bacteria live in symbiosis with the squid. The squid uses the light to avoid predators, attract prey and
find a mate. In exchange, the bacteria are provided an environment with a steady supply of nutrients in
a specialized compartment of the squid. Within that microcosm, the bacteria are able to multiply in
number and emit light. But when they are freely living in the ocean outside the squid, they do not emit
because the ocean dilutes out the concentration of the quorum sensing signal called acyl-homoserine
lactone (AHL) or generally known as autoinducers. AHL triggers the expression of the set of genes for
bioluminescence called lux. The expressed enzyme or protein called luciferase catalyzes the chemical
reaction to produce light.

Objectives:
At the end of the exercise, you should be able to:
1. understand the concept of bioluminescence;
2. isolate bioluminescent bacteria from Philippine fish and squid; and
3. appreciate the applications of bioluminescence in biotechnology.

Department of Biology, School of Science and Engineering

Ateneo de Manila University
Bi 6 Biotechnology for Everyone, Laboratory

Exercise 6. Microorganisms in the Environment

Name:___________________________________________ Date: _______________________

Group No.: _______________________________________ Instructor: ___________________

Materials:
Squid or fish (fresh from the sea) – transport to the laboratory with ice
Inoculating loop/sterile cotton swab
Pair of gloves
2 agar plates
Match/lighter
Marking pen
Alcohol lamp

Procedure:
1. Label the agar plates with your group number and name of sample on the periphery of the
bottom plate. Divide the plate using a marking pen depending on the number of samples.
2. Swab the body of the squid using sterile cotton swab or inoculating loop and inoculate in
zigzag motion on one part of the agar plate.
3. Open the ink sac of the squid and inoculate the black ink in zigzag motion on the other part of
the agar plates.
4. Swab the body of the fish and inoculate on one part of the second plate.
5. Open the belly of the fish and swab the sample on the other part of the second plate.
6. Wrap the inoculated plates with paper and incubate the plates in inverted position (upside
down) at room temperature.
7. Check for growth of bioluminescent colonies in the dark room after 8 h of incubation. Incubate
for 12 h or further if bioluminescence was not observed.
8. Take photos of the plates with bioluminescent bacteria under bright light and in the dark. Store
the plates in the refrigerator if positive (not in the freezer).

Department of Biology, School of Science and Engineering

Ateneo de Manila University
Bi 6 Biotechnology for Everyone, Laboratory
Results and Observations:

Figure 1: ____________________ Figure 2: ____________________

Magnification:________________
Description: __________________
____________________________
____________________________
____________________________
____________________________
Magnification:________________
Description: __________________
____________________________
____________________________
____________________________
____________________________

Guide Questions:
1. What is bioluminescence?
____________________________________________________________________________
____________________________________________________________________________

2. Why is quorum sensing unattainable by a single bacterium?

____________________________________________________________________________
____________________________________________________________________________

3. Describe the benefits that the squid and bioluminescent bacteria get from each other.
____________________________________________________________________________
____________________________________________________________________________

4. How does bioluminescence occur? Show the chemical equation.

____________________________________________________________________________

5. Describe a biotechnological application of bioluminescence.

____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________

Department of Biology, School of Science and Engineering

Ateneo de Manila University