The original hypothesis that luminescent bacteria could be isolated from
squid was proved using enrichment methods. Though several alterations
were made to the original protocol, luminescent bacteria were successfully
isolated using NaCl Agar and seawater complete liquid medium. It
was originally intended to use luminous media; however, no growth was observed
on the plates. Therefore, it was decided that the use of luminous
media should be omitted from the final protocol. Luminescent bacteria
were not visible on squid incubated at 4°C; however, once the squid
were swabbed and the samples were plated, luminous colonies were observed
at room temperature. In order to account for this observation, the
squid should be incubated at room temperature rather than at 4°C.
Furthermore, growth was not observed in the seawater complete liquid medium
until day 5. Therefore, seawater complete liquid medium should be
incubated for at least 5 days. While luminescent
bacteria were not visible on the squid, colonies appeared after incubation
of NaCl Agar overnight at room temperature. Five days after the inoculation
of the seawater complete liquid medium, luminescence was observed.
From here, growth of the organism was maintained and characterization was
possible. Initial research led to the belief that the organism that
would be isolated would be of the genus, Vibrio. Results proved
this incorrect. The
Gram stain proved the isolated organism to be Gram-negative, bacillus.
Although it was predicted that these results would be obtained, long, thinner
rods were expected. Instead, the rods appeared short and plump.
Most luminescent species are motile thus leading to the prediction that
the isolated organism would be motile regardless of the species (Holt 1984).
After performing a motility stab and observing a wet mount, results proved
the organism to be motile. According to research on Vibrio
species, organisms were expected to ferment both mannitol and glucose
(Holt 1984); however, fermentation of
mannitol was not observed. Fermentation of glucose indicates that
the organism has both a respiratory and fermentative type of metabolism.
This led to the belief that the isolated organism may not have been of
the genus, Vibrio. The oxidase test further supported this
belief.
Vibrio species are oxidase positive (Holt
1984); however, after repeating the oxidase test 3 times, a positive result
was never obtained. This indicates that the organism may have an
electron transport chain that does not use cytochrome oxidase. These
inconsistent results for Vibrio led to further research of the other
genus of luminescent bacteria,
Photobacterium. While Vibrio
and Photobacterium exhibit many similar characteristics, they differ
in fermentation of mannitol and in oxidase (Holt 1984). The results
obtained suggested that the isolated organism was from the genus, Photobacterium.
Three species of Photobacterium exist, two of which are luminescent.
Of the two luminescent species, P. phosphoreum and P. leiognathi
, it was apparent that P. leiognathi was the organism isolated.
This is because P. phosphoreum exhibits growth at 4°C and produces
gas upon fermentation of glucose, neither of which was observed.
On the other hand, P. leiognathi does not grow at 4°C and does
not produce gas upon fermentation of glucose (Holt 1984). Therefore,
it could be concluded that the isolated luminescent organism was P.
leiognathi .
Figure 9. Maintained growth and isolated colonies of luminescent
bacteria visible on day 4 after incubation of squid. Colonies present
on NaCl Agar incubated at room temperature.
The glowing charactistic of luminescent bacteria occurs when the bacteria
colonize specialized light organs in fish or squid. They can exist
freely or in a symbiotic relationship with these marine organisms.
Mutualism is observed in the symbiotic relationship as the squid benefits
because the bacteria play a large role in attracting prey and in camouflage.
The light radiating from the fish or squid matches the intesnsity of moonlight
in order to prevent a shadow from appearing on the seafloor and thus warning
prey (Farmer and Hickman-Brenner). In return, the host provides a
niche and nutrients for the bacteria. Luminescent
bacteria have been used in recent years as detectors for carcinogenic compounds
and to monitor toxic activities because light emission can be easily and
quickly measured (Quinto 2001). Since luminescent bacteria have ecological
and clinical applications, it is important to isolate this organism using
a consistent set of procedures. The organisms on which these bacteria
can be obtained may contain a wide variety of microorganisms, including
pathogens. Therefore, a clear step by step enrichement protocol is necessary
to select against undesired microbes.
Return Home
Back to Results 
Final Protocol