Isolation of Vibrio Fischeri From Seafish

I did a few biology (specifically bacterial) projects this summer.  The first was an enrichment of V. Fischeri or V. Phosphoreum from squid.  Vibrio Fischeri and Vibrio Phosphoreum are bioluminescent bacteria that are commonly found on fresh fish or other sea animals.  The reason you don’t normally notice them in water is because they are very small, are at a low concentration and only glow when there are a lot of similar bacteria around it (in another post I will explain how that works!).  The trick then, is isolating the target bacteria from all the other bacteria in the ocean.

The first step in any enrichment is to do background research.  The first thing almost anyone stumbles upon is this article from Indiana Biolab.  It turns out that the bacteria I was looking for grew at low (4C) temperature, in salty water.  That is the kind of temperature that is easily achieved with ice and a cooler, and a salinity that is easy to achieve with table salt.  I used a handful of salt and about half an inch of ice (measured from the bottom of my cooler) for experiment.  The “proper” amount of salt would be 30g/L, or 2.8 tsp/L.  The squid I used was from one of the butchers in Haymarket in Boston.  I placed it on the ice so that it would never be completely submerged.  The squid can’t be submerged because the bacteria require oxygen to bioluminesce, and to pick the colonies of bacteria out, you need to be able to see them glowing.

Once the squid is in the cooler, the next thing to do is to let them incubate in a cool room for a few days.  Basements and garages are good places to do this.  You could probably do it in your house/fridge, but there is one caveat: rotting squid smells TERRIBLE.  It also attracts flies like no other.  When you throw the squid away, be sure to double, triple, quadruple bag, do it the day your trash gets picked up, and bleach EVERYTHING, because this will attract flies like a magnet and stink to the high heavens.  Also, be careful of the squid-juices that will form in the cooler.  They smell bad.  The best option may be a disposable cooler that you can just tape up and throw away after.

During those few days, you should check on the squid every 6-12 hours.  Don’t worry about missing the window, but definitely throw it away after 2 days if you haven’t seen anything (see rant above about smell).

Eventually, you may see some glowing.  If you want to continue culturing the Vibrio, I recommend trying to pick off individual colonies and spreading them on plates and continuing the isolation of colonies there.  Here are some pictures I took of the glowing squid!

Taken with long exposure

Taken with long exposure

A note on brightness:  It is hard to capture the light produced by these bacteria in a photograph.  To give you an idea of their luminosity, I would say that a few colonies roughly .25mm in diameter are are comparable to a firefly.  I would also say that in complete darkness, the light from the colonies was enough to illuminate the inside of the ice chest I used, which was surprising.

Even though I never managed to get these growing in culture, it was amazing to see them glow in the dark.  I would recommend this experiment to anyone who is interested in biology.