GLOWING BACTERIA GEN by Amanda Olesen

Always good to start with Kombucha

Always good to start with Kombucha

What we did, and how we did it...

 

Steps we did two days before the experiment night

1. We bought fish from the market (squid, herring and shrimp). Ideally the fish should be as fresh as possible, as little cleaned as possible (not with chlorine water) and also, coming from the Nordic Sea which is more favorable for Aliivibrio Fischeri (the glowing bacteria) to be found (in comparison to the Baltic Sea, which is more polluted).

2. We made the sea water complete (SWC) media using the recipe we found online here. The SWC is the food that Aliivibrio like to nurture on. We replicate the recipe for SWC here:
500 ml water
12 g salt (instant ocean mix/aquarium salt)
1.5 g dried yeast
2.5 g peptone (found in most protein mixes from bodybuilding shops)
1.5 ml glycerin

3. We poured the SWC over the fish in plastic containers and left them to incubate at the room temperature in our lab. If you have ways to control precisely the temperature, then ideally the temperature should be 15 degrees Celsius. We placed the lids on the containers (the fish starts to stink super badly after a few hours), but we did not close them tightly, because Aliivibrio needs and likes oxygen.

4. We added the agar agar (7.5 g) to SWC and poured the mix into larger glass jars. The jars went into the pressure cooker to sterilize the mix. After boiling them for 45 mins (20 mins is usually enough, depending on the type of the cooker), we poured the hot mix in the petri dishes. The agar can be found in chinese shops. Agar is extracted from algae and it is used to gelify the mix. You can find agar in chinese shops.

5. After cooling, the mix has set in the petri dishes (i.e., it became like a gel), and the resulted agar plates (petri dishes with SWC mix in them) were stored in the fridge. The agar plates are stored upside down to keep the SWC mix clean by preventing eventual bacteria from falling from the lid into the SWC gel. We also made some SWC using sea water (that means that we did not add the salt in the mix) just for investigation purposes.

Preparing the SWC

Preparing the SWC

The stinky squid

The stinky squid

Incoculating the agar plates with Aliivibrio

Incoculating the agar plates with Aliivibrio

On the day of the GEN

6. We learnt to make a simplified SWC media. For that, we cut into pieces a frozen fish and boiled it until the skin came off. We strained the fish and kept 500 ml of the resulted water. By boiling the fish we extract the nutrients from it, so we can use the water to replace the peptone and yeast in the SWC recipe. We added the 12 g of aquarium salt and 7.5 agar in the fish water. Please mind that the agar needs to be boiled in order to disolve properly and gelify the mix. In case you do not have aquarium salt, but you live in Copenhagen :), take a walk to the beach and take some water from the sea. If you use the sea water, the salt is not needed anymore. So, the resulted mix (fish water, plus salt, plus agar) had to be brought to boiling point for a few minutes.

7. We used the home-made version of SWC to learn how to plate the petri dishes. We did this step for demo purpose, so we use some plastic containers (used previously for modeling clay), we sterilized them (with boiling water and alcohol) and learnt how to pour the agar mix. However, because we did not boil the mix, the agar did not dissolve and the mix did not set (it did not transform into a gel). Hence, do not forget to bring to boil the agar mix. Also, in ideal case, the mix should be sterilized, but since the salt concentration is quite high, the probablity of growing other types of bacteria than Aliivbrio is quite low.

8. We learnt how to inoculate agar plates. We used the plates that were prepared in advance (see steps 1-5) and inoculated them with Aliivibrio strains from the lab (see Picture X). For that, we used some pipette tips (in the lack of inoculating loops). In the absence of sterile pipette tips, you can make an inoculated loop from a metal (like a paper clip unwraped) as explained here. Or you can sterilize some Q tips. The plates should be streaken in a zig-zag pattern (large zig-zags for Aliivibrio), and with care not to contaminate (keep it away from mouth area) and not to tear the agar gel. The lab strains of Aliivibrio stopped glowing the day before the GEN, so we could not see them glowing. That does not necessarily mean that the bacteria are dead. Aliivibrio are social bacteria, i.e, they glow only when they detect that they are enough (quorum sensing). However, if they are too many, and they sense there is a lack of food, they shut down their glowing properties. Like in a party: it has to have a certain number of people to be a good party, too few or too many kill it :).

9. We checked our incubated fish for some bacteria. We could not find any. It could be that the fish were too well cleaned and washed, or that it was too soon (ideally, the bacteria needs 4 days to glow). I kept the fish in my room, in a dark place and I will check them again in a few day to see if anything is glowing. We also tried to dissect the squid and take the ink out of the sack. Aliivibrio like to stay around the ink sack and there is a big chance we could have found something. However, we learnt that a mushed squid is not useful, so that step had to be done before …. We inoculated some of the plates with the liquid from the squid just for fun.

10. Everyone taped and properly labeled they agar plates. The plates will be kept in the fridge and in around a week we will check if any bacteria grew and if it glows. Also, Aliivibrio needs oxygen to glow, so, when open the plate for a short time to expose the bacteria to oxygen. It might glow stronger. However, be quick, as you do not want to contaminate the plate.