Dr Susie Wood of Cawthron, Nelson describes the process involved in testing sea slug bacteria for tetrodotoxin. She explains how bacteria are removed, isolated and grown in agar plates and then marine broth. The broth is then removed from the bacteria. Chemicals are used to break up the bacteria, which is then analysed for tetrodotoxin using liquid chromatography-mass spectrometry (LC-MS).
Jargon alert: Liquid chromatography-mass spectrometry (LC-MS) is a process using specialised equipment to detect toxins in substances. The process can accurately determine the molecular mass of different toxins, giving scientists the clue to work out what toxin it is.
TTX – abbreviation of the toxin tetrodotoxin.
DR SUSIE WOOD
We take a slug that’s got, hopefully got high levels of TTX, so we select slugs from sites where we know the levels were really high. We’ve then been dissecting the slugs. We take out the gonads, the stomach and usually the mantel, so the outside of the slug. Our previous research has shown those are the areas where the levels of TTX are highest. We take a little piece of each of those organs and we homogenise it, we blend it up with a little hand-held blender. We then take a subsample of that homogenised organ and we spread it out onto an agar plate
We leave those plates at 18–20 degrees, which is a similar temperature to the environment that the slugs would be in. And hopefully after 24 hours or maybe up to 3 days, we get individual colonies of bacteria growing. And we look at those and we try and pick different colonies which might be different shapes or different colours to try and capture as much diversity in different species as we can.
Then we take a little scraping of one of those colonies and we put it into a litre of what we call marine broth, and so marine broth is basically just seawater with nutrients added and so it just increases bacterial growth. And that marine broth sits on a shaker, which just keeps the liquid swirling around and in suspension for about 48 hours. And at the end of the 48 hours, hopefully we’ve got a broth that is nice and thick and full of billions and billions of that one bacterial species.
And from there, we take the broth and we centrifuge it. So centrifuge is spinning the sample round really, really fast in a machine until we get a pellet at the bottom of the sample bottle. So we separate the pellet, which is all the bacteria from the media, and we get rid of that extra liquid cos we don’t want to analyse that, and we just take the bacterial pellet, and we try and extract any TTX out of that. And that just involves a series of adding different reagents, different chemicals to that pellet to break it up. And from there, the sample is analysed by our chemical method that we’ve got set up here, the liquid chromatography-mass spectrometry