Preparative HPLC at Plymouth Marine Laboratory

I recently had the opportunity to visit Dr Ruth Airs at Plymouth Marine Laboratory (PML) to learn some preparative HPLC techniques and fractionate some of my seaweed extracts. We wanted to try and split the ‘soup’ of compounds in the crude seaweed extracts into distinct groups, which I could then re-test to determine which fraction(s) contain the active compounds.

We first decided to try liquid-phase chromatography. However, we didn’t have a clear idea of the kind of compounds we were looking for (which makes it difficult to select the correct parameters for the extraction). Moreover, over 20 fractions were generated per seaweed extract, limiting the number of samples we would be able to process. After additional reading, we decided that we could be dealing with some very polar compounds- possibly beyond the remit of the column we were using.

With this in mind we switched to solid-phase extractions using a Waters column(more info here). This enabled us to cut the extract into broader ‘chunks’ and to more effectively target highly polar compounds. In short, the extracts are ‘loaded’ onto the column and then washed through with increasing concentrations of methanol. The more polar compounds are eluted first (in the more aqueous concentrations) and are then followed by non-polar compounds.

There were some pretty pigments visible in the extractions!

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Dr Hannah Florance has been doing some further HPLC at Exeter to try and dig even deeper into these fractions (no mean feat!) with the hope that we can get some more clues into the nature of our active compounds. I will be joining her in a few weeks to look through the data and will post again when I understand more!

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Why you should do a year in industry

Doing a year in industry has been the single best decision I have made during my BSc.

I was always sold on the idea of doing a lab placement, even if I didn’t understand what it would entail. In fact, I would go as far as to say that I hated labs with a passion and definitely could -not- see myself where I am now. If you get the opportunity, grab it. What’s the worst that could happen? You do a few months/year and hate it, but then you still have an awesome addition to your CV. You never know, you might just love it..!

Doing a lab project taught me that it is okay to fail.. and it happens all the time. Things go wrong and you have to go through everything until you find the problem. Science is full of highs and lows, and labs are often moving at 100mph or struggling to get off the ground. I have learnt so much more than I though I would, and have managed to learn loads of techniques and am even dabbling in a bit of chemistry next week!

So, thinking about applying? Here are some things to look for…

(1) Find a supervisor who will give you ‘guided freedom’
At first, freedom to shape your own project and design experiments will feel overwhelming, but by going through this process you learn 1000x more than if it was all handed to you on a plate. Although initially terrifying, I have learnt so much more by having the freedom to think critically and plan my own experiments with the knowledge that I have the support of my supervisor and can ask for advice whenever. Make contact with potential supervisors and hear what they have to say- remember the selection process works both ways!

(2) Location. Do you prefer being near the sea or does boredom strike when you are away from the city? The location will determine your social life and how happy you feel when not on placement.

(3) Subject. Think carefully about which area you want to do research in. If a project comes up that is really interesting to you but you are still indecisive about even doing a project, then think hard before turning away from the opportunity!

Above all, I have made some great like-minded (cray-cray) friends this year and have had a blast.. take a look for yourself!!

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New camera!

I had a dilemma last week.. to spend my savings (accumulated birthday money and the likes) on a camera, or to move it into my student account and start frittering it away on baked beans and southern comfort..

The pros and cons list went something like this..

Camera
Pros- FUN
Cons- Inedible

Food
Pros- Pretty tasty
Cons- Only provides short term gratification

So in the end I opted for the Nikon D3200.. which is an awesome entry level DSLR. I have already got some great pics from the camera (when I remember to take the lens cap off *cough*)! I am tempted to do a project similar to Humans of New York, but I am not sure how the good people of Falmouth would react.. Would be interested to hear your thoughts!

In other news, I collected some Wakame today. I started off at castle beach to have a quick look in the rock pools and have lunch before heading off to Falmouth Marina:

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Wakame

Wakame

Weather was lovely!

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Lush lunch at castle beach!

Lush lunch at castle beach!

Without further ado, here are a few general pics from my new camera!

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Washed up kelp

Washed up kelp

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Ceramium! Ceramium! Ceramium!

Finally.. a blog post that starts with ‘Today!’

Today, I went rock pooling with my flatmate and budding photographer Sonia Stokes (see her photography website here). It was another lush day in Falmouth and the rock pools are starting to look colourful again:

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Diversity in the rock pools! Including wireweed, ulva spp., and lots of coral weed (bright pink!).

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We were stumbling around the rocks (me in my newly acquired wellies, Sonia in trainers) when I spotted something red out of the corner of my eye.

I found a big clump of Ceramium (unmistakable due to the banding on the thalli). Upon closer inspection back at home, I am pretty sure it is Ceramium rubrum. My macro lenses for my iPhone (of which Sonia is intensely jealous), helped with the identification, and although this doesn’t replace microscope identification, it helped me to rule out a large number of Ceramium species due to the lack of ‘hooks’ along the thalli. Either way, I have collected a voucher specimen which is currently being pressed for future reference.

The Ceramium was actually growing epiphytically on some kind of brown seaweed:

Ceramium rubrum in shallow rock pool

Ceramium rubrum in shallow rock pool

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A x15 macro shot of the C.rubrum, showing the banding and distinctive hooks. You can also see what looks like a coating of epiphytic algae on the C.rubrum itself.

Here’s a pic of us enjoying the day:

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Rock Pooling in Penzance

Had a great day rock pooling in Penzance a few Saturdays ago with David Fenwick Snr and his partner Carol (their website aphotomarine contains a wealth of marine photography and information), as well as my supervisor Mick Vos and family (See Mick’s blog An Bollenessor, which documents both his rock pooling adventures and evolving marine tank). It was a lovely sunny day and it was great to talk to David and Carol about marine life and their passion for the conservation of the stalked jellyfish, a member of the Stauromedusae order.

We collected 15 new species to test, including two different Codium species (C.tomentosum, native species, and C.fragile, an invasive species). The antimicrobial activity of both species has long been reported, with no difference observed in antimicrobial potency and both having good activity against S.aureus (Hornsey and Hide, 1974). It will be interesting to see if these results will be replicated in my own experiments. I am also trying to collect more red seaweeds (especially Ceramium spp.) after some promising halos (see my last blog post).

Some pics from the day:

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A Seaweed Rainbow!

Going to do a few posts in quick succession to catch up on my blogging backlog!

I did my first large experiment, testing 29 extracts against 3 of my ‘weakest’ (aka. least resistant to antibiotics) S.aureus and K.pneumoniae strains. I evaporated some of the excess methanol away to increase the concentration of my extracts, which looked very pretty in the fume hood!

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The final extracts looked like this after evaporation:

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8 extracts were active against S.aureus and 2 against K.pneumoniae. I will be focusing more on S.aureus in future assays, as it is generally more susceptible to the seaweed extracts than K.pneumoniae. The largest halo was produced by a species of Ceramium (unfortunately not identified). Both the Ceramium sp. and the Bushy rainbow wrack extracts had activity against both bacteria, although the halos were always smaller against K.pneumoniae.

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Disc impregnated with Ceramium sp. inhibiting growth of S.aureus.

Ceramium sp. produced a smaller halo when tested against K.pneumoniae

Ceramium sp. produced a smaller halo when tested against K.pneumoniae

I am now refining my methods to try and standardise the concentration of bacteria (notice the swirly uneven distribution in the agar) and am making sure my methods are sound before I continue with any more assays!