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Lab work

Highs and lows and highs

This week was filled with anticipation; ELISA kits due to arrive, antibodies due to arrive, tissue models ready to be used for infections.. However, it quickly became apparent that this week was going to be a tough one!

After chaining the medium of the tissue models (all 36 of them..) on Monday, all was well. Until Wednesday. During the medium change on Wednesday, I noticed the medium was cloudy, and upon a quick look under the microscope I found the wells packed with what look like fungal spores, or yeast cells (Candida?)! Such a shame as these had been growing so well for about 3 weeks, and only now, two days before they were needed, they were infected. I collected some and took them for pathological analysis anyway to check the structure, and this is where the good bits come into play – the structure seems pretty good for a first attempt at tissues (and with cells that are known to be poor at tissue models!). H&E staining was great, and the thickness looks pretty good, just need to wait for a proper pathologist to take a look and critique!

Another high point was the arrival of the ELISA kit which now works properly! Thursday was a great day for this – the protocol finished, and showed a brilliant set of results – nearly exactly what I wanted to see – LPS influences pro-inflammatory cytokine release of monocyte cells :D. More work to be done..but watch this space!

And.. after 2 1/2 years here, I had my first fire alarm experience…and then another today! Two in two days, but nothing for the whole time I was here before. Thankfully no fire or damage phew, and all is well!

Busy week ahead with more monocyte priming/challenge work to be done, flow work, ELISA, qPCR and biofilms!! Busy busy 🙂
DM

Back to it!

So..been a while since my last post!

Whats happened since?
2014:
– I attended OMIG at Gregynog, which was my first conference. I presented a poster and won the poster prize! #Win
– I completed the MEDIC SSC project with grate success
– I attended the MITReG Conference in Cardiff and won second prize for my poster
– I passed my first year progress monitoring with no problems 😀

2015:
– A paper on which I was second author was published int he journal Biofouling. It encompasses my first years work and having spent an amazing year working with Yuri Cavalcanti, we managed to get this done.
– I went to Boston, MA, USA for the IADR conference and was accepted for an oral presentation
– I had another son – Oliver!
– I passed my second year progress monitoring with no problems
– I attended 3 conferences in 3 weeks; BSODR, Cardiff (oral presentation;) CITER, Bristol (oral) awarded Highly Commended; and GSK symposium in Weybridge (oral).
– I co-founded the Early Stage Researcher Peer Support Group (ESRPSG) with Kirsty which is going well
– I helped organise a conference (PGR Day) and was tasked with organising the external speaker (Robin Ince – what a guy!!)
– I spent 3 weeks in Sheffield University with Craig Murdoch and his group (Lucie, Luke) learning tissue model growth, which I’ve just started back in Cardiff too 🙂

Theres probably a lot more that I’ve just forgotten…but thats enough for now!

2016 has started slowly..with lots of things taking forever to get established, and things not working, but onwards and upwards so they say!
DM

Teaching and Learning, OMIG 2014

I really dislike PCR. There. I said it.

OK, maybe I don’t really dislike PCR, but I don’t like when experiments don’t work. I find it hard to acknowledge the fact that doing a PhD is 10% intelligence and 90% persistence..life is very different in industry; if it doesn’t work, you leave it, or try one other thing to make it work.

Academic research is very different.. it brings to mind the phrase “if at first you don’t succeed, try, try and try again”. However, I had made a monumentally ridiculous transcription error (see what I did there!). Having had the PCR master mix recipe in my book (written by my PI for me), I had somehow managed to re-write this but during the copying over from one page to another, make a mistake in the volume of MgCl2 to add.. It went from 30uL to 5uL although I have no idea how I could have possibly done this..

Anyway, long story short, four failed PCR runs later, I decided to double check the master mix total volume..which didn’t add up, so decided then to check my PIs master mix and found the potential problem. the only other problem is that I have now spent hundreds of pounds on new reagents that I might not have even needed in the first place!!! My fingers and toes are crossed for tomorrow when Lucy kindly runs a gel for me to confirm amplification..picture to follow!

On a brighter note, I was accepted for a poster presentation for the OMIG meeting at Gregynog on the 12-14 March (tomorrow!!). So, five drafts of my poster later, and I picked up my final copy, perfectly printed (as far as I could tell after a quick glance) in A1 for presentation on Thursday (picture to follow too!). The hire car will be with me about 9am, leave mid-day and pick up Kirsty on the way through to Gregynog..looking forward to getting there and having the two course dinner 😀 (simple things 🙂 )

I finally met my first year undergrad MEDIC students this week too – in four groups, I gave a presentation of my PhD project in brief, and what the students are going to be doing as part of their practical project that I proposed a few months back. They all seem really excited to be doing it which is great, although not many of them knew much about microbiology, or candidosis..they will learn pretty quickly though I’m sure!

DM

Monday blues through to TGIF!

Monday morning came too quickly this week. Sat (what I hope to be) the last exam for he foreseeable future; Biomedical methods research techniques, and honestly..it was hard work! A lot about protein work, cell culture, bioinformatics but very little microbiology (one page of twelve total!) so not a particularly equal spread of questions over the course! Such a shame..but fingers crossed now until the results.

With the dreaded exam over, the rest of the week should really have been easy going – just get on with in vitro biofilm growth, confocal analysis on Wednesday, start again on Thurs and do adherence assays with more fluorescence analysis.. but no, this week had to be difficult!

Tuesday and wednesday came and went, the biofilms were analysed via confocal down at BioSci with Mark (who I hadn’t met previously..lovely guy, very helpful). Candida samples were great, worked well and got a few good images .. Streps however..grr! Managed somehow to get some kind of (what looks like Candida) contamination!, and there were some issues with the acrylic too, so couldn’t really get particularly great confocal images. On a good point though, biofilm thickness was about 25 microns again which is what I had hoped for (consistent with previous results, and some papers I have found since) :).

Thursday..what a pain, Cardiff Uni (dental school) have something called TESS..its a seminar series with invited speakers and internal speakers. now it hasn’t been running for quite some time, but this week was a visiting speaker looking at something completely outside of my research area, and of no real academic interest whatsoever to me personally (for others, it was very close to their projects so for them it was perfect!). But this seminar was compulsory for PhD students…compulsory! right in the middle of the day..when I should have been preparing to inoculate my acrylic..so that was scrapped for continuation today. Then today I realised that I said I would take Yuri to the confocal lab for his samples..so again, I couldn’t do an awful lot with my experiments! I did manage to get quite a few words written for my first year substantial report, but still..so glad this week is over and can hit the ground running Monday morning! Cannot wait!!!

DM

Oh, on another side note, I had an abstract accepted for a poster presentation at Gregynog in March 🙂 (didn’t get the oral presentation, but a poster is good none the less!!) So excited to give my FIRST poster presentation in this PhD, really looking forward to it 😀

Catching up!

Wow, over three months since my last post..sorry about that!

So what have I been up to in the past three months?
– I’ve gotten stuck in with lab work; making acrylic pieces, doing adherence assays and growing biofilms on them (and looking at them using fluorescence confocal microscopy to determine depth and arrangement!). Also have done DNA extraction, run PCRs and purified samples (ready for sequencing) of my stock cultures.. all good fun but very important work for when my clinical samples start coming in!..
– Talking of clinical samples, IRAS (ethics) forms have been submitted and we are ready for the panel meeting which is going to be the start of Feb.
– Literature reading..and lots of it!
– Cardiff University have a yearly review for each PhD student, but instead of being at the end of the year (as it is called an End of Year Progress Report), its requested at 8 months!! This is a substantial report (9,000 words), including a significant literature review and details of my methods, results and conclusions to date.. all the hard lab work will be put on trial!

Since my last post, we’ve had Christmas and New Year – so happy new year to everyone that I haven’t yet seen/spoken to :). Christmas was lovely – spent a lot of time with the family which was great, Ruben had a blast with all his presents and it was really nice to be able to spend as much time with him as I was able to!

They say doing a PhD is 90% perseverance, 10% intelligence..and that is most definitely the case!! The trouble with growing biofilms, which grow for 7/14 days or more, is that if you get a single contaminant..that’s it! I found out the hard way.. one week in, I noticed that the media was changing colour by the end of each day and wondered why that was but just thought it was significant growth..no.. turns out I had contamination and all sorts grew when I enumerated my experiment! Three weeks worth of work (as I staggered the experiments) down the drain..just before Christmas!!! These experiments start up again this week, so fingers toes and everything that will physically cross are crossed in hope that this works (and I’ve stepped up my aseptic technique to avoid any issues 🙂 ).

One of my goals when starting the PhD was to do demonstrating/teaching at the undergraduate level. Well, my luck is in! I’ve managed to take part in a first year undergraduate medical students demonstrating scheme. I get a collection of 12 undergrads, and get them to do some practical work with me which is part of their curriculum (and it gives me teaching experience too!). I’ve chosen an adherence assay with some variables that are of interest to me, so they may even contribute toward my work progress in some way. I have to give them a presentation in March sometime, then they join me in May for a morning (in three groups of four students). Then they go away and analyse the results, and prepare a presentation of their work. They do a lot of the work, but I’m there to help point them in the right direction..should be good fun!

I think that’s enough rambling for now..I now have a reminder to post more frequently! 🙂

Ooh, and I got my first tattoo today!! #TattooTimes

Tattoo picture: 'If I have seen further, it is by standing on the shoulders of giants"
‘If I have seen further, it is by standing on the shoulders of giants”

DM

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