Doctor in training, teaching doctors in training

And so the practical SSC for undergraduate medics started and finished this week. Such a lovely group of students – very knowledgable, inquisitive and what I think was genuinely interested in oral microbiology!

The adherence assay is not a particularly challenging experiment to do, but for those that have no lab experience, it can still be quite exciting. The intro lecture was pretty good too – covered quite a lot of background, and hopefully passed on some knowledge and expertise of my own. Now they have a presentation of their own to think about and prepare for..

Final preparations are underway for OMIG which starts this Wednesday! Presentation is 95% finished, awaiting supervisors final input and then think about packing and the trip up :).

DM

When the PhD gives you lemons..

..make sure you get at least n=3. 

They say s PhD journey is 10% intelligence and 90% persistence and perseverance, and to be honest, I’m very lucky to have the project and supervision that I do. However, I would liked to have had this the other way around – not being able to get things to work in the first year, only to then generate a ton of data in the following years. It’s also times like these that the ‘imposter syndrome’ begins to rear its ugly head; why am I here when I can’t get s simple experiment to do what it should?! Why is cell biology such that cell lines appear to change in behaviour the more they’re passaged..that’s the whole point of an immortal cell line, it’s consistent! And thinking about it, it’s been consistent in my case but not the way it should be! Oh well, n=3 so whatever is, is.

On the positives, medic students are joining me next week for a dabble in the tiny world of microorganism and adherence,.going to be good fun and they’re really looking forward to it which is always nice :).

Preparation for OMIG well and truly underway too, car hire being sorted, presentations being written, posters being designed..and lots (and lots!) of lab work to boot! Looking forward to catching up with friends over a few drinks (soft, of course) πŸ˜‰

DM

‘Flow’ing like a river

So the plan for the past few days was to keep my cells growing (enough to use them soon if they grow quick enough!), do a bit of monocyte work and set up and maintain biofilms.. and it has once again proved to be a somewhat challenging but rewarding week at that.

Cells have established themselves very well indeed, and I’ve managed to grow enough to have two sets of freezer stocks, and two flasks actively growing, and the keratinocytes are doing pretty well too πŸ™‚ (albeit significantly slower growing than the fibs!). Monocyte work didn’t really go to plan..as ever..but still, learning how, and having the confidence to use the flow cytometer on my own is somewhat an achievement as far as I’m concerned – a mere microbiologist using some fancy tech haha! Still haven’t got the result I was hoping for, but it seems as though it is getting close to being statistically significant in it’s own right..so watch this space!

Back to the biofilm work that I’ve missed for some time while developing my ‘skills’ in tissue culture.. I fixed some for later (CLSM) and took some for RNA extraction, using the same method, but pooled three biofilms together..and managed to get more than 10x as much RNA yield!! Purity is amazing..but yield is just incredible. Considering for the past 2 years I’ve been getting yields of 40-80 ng/ul, to get 500 as the lowest is a pretty good achievement!! Fingers crossed for the next lot of batches too! I’ll have more DNA than I know what to do with πŸ˜€ (at least now I can do some proper qPCR optimising too..primer checks and all that jazz with consistent samples of DNA). Super chuffed to say the least!

Next week is the last before preparations for OMIG begin (9th-11th March, Gregynog (so excited)), and I have medic students with me Monday pmΒ and Wed (am), Thursday and Friday all day..should be fun (I think!). Anyway, today is Thursday which means Friday tomorrow, and I’m looking forward to spending a lovely day with my two boys at the museum, Ruben is really looking forward to seeing the dinosaurs, and Oliver will be too I’m sure if he knew what was going on! Fun times ahead πŸ˜€
DM

This week in review

What a week!

It didn’t feel particularly busy, because working with cells means a lot of waiting for them to…well, grow. It’s all the little bits that seem to add up and make what seems like it should be an average week, into a very not average week.

Did some flow work at the start of the week which showed the new antibody I bought in worked :D. I also had another clinical sample – a stomatitis one this time too, so excited to see what comes back off the plates for isolated bugs..and need to sort the DNA extraction for that too.. good times! Nearly half way with these samples now.

My supervisor had his reviewers comments and scores for a grant application which he needed to get on with, and has taken such a long time to get this far (fingers crossed David!), so this week was mainly focussed on establishing cell lines after the infections, making sure my bugs were viable and growing ok, trying to ship cells from Sheffield and preparing abstracts and presentations! The cells did finally arrive this week after a nightmare with the shipping, but they’re now established and growing (hopefully well!). I managed to get images of all the tissues from the batch that were infected, which took forever to do…the bloody microscope is not very easy to work with, and the x40 mag sense is awful!! Does not work at all..

Wednesday was a good day…gave a presentation at the School of Dentistry Research Day – all about biofilms and my work. Went pretty well, had some good questions (which I think I answered ok!), and some great feedback from people after the talk which is always nice to get.Β Also submitted an abstract for Biofilms7 in Porto, Portugal – a biofilms related conference (it’s in the name!) so fingers crossed for the tone..next I’ll be applying for some money to actually go haha! Priorities πŸ™‚

Next week is going to be a busy one..as it’s half term too, so staggered days of doing things, but should work out quite well I’m sure!
Time to go and pick up the kids now though!

DM

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 πŸ™‚
DM

An early (or late) present-ation

Today I headed downstairs to the labs and bumped into Josh (friend of mine in my research group), and as we’d both submitted an abstract to OMIG for this year’s meeting, I suggested that we may hear sometime this week – as last time the response was about a week after the submission deadline.
Anyway, we had a chat and I said for him to keep an ear out toward the mid-and of the week for any news. Low and behold, literally two hours later, we received the notification email!

Both of us had received the same email stating we have accepted for oral presentation at the meeting, meaning our registration fee is to be waived! A great meeting, now made much more affordable for the group!!

The meeting is 9-11th March at Gregynog Hall, Newtown, and proves to be another great success. Looking forward to it!
DM πŸ˜€

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 πŸ˜€

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 πŸ™‚

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

PCR – Positive clear result

FINALLY!!!

So, I’ve been doing DNA extractions of planktonic microbial samples, and biofilms…running PCR and then a gel as you do. I’ve seen amazing bands from my reference ladders, but absolutely nothing from my samples unfortunately. Now the reason why has somehow eluded me until now. Ok,Β Candida is different to bacterial samples, and is a bit more difficult to actually get at the DNA, but surely I should have been getting something to amplify!?

No PCR Bands :(

Well, I’ve been changing the variables bit by bit over the past few months, MgCL2 concentration, new and old primers, primer concentrations etc…but yesterday I changed the polymerase. I’ve been using the new G2 Taq polymerase from Promega, but for some reason haven’t been getting any results..however, I switched back to the original Taq and boom, clear bands!!

 

photo 2 (1)Now, I just need to repeat this so that I can say for sure that it is working, then get in touch with Promega for a refund of my 500U Taq purchase!! Ooh, also run someΒ CandidaΒ PCRs..

On the other hand, good progress has been made with the clinical samples – we’re basically ready to go (when I get this DNA extraction sorted at least!!). Exciting times ahead in the very near future πŸ˜€

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 πŸ˜€ (simple things πŸ™‚ )

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 πŸ™‚ (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 πŸ˜€