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Lab work

Hard at work, or hardly working? 

That has always made me chuckle. And for those who are concerned, hard at work, all the time ;). But the past two weeks have been particularly super busy! Mainly in the lab. That’s a good thing I guess, in some ways at least. It means you can’t get distracted, but by working in the lab you are by definition distracted from writing.

This week, Josh and I had some tissue models come in for infections, so we grew biofilms as we do for a few days, and then infected the tissue models. All 72 of them!!

Luckily we can do the analysis in stages, so we did LDH, and then the RNA extraction, and Josh has since done his RT, which I’m yet to do. We also had some. Time in pathology, embedding the models in wax, with some help and training from the lovely lovely path guys!

So, that doesn’t sound like much but it’s taken a week to do that! And ne t up we have the sectioning and staining, microscopy, qPCR and analysis of the data then from the stained sections, more sections and use of fluorescent probes and more microscopy! Much fun to be had.

And in the background, I’ve been writing the thesis. It’s been difficult, and I wasn’t able to get as much done foe my first section (which was the lot review, and was incredibly difficult to do!) but i did get to hand in a draft of another section of a chapter, and I’m ahead of schedule (a day early of my weekly targets!) for this next one! These may only be materials and methods, but are still important and a necessary part of each chapter.

I’ve made some changes to my IADR poster and sent that for printing (paying £50 for the privilege! £30 of which was the rushed delivery haha!). Looking forward to receiving it and taking a look, fingers crossed for no typos haha. Onwards!

I can see clearly now

The leading statement ‘I can see clearly now’ has dual purpose. Firstly, I have new glasses 😀 20161019_165146

I haven’t had glasses since I was really young – probably 20 years ago now (man that makes me feel old..). I should have continued wearing them through my childhood and maybe I wouldn’t have needed them now!! Oh well. Ruben has glasses, and if he can pull them off as well as he does, I’ll give them a go. So I went to have my eyes tested, and sure enough I still need them (believe it or not). Heres the technical part – my eyesight is 20/20 apparently…but my prescription is longsighted; a 1.75 in both eyes but they gave me a 1.25 (to make it easier on my eyes first time round), theres also a 2 point astigmatism, and very slight turn in for my right eye. No, I have no idea what it means either, but I need glasses for concentration work. So I chose these – nice eh?

Anyway, I digress.

I have had a ton of NGS data back form the sequencing company, and the lovely Ann has done a load of analysis for me on it. The data is really good quality apparently (which is always nice to hear of course!), and the analysis has started. Trying to make head or tail of the hundreds of thousands of cells of numbers, OTUs, proportional data, phylogenetic stuff and diversity/abundance…its all very new, very daunting but very very interesting.

Ann has done such a great job of answering the questions I have for my project so far, with a few more to come, and the data was presented at the recent GSK symposium, and after a supervisory meeting this afternoon, is beginning to make much more sense. It turns out, its actually a pretty good result overall! #LetsGetThisPublished

More to follow when I can! But lots more to do for now, so bye!

Summertime!

And so the summer is here, kids are off school for summer, people going on summer holidays, the sun is out (or was for a few days at least), and people are generally in a happier mood :). And for those budding microbiologists of you out there, you know the time where you can grow your bugs on the bench instead of the incubator 😉 Come on, you’ve all done it….

Win win, right?

Unfortunately, science doesn’t know what summer is, and doesn’t know what a holiday is. But it’s not all bad! Currently I’m at all systems go! My NGS data first stage analysis has come back from the lovely Ann in BioSci (woo!) and it is pretty good actually. Some great looking data which just needs more analysis, questions being asked of it and answers hopefully coming out of the other end of the pipeline..with plenty of graphs and pretty pictures to explain them! This is the hard bit!! Staring at a spreadsheet of numbers, making slight adjustments to species names based on BLAST info..and making general sense of the just of what is going on. *sigh* PhD..keep thinking, it is for a PhD! There is soon much to learn about this bit of work, and too much to do in the other bits that I haven’t finished yet! And it I want to be out of the lab in October time, I need to move relatively quickly..

So, with that in mind, I have tissue models growing, biofilms growing, RNA ready for extraction, qPCRs ready to run, cells growing, bugs growing, hundreds of PMMA coupons made and soaking..the next few weeks are going to be busy, but all in the good name of science. The light in this tunnel is nearly closing in…! I am looking forward to spending some time with Lauren and the boys for sure!

It is nearly time, but there is never enough time!

This week has been a hectic one to say the least, and we’re only half way through!!

The week started with poster printing for the Biofilms7 conference this weekend (25-28 June) in Porto, Portugal. All sorted and poster collected, I then had another run of host cell responses to microorganisms underway. Seeded the cells ready to go..we’ll see how that goes in the next couple of days..

Tuesday came and went really quickly, Get into work and then straight on with challenging my cells with various conditions for the responses expt. Had a course booked, but with everything that was going on in the lab, I couldn’t go..but it was a presentation skills one (and having won awards for presentations already, I’m not too disappointed!). Antibodies have arrived to do some tissue section staining int he coming weeks/months too…so exciting times ahead, watch this space!

Wed was another super busy one. Dropped Roo off at nursery and rush into work to get everything done that I needed to. Check insurance/MOT docs for travel, sort passport details for flights, check in online and sort boarding passes, start the ELISA for cell responses and collect 24h samples for it! All while trying to give some guidance to PhD friends, and somehow fit in a coffee…! Preparations are well underway for my trip to Porto this weekend..and lots of fun to be had I’m sure. However, thinking closer to home, things may well be very different in the future if I wanted to travel to Portugal if the UK make any mistakes during the referendum tomorrow….

All this rushing has got me thinking. Imagine working in a country that didn’t adhere to the EU working directive, of no more than 48 hr/wk. Now, this is a real possibility if we are not governed by the EU. As in, if we left the EU. What a stupid mistake that would be, not only for the working directive, but for financial, economic, collaborative (particularly scientific), and security implications. We as a country need to REMAIN as part of the European Union, and give the finger to those that are on the side of selfishness, and in my opinion, bordering racist. Those who claim that immigration is the biggest issue with our relationship with the EU. Those that make worst of factual evidence that simply doe not stand on it’s own. It is such a shame that these idiots are in a powerful position of scaremongering the public (particularly those who are led by the media), and poisoning the minds of those that are unsure, or less well informed of the real facts.

Sure, our relationship with the EU is not perfect. No relationship ever is. But we cannot, and should not want to, walk away from this relationship as a result of these poisoned ‘facts’ that are being thrown around. We have one more sleep(less) night ahead before the biggest decision the UK will make in a generation, which will affect the next generation, and even the generation after that. Dint make a stupid decision, Vote Remain

No more hand-me-downs

NEW PIPETTES! Most often is the case that researchers join an already established group, where they inherit equipment, consumables, reagents etc, and included in this are pipettes. They may be old, uncalibrated, unchecked, but still used. In a scientists day to day life, here are few things quite as exciting as getting new pipettes! Not very often do you get an opportunity to browse, test and evaluate pipettes and then get a new set! Of course they belong to the group, and I won’t be the only one using them but they’re amazing and I’m so chuffed 🙂 I can’t wait to get my qPCR on and make use of them!
DM

If it looks too good to be true

Then you’ve probably done something wrong! Like picking up a pack of chicken breasts at £1.15 thinking you have a real bargain, when they are actually £1.15 for each breast…so £4.60 in total!! But you buy two packs because you’ve no idea, and get home to be told what an idiot you really are!! Or adding too high a concentration of standard! In this case anyway…
Looking at host cell responses for cytokine production of cells after microbial challenge, the ELISA to quantify this cytokine worked all too well. The concentration of the standard was way too high for the samples added..so a few amendments needed but nearly all good to go – great news!
DM

Aseptic technique..

This is a pretty important aspect of microbiology: necessary to avoid contamination, or cross contamination; good old observation of strict aseptic technique.

Now you don’t necessarily have to observe strict protocols or even adhere to ‘strict’ aseptic technique, but if you are advised to work around a bunsen, flame tweezers before use, flame lids and keep tip boxes closed between use, then you’ll probably avoid most contamination risks. Well if you don’t listen, this is what you end up with.. 

 I’m pretty laid back about working habits and don’t get contamination, but I’m also impressed that it’s even possible to get this much contamination.. Oh well, positivity..that’s what they say right?!

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) 😉

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 🙂 (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 😀 (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 pm 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 😀
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. 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 🙂

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

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