3RD WEEK

Another week upon my quest to become the Media Queen! (It’s actually going quite well). Started out the week by banging out a few Falcons of Piggy IVM media not only faster than before, but remembering that they live in the fridge (as opposed to the incubator) and end up in Nunc plates, making themselves useful (instead of being chucked in the bin). YEY! The incubator we have been using decided it wanted to break so kept making a joyous noise (see video) which probably just put me off going anywhere near it! The most fantastic bit about the IVF lab is that the pig stuff doesn’t like aircon..fabulous when it’s almost 100,000 degrees thanks to the heatwave!

Silly incubator 

This week was a big ovary week with a nice(?) flaskfull arriving on Monday and a few more (not as many as expected L) coming on Wednesday! On Monday afternoon I spent 2.5/3 hours sucking oocytes out of about 70 ovaries (delicious) but on Wednesday had to stay at home thanks to a bit of food poisoning (could have been interesting trying to aspirate stinky pig ovaries in that state).

Piggy ovaries in a jar, piggy ovaries in my hand and piggy oocytes and goo.

Towards the end of the week I started getting things ready for some mini-prepping and labelling. First step was making up a couple of plates (haven’t done that in a while so had absolutely no clue what I was doing although I definitely should have! Bad scientist L) which were then streaked with BAC clones and incubated overnight. The BAC clones (called Pig-E BAC…piggy BACK..piggy-back, gerrit? tehe) are stored in an extremely cold freezer which makes it feel like you’ve stepped into the arctic when you open the door – which oozes a whole load of condensation/fog (pretty epic)!

Frozen freezer

In an attempt to fix the extremely annoying incubator the CO2 canister was changed to see if that’s what it was moaning about. Kate and I had the fun task (I say I but I was just there for support and door opening ;) ) of going to stores to get a new one. This is the furthest lab away from stores, CO2 canisters are heavy and their trolleys are a bit pants. Managed to drag it back and get it refitted just to find out that the canister wasn’t the problem at all.

Fertilisation Friday came about really fast! The morning was occupied with exercising some sperm while separating the “boys from the men” using a swim-up tube which is pretty much a spermy race. Some pretty photos were taken of the nicely matured eggs (I’ll put them up next week) and then we all headed off to Pizza Express for Kara’s birthday lunch J (she’s nearly finished her PhD, woohoo!). A full (both through busyness and food) afternoon was compiled of fixing and mini-prepping. We fixed some embryos (stuck them to a slide) so that we can do some FISH on them to test our sexing stuff (not as naughty as it sounds) however, apparently this rarely actually works- I was told the embryos “float off into the sky” but I think that was a fib. The mini-prepping was done to get ready to make the probes for the FISHing (busy busy busy).

Nicely mini-prepped and ready for next week!

  

WEEK 2

What a busy busy week! (But also a really cut up and inbetweeny one). No ovaries this week because of the lab away days but there are loads coming next week so a lot of my time was spent making media (or cacking it up as the case may be) and learning a bit about PCR! I also had all of my old housemates staying with me so they could graduate (don’t leave me!) CONGRATS TO THEM ALL!

So, at the start of the week I went about learning what the PCR fragment analyser actually is/does (with the help of Evi and her notes, obviously). Basically, what this massive noisy machine does it estimate the size and amounts of DNA fragments as well as the alleles that they represent. What we’re using it for is to see if we can determine the sex of a pig embryo based on the presence of a specific gene.  To get it going you have to pull out loads of fragile looking things and replace them with even fragilerer things! You then load your samples and a ladder which you’ve previously PCR’d and voila, after a few hours you’ll get some nice graphs showing you what was inside.

Why so sad Beckman? (Kinda looks like a sad/happy snowman)

Making the media was a fun experience…it was all going fantastic just chilling in the IVF lab with no aircon (the piggies don’t like it) during some of the hottest days ever until it got to the bits where I needed to measure the pH of the stuff. Stupid pH machine absolutely hates me (yes, that is possible) and it took an age for it to decide to tell me what I wanted (this is after 3 people recalibrating it!). If it wasn’t so useful and expensive I would’ve hurt it but instead I just gave it a piece of my mind and moved on. Naughty pH meter. To top it all off, clever Daniella over here thought she knew what she was doing so went straight ahead and put all of the media she’d spent the last few hours straight into the incubator…it lives in the fridge…D’OH. And that day’s life lesson was that Daniella knows nothing. I joke, it’s to thoroughly read instructions.

In the bin!

On Friday I spent the day remaking everything (the silly pH meter lied to me and forced me to have to remake one or two agaaaain) but practise makes perfect and I WILL become the Media Queen.  The media are now all nicely resting in the fridge and are ready and waiting for their time to shine when the ovaries are delivered next week (Woohoo more hoovering)!  

I hate you.

The lab away days were fantastic! A great opportunity to get out of the lab and discuss what’s been going on, future plans and, of course, do some “team-building” (also known as a fantastic excuse to play ridiculous games like blindfolded maze and the box game). It was crazy sunny and absolutely beautiful. It was great seeing everyone a bit more relaxed than their usual crazy lab-selves and I’m very grateful to have been invited along! 

Just down the road from the Lab Away Day, so puuuurdy!

WEEK ONE!

WEEK 1

This summer I have been given the opportunity to spend 8 weeks working in the Griffin Lab at the University of Kent working on Pig IVF and embryo sexing thanks to the Biochemical Society summer studentship (woohoooo science!). After already spending last summer here as a volunteer I have the advantage of knowing everyone (I'm part of the furniture) which got rid of some of the nerves. However, since last time I was here I was working on a different project, I have absolutely no idea what I am doing! The first job of all was to get reading! I was given a pile of relevant research papers and I spent a good amount of time highlighting and asking loads of ridiculous questions. For the first week I have been focused on getting to grips with the pig IVP protocol which involves a vast amount of pipetting, a huge amount of waiting and then (the fun bit) using the micromanipulator!

After a couple of days delay the pig ovaries finally arrived, albeit slightly too warm for comfort at a toasty 39˚C (they should be a few degrees less after their little journey- probably a bit hot going in!). Opening the flask released the stench of pig juice that soon filled the room (I could even smell it through my cold!). After a bit of getting used to the smell, the ovaries were taken out and an aspirator was used to extract the oocytes from the follicles. Only the larger, juicy follicles were pierced and the dark, nasty looking ones were left where they were. Some of the warm handling medium needed to be aspirated regularly to maintain the warm environment for the removed oocytes. The handling medium and oocytes were then poured onto a pre-warmed petri dish and, using a really cool EZ grip pipette with a bendy needle on the end, about 200 good oocytes (with 2/3 layers of compact cumulous cells) were moved to a nunc plate. Porcine oocytes differ from human oocytes in that they are much darker and thicker in appearance. These cells were then transferred to a maturation plate containing some hormones, along with some other bits and bobs, and placed in a CO2 controlled incubator until the next day.       

Evi (Masters) being a scientist and some cute piggies!

The next few steps involved some moving of media and oocytes into different incubators but, seeing as this was quick to be done, I got to spend the rest of the afternoon on the micromanipulator! Just for a bit of practise, I spent some time figuring out the different contraptions such as the fine and coarse controls as well as the micro tool holders and then played around sucking dead pig oocytes into the holding pipette, moving things around with the biopsy pipette and making holes in the zona pellucida(s/ae?) with the laser (which had a fantastic huge red “FIRE” button on the remote). The coarse controls let you move the micro tools further across the field of view than the fine controls, which are used for the fiddly bits. I tried my hand at then sucking out the cytoplasm of some of the cells (pretending that I was extracting a blastocyst) by pushing the biopsy pipette through the hole created by the laser and then using the suction control to pull out the contents of the cell. This was really tricky because of the rubbery texture of the cells and it took a few stabs with the pipette and a couple of extra laser blasts to break through. My supervisor, Kate, is going for a lecturer interview tomorrow so we all spent a bit of time in the afternoon quizzing her and making her practise her presentation!

Micromanipularator

Fertilisation day arrived! Yet to see how this has panned out but the sperm motility was pretty poor (they were mostly dead or just having a little wiggle on the spot). Lots of testing of the media had to be done to ensure good pH and osmolarity levels. After a small mishap with a slight pH overshoot after adding too much NaOH all was forgiven and both levels were on track for success! The only other issue was a slight spill of half of the sperm wash stash (it was definitely one of those days) but we luckily still had enough for what we needed to do! One of my favourite aspects of today’s protocol was that you had to give the sperm their morning fix of caffeine so that they would get a bit of a move on, you can do it boys! And also…Kate got her job, YEYYYY!

Kate being happy about her new job!

Next week: PCR, FISH and more Piggiessssss!