Progress report #3

What has happened since the last post?

I’ve completed the first experiment, come up with a new idea, flown to Europe, fleshed out said idea, back to Sydney, finished developing the idea, come up with a new path for the PhD, moved out of the old building, moved out.

It has been an eventful few months to say the least.

I suppose biggest difference. Change in the PhD.

My previous question was incredibly function-based. Not that that itself was a problem, but it was built on a premise that was rickety to begin with. The trip to Europe was fantastic to clear that up. But at a fundamental level, the field lacked a cohesive conceptual framework to test hypotheses. Lots of research was coming out, but it lacked a central point to gather into theory.

Here comes the change.

I had realised that I was attempting to build upon existing observations, but those observations had little general theoretical underpinning. It’s difficult to make sense of data, when the data doesn’t build up to something. So now I’m attempting to build up the conceptual framework. To create a thread of theory to attach observations and data to.

Now my background wasn’t in community ecology, and it comes as a bit of a shock to myself to find myself theorising about the drivers of community assembly. It’s an incredibly hot field currently, and to ride that particular wave is exciting.

Now comes the hard part of finding a place to accept that idea.

And back off to writing i go.

 

 

 

 

 

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The Second Pilot.

Because the first one didn’t go as well as it could have.

Pilot studies are pretty integral when you go exploring in uncharted territory. Doubly so when you’re borrowing a technique and then changing bits and pieces of it. While the technique is pretty established and relatively fool-proof, changing it up has had it’s issues.

Now I had set this up on Monday this week. It’s currently Friday and two days behind my intended schedule for my first data point collection. And that was because I had set it up Monday morning, then promptly developed a fever Monday night. After spending three days in bed running a 38-39 (102-103?) degree fever, I hoisted my ass out of bed dosed on enough drugs to make sure I don’t screw up a 10 minute plate read.

This entire shebang would’ve been a lot easier if I didn’t live a 2 hour train ride away. Even now as I type on the train I can feel the cold sweat and clammy feeling on my skin.

And im going to try my hardest not to sneeze onto my indicator plates.

Nine Month Mark

Nine months.

That went by quick.

Of course it’s closer to 10 months by now, but i’ve had this draft open for the past few weeks with every intention to get some words on paper. Not to be of course. But I have given myself the goal to write some sort of a progress report and unlike the previous few posts, boy do I have things to report.

After much wrestling with PCR and mite infestations, I am happy to report that yes, I have put that chapter behind me. Not to say I won’t revisit said chapter, but for the foreseeable future, I am working on my first experiment!

Which has been put into logistical limbo for the past few months.

If it wasn’t delayed meetings it was reagents taking forever to arrive. Then it was a failure of the agar to set. But all my fears and anxieties were allayed by the reassurances of everyone from fellow PhD students to my review panel going “oh yes, shit happens.” Though that does little to halt the feeling that I haven’t achieved much this year. Which I know at least on a logical level is complete bollocks. I have extracted a library full of fungi,make agar, make antibiotic agar, learnt how to PCR, clean DNA, prepare sequencing reactions, reviewed the literature, actually review two papers (i’m so sorry), plan my papers and have had several successful meetings with the post docs of my supervisors.

Having written that all down i can conclude that yes. It has been an eventful year.

Yet I still don’t have data.

Which is a bummer, yes, but in the meantime I’m working on an old manuscript.

And wrestling with R.

At least i think i’m winning in that altercation.

I think.

 

 

 

Molecular Madness

I suppose it comes as little surprise to people that molecular work is finicky. I thought I knew that until I started working on it. I’ve managed to contaminate my negative control 3 out of the 5 times I whipped up the master mix and somehow, just somehow, I managed to get a clean sequence out of it.

Jess, the amazing human being matched with equally amazing patience finally deduced that one of my core PCR ingredients was contaminated.

Hur Dur.

So of course when I mixed up another batch, the contamination persisted.

Hur Dur^2

Jess then suggested that maybe I’m getting spores from the outside of my tubes. And considering the fact that many of my fungi are sporulating like mad, it was very likely true.

So now I’ll be redoing some samples and hopefully I’ll have clean PCR products to run sequences.

And then begin the next chapter of my molecular madness journey and learn a brand new technique!

I would say I miss the greenhouse, but honestly, i’m kinda digging this white lab coat business.

Writing.

is bloody hard.

It always seems to come as a shock when my hands do a fantastic impression of spirit-fingers over the keyboard; they never quite touch it and yet when they do, it’s normally followed with the ferocious tapping of the backspace key.

I wonder if they intentionally change the pressure plate of the backspace – it always seems to sound louder. That or maybe I have more conviction in what I know to be wrong compared to the tentative stringing together of words to form sentences I’m proud of.

I write slowly.

Maybe it’s the fact that I’m not 100% on facts. Maybe it’s because I’m neurotic and if my sentences don’t flow perfectly, they’re not good enough.

I also don’t draft nearly as often as I should.

All of the above basically means that come writing season, I’d be lucky to get more than a paragraph a day.

That’s almost a win.

 

also.

 

#yesoxfordcomma

 

Progress Report #2

Can you believe it’s already been one week.

No, I’ve been slack and need to pick this up again. But alas, it’s been an eventful few weeks. I had completed all my subculturing, submitted a manuscript, gotten my manuscript rejected, started my proposal, went to the wedding of one of my favourite people, and chucked out almost a quarter of my subcultures due to a mite infestation.

And here I thought I’d left the mites behind since leaving the greenhouse. But nope. No luck.

image

But it’s not all doom and gloom.

On the plus side, I’m fair certain I’ve gotten at least an analogue for all the cultures I’ve made. And means less PCR samples. But minus side. Maybe I lost a few.

Which brings me to PCR. Last time I did that, I was in my second year doing a genetics course. Of course all I rememeber/took from the course was how to characterise Drosophila melanogaster bits under a microscope. But the skill I mainly developed was using just the right amount of pressure to anaesthetise the flies on ice and not turn them into fly putty. All this meant that I hadn’t the foggiest idea how to PCR.

Though I’ve been comforted with the fact that someone will be walking me (holding my hand) through the motions.

I mean there’s definitely a limit to how badly I can screw up PCR right.

Who knows. Maybe giving myself a sixth finger won’t be too bad.

Progress Report #1

Long overdue – but I’ve come to realise that there’s a limited amount of things to actually write about. So forgive me if the “things” become more abstract.I began this venture as an attempt to get myself writing more and I’ll be damned before I drop out after three weeks! So before I start abstracting, I’ll start with some more things that have happened.

I’ve now completed all my fungal isolations.

That was a monumental moment for me that lacked any fanfare and kind of fizzled with the dying wheeze of a deflating balloon. I remember chipping up my last wood block and clapping my hands together and thinking “done!” before thinking about catching the bus home.

I suppose realising that it was the first of many milestones to have happened kinda dulled any celebration. I expect there to be a lot of steps to be done before I can step away from the lab for a bit. Being woefully behind on reading isn’t inspiring much confidence in me.

The next few steps involve further subculturing by placing fungi on one plate to another.

Then the massive task of identifying all the plates I have. I think i’ll let myself have a party when that finally rolls along.

In the meantime, I’m trying to submit a manuscript – but the process is just a hoop after a hoop after a hoop and I just wish they could streamline the process and make it just a little friendlier.

Also I’m going to attempt to write every week again. Just to get into a rhythm again.

Even if the posts are disjointed and short. It’ll be good to pick up the habit.

 

 

 

 

 

 

Plates vs. Plants

I’ve made a point to say that for the most part, I’ve worked in a greenhouse. I collect seeds from weeds, germinate them in soil, and then transplant them into my test pots. I don’t normally get results for weeks on end.

Which makes plates somewhat a novelty for me.

Plants grown in the greenhouse are stationed there. They get regular watering from automated sprinklers (apparently not a universal greenhouse feature I’ve been told), and the temperature is regulated*.

Meanwhile, I’ve got 15 plates with malt extract agar sitting in my shelf wrapped in foil.

Bringing my experiment home is strange, but it affords me the option to see how my fungi are growing. With 4 hours of commute by train, I’d much rather not waste a day way out west by seeing how my fungi are doing at home.

But in saying that, I’m also a way out of my depth. I have yet to figure out how to ID fungi from plates – and cursory searches online and meetings with my supervisor have allowed me to come to the conclusion that short of PCR, ID may be impossible.

Microscopic differences also require some delicate measure, using sterilised cover slips and a water agar to get a sparse fungal growth over the slip. It’s all getting quite complicated.

As of last Friday, I have completed all the isolations on MEA + RB media. Which still leaves me with a lot of subculture plates to go. I fully anticipate drowning in plates before the start of March.

And then I’m reminded that I’ll be doing more isolation using a few other agar plates. Which means I’ll be doubling what I’ve already completed.

Glutton for punishment indeed.

 

The Lab

“…ple coloured bin. While making sure you put the used tips into the bag.”

Lab inductions are a necessary evil, even if the majority of what is said can be summarised with “don’t be an idiot with things that could hurt you”. But then if you’re like me, you zone out at inopportune times, which means you may have to scrounge around for context clues to figure out which colour bin you should be putting those used tips in (the fact that you wouldn’t be touching the chemicals is irrelevant, though it may have contributed to the aforementioned zone outs).

To continue, I haven’t been trained to work in a proper microbiology lab. I knew vaguely of the procedures required from having done a course or two on microbiology (or was it molecular biology?). My undergrad experiments were primarily done in a greenhouse. There were rarely any hazardous chemicals and the level of precision required for the chemicals I did use were measured by the “capful” and “one more for good measure”. The safety paperwork involved drinking water and avoiding that slippery patch of algae between rows three and four.

Suffice to say, coming into lab with standard operating procedures for every conceivable thing was a culture shock.

(Dirty glasses on the bottom of the ORANGE trolley.)

I’d like to think I’m adaptable – which is why I found myself studying fungi perhaps, that or a deep seated sense of masochism – so I made sure I paid attention to the induction. Except when it ticks past 73 minutes, which is the extent of my attention span. Though as I scramble to refocus my attention, the lab safety person goes to the last slide. Looking down, I realised I had still managed to write down a page of notes. I figure I’m good enough to not set myself on fire.

The first time I entered the lab, I was met with the stark realisation that it really was just a small room (cramped to be honest) filled with expensive equipment and glassware. I pointed out the plentiful glassware as a stray observation and the person showing me around told me that there were never enough and that there is a store downstairs for more. My mind boggled. My legs followed.

In my first week working in the lab, I had learnt how to make my own agar media, how to autoclave said media without it blowing up, how to use three different kinds of fume/flow hoods and how to crack open a piece of decaying wood (though the last point was mainly through trial and error). Really, it wasn’t as bad as I thought it would be. It wasn’t a leaky greenhouse where I could (would) dance and sing loudly to whatever was playing on the radio that day, but I could see myself doing the work.

Based on my timeline, I should have pure cultures of my fungi in a few months, so I’ll be here plenty yet. And now at three weeks in, I’ve yet to set myself on fire despite working with 100% ethanol.

Which is of course when my supervisor sends around an email saying that one of the bins caught fire. He didn’t specify which colour it was.

I’m fairly certain I didn’t set something on fire.

An exercise in writing/distraction

My first (proper) meeting took place somewhere an hour and 45 minutes train ride away. I was dressed to impress in jeans and a button up while my supervisor was dressed casually in shorts with mismatched socks. At this point I was reminded that I had, in fact, chosen to pursue a life of tweed, elbow pads and socks with sandals.

It was always present somewhere at the back of my mind that I would do a PhD. Despite all the doomsayers and bleak prospects, I had still managed to miss the clue train and signed up for another three (let’s be honest, four) years of study. Some friends knew it all along and sighed their sympathies. Others would bawk a little, to which I replied that I was a glutton for punishment. But I always appreciated multiple perspectives, it stops me from becoming complacent with my own. Like this one student who shared a room with me while I was looking at leaf hairs of a beach daisy under a microscope. She hated her project and dissuaded me from doing a PhD as we did our small talk. Over the course of small talk my internal dialogue went as follows:

Firstly. You chose to work on snails that required the TLC of daily visitation.

Secondly. You’re putting vials of desiccated snail poop through a spectroscopy machine with a look of abject boredom on your face. Dig deep and remember what you’re doing.

Now I may be bright-eyed with naivete, and perhaps I’ll one day become so disillusioned with my own project, but the small talk taught me a few things. One, work on something that really excites you. Two, don’t work on moving, living things that need daily feeding and can die at any moment.

Which brings me back to my meeting.

It was probably absent-mindedness that made me forget that the further from the coastline you go, the hotter it gets. So as we pull up to the lab, I’m fidgeting a little, edging away from the solid slice of pre-noon sun filtering in through the car window.

The meeting with my other supervisor proceeds swimmingly though, with me dutifully taking notes and sharing ideas of what I could be doing, most of which were met with agreeing nods. A good start.

At some point, we take a small trip to the study site.

Now both my supervisors clearly had their wits about them, as they wore shorts and a short sleeve shirt, chatting amiably and pointing to the large ring structures that look out of place in the Eucalypt forests. Meanwhile I’m simmering a pace behind them.

At least I looked good.

Though my suede shoes were really not liking me.

The day finishes quite quickly after that. With a final skype meeting with a collaborator where I took notes for things I had no idea for. But it provided the necessary illusion.

Smoke and mirrors.

All I had left that day was to catch a train home. Another hour and 45 minutes. Of course my supervisor made every moment count and typed up a letter in that time. I slept, because the sun always makes me sleepy. And I figured that I’ve committed a few faux pas at that point with my dress, that one more one less wouldn’t hurt.

But before I dozed off in the warm carriage, I couldn’t help but to remember that my other supervisor wore sandals.

At least he wasn’t wearing socks with them.