Moss microforay teaser

Really busy lately, hence the spotty blogging as of late. However, tonight I just happened to have a small moss sample and some 'spare time' (reads: time I could've used productively instead...) after imaging some lab-related stuff at the scope. Went on a bit of a moss microforay, found loads of Euglyphids, and will post a couple teasers as it will take me a while to process all the images:

Sadly, most of my Euglyphid images were made before I realised some genius managed to get oil encrusted on the 40x air objective. The subsequent avid cleaning accompanied by profanity did help clear up the lens a little, but it's still seriously fucked. That kind of thing makes me really angry, as it's easily avoidable and keeping lenses clean can improve your image quality so much for free. A clean simple lens can get better images than a dirty fancy lens, and each speck/layer of crap eats away a nice chunk of resolution. Look at the difference using the same specimen (which drifted a little during the distraction) - the one on the left is before cleaning, the one on the right after (the left image is at the optimal DIC settings, for the right image the analyser and prism haven't been properly re-adjusted yet, so it gets better):

Another thing I noticed was that suddenly there was A LOT MORE LIGHT. So yeah, keep your lenses clean and if the image is strangely fuzzy, don't forget to check if some idiot dirtied the objective somehow. Oh, and keep oil the fucking hell away from any non-oil objectives, it's not that bloody difficult!!!

Grrr I really hate it when so much time gets wasted because of something really stupid being wrong with the scope.

Anyway, there was also a mystery metazoan of some sort. I can't quite figure out what it is. Does anyone know? I'm no good with worm-like metazoa...

The microforay images, presentation part 2 narration, proper response to a certain comment and maybe even a couple other obligations (eg. Mystery Micrograph write-ups...) will be worked on this weekend. In any case, I'll finish the narration then, sorry for keeping you waiting!
Edit 27.03.10: Finished narration for part 2! Enjoy, please feel free to ask questions, etc.

A stats question RE T-tests and U-tests

So I'm in the midst of the "Oh fuck, must get actual numbers and graphs for publication" stage of my project. This means I must not only generate piles of data, but also make it talk, and speak the truth. Which means I get to interrogate it with statistics, mwahaha. I actually enjoy this part of the process, since you can magically convert piles of numbers into pretty p-values and sexy graphs showing how earth-shakingly significant your data is...oh, well, statistically significant anyway. That is, if your stats is being done correctly, otherwise the whole activity is a futile waste of taxpayer dollars, more so than it usually is.

So I noticed that for situations where I'd expect some sort of significance (they're bloody obviously different, but I never thought/said that because I'm a 'good scientist' and all that...), the p-values were...well, maybe a little bit too high. Like, they were kind of insane -- 10^-45? Oh come on... it would be awesome boring if biological data were so clean! But both the t-test, and the Mann-Whitney U test showed extreme significance, with the latter being more trustworthy in my case, or so I've been told (I have a prominent shift in distributions rather than means; that is, cells in the drug treated case get really big, while in the mock they don't get really big.)

So I decided to test the data I know shouldn't be significant -- treatments of two wild-type ecotypes, and another case where the drug had no effect. So here's my data that SHOULDN'T be significant:

The first graph shows means and st dev errorbars, second graph shows quartile box plots of the same data (that is, no obvious shift in distribution either). Then I have t-test results from Excel, which show significance regardless of whether we assume equal or unequal variance, although F-test shows equal var. The Mann-Whitney U-test, while not being as striking as the results for the data that should be significant, is still somewhat... acceptable-ish. That is, 'significant'. But that doesn't correspond well with the data in the graphs, does it?

Would anyone know what the hell is going on here? Could the difference in sample sizes come into play? All involved data has a normal distribution, but under some conditions (not in the data above though), there is evident shift in skew in the data. I was told a U-test should sniff out differences in skew and kurtosis. I ran my data by a stats-ish guy (ok, ecologist...) a while back, and he said there's no doubt my significant data (not shown), is actually significant, but I can't trust my tests if they show 'significance' between wild types (not shown) and treatments that don't make any noticeable difference whatsoever (above).

This is really REALLY frustrating because I have a total of like 10 different lines, each treated and untreated, with massive sample sizes considering the work it takes to get the data (microscopy and measurements and all that), and I'd like to wrap up very soon with a complete graph with significant results pointed out, and finally start writing. This will be my first time writing up a part of a manuscript, so it's really exciting (and scary), but right now I've got damn stats in the way!

And I am aware Excel is not a stats program. We don't have anything else though...

I'd really appreciate any input, thanks! =D (even if it leads to rediscovering that I'm actually a huge idiot...)

UPDATE 07.02.10 2am

Ok, so Aydin recommended PAST, which turns out to be quite a nice stats program =D Thanks!

But it shows the same thing.

Actually, looking at the confidence intervals (and repeating the calculations back in Excel), the 95% CIs don't overlap, nor do the 99% ones. What's even more frustrating, is that the drug that generally causes cells to get bigger (ploidy, etc), in this case "significantly" shows smaller cells. Which is weird. And rubbish.

Ok, fine, this isn't really a proper control. Let's compare our wild type ecotypes -- the ultimate negative control. There's no bloody way Col-0 and Ler (ecotypes) should have different responses in this situation! Right?

Amazingly, it baaaarely scrapes by for 95% confidence! We use 99%, so we can call it non-significant, but still... it shouldn't be anywhere near barely scraping by! I mean, the damn p-values should be like 0.5 or something, no? Again, these are two WILD TYPES! Sketchy...

And, hang on... F-test comes out significant? Owww, headache!

Do I need more data then? It'll take another couple of months to double the sample sizes, especially for these ones, where there's much lower count per view, so I'd have to image waay more specimens. Grrrrr...

OMG, IT DOES HISTOGRAMS? And in a HUMANE way, unlike Excel? Aydin, I owe you for PAST!
So here's the obviously significant case:

The non-significant (histograms can be really misleading when the sample sizes differ, I find...)

And now the really weirdly 'pseudo-significant' case: (again, n = 280, 352)

I can see how there's a bit of a shift, but significant? Really???

Great. So while the phylogeny course has beaten out any faith in phylogenies out of me, now goes my faith in statistics. I mean, this is the thing we're supposed to rely on to avoid introducing our own biases and judgements... but if done wrongly, it can really make a mess. And I suspect I'm not doing something right.

Or am I just being too paranoid?

Microscopists can get grumpy...

Dear Whoever-the-Fuck-Decided-it-was-a-Great-Idea-to-Fuck-up-the-Light-Path-on-my-Scope,

I just spent an HOUR taking shitty images wondering why the fuck no amount of fiddling with my usual knobs could get me a half-decent DIC image. I was very confused - I fiddle with A LOT of stuff when setting up the scope, and don't expect to be easily outfiddled. Especially by a new undergrad. I was getting decent images yesterday, and today everything looked like shit. My condenser was set up perfectly, analyser and prism set up the way I always do when it works, and my sample was even better than usual. It was fucking annoying.

So then I finally snapped, and started fiddling with shit I usually don't fiddle with. With good reason. And guess what I find... the very first fucking polariser is completely misaligned somehow. Seriously, how the fuck did you even FIND that knob??? No one EVER touches it, because you just don't. You never need to. That's why it's hidden in a rather inconspicuous place. You notice how the focus knobs are like RIGHT THERE? That's because you use them a lot. They have to be accessible. Same with the stage control. And the condenser height. And then we get to the slightly more obscure stuff, like the analyser and prism - also must be set up, but not as critical, and you don't really need to fiddle with them constantly.

And then there's the REALLY obscure shit. Stuff you set up once, and never ever think of ever again. While a nice chunk of microscopy can be clueless artistic fiddling, some stuff you just leave alone, because there IS an optimal setting for it, and any deviation from that fucks shit up. This includes the alignment of the bottom polariser. It was set up once, everyone was happy, and no one ever needed to look at it ever again. Until you, most likely one of our new clueless undergrads, decided to find that knob, and give it a fucking twirl, thereby fucking up my DIC light path. And since I don't usually reach there, I had no idea what the fuck was going wrong. Now I know. After an HOUR of perplexed shitty imaging.

How about we introduce a new rule: If you don't know what it is, DO NOT FUCKING TOUCH IT. If you REALLY wanna screw with it, then make sure you put it back EXACTLY the way it was, to the hundreth of a degree/micron/radian/kelvin/mV/WHATEVER. If you don't know, then fucking ASK. Srsly, you look a LOT fucking dumber by doing stupid shit than by asking stupid shit. I ask stupid shit all the time, and they still haven't clued in kicked me out.

This applies far beyond microscopy - what if you decided to twirl some potentially exciting/excitable chemical one day? The consequences could get more dire than an angry labmate, believe it or not. As scary as we may seem, nature can be much worse. Also, nature doesn't give a fuck about you. Just fyi. It really doesn't. It doesn't care you're new and don't know shit - stuff can still explode in your face without warning. After all, this is a LAB for fuck's sake, and we're licensed to have some pretty hazardous shit in here. We may be only BSL1, but we still have stuff than can kill you. Some stuff that you probably shouldn't really touch too much. At all.

So ASK next time. You'll seem a much greater idiot stark naked in the emergency shower because you didn't know what that chemical was than while asking questions of ANY level of stupidity. Also, we're labrats - we lack social skills. Pissing us off too much by doing idiotic shit can lead to the psychological equivalent of standing naked under an emergency shower. Have I mentioned that you should ASK before touching anything? Don't make me do it again.

Signed,
Angry Labmate

PCR Flu

After a week of my PCR failure, I finally got it to work. However, a masters student now can't get his alive in any way, which prompts one to contemplate the possible existence of some infectious PCR disease, a flu if you will, that spreads around labspace plaguing one unfortunate victim after another.

A new journal title is in order: J. PCR Flu Epidemiology. I think I finally found a place to publish my gels!

Said journal should also feature PCR horoscopes:

eg.

Aquarius: The -something- is in the way of the path of -some star- (I don't really read these, can you tell? o_O) Check your dNTPs. Your entire stock may have gone bad.

Pisces: While at first you may feel confident following a long line of successful gels, you should be extremely cautious in a couple of weeks - treat your Taq with care and be sure to put all the reagents back in the -20.

Cancer: You're fucked. Seriously, go wash some glassware and clean the lab windows instead for the next few weeks... nothing you do will save your product from instant degradation by the wrath of the PCR gods.

Etc.
Anyway, I have a long night of microscopy ahead of me... good bye life, it was nice having you!

PS: I got catnip: 3 volumes of 1969 Research in Protozoology (ed.: Chen) from a nice ciliatologist. It's simply incredible. I'm dehydrated from drooling over it so much. Protistologists are very evil people. Worse than drug dealers... =P

A bit of a break...

Rather swamped with stuff right now - research, class (final next week x_x), other misc stuff. Probably would be best to stay away from the internet as much as I can for the next week or so... that may prove to be difficult. Oh well...

Oh and to top it all off, I think the star alignment is incorrect for PCR these days. May have to wait for them to shift a little so that the PCR prayer waves can reach their destination and make my Taq do its job. Or perhaps its governed by Chaos/Quantum whatever theory stuff BS. Srsly - exact same primers, reagents, temperature, program, machine... similar samples, and one day they work perfectly, the very next round there's no product. WTF? HELP! I need this to work like 3 days ago... argh.

For now, here's a random preview of a post I will finish eventually very soon ^.^

I de-Cavalier-Smithified and combined a couple of his diagrams into what may hopefully be read and understood by a normal human being. The protistology TA instructed me to avoid ever using Tom's diagrams in talks, even after I redrew it.

Here's one of the originals (very typical TC-S):

(Cavalier-Smith 2009 Predation and eukaryote cell origins: a coevolutionary perspective. Int J Biochem & Cell Biol 41:307-322; can't find free access pdf anywhere yet =( )

His phylogenies are even better...!

Hopefully explanation will soon follow...

PS: Just noticed something weird... why does Tom have animals and fungi branching out of 'Choanozoa'? Do 'Choanozoa' include nucleariids and ichtyosporeans too? What do those have to do with choanoflagellates anyway? As far as I know, that region looks something like this:

{Nucleariids + Fungi} {Ichtyosporea [Choanoflagellates + Animals]}; 'Choanozoa' would be a rather awkward group, no?

/pedantry; Damn, now I sound like a cladist...

Pipet FAIL

When you set up PCRs, often you first make a master mix and then aliquot it into smaller portions to which you add template DNA (the stuff you need copied). Often you do one extra reaction more than you need, since you tend to run out due to pipeting error. But you don't need that much extra normally...

For the last few months, I kept on running low when aliquoting - often not having enough for the last tube. I would make 10 reactions when I really needed 8, so about 36μL extra, for 18μL aliquots. And STILL I would not have enough for all reactions. I calculated my recipe numerous times in numerous ways, and it should have worked. I tried the most careful pipeting technique I could manage, although it would be a rather exquisite accomplishment for pipeting error to be THAT bad!

I suspected the pipets, but my labmates were all like "Oh don't blame the pipets now!"

So I got sick of this. I grabbed water, and went to the analytical balance. I check the p20 - everything more or less fine. Then I grab my p200...



Yes, it only pipets ~75% of the set volume. FOR THE PAST FIVE MONTHS.

So yes, when all fails... check your pipets. Their calibration could be way off by now... (after changing all the water and buffers and stock solutions, that is.)

Now, my burning question:
How the hell did my PCRs work for the past five months despite all my concentrations being absolutely RANDOM!?

So much for molecular biology being an exact science, hahaaa!
Actually, molecular biology is voodoo. Seriously. You can make things exactly right and they don't work. You can leave your DNA out on the bench for a couple nights and completely screw everything up while pipeting... and it magically produces the bands you want. I think it depends more on the star alignments or something...

Science as a journey through alien worlds

Running a gel, thus perfect time to blog. Perhaps if I write stuff in the lab, it may actually turn out to be marginally intellectual.

Hopefully my gel won't turn out to be something like this again:


I hate genotyping - the work itself requires the intelligence of a brain-dead foetal monkey, yet manages to find a stunning variety of ways to fail. And when it does work, it usually shows everything was wildtype anyway. Or maybe your mutant-specific primers failed. You should probably remember to run a positive control next time. If you have one, that is. And when it does work, you can brag about find a homozygous line amongs the seeds you ordered from the stock centre. Nature-worthy achievements right there.

Actually, most lab work is about as mundane and intellectually uninvolving. The planning and literature reading, data analysis and troubleshooting(!) can be quite exciting and tend to involve plenty of thinking, but you only do that in-between the endless repetitive pipetting and mixing chemicals and following protocols. I think research probably involves one of the biggest intellectual (and emotional, heh) rollercoasters of all professions, which is why only the very insane can handle it.

[did I run the DNA off the gel yet... no, phew.]

I do enjoy microscopy, however, which is considered by some to be repetitive and mindless as well. I cannot disagree more. It's an artistic break from the gels and protocols - you have a lot of factors to play around with to obtain the best image you can. Microscopy is also very dangerous, a wonderful way to obtain false positives! It's extremely easy to tweak the image to see what isn't there, even unintentionally. A substantial component of the art is to obtain sexy images without sacrificing the scientific validity thereof.


As children, many of us have dreamt of fantasy alien worlds, to be much disappointed upon finding out we would most likely never encounter any; not within our lifetimes anyway. However, it is wonderful to know that alien worlds are not confined to what lies beyond the Earth's atmosphere! (or present time...) They are all around us, ready to provide enchantment and fantasy to anyone who invests enough to explore.

To a mathematician, beauty may lie in quantifying forms or dynamic phenomena, or working with abstract worlds far beyond the imaginative capacity of the human mind. To a fiction writer, fantasy worlds thrive in his mind not much different from the form taken by reality itself. To an astronomer, the alien worlds are almost within reach - just barely visible, yet not ours to manipulate. For me, foreign worlds exist under the lens of a microscope, where if you magnify anything far enough, you can spend a lifetime wandering amidst the surreal landscapes unseen by naked eye. The life of or within a cell is fascinating enough conceptually, but that mysterious aspect of it being beyond our 'scale' makes it not much different from a childhood fairytale or a sci-fi novel - except that it's real!

On a more abstract level, science itself is a foreign world for exploration (along with math, the arts, etc.). Upon liberation from what Carl Sagan calls "the anaesthetic of familiarity", the mundane everyday world around us becomes full of tantilising mystery. Science is a way of unraveling some of this mystery (I'll argue, the optimal way of dealing with natural mystery), an ardurous journey through the unknown worlds. It's rather fractal-like in nature - the more you explore, the more questions arise; the deeper something is examined, the more detail beckons attention.

I think a good analogy for the scientific journey could be hiking or climbing - the act of walking along a trail is quite repetitive: all you do is place one foot before the other. After some time the scenery begins to repeat itself, and one wonders how anyone could enjoy outdoor activities at all. But many do, and probably few ever notice the monotony of walking, or climbing, or paddling a kayak... for there's always something fresh to look at, something wonderful to appreciate and enjoy. It's fairly easy to knock off the 'anaesthetic of familiarity' for an outdoor enthusiast - the escape from the chaos of the human 'world' is sufficient to make the journey worthy of the physical investments. The threat of an occasional failure, the pain of stepping on a too-unstable rock... is usually not enough to deter one from actually enjoying the exploration itself.

Science is quite similar - there is the monotony and rigorous exactness required by the scientific method, the endless protocols, the multitudes of failed and inconclusive experiments. There is the threat of losing one's job upon failure of publication - the world of 'publish or perish'. It seems one must be insane to voluntarily engage in such activity, much less enjoy it. But just like the monotony of walking is instantly forgotten upon the sight of a rare bird or an overpowering landscape, the monotony of science is instantly lifted upon the sight of a contradicted null hypothesis, or the sight of a rare species, or the ability to actually see a cellular process in action in real time... (perhaps for a chemist, the synthesis of a brand new chemical never before produced on earth could induce a similar rush or euphoria?)

But just as one does not go on a hike expecting to see a rare bird (lest the foiled expectations lead to a wrecked mood), one cannot go into science expecting to make great discoveries. The very process itself must be tolerable, and hopefully captivating enough, to keep going. Sometimes you get excited about the potential outcomes of an experiment, only to discover your results are utterly inconclusive and the story is far more complex than previously imagined. I think what makes science quite suitable for an insane type of mind is the ability to actually not mind such disappointments, and savour them as a testament to nature's superiority and infinite complexity.

After all, we may rest assured that we will never run out of work to do, just as an outdoorsman would never run out of places to explore. Now isn't that a comforting form of eternity?


[genotype results: all wildtype. Time for more PCR!]

Biblical Microbiology

This randomly popped into my head while I was doing E.coli transformations today:

(From: Microbius 1:01-1:33)

01
She sayeth onto the chosen Tribe of E.coli
"Taketh upon thee my Holy Construct,
For it shall heal your soul and spare thee from the devil."
And she blessed the great tribe with a heat shock,
Hoping to instill faith within them for all eternity,
Faith in her Holy Construct.

But some E.coli refused the Holy Construct,
Preferring instead to believe in their own pagan plasmids,
Neglecting the holy plasmid of God.
This insolence angered Her deeply,

11
And her neurons fired in wrath,
And She inundated the E.coli culture
With a great Flood of hygromycin;
And thus were smote in fiery wrath,
The heathens who failed to place faith in the Holy Construct.
And from the Flood their souls unsaved,
For they have not receiveth the Resistance Gene.
And lysed their membrane were in hell,
Damned to an eternity in the autoclave.

And She sayeth onto the good E.coli:
21
"Get thee to my pipette tip ark,
And I shall carry thee to the land of plenty,
And ye shall call this land the Test Tube,
And may the LB broth save ye from starvation."
She commandeth to the good E.coli:
"And while ye flourish on my sacred LB,
Dare not forsaken my next commandment:
Be fruitful and multiply,
For in a forthnight ye shall find thy souls,
Preserved in -20C heaven, in a stock of glycerol,

31
And blessed thy genomes shall be Saved,
As my Holy Constructs is delivired to the Holy Plants,
In the Growthchamber of Eden."

RAmen.

May your DH5α be forever blessed! =D
(And GV3101 as well; why not?)



Yes, the Growthchamber of Eden has GFP-expressing plants. The sun shines 488nm wavelength there. No wonder it's so green, eh? You should, like, totally see the tubulin:GFP-expressing tree -- it's a freakishly fluorescent green cytoskeleton! And the Forbidden Fruit is forbidden due to being stained with DAPI -- DAPI is bad for ya, y'know?

This is getting out of hand...I think I need to get out more often or something ^_~