For want of a nail

Ours is a relatively new lab. Which means it's constantly in the process of being set up. And that means there's new equipment/chemicals/supplies coming in nearly everyday. Right now, my workbench is filled with boxes of pipettes that need to be moved to the store room. And honestly, it isn't comfortable typing with a laptop on your lap. Which kinda defeats the purpose of a laptop, but anyway.

Part of the problem with supplies coming in is that more often than not, somebody messes up somewhere. In the last three months we got reagents we didn't need, equipment we couldn't use and, of course, cells that kept dying on us :) More often than not, it's like that nursery rhyme:

For want of a nail, the shoe was lost;
For want of the shoe, the horse was lost;
For want of the horse, the rider was lost;
For want of the rider, the battle was lost;
For want of the battle, the kingdom was lost;
And all for the want of a horseshoe nail

Case in point, the big-ass centrifuge we had sitting idle for two months in our lab. We couldn't use it because it didn't have the right kind of holders for the tubes we were using. It was pretty frustrating to see that big hunk of metal just sitting there, taking up space.

But that's nothing compared to the trouble we've had getting laminar flow hoods for our cell culturing work. A laminar flow hood is a metal bench which is enclosed from three sides has a door on the fourth and a blower on top. The blower blows finely filtered air through the enclosed space, to prevent contamination by organisms floating in the air. We use two different kinds of hoods for our work. A simpler, more robust kind for working on bacteria (because bacteria are robust), and a more sterile, more expensive kind for animal cell cultures. And right now, we need four of the bacterial ones and two of the animal cell ones. Here's what's happened so far:

Initially, we'd ordered one of the animal cell ones from a local manufacturer. It arrived on time, and did everything we wanted it to do. It wasn't great, but it did the job. We then placed orders for two animal cell ones with an MNC and four bacterial ones with an Indian manufacturer.

The Indian hoods showed up first. And it was pretty messed up. The blower was at the bottom, although the vent was on top, There were holes on the metal workbench, which meant that if we worked with liquid media and if any happened to spill over, it could pour right into the blower mechanism. Plus, the door in front didn't close the way it was supposed to. There was no way we could use these, so we sent them away, and ordered new hoods from another manufacturer.

Next came the MNC hoods, all the way from the U.S. of A. Messed up even more, if anything. It had an annoying alarm that went off if the door was even a millimetre off the "correct" open position. It had no electric sockets (we use mechanical pipettes that need electricity) And even though it was of a different design, the door didn't close properly! Plus, only one showed up. The other hood is still missing.

And today, the hoods we ordered to replace the first bunch of bacterial hoods showed up. Weirdly, these have holes on the back wall, which means your workbench won't be sterile when the blower is off (in case you want to leave your media inside the hood overnight to set, for instance) But the worst part is that they didn't come with legs! That means the workbench is just 6 inches off the ground. Maybe they expect us to work sitting cross-legged on the floor...

And yet, in spite of all this we still manage to get work done. When somebody tells you that life in science isn't easy, believe them!

Go west, young man

I’m back, after a break from the blogosphere, and the lab. I was home for an extended Dusshera-Diwali break. Plus, there wasn’t much happening in the lab over the last month anyway, barring the usual culturing and maintaining of cell stocks. By the way, HeLa has been a bit of a temperamental cell line. I had two sets die on me last week, and that shook me up a little. (My guide says you can never take it easy when it comes to cell culture. Even if you’ve been working with the same cell line for years, decades even, and you know all the ins and outs of your cell line, you can never let your guard down)

 

Anyway, I’m going to run my first western blot today. A western blot is an assay to check for the presence of a specific protein, first by separation based on size and then getting antibodies specific to that protein to bind with it. We’ll know that the antibody has bound to the target protein because it fluoresces on binding. I’m excited because:

a) It’s my first time

b) This is where my project really begins

 

If I haven’t already written about what I’m working on, here goes. My guide is a cancer biologist. She studies the proteins that are activated due to DNA damage. Some of these proteins arrest cell division, till the damage is repaired. It is suspected that these proteins don’t work the way they’re supposed to in cancer cells. We’re trying to confirm that that is what’s happening. Currently, I’m trying to induce DNA damage in HeLa and 293T and then see how expression of these proteins varies from a normal cell from either line. That is, of course, when my cells aren’t dying on me :(

 

I’ll be back later this week with a complete post on how my first western blot went. For now, I’m going to leave you with a bit of trivia. The reason western blotting is called western blotting is because it’s a play on Southern blotting (always a capital ‘S’). Southern blotting was developed by Edwin Southern in 1975 to check for specific DNA. When a technique to quantify protein was developed some time later, they called it western blotting. For RNA, it’s called northern blotting. No, there’s no such thing as an eastern blot, even if it seems unfair. Yes, I know, scientists are a very imaginative lot (rolls eyes)

Little drops make a... what?

My regular coursework is going on side-by-side with the project. And we’ve had guest lectures this week by a professor from the National Institute of Immunology, Delhi. A few of us were talking to her after class, and she told us that there was such a thing as too much vaccination. 

When a bug infects you and you’re able to fight it off, your body keeps some immune cells in your body which are capable of identifying the bug, should it ever infect you again. That way, your immune system will know that there’s been an infection, and it will also know what caused the infection. It will be able to tackle the infection that much more quickly. That’s why you never get the chicken pox more than once in your life.

When the second infection occurs, the cells that “remember” the infection (called memory cells) have to multiply and change into cells that can actually tackle the bug. But there’s a catch. Cells in your body can multiply only a certain number of times before they stop. (Incidentally, that limit is one of the reasons cells don’t turn cancerous very easily) So, if you have repeated infections, your memory cells will hit the limit and once they die, they won’t be replaced.

Now, vaccination is like giving you a mild infection that you can recover from, but which will also equip you with memory cells. That way, the body responds much quicker when an actual infection takes place.

Repeated vaccination is like repeated infection. There’s a risk that you could lose your immunity against a bug if you get too many booster shots too often. Which apparently is what is happening with the oral polio programme. In Delhi, oral polio vaccine campaigns are conducted nearly every month. Children under 5 are given oral polio drops. Which means if a child has very conscientious parents, it could get up to 60 doses in 5 years. That’s more than the limit on multiplication of cells. And that potentially defeats the whole purpose of the programme. There isn’t any experimental evidence yet to show that children who’ve gone through such a regimen have impaired immunity against polio, but there’s a risk that it could happen.

So, here’s the take home advice. If anyone tells you that you need booster vaccinations for the most common bugs more often than once in 6 months, check again somewhere else. Otherwise it might be as good as not getting a shot at all.

Back to the lab again yo, this whole rhapsody...

The project is back on track! We received new stocks of 293T on Friday, and HeLa on Tuesday. And I split my batch of 293T on Monday. Damn, it feels good to be back in the lab and get some real work done. And I mean handling something live, not just making solutions.

Not that making solutions is boring. We messed up the proportions of one of the buffers we were making last week, and ended up making 50% more buffer than we intended to. It was a nail-biting affair all right, trying to judge how much water we could add to get the ingredients to dissolve without the beaker flowing over!

So far, both cell lines seem to be healthy. The HeLa cells, especially, seem a whole lot healthier than the ones we got the last two times. My guide thinks it might be because the last two lots spent 16 odd hours in transit, and HeLa doesn’t like long train journeys. The new lot was in transit for 4 hours. I guess it makes sense. You do feel more out of sorts after a 16 hour journey than a 4 hour one.

It’s kinda exciting to think that I will work with the world’s oldest cancer cell line in about a week’s time from now. HeLa was first isolated in 1951, from a cervical tumour in a woman named Henrietta Lacks (hence HeLa). She died soon after, because the cancer had already metastasized by then. But a little bit of her lives in labs all over.

HeLa is very different from most other human cells. It has a chromosome number of 82, as against the normal human number of 46. This is partly because it has genes from the Human papilloma Virus (HPV), the virus that caused Henrietta's cancer. In fact it’s even been characterized as a different species, called Helacyton gartleri, after Stanley Gartler who discovered some of the many ways in which HeLa differs from human cells.

HeLa is so commonly used the world over it’s estimated that the total number of HeLa cells alive outnumber all the cells that were in Henrietta’s body. And soon enough I’ll be one of the thousands who’ve used this cell line. It feels good, it really does.

Up above the world so high...

Excuse me while I get into my Ivory Tower.

 

There, that’s better.

 

As you might have guessed already, my project is still at a standstill. I have started preparing the solutions and buffers that I need, just that there aren’t any cells to use them on. Although new cells are expected to arrive this weekend.

 

So what am I doing in my Ivory Tower? I’m going to talk about my experience with high-level research. And by that I mean the projects I have done in labs located on the 3^rd floor and higher.

 

Yes, that was a very lame joke. But I have to do whatever I can to amuse myself. Surprisingly though, all the projects I’ve done so far have been in labs located on the 3^rd floor and higher.

 

I guess it makes sense to put labs high up and the admin offices lower down in a building. That way, mail reaches the office faster. The scientists get a good view, which can do a lot for your morale. Plus, if your lab is high up, it makes going anyplace else seem like too much work. That way, you ensure that when people get to the lab, they work.

 

Of course, the flip side is that the lab may seem too far away and you skip going to the lab altogether. But that’s where the guilt comes in. You tell yourself “I’m getting so much in grant money. There’s so much shiny equipment up there. The people in my group depend on me being there. This is no time for feeling lazy!” And march off to the lab.

 

That’s when the feeling of being trapped up there hits you. And you come up with theories of being trapped. Like this one.

 

Okay, I’m done. Have a class now which thankfully is on the 1^st floor, so I’m going offline. And I’m leaving the Ivory Tower for now :)

Geeky cool

Since the project is still on hold, I’m reading papers (as if!). I’ve said that enough times already, so I’m going to shift track to things that are happening to me in college.

I’m a reporter with the college newsletter. My beat is “celebrity interviews”. Now, I don’t mean I’m the typical celebrity interviewer who has loads of contacts and blends seamlessly with the Page 3 crowd (ha ha!) Please, I’m doing a grad course in biology. I interview scientists who visit our college (we have a fair number of visitors) Yes, I know. I wear my geekiness on my sleeve.

 

So far, I’ve interviewed an evolutionary biologist from Bangalore and an astrophysicist from Cambridge. And one thing I’ve found in both of them is that they have a way with words. They aren’t great orators, mind you (I nearly fell asleep during both their lectures). But when you’re talking to them on a one-to-one basis, they seem very willing to talk and they put you at ease very quickly.

 

I don’t know. Maybe it’s just that I’ve been meeting the wrong kind of people so far, but I’ve hardly ever seen any scientist who lives up to the stereotype of a hard-nosed, hawk-eyed brainiac who’s miserly with both words and time. The kind of person who you can bet spent far too much time with his textbooks as a young adult, got picked last during games… you get the drift. The only one who are remotely like that are the ones who spent a lot of time in academia, and less in active research.

 

I guess this underscores two things. Scientific research today requires a great deal of cooperation. And the only way you can get other people to cooperate is by coming across as a decent human being. Besides proving that you can do solid research yourself.

 

The other thing is that scientists are in fact really cool people inside. Approachable, laidback and capable of frivolous conversation if need be (some of my questions are kinda frivolous. I try to maintain the “human interest” aspect, whatever that is). For instance, every professor in the bio department, up to and including the HoD, wears jeans to work. You’d only see that in a start-up, or in some kind of a media establishment. Makes me feel a whole lot better about what I’m getting into.

 

And hey if we scientists don’t call ourselves cool, who will?

Grand unification

I'm waiting right now for a lecture on high-energy physics to begin. We have weekly lectures in my college, where speakers are invited to give general talks in their fields of specialization. This one is about the existence of a unified theory that brings all the fundamental forces together. What's a biologist doing at a physics lecture? I'm bored, and we still haven't got any new stocks of cells yet. And there are only so many papers I can read before I drift off.

Since Monday, we've been having lectures in Immunology. The person conducting the lectures is doing a bloody brilliant job. And well, he should. He's on the committee that writes the NCERT bio textbooks, so he should be able to get ideas across well. Although the lecture on Monday was just a refresher, he still did everything from first principles and basic assumptions. And then brought up what difficulties and advantages a multicellular host would have over a single-celled host in dealing with a parasite. All of it makes sense. And since it comes up in such a logical fashion, I didn't have to take notes at any point in the last three days.

Honestly, if I ever get into academics as a career, I want to be able to teach like him.

That's probably a target worth working towards, even if I'm not in academics.

When it rains, it pours

It's surprising how things can change so quickly. A week ago, I wrote about how we tried to rescue our 293T stock with my cells...

On Monday, all our 293Ts croaked (if you can call it that. Maybe individual cells actually do make tiny gasps when they die) Talk about cockiness. The medium changed colour completely, and all the cells detached themselves from the bottom of the plates. And we have absolutely no idea why that happened. The cells may have grown too fast, used up all the medium, run out of nutrition and starved to death. But that's just my theory.

To make things worse, on Thursday all the HeLa cells died. Every last one. They were in trouble right from the beginning, so it wasn't entirely unexpected.

Now that there aren't any cells around, most of my work over the next few days will be reading about checkpoint proteins. When DNA in cells is damaged (by UV or some chemical agent) and if the cell's DNA replicating machinery detects the damage, the cell is prevented from multiplying till the damage is repaired. Checkpoint proteins bring about this arrest. And I will be studying one of these proteins over the course of my project.

Of course, to study these proteins, we need to kill the cells and extract them. And we need to prepare 30 plates of cells each time. So far, we haven't even been able to sustain cultures of 3 plates. Apparently, this kind of trouble is always there while starting a new lab in India. So wish me luck. I'm going to need lots of it to get through.

Errare humannum est..

So, as it turned out, my guide's cells were contaminated. There was zero growth in one of the plates, and very patchy growth in the other one. One of the other professors said it looked like there was a lot of debris in her plates. My cells, on the other hand, grew quite nicely. Almost 90% confluency (almost the whole plate was covered with a lawn of cells) and they looked pretty healthy.

So, on Friday, we split my 293Ts into two plates, in case we aren't able to revive the one other plate. And when I checked on Saturday, cells in both plates seem to have settled down quite nicely. We still haven't figured out what went wrong on Monday, though. Just hope it doesn't happen again.

A second batch of HeLa cells came in from Hyderabad on Thursday. The first lot came in two weeks ago, but they were in pretty bad shape when we received them. They were contaminated and we couldn't salvage anything. We tried splitting the second lot of HeLa cells as well on Friday. Come Saturday, and it looked like the same contaminant which hit the first lot had hit these cells too. There were clumps of white cell-like structures floating in the medium ( I couldn't see a distinct nucleus in any of 'em). Funnily though, HeLa were growing just fine at the bottom of the plate. I really hope we can fix this soon.

Proudest moment this week: The prof who was supervising my work while my guide was away told me I had good hands, and that I'd taken to cell culture like a duck to water. Granted he didn't watch me for more than 20 mins, but it still felt good to hear stuff like that. Makes me feel much more at ease with what I'm doing.

The spy

My guide’s gone out of town. So I had to split not just my cells, but her cells as well today. I followed the protocol correctly for the most part, but I may just have messed up splitting her cells. I think I added too many cells to her plate while splitting them. As usual, I’ll know only two days later when I change the medium.

Lately, I’ve been thinking about what it means to be a biologist. Back when I was picking a college to join, a lot of people expected me to take up medicine. That’s how it works in India. If you’re good in bio, you’re expected to become a doctor. If you’re good in math or physics, you’re expected to become an engineer. If you’re good with people and talk well, you’re expected to become a lawyer. And if you do none of these, you’re expected to do an MBA and get into management. Thankfully, attitudes are changing now.

I don’t think I would’ve been happy as a doctor. I’m using a very crude analogy here, and I don’t mean to offend anyone, but a doctor is a little like a car mechanic. He doesn’t have to be involved in the design side of things. He needs to know how to patch things up. As a doctor, most of your day is spent looking at things that aren’t working right.

I’m not saying I’m on the design side. No sir! We’re nowhere near design, as far as life is concerned. No, I’m kinda like the industrial spies car companies employ to find out how a rival’s new thingamajig works. And when your rival car company is natural selection (which produced you in the first place), boy, do you have a lot of spying and analysis ahead of you!

Oh, and in case you’re wondering about the car analogies, I’m a car nut. I have a soft spot for Italian and German cars. And I think most American cars are just a waste of money and metal.

Every small step...

The cells are okay! They’re not growing nearly as quickly as my guide’s. But it’ll do. Somehow, I already feel an attachment of sorts to them. That I’m responsible for them. Yes, I know it sounds lame, and very unprofessional. I guess I’m just completely caught up in the rush of it all. New lab. New equipment. The gloves and extra sterility. Plus, it’s been a full year since I worked with cells of any kind. And those were bacterial cells [S.aureus, to be precise. They’re the bacteria you find in pimples :D]

After checking them, we changed the medium in the plates. The cells should have fresh nutrition available now. Remember, TLC.

Oh, and in case you’re interested, here’s the protocol to dilute (or split) a culture of 293T. Note that I’m using 10ml of medium and 10cm dia. plates.

1) Aspirate (remove using a vacuum pump) the media using vacuum
2) Rinse with 5ml PBS (Phosphate Buffer Saline) gently
3)Aspirate PBS. Add 2ml trypsin. Leave for max. 2 mins. (This is to loosen the cells from the plate. Trypsin is an enzyme that digests protein)
4) Add 5ml of medium to stop the reaction.
5) Loosen the cells by tapping the side of the plate
6) Transfer 7ml of medium + trypsin to a 50 ml Falcon tube
7) Centrifuge for 10 mins at 1000 rpm, 25°C . Use balance.
8) Aspirate supernatant.
9) Add 1 ml medium to tube. Re-suspend cells.
10) Add 10ml of medium to fresh petri dishes. Add required dilution of re-suspended cells.
11) Move cells around in plate to spread evenly. Keep in incubator.

And that’s how it’s done :)

In splits

I have a small confession to make. When I said worked with, in the previous post, it’s not entirely true. I just stood by and watched while my guide separated them out into culture plates. I actually handled the cells on my own today. I diluted the cells and plated them all by myself. My guide was there to make sure I didn’t mess up, though.

Remember what I said about 293T needing TLC? It’s like this. As the cells grow, they multiply. And when they multiply it leads to crowding. See, 293T grows best when it has some surface to cling onto. But there’s only so much area on a single culture plate for them to grow on. Secondly, when crowding happens, there’s more waste products being released into the culture medium, and less nutrition available to each individual cells. That’s why we need to dilute the cells, to make sure there’s enough room for all of them.

Dilution isn’t a simple matter of just pouring more medium into the plate. It’s a cell culture, not orange squash. First, you have to remove the old medium, peel the cells off the plate (not literally) and then put a fraction of the cells you’ve recovered into a fresh plate with fresh medium. And all of this has to be carried out under sterile conditions. 293T is particularly sensitive to bacterial infection. (FYI, most culture media are banquets to bacteria and fungi. The media are so chock-full of nutrients it’s almost as if they beg to be contaminated)

The whole procedure took me around an hour and half, from start to finish. And I think I’ve done a reasonably good job. I won’t know till the 16th, though. That’s when the cells should have settled on the plate and started multiplying. Hope they’re OK. Fingers crossed!

TLC for 293T

I started cell culturing today. Okay, I know that that isn’t the correct term, it’s tissue culture. I’m really excited about it though. This is the first time I’m going to handle mammalian cells, barring that silly experiment in the 10th grade where they make you scrape out cheek cells and look at them under a microscope.

No, this time it’s proper human cells from a proper cell line. A cell line is a strain of cells that are unique in their genetic composition. And they’re stable, as in they don’t mutate or change properties in any way easily. Stable bacterial cells are called strains. Stable plant and animal cells are called cell lines.

The cells I worked with today are from a line called 293T. These cells are mutants from embryonic kidney cells. They’re not aggressively invasive, like a lot of other lines. The HeLa line, which I hope to work with some time down the line, is aggressive. Also, 293T is kinda sensitive to temperature and nutrients. It needs a lot of TLC (37°C, perfect osmotic balance, the works), which is probably why it isn’t very aggressive either. Can’t really survive outside the body.

And since they need so much TLC, we’re gonna focus on just keeping them alive and getting them to proliferate, for the next two weeks. After that, I’m not quite sure where we go. I’ll keep you posted, though.

Number one

When I started this blog, I meant it to be a site that explains biology, in its broadest sense. But then I realized that, for one, I’m just an undergraduate student and that there are far, FAR more qualified people besides me out there in the internet. And a lot have done an admirably good job of explaining things. For another, the field is just so goddamn vast and varied that I don’t know where to begin.

So, Not Quite Alchemy will be the diary of a biology grad student. Follow me as I go about another semester of my course.

Just to get you up to speed, I’m doing a newfangled five year course, from which I will graduate with an M.Sc. And I’m currently in the third year of the course. Now that that’s settled, wish me luck. A new semester, new challenges. And new insights, hopefully. Keep your rubber gloves handy :D