by PhilipJ on 9 November 2008
Dr./Mrs. Jekyll/Hyde has been teaching a new laboratory tech the tricks of the trade (or, as it turns out, some basic algebra), and in one of the posts mentioned something that we often don’t think about in science education but is critically important when you’re actually in a lab: having good hands.
This is important for a variety of reasons: pipetting solutions, filling the wells of a gel before running electrophoresis experiments, or even simpler things like not stabbing yourself with a needle or not dropping expensive samples. The shakes can be the difference between having to do experiments only once and wasting an entire week repeating them, with questionable results each time.
Not drinking coffee is, for me at least, not an option. Thankfully, my Ph.D. work involves very little that explicitly requires extremely good hands (mind you, pipetting solutions in a dark room under dim red lights* is annoying no matter how good your hands are…). I was not so lucky for my Master’s work, where, you may recall, I dealt with micropipettes.
Making the micropipettes was annoying for consistency reasons, but actually getting a micropipette into a flow chamber was the real fun part. Our flow chambers looked like this:
Brown tubing, having an outer diameter of ~160 microns, and an inner diameter about half that, was squished between melted Nescofilm between two No.1 coverslips, and was the means of inserting micropipettes into the active flow area.
The micropipettes, shown on the right, were tapered to ~1 micron at the very tip (the polystyrene bead held by suction at the tip is 2 microns in diameter), and for practical reasons were usually 3-5 cm long. They were connected to a length of thin tubing so as to allow for easy connection to a syringe, and the whole contraption was mounted on an aluminum clamp, with a separate clamp specifically to hold the micropipette in place.
As I mentioned above, I drink a lot of coffee, and while I don’t have a serious case of the shakes, I don’t have a particularly steady hand. Despite this, I would insert the micropipettes into the brown tubing by hand. We had a 20x optical microscope with two-dimensional motion on the microscope stage. I would mount the flow chamber to the stage, centre everything such that it was all in focus, bring a micropipette tip into the field of view by hand, and, once also focussed, try to move the stage so that the pipette tip ended up in the brown tubing.
This would (obviously) often result in disaster, as any time the tip of the pipette would touch the brown tubing it would be ruined, no matter how incidental the contact. For a while I was keeping a log of successful tips to ruined ones in my lab book, and the ratio got so depressing I had to stop. By the end of my Master’s though, despite my regular multiple cups per day, I had developed a couple of tricks to raise
the success rate. The most useful of which was to introduce water into the system.
Flushing the flow chamber with water would produce a large (under the microscope, anyway) bead at the opening aperture of the brown tubing, which, after Kimwiping it away, would leave a slight meniscus of water right at the entrance. When the tip of a micropipette was nearing the entrance to the brown tubing, the pressure difference in the pipette tip would result in rapid suction of water from the meniscus into the pipette. With much practice, rapidly moving the stage once observing water in the pipette led to, near the end anyway, maybe as high as a 50% success rate in getting tips into chambers.
While my Ph.D. work has it’s own series of issues (this week’s: overlap two 200 femtosecond pulses focused into a ~100 micron diameter area. Spatially is simple, temporally, less so), I am very happy I don’t have to do anything like that anymore. I think I’ll have another espresso to celebrate!
* I’m working on the most-obviously-photoactive protein you can imagine. Take a guess which!