In light of the approaching -le cough- EVENT tonight, I have decided to describe to you some of the many things I have learned this semester in my BIOC course (disclaimer: all of this information may be found in the OWL-SPACE and OWLNET sites for the course.)

Polarography

Polarography, as applied in this course, is the measurement of dissolved oxygen concentration in a certain solution containing  various substrates and/or poisons and mitochondria.  As the mitochondria continue to respirate, they consume O2 gas, combining it with H+ to form H2O.

What I’ve said above is completely unnecessary to understanding the principle of the polarographic system, which consists of a sealed chamber with the medium inside, a Clark oxygen electrode, and an oxygen monitor, and probably also a stirring apparatus which helps to ensure consistent oxygen diffusion.  As Caprette writes, “The presence of oxygen causes the electrode to deliver a current to the oxygen monitor, which amplifies the current and converts it to a voltage output that is directly proportional to the concentration of oxygen in the chamber. The recorder moves a paper chart at constant speed, so that when the recorder pen moves in response to voltage changes, oxygen content is recorded as a function of time.”

Simple, yes?  Even elegant.  But the principle of the Clark electrode (named after Dr. Leland Clark) is where some gen chem background comes in handy.

That’s Dr. Clark.  He’s obviously awesome.  Let’s move on.

A Clark electrode, like most electrodes, consists of two half cells connected by a salt bridge.  The anode is made of solid silver (shiny!) and the cathode is made of solid platinum, which reacts with dissolved hydrogen and oxygen ions to form water.  As the platinum’s electrons are used up, electrons flow across the salt bridge from the silver anode, which produces silver ions, which combine with dissolved chloride ions (salt, baby!) to produce AgCl buildup on the surface of the anode.  UNfortunately this leaves the positive half the salt molecule behind in solution, feeling sad – but since H+ ions are being consumed by the production of water, the charge remains balanced and everyone is happy.  Good job, Dr. Clark!

That flow of electrons from the anode to the cathode is what is measured in current (proportional to the amount of dissolved oxygen remaining) by the oxygen monitor.  The solution that we used in lab contains approximately .237 micromoles dissolved oxygen per mL at room temperature, which when used with the known volume of the container, can be used to calculate total dissolved oxygen, and even how much oxygen is consumed per unit of time given the slope of the graph!

So, knowing this, here are some rhetorical questions for you: what do differing slopes mean on a polarography graph?  How about a flat or very steep slope?  Additionally, sometimes short dips in the graph may be seen – what does this indicate?  (You might need to use your prior knowledge of biology for that one!)

Microscopy

Your basic compound light microscope looks like this.

The condenser (called the “diaphragm” on the above diagram – hehe) focuses the light from the light source onto the specimen, which would be on the stage.  The condenser is what you control with the coarse and fine focus.  The eyepiece, or ocular, actually magnifies all on its own – usually about 10x.  This, combined with the objective lenses arranged on the turret, makes it easy to see just about anything – depending on what condenser/stage setting you are using.  Common settings include bright field (white background), dark field (black background), and phase contrast (which converts phase shifts in light to different levels of brightness so we can see them.)

It’s important to remember that things don’t always work logically when you’re looking through a microscope.  If you want to move the image in the microscope up, you must move the stage down, and vice versa.  Additionally, the higher power the objective is, the more difficult it is to focus.  I don’t know if you’ve ever stepped back from a microscope that you were using 40x objective on, but if you do you’ll see that the objective is basically touching the slipcover of the specimen.  Use the coarse focus and you might very well crack the objective, which will probably cost 500-1000 dollars to replace.  Always use fine focus when you are using high power objectives!  “Empty” magnification means the enlarging of a blurry picture.  Too bad, you’re probably too close to the specimen to begin with, so back up!