 The Fundamental Device |
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The basic non-electronic part of a Coulter counter unit, as shown above, consists of a reservoir into which a finger-like tube is partially immersed. Near the lower end of the tube is a very small hole of known diameter. (Assortments of tubes are available covering a large range of hole sizes.) If the “finger” is filled to some point above the level of the outside reservoir, the contents of the finger will slowly flow through the hole until the two levels are equalized. This means that any particles (e.g.: blood cells, silt, bacterial cultures) will get flushed through the hole also.
Adding some electronics: If we place electrodes in the finger and in the reservoir, an electrical current will then also pass through the hole (aperture) along with the fluid (see figure to the right). This current can be monitored for irregularities by an oscilloscope type of device. In the picture next to the title, you can see the grayish “+” and “-” leads. The “+” goes into the reservoir, and the “-” goes into the finger. These are made of platinum, which, being a noble metal, does not electrolyze into solution.
Also in the top figure you see that there are two other electrodes in the finger. These detect the fluid level in the finger, and are usually spaced such that 0.1 ml (100 μl) is between them (or some other known volume as printed on the finger). Thus, after you fill the finger to above the upper sensor, the fluid will begin to drain through the hole into the reservoir. When the meniscus breaks contact with the upper sensor, the counter begins counting, and when the meniscus breaks contact with the lower sensor, the counter stops counting. Thus you know just how many particles were in that 0.1 ml.
Counting: The device counts because whenever a particle (here represented with a crystal of something) goes through the hole, the electronic system detects a sudden and momentary increase in resistance (a partial interruption of current flow). and a green vertical line appears on the screen, as shown at the top. The oscilloscope is able to detect up to about 300,000 partial interruptions per second before Beer’s law kicks in and you get out of linearity of true numbers and counts. This is because as concentration increases, the likelihood that two particles are going to try to squeeze through at the same time increases. Thus it might be that you must dilute your in some known way with buffer. (Why a saline buffer? To conduct the current, of course!)
Before continuing, there is one more practical point. This device is extremely sensitive to invisible air bubbles, lint and anything else that might clog the hole. All suspensions must be made in pre-ultrafiltered buffer. And even then cloggings occur. Coulter counters have small preset microscopes that look at the hole so you can see if it is plugged, and specially miniature “belly-button” brushes are available for wisking away stoppages.
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Sorting by Size: In the example here, we are using blood cells as our particles. In the sample are smaller red blood cells and larger white blood cells. If a small RBC enters the hole (left figure), the current is diminished much less than if a large WBC enters (right figure). Thus the passage of an RBC makes a short green spike, and that of a WBC makes a tall green spike on the oscilloscope. Thus if we judiciously place the horizontal red line on the screen, the counter will count all those that spike above the red line and put that number into one storage scalar for later retrieval by the computer that is usually connected to the device. In another storage scalar might be the total counts, and in a third scalar all the “short” counts. Thus, with a little practice in adjusting the red line, you can get very reproducible blood counts. |  |
WARNING! Note that you are counting particles. The machine does not know whether they are alive or dead.* Thus for those of you needing to count bacterial or yeast cultures, you will not only need to use different hole sizes than for blood counts, but you will likely also have to do some “viable cell counts” on petri plates. This bit about particles and viability counts is a classic on GRE exams. A Coulter printout is given and the test-taker must decide if this represents the same as a “plate count.” The answer is NO! The plate count is lower than the Coulter count because there are a few microbial corpses mixed in. The corpses get picked up by the Coulter counter, but not by the plate counting methods. In some other words: the plate count can NEVER be higher than the Coulter count.
Another minor concern, that some of you might have when you actually do this, is about why all the WBC spikes are not the same height, and why all the RBC spikes are not all the same height. The reason is that these cells are not spherical. Sometimes the disk-shaped RBC’s go through the hole like a frisby (sideways), and they don’t interrupt much current flow. At other times they might go through like a lemon-custard pie aimed at a clown, and that means a greater amount of momentary interruption of current. The same goes for WBC’s.
Yeast, which are more spherical, might be expected to be more uniform in size, but this is not so because of they way they bud and sometimes cling together for awhile. Also newly budded cells are smaller than the mother cells. With bacteria there is much more variability which is generally unknown to most bacteriologists because they are not familiar with results from Coulter counters. Most bacteria, such as E. coli, when dormant have very small cells. In the most rapidly growing cultures, the cells are about 500 times larger in volume. Thus cell size is a method for monitoring a culture’s generation time.
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