hemocytometer

A device for counting blood cells, originally, but now used to count any free-floating cell. Variably spelled hemacytometer, haemocytometer, and haemacytometer, though I believe hemocytometer is correct, at least in the USA.

The device is basically a special microscope slide, made to high tolerances. There is also a very flat, reusable cover slip. Basically, the hemocytometer is made such that the thickness between the slide part and the cover slip is constant. And the slide part is etched carefully, so that when one looks at the slide through a microscope, you see squares. Because the height is constant, the volume of fluid in the square is known.

It is used by putting the cover slip on the device, and filling the space with a liquid containing the cells you want to count. The fluid is usually drawn into the space by capillary action. Then one counts a number of squares, and you work out how many cells you counted per volume counted. This gives you the number of cells per volume, and you multiply by the total volume of fluid containing the cells.

There are also versions made for counting much bacteria, which are much smaller. These devices are made such that the height is much smaller than in normal hemocytometers.

The set up of the hemocytometer is a mirror-like surface, divided into two parts. Each part has a small grid on it. The mirrored surface is surrounded by a small "moat" so allow any excess liquid to drain away. The rest of the slide is just thick frosted glass. The glass edges are a constant 0.1mm higher than the mirrored surfaces.

______________________________________________
|             |   |   \/   |   |              | 
|             |   |        |   |              |
|  brand      |   |   #    |   |              |
|   name      |   |________|   |              |
|             |    ________  M |              |
|             |   |        |   |              |
|             |   |   #    |   |              |
|             |   |        |   |              |
|_____________|___|___/\___|___|______________|

M - The moat surrounding the grid surfaces.
# - The suface where the grids are. Proportionately they are bigger than shown, about 3 times.
The V shapes are little divets that the cover slip barely over laps, where the liquid containing the cells would be dropped, to be sucked up by capillary action.

How to Use a Hemocytometer:

1. Wipe the mirrored surface and the cover slip gently with a dust-free wipe, such as Kim Wipes. Never use Kleenex or anything similar, you will add more dust than you remove.

2. Make sure your cells are mixed very well in the solution, so that your counts are an accurate representation of the whole sample. This can be done by pipetting up and down.

3. With the cover slip on, use a clean (autoclaved, if necessary) Pasteur pipette to load the hemocytometer. To do this just place your finger over the end of the pipette, and when you dip it in remove it. This small amount is enough. Just lightly touch the end of the pipette to the V shaped divets, and the capillary action will suck up the liquid and cells.
Note: No matter how much of the solution you add the volume taken in by the hemocytometer will be constant, since the available space is constant.

4. Count the cells. a)There are 9 large squares broken into smaller squares on each of the two grids. The large squares tell you where to count. One large square should take up most of the field of view on the microscope. The smaller squares inside help you keep track of where you are in the field of view. b)On each grid count the middle square (which is dived into smaller pars) and two corner squares. This gives you 6 counts in total. A desktop or hand held counter is helpful, because often your counts will be to high to count in your head.

5. Clean off the hemocytometer by rinsing with water, or dipping in warm soapy water. Try not to scrub, as you may damage the mirrored surface. Dry with Kim Wipes. Do not leave the hemocytometer lying around with cells on it, as the solution may start to dry up, leaving permanent marks on the surface.

How to Calculate your Cell Concentration:

1. Average all your counts.

2. Your concentration is Average x 10⁴ cells/mL. The number of cells you have in total is Volume(in mL) x Concentration.

This simple calculation makes the hemocytometer an easy, and quick way to count a sample of cells. However, it is not practical to use for many batches of cells in a counting experiment. It is very time consuming when compared to other methods, which are often more systematic and automated, which removes some of the element of human error. Such methods include Coulter Counters or using a radioactive tag.

Hemocytometers are sold by most laboratory supply companies. They are generally priced from $80-200 American, depending on the quality of the grid and the uniformity of design.