What should I count?
Blood irrigates our whole body and therefore contains many types of cells that effect different tasks: red blood cells that carry oxygen, lymphocytes that fight infection by adaptive mechanisms (i.e. they find a tailor-made “cure”), leukocytes that constitute the innate response to infection (i.e. they display a universal “cure” and contribute to the healing process of wounds etc…). When we do a blood cell count, all of those will appear. However, because of their size, red blood cells will be distinguishable from white blood cells (all the others). That’s the way we are going to tell the difference between them when we count.
Diluting the blood
You can count blood cells with as little as a drop of blood. Because the cell density is very high, you have to dilute so much that you could do over 200 cell counts!
The dilution that is usually performed is 1:200 blood:isotonic solution. So for example, we could take 1μL of undiluted blood and add 199μL of isotonic solution (or do serial dilutions).
Loading the sample in the hemocytometer
To prepare the hemocytometer, make sure that you clean it properly with a tissue and ethanol, and place a clean glass slide on top. Once you have diluted the sample, you can additionally add a viability dye such as erythrosine B or trypan blue, on a 1:1 proportion. So you can take 10μL of the diluted blood you already have and add another 10μL of erythrosine B. This is going to be your counting solution. Introduce it with the pipette in the gap between the hemocytometer and the slide, taking care not to overfill the chamber while covering all the elevated surface of the chamber.
Counting the cells
White blood cells: because they’re bigger, you are going to count those in the four corner squares. As a reminder, you should establish a rule for the cells that are touching the peripheral lines: you can count the ones touching the top and left and skip the ones on the bottom and right, or any other combinations of two consecutive lines that you want. Note down your counts (discriminating between live and dead if you added a dye). Red blood cells: zoom into the central square, where smaller squares have been drawn. Count the cells in the four small corner squares and the small central square, and do as with the counts of the WBC.
Calculating cell density
You can proceed with the counts in the same way as in here, but this time remember to multiply by 200 due to the initial dilution you made, and additionally by 2 because of the viability dye. For faster counts, check out HemocyTap, the hemocytometer app.