This thing is awesome for counting microorganisms.
The full grid on a hemocytometer contains nine squares, each of which is 1 mm2. The central counting area of the hemocytometer contains 25 large squares and each large square has 16 smaller squares. When counting, count only those cells on the lines of two sides of the large square to avoid counting cells twice. Suspensions should be dilute enough so that the cells or other particles do not overlap each other on the grid, and should be uniformly distributed.
To distinguish between dead and viable cells, the sample is often diluted with a particular stain, such as Trypan blue. This staining method, also known as dye exclusion staining, uses a diazo dye that selectively penetrates cell membranes of dead cells, coloring them blue, whereas it is not absorbed by membranes of live cells, thus excluding live cells from staining. When viewed under a microscope, dead cells would appear as dark blue.
To perform the count, determine the magnification needed to recognize the desired cell type and systematically count the cells in selected squares so that the total count is approximately 100 cells, a minimum number of cells needed for a statistically significant count.
For large cells, you can simply count the cells inside the four large corner squares and the middle one. For a dense suspension of small cells you may wish to count the cells in the four outer and middle squares of the central square or make a more dilute suspension.
Remember if a cell overlaps a ruling, count it as "in" if it overlaps the top or right ruling, and "out" if it overlaps the bottom or left ruling.
The area of the middle and each corner square is 1 mm x 1 mm = 1 mm2: the depth of each square is 0.1 mm. The final volume of each square at that depth is 100nl.
Once you have obtained the total cell count, cell concentration can be calculated from the following formula:
So, for example, if you diluted your sample 1:1 with Trypan blue, and you counted 325 cells in 4 corner squares plus the central big square, total cells per ml =
If you want to know how many cells you have in your original sample, just multiply the cell concentration by total sample volume. For example, if your original sample volume is 5 ml, then your sample has a total =
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