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 (Figure 3A) or make a more dilute suspension. 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.įor large cells, you can simply count the cells inside the four large corner squares (Figure 3B–E) and the middle square (Figure 3A). 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.įigure 2. When counting cells that overlap an exterior line or ruling, count only those cells on the top or right-hand line of the large square to avoid counting cells twice. The central counting area of the hemocytometer contains 25 large squares and each large square has 16 smaller squares. The full grid on a hemocytometer contains nine squares, each of which is 1 mm 2 (Figure 2). Allow the sample to settle for a couple of minutes and avoid moving the coverslip as it might introduce air bubbles and make counting difficult. The loaded hemocytometer is then placed on the microscope stage and the counting grid is brought into focus at low power. You can load two samples on one hemocytometer, one into each of the two grids. The area under the coverslip fills by capillary action.Įnough liquid should be introduced so that the mirrored surface is just covered, usually around 10 µl, but don’t overfill the surface. Then place the pipette tip with your sample into one of the V-shaped wells, and gently expel the sample. ![]() Make sure you place the coverslip over the counting surface before loading the cell suspension. Coverslips used for mounting on hemocytometers are specially made to be thicker than conventional microscopy coverslips because they must be able to overcome the surface tension of a drop of liquid. Loading the Hemocytometerīefore you get started, ensure that both the hemocytometer and its coverslip are clean by removing any dust particles with lens paper. Whatever dilution you use, make sure to note it down as you’ll need this for your final calculation. You can dilute your sample with trypan blue at any ratio, but a 1:1 ratio is the most common. When mixed with your cell sample, any dead cells will be stained blue by the dye, meaning that you can count only those cells that are living and viable. Trypan blue is a stain that allows you to distinguish dead cells from living cells. Using a Hemocytometer in Four Simple Steps 1. Here, we’ll talk you through using a hemocytometer and calculating your cell concentrations accurately. A classic hemocytometer (Image credit: Jeffrey M.
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