Cells observed on a slide placed under a microscope are counted using a common laboratory tool known as a hemacytometer, or a cell counting chamber, in conjunction with an exclusion dye called Trypan blue. The dye stains dead cells blue but cannot enter live cells, which will appear as bright, white spheres through the microscope eyepiece. Scientists need to count cells in order to place the correct number of cells into a cell-culture medium or experimental reagent for quantification and analysis or simply to prevent cells from overgrowing.
Determine the type of hemacytometer (e.g. Neubauer) being used. These are basically thick microscope slides containing a central area etched with counting grids. Place this under the microscope set to a low magnification (10-20X) and adjust the focus of the lenses until invidivual counting grids are visible.
Prepare a cell suspension. Use the enzyme trypsin to break up the tissue into single cells and then neutralize the trypsin with serum. Wash and pellet the cells, and resuspend in media or saline. Eliminate cell clumps by pipetting gently up and down. If the suspension appears cloudy, dilute this further with more saline.
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Use Trypan blue to exclude dead cells. Transfer 10-20 microliters of cell suspension into a sterile tube and mix with the exact same volume of Trypan blue dye by pipetting gently. This dilutes the cell suspension 1:2 in Trypan blue.
Prepare the hemactyometer. Wipe with 70% ethanol, air-dry and then place a coverslip over the counting grids. Into the \"drain\" at the top of each grid, place 10 microliters of cell suspension and allow the \"drain\" to pull it beneath the coverslip. Repeat for this other counting grid until both have an even layer of cell suspension.
Count the number of cells using the manual counter. Live cells will reflect light and appear shiny, whitish and spherical. Cells that are blue are dead or damaged and should not be counted unless non-viable cell numbers are required. Repeat for all 8 counting grids and then take the average. For example, if 800 cells are counted in 8 grids, then the average per grid is 100 cells.
Calculate the cell density in the original suspension. Apply the average number of cells to the following formula: Cell concentration = Average number of cells X 10,000 X dilution factor (=2, if dilution is 1:2) X volume of suspension (in milliliters, adjust as required for microliters and so on).
Clean the hemacytometer. Wash off the cell suspension, wipe the hemacytometer down with bleach and/or 70% isopropanol, or flood with 70% ethanol. Allow to dry or wipe dry with a lint-free cloth and discard all unwanted cell suspensions into biohazard containers.
Trypan blue is a mutagen and stains permanently, so ensure that protective clothing and gloves are worn before proceeding.