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- Place the cover glass on the hemacytometer, and load 10 μl of the cell suspension.
- Observe the entire grid of the hemacytometer in a phase contrast microscope. Focus on one quadrant of the grid (such as the one in red).
- Dead or dying cells will appear dark because they absorb more light in comparison to viable cells. Viable cells will not appear dark and will be surrounded by a “halo” of light in phase contrast (will be “phase-bright”).
- Count the number of viable cells in one quadrant (area 1 mm2). You can also determine cell viability by dividing the number of viable cells by the total number of cells. Count cells in 3-4 random quadrants and determine the average number of cells per quadrant. Since the volume of a quadrant is known to be 10-4 ml or 0.1 μl, the concentration of cells can be determined
- You can use the following shortcut to determine the #cells/μl:
- Total # of cells in tube =# cells/μl X μl in tube
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