Lab 7.    Cell Counts

            In this lab exercise, you will learn how to use a hemocytometer to count cells.  This method allows you to determine the number of cells per milliliter and the total number of cells in a given volume.  Cells will be stained with trypan blue first.  Trypan blue is a dye that cannot penetrate the cell membrane.  Only dead cells, where the cell membrane becomes permeable to trypan blue, will be stained.  Using trypan blue, the percentage of viable cells can be determined.

Procedure

            Cells to be counted are in two eppendorf tubes.  The concentration of the cells in one tube is known, 5 X 10⁶, and the concentration of the cells in the other tube is unknown.

1. Make sure the cells you are going to count are completely resuspended by pipetting.  In an eppendorf tube, mix 20 ml of the cell suspension with 20 ml of trypan blue.  Mix thoroughly by pipetting up and down in the pipette tip.

2. Place the coverslip on the hemocytometer.  Transfer 10 ml of the cell/dye mixture to each chamber, loading at the edges.

3. Starting with one chamber, count all the cells in the four 1 mm corner squares.  Keep a separate count on the number of viable cells and dead cells (dead cells stain blue).  For cells lying on a line, count those that lie above or the left of the lines of the squares, but not those below or to the right lof the ines of the squares.  If there are too many cells to count accurately, you must start again and dilute your sample.  For example, you could do a 1:10 dilution by mixing 10 ml of cells with 90 ml of trypan blue.

4. Count the second chamber in the same manner as the first.

5. The hemocytometer chamber represents a volume of 1 X 10^-4 ml or cm³.  The cell concentration per ml can be calculated using a chamber volume conversion factor of 10⁴ in the equation below.

# cells/ml = average count per square X dilution factor X chamber conversion factor

6. If the cells appear clustered or there is more than 10% variation in the cell counts between chambers, repeat the count after dispersing the cells in the original suspension and then the dye dilution by vigorous pipetting.
 

Study questions

1. What is trypan blueused for?

2. You are counting cells.  First, you mix an equal volume of trypan blue with the cells.  If the average count per square is 30, how many cells are there in 1 ml?
If you need 1x10⁶ cells for your experiment, how many ml should you take from the original cell sample?