Cell Counting with a Hemocytometer

The hemocytometer is divideded into 9 major squares of 1mm x 1mm size.  The four coner squares (identified by the red square) are further subdivided into 4 x 4 grids.  The height of the chamber formed with the cover glass is 0.1 mm, so a 1 mm x  1 mm x 0.1 mm chamber has a volume of 0.1 mm3 or 10-4 ml.

To count cells using a hemocytometer, add 15-20μl of cell suspension between the hemocytometer and cover glass using a P-20 Pipetman.  The goal is to have roughly 100-200 cells/square.  Count the number of cells in all four outer squares divide by four (the mean number of cells/square).  The number of cells per square x 104 = the number of cells/ml of suspension.  This protocol works well for either adherent mammalian cells that have been trypsinized or for suspension cells including Sf9 insect cells.  Red blood cells are typically too small and numerous for this protocol and utilize the middle square instead.

When finished, spray the hemocytometer and cover slip with 70% ethanol to kill the cells.  Wash both with deionized water and wipe dry with a Kimwipe.  Wrap in a clean Kimwipe and return to the storage box.  Note, the cover slips for the hemocytomer are made of a special thicker/flatter glass.  Please try to avoid breaking or losing it.  If you do, reorder hemocytomer cover slips, not regular cover slips.