Technical Reference Guide

Cell Counting and Determination of Viability

via Hemocytometer
The hemocytometer is a device originally used to count blood cells
(as the name suggests). It is now used to count other cells and
many types of microscopic particles. It consists of a thick glass
microscope slide with a rectangular indentation that creates a
chamber of certain dimensions. This chamber is etched with a grid
of perpendicular lines.
The device is carefully crafted so that the area bounded by the
lines is known, as well as the depth of the chamber. Therefore, it is
possible to count the number of cells in a specific volume of fluid
and calculate the concentration of cells in the fluid overall.
When a liquid sample containing cells is placed on the chamber, it
is covered with a cover slip, and capillary action completely fills
the chamber with the sample. Looking at the chamber through a
microscope, the number of cells in the chamber can be determined
by counting. There are chambers of various compositions avail-
able (e.g., Neubauer chamber, Thoma chamber, Fuchs-Rosenthal
etc.). All hemocytometers consist of 2 chambers, each of which
is divided into 9 squares with the dimension of 1 x 1 mm. A
cover glass is supported 0.1 mm over these squares so that the
total volume over each square is 1.0 mm2 x 0.1 mm or 0.1 mm3, or
10-4 cm3. Since 1 cm3 is equivalent to 1 ml, the cell concentration
per ml will be the average count per square x 104. Any dilutions
have to be taken into account for the calculation of the cell
concentration.
Overview of Counting Cells via Hemocytometer
— If necessary, trypsinize cells.
— Pipet 10 μl of well resuspended cell suspension into Neubauer
counting chamber. (A)
— Count cells in 1 quadrant (blue). Alternatively as a general rule
2 – 4 quadrants are being counted. (B)
— To determine cell number per ml, multiply cell number by factor
of dilution and 104 (disregard trypan blue positive dead cells). If
you count 2 – 4 quadrants you need to divide by the number of
quadrants.
— Example
50 cells in quadrant (C)
50 cells x 104 = 0.5 x 106 cells/ml
To get the total cell number, multiply the cells/ml by the total
volume of the cell suspension.
A

The cell suspension should be diluted so that each such square

has between 20 – 50 cells (2 – 5 x 105 cells/ml).
B C
1 mm 0,2 mm 0,05 mm 0,25 mm

Accuracy of manual counts with a hemocytometer depends on:

— Accurate mixing of the sample (if bubbles are introduced into

the chamber, the chamber will need to be emptied, cleaned, and
filled again).

— Number of chambers counted; by performing a redundant test

on a second chamber, you can compare the results .
0,05k mm
k
0,2 mm
— Number of cells counted; appropriate dilution should be used. 1 mm 0,2 mm 0,05 mm 0,25 mm Count cells on top and left touching middle
line. Do not count cells touching middle
line at bottom and right.
 Technical Reference Guide

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