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