USE OF COUNTING CHAMBER (HAEMOCYTOMETER) FOR CELL COUNTING, Improved Neubauer counting chamber, Fuchs-Rosenthal Counting Chamber, Charging the Counting Chamber for cell Counts

USE OF COUNTING CHAMBER (HAEMOCYTOMETER) FOR CELL COUNTING

A haemocytometer consists of a counting chamber, a coverglass for the counting chamber and the diluting pipettes. Many types of counting chambers are available. Improved Neubauer and Fuchs Rosenthal are the two most commonly used chambers in laboratories.

Improved Neubauer counting chamber

The improved Neubauer counting chamber consists of a thick rectangular glass slide with an 'H' shaped trough, forming two counting areas (Fig.3.4(a)) .Beyond the two vertical arms of the trough are two raised shoulders which support the specially made thick, optically flat coverglass. The space between the coverglass and surface of the counting area (i.e. the depth) is exactly 0.1 mm if the coverglass is correctly applied (Fig. 3.4(b)). Each counting area is 3 x 3 mm. This produces a total counting volume of 0.9 cu.mm.

When the ruled area is viewed under the 10 x objective of the microscope, it shows 5 squares each having 1 mm2 area (Fig. 3.5). These 1 mm2 sections in the four corners are divided into 16 equal squares. The square in the centre of the ruled

area is divided into 25 equal squares. In turn, each of these 1/25 mm squares are further divided into 16 portions, each having an area 1/400 mm2. The area can be selected according to the cell type to be counted. Figure 3.6 shows the various areas and their dimensions.

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Fuchs-Rosenthal Counting Chamber

There are two types of Fuchs Rosenthal chambers available. The older type has 16 squares, Immeach, bounded by triple lines (Fig. 3.7). Each of these squares is further subdivided into 16 squares. Thus the smallest square has an area 1/16 mm'. The depth of the chamber is 0.2 mm.

The other type of Fuchs-Rosenthal chamber has a total ruled area of 9 mm' which is divided into 9 squares. The depth is 0.2 mm. Fuchs-Rosenthal chamber is more commonly used for counting cells in cerebrospinal fluid.

Charging the Counting Chamber for cell Counts

The counting chamber should be set up correctly so that the depth of the counting chamber is uniform. To facilitate this, moisten the raised shoulders of the chamber and slide the cover slip onto the shoulders with both thumbs. If correctly placed, the coverslip should not fall off even after inverting the counting chamber and should show rainbow colours (Newton's rings).

To fill up the chamber, care must be taken that the diluted blood does not overflow into the 'H' shaped trough around the counting surface. Fill the

counting area by touching a drop of well mixed diluted blood to the edge of the coverglass and the fluid is drawn in by capillary action. It is necessary to charge both the counting areas. Before counting, allow the cells to settle in a moist chamber. A simple moist chamber can be prepared by placing a wet cotton pad or filter paper in a petri dish.

Counting of Cells While counting the cells in the squares include those that touch the lines on the left side or on top of the squares, and exclude those that touch the lines on the right side and at the bottom of the square (Fig. 3.8). For squares outlined with a triple set of lines, the central line denotes the boundary of the square. This system of counting avoids the chances of a cell being counted twice or of being omitted.