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Hemocytometer (Counting of Cells)
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Objective

 

To determine the concentration of cells in a given sample. 

 

Theory

 

For microbiology, cell culture and many  of the applications that require use of cell suspensions, it is necessary to determine the concentration of cells. The  device used for determining the number of cells per unit volume of a suspension is called a counting chamber. It is the most widely used type of chamber, since it was mainly designed for performing blood cell counts. It is now used to count other types of cells and other microscopic particles as well.

 

The hemocytometer was invented by Louis-Charles Malassez. It is a  special type of  microscope slide consisting of two chambers,  which is divided into nine (1.0mm x 1.0mm) large squares which are separated from one another by triple lines. The area of each is 1mm².  Cover glass is supported over the chambers at a height of 0.1mm. Because of that  the entire counting grid lies under the  volume of 0.9 mm² on one side. The cell suspensions are introduced into  the cover glass. The hemocytometer is placed on the microscope stage and the cell suspension is counted..

 

The glass microscope slide has a rectangular indentation that creates an 'H' shaped chamber at the centre. This chamber is engraved with a laser-etched grid of perpendicular lines. Two counting areas with ruled grids are separated by the horizontal groove of the 'H'. There is also a very flat, reusable cover slip. The glass cover slip is held at 0.1 mm above the surface of the counting areas by ground glass ridges on either side of the vertical grooves of the H shape. The device is carefully crafted so that the area bounded by the depth and  lines  of the chamber is also known. Because the height is constant, the volume of fluid above each square of the grid is known with precision.

 

The hemocytometer is used by putting the cover slip on the device, and filling the space with a liquid containing the cells you want to count. There is a "V" or notch at either end which is the place where the cell suspension is loaded into the hemocytometer. The fluid is usually drawn into the space by capillary action. A cover glass, which is placed on the sample, does not simply float on the liquid, but is held in place at a specified height. In addition, the grid arrangement of squares of different sizes allows for an easy counting of cells. It is possible to identify the number of cells in a specified volume by this method..

 

   

 

The ruled area of the hemocytometer consists of several large 1 x 1 mm (1mm² ) squares, which are subdivided in three ways; 0.25 x 0.25 mm (0.0625 mm²), 0.25 x 0.20 mm (0.05 mm²) and 0.20 x 0.20 mm (0.04 mm²). The central, 0.20 x 0.20 mm marked, 1 x 1 mm square is further subdivided into 0.05 x 0.05 mm (0.0025 mm²) squares. Hold the cover slip(  0.1 mm)  at the raised edges of hemocytometer, which gives each square a defined volume.

 

Dimensions

Area

Volume at 0.1mm depth

1 x 1 mm

1 mm2

100 nl

0.25 x 0.25 mm (1/16)

0.0625 mm2

6.25 nl

0.25 x 0.20 mm (1/20)

0.05 mm2

5 nl

0.20 x 0.20 mm (1/25)

0.04 mm2

4 nl

0.05 x 0.05 mm (1/400)

0.0025 mm2

0.25 nl

 

 

 

 

 A number of stains have been employed to distinguish between viable and nonviable cells. This is based on the principle that live cells contain intact cell membranes that eliminate certain dyes, like trypan blue, Eosin, or propidium. In dead cells, the stain enters the cytoplasm and the cells take on the stain. If more than 25% of the cells are stained, the cell suspension is most likely not a viable one.

 

To prepare the counting chamber, the mirror-like polished surface is carefully cleaned with 75% ethanol and the cover slip is also cleaned.. The cover slips used for counting chambers are specially made, and are thicker than those cover slips used  for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid. A cover slip is placed on the counting surface prior to putting on the cell suspension. Introduced any of the cell suspension  into any of the V-shaped wells with a  micropipette. The area under the cover slip fills by capillary action. Sufficient liquid should be introduced, so that the mirrored surface is just covered. The  charged counting chamber is placed under  the microscope stage and the counting grid is brought into focus at low power.


 

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