Enumeration of Red Blood Cells

Enumeration of Red Blood Cells using Haemocytometer.

1. ENUMERATION OF RED BLOOD CELLS VIT UNIVERSITY 16BBT0058 NIKITA SREENATH BIT1006

2. RED BLOOD CELL COUNT A red blood cell count is typically ordered as part of a complete blood count(CBC). RBCs circulate in the blood and carry oxygen throughout the body. A red blood cell count is used to help diagnose and monitor a number of diseases like anaemia, kidney disease, etc.

3. HAEMOCYTOMETER Haemocytometer is a counting chamber recommended for cell counting. The haemocytometer was invented by Louis-Charles Malassez. It consists of a thick glass microscope slide with a rectangular indentation that creates a chamber . This chamber is engraved with a laser-etched grid of perpendicular lines.

4. The device is carefully crafted so that the area bounded by the lines is known, and the depth of the chamber is also known. The gridded area of the haemocytometer consists of nine 1 x 1 mm (1 mm 2 ) squares. These are subdivided in 3 directions; 0.25 x 0.25 mm (0.0625 mm 2 ), 0.25 x 0.20 mm (0.05 mm 2 ) and 0.20 x 0.20 mm (0.04 mm 2 ). The central square is further subdivided into 0.05 x 0.05 mm (0.0025 mm 2 ) squares. The raised edges of the haemocytometer hold the coverslip 0.1 mm off the marked grid, giving each square a defined volume.

5. USING THE HAEMOCYTOMETER T he special coverslip provided with the counting chamber is properly positioned on the surface of the counting chamber. When the two glass surfaces are in proper contact Newton's rings can be observed. The cell suspension is applied to the edge of the coverslip to be sucked into the void by capillary action which completely fills the chamber with the sample. The number of cells in the chamber can be determined by direct counting using a microscope, and visually distinguishable cells can be differentially counted. The number of cells in the chamber is used to calculate the concentration or density of the cells in the mixture the sample comes from .

6. CONCENTRATION CALCULATION We apply the formula for the calculation of the concentration The number of cells will be the sum of all the counted cells in all squares counted. The volume will be the total volume of all the squares counted. Since the volume of 1 big square is: 0,1 cm x 0,1 cm = 0,01 cm2 of area counted. Since the depth of the chamber is 0.1mm, 0.1 mm = 0.01 cm, 0.01 cm*0.01 cm = 0.0001 cm 2 = 0.0001ml = 0.1 µl In case a dilution was applied, the concentration obtained should be converted to the original concentration before the dilution. In this case, the concentration should be divided by the dilution applied.

7. APPLICATIONS Blood counts: for patients with abnormal blood cells, where automated counters don't perform well . Cell culture: when subculturing or recording cell growth over time. Beer brewing: for the preparation of the yeast. Cell processing for downstream analysis: accurate cell numbers are needed in many tests (PCR, flow cytometry ), while some others require high cell viability. Measurement of cell size: in a micrograph, the real cell size can be inferred by scaling it to the width of a haemocytometer square, which is known.

8. REFERENCES http:// celeromics.com/en/resources/docs/Articles/Cell-counting-Neubauer-chamber.pdf https:// en.wikipedia.org/wiki/Hemocytometer

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