Microscopic technique – application of neubaur chamber for cell count

/Microscopic technique – application of neubaur chamber for cell count
Microscopic technique – application of neubaur chamber for cell count 2018-02-23T16:21:20+00:00

Microscopic technique – application of neubaur chamber for cell count (lecocyte cell concentration)

Introduction

The number of leukocytes contained in 1 litre of blood is called the leukocyte number concentration or leukocyte count. In certain diseases the number of leukocytes in the blood is altered. For example, in infectious mononucleosis and bacterial infections there is a marked increase, whereas in typhoid fever there is a marked decrease. The blood is diluted in a leukocyte diluting fluid which haemolyses the erythrocytes, but leaves the leukocytes intact. The leukocytes are then counted in a counting chamber under the microscope, and the number of cells per litre of blood is calculated.

Requirements

  1. Ruled counting chamber — preferably the improved Neubauer chamber
  2. Graduated pipette, 1ml
  3. Diluting fluid (prepared by adding 2 ml of glacial acetic acid to 1ml of gentian violet, 1% aqueous solution, and making up the volume to 100 ml with distilled water).
  4. The dimensions of the Neubauer ruled chamber are as follows:

area = 9mm2
depth = 0.1mm

Procedure

  1. Make serial dilutions of the bacterial culture.
  2. Pick 0.1 ml of the appropriate dilution, using a micropipette.
  3. Attach the coverslip (supplied with the chamber) to the counting chamber, pressing it carefully into place. When the coverslip is properly attached, coloured bands called Newton’s rings appear between the two glass surfaces.
  4. Load the counting chamber with the diluted culture. Take care not to overfill beyond the ruled area.
  5. Important: If the liquid overflows into the channel between the two chambers, you must start again: remove and clean the coverslip; clean the counting chamber; and refill with another drop.
  6. Leave the counting chamber on the bench for 3 minutes to allow the cells to settle.
  7. Place the chamber on the stage of the microscope. Use the 10X objective and the 10X eyepiece. Reduce the amount of light entering the condenser by adjusting the iris diaphragm. Focus the rulings of the chamber and the leukocytes. Do not mistake pieces of dust for leukocytes.
  8. Count the cells in an area of 4mm2 of the chamber, using the corner squares numbered 1, 3, 7 and 9. This square represents one of the four counted.
  9. Calculate the number of cells per litre by multiplying the number of cells counted in the four squares by 0.05. Report the result as “number by 109/l)”.
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