STUDY OF NEUBAUER’S CHAMBER Fahareen-Binta-Mosharraf MNS

HEMOCYTOMETER Hemo: blood Cyto: cell Meter: measurement/counter Thus, it is an instrument used to count the blood cells.

THE NEUBABUER CHAMBER, OR HEMOCYTOMETER The Neubauer chamber is a thick crystal slide with the size of a glass slide. (30 x 70 mm and 4 mm thickness) In a simple counting chamber, the central area is where cell counts are performed. The chamber has three parts.

It includes: a) Neubauer’s slide b) Cover slip c) RBC pipette d) WBC pipette

NEUBAUER’S SLIDE It is the name given to a thick glass slide. In the centre of the slide, there is an H- shaped groove. On the two sides of the central horizontal bar, there are scales for counting the blood cells. The depth of the scales is 1/10mm or 0.1mm.

Materials The necessary elements to perform a cell count with Neubauer chamber are as follows: a) cellular dilution to measure b) hemocytometer, or Neubabuer chamber c) optical microscope d) cover glass e) pippette / micropippete with disposable tips. f) Dilution buffer / PBS (if needed)

NEUBAUER’S CHAMBER Neubauer’s slide with a cover slip over it, is called a Neubauer’s chamber.

Each scale is 3mm wide and 3mm long. The whole scale is divided into 9 big squares. Each square is 1mm long and 1mm wide.

Hemocytometer Chamber

The four corner squares are further divided into sixteen smaller squares and are used for WBC or microbial cell counting.

Four corner squares are meant for WBC counting. Total = 64 small squares W W W W

The central square is subdivided into twenty five smaller squares and each of these smaller squares is further subdivided into sixteen smallest squares. These are meant for platelet and RBC counting.

The platelets are counted in all the small squares of the central square, while the RBCs are counted in five small squares, four of corners and one of center. (total of 80 smallest squares)

RR R R R

CALCULATION OF THE VOLUME OF WBC SQUARES “Cells touching the upper and left limits should be counted, unlike cells touching the lower and right limits which should not be taken into account” 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.

Counting Rule Do not count cells touching Bottom line Right line  This is to avoid double counting.

CALCULATION OF VOLUME OF RBC SQUARE Total no. of cells in 4 big squares = X Average No. of cells in one big square = X/4 Since the volume of 1 big square is: 1 mm x 1 mm= 1 mm 2 of area counted. (0.1 cm x 0.1 cm = 0.01 cm 2 of area counted. ) Since the depth of the chamber is 0.1mm (0.1 mm = 0.01 cm ) 0.01 cm 2 *0.01 cm = cm 2 = ml = 0.1 μl (0.0001ml) So, for the Neubauer chamber, the formula used when counting in the big squares.

0.0001ml contains =X/4 cells 1 ml contains = X/4x10000 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