By: AISHATH SIYADHA SAEED, ROLL: 10

Date of Submission: 18th April 2013

WBC count by Hemocytometer

AIM

To accurately count WBC in Chamber.

To perform reliable dilution of blood cells

To calculate the number of cells/µL

PRINCIPLE

The white cell count (WBC) is the total number of leukocytes in a volume of

blood, expressed as thousands/•l. As with the RBC, the WBC can be done by manual

methods or by automated cell counters. The WBC by any method is a count of nuclei or

total nucleated cell count. If nucleated red blood cells (nRBC) are circulating in blood,

they will be included in the nucleated cell count whether the count is done by manual

methods or by automated analyzers. In such cases, the WBC represents the leukocyte

count only after it has been corrected for the nucleated red cells (nRBCs). The number

of nRBCs per 100 leukocytes is recorded during the differential leukocyte count. For

microbiology, cell culture and many applications that require use of suspension of cells,

it is necessary to determine the cell concentration. A device called a COUNTING

CHAMBER is used which determines the number of cells per unit volume of suspension.

The most widely used type of chamber is called a HAEMOCYTOMETER since it was

originally designed for cell counts. The hemocytometer was invented by Louis-Charles

Malassez and 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. The device is carefully crafted so that the area bounded by the lines

is known, and the depth of the chamber is also known. It is therefore possible to count the number of cells or particles in a specific volume of fluid, and thereby calculate the

concentration of cells in the fluid overall.

Counting of WBC is done under a microscope in the 4 corner squares of 1mm²

areas each. Each corner square is divided into 16 smaller squares of 1/10mm²(1/4 ×

1/4) area each. The depth of the counting chamber is 1/10mm; hence the volume of the

fluid in each smaller square is 1/16 × 1/10 or 1/160cumm. Therefore, the volume of

each corner square is 1/160 × 16=1/10mm³. From the average number of WBC counted

in 4 corner squares, number of WBC is calculated.

The blood after being sucked into a WBC pipette upto mark 0.5, is diluted 20

times with diluting fluid which destroys RBC and allows even dispersion of WBC

without changing their conformation. The diluting fluid contains glacial acetic acid

which haemolyses the red cells and gentian violet slightly stains the nuclei of WBS

which may therefore be recognized.

Normal range of WBC in humans is 5000-10,000/mm³.

MATERIALS NEEDED

Microscope Neubauer hemocytometer Coverslip Disposable sharp lancet Disposable quantitative blood collection tube Small test tube 1ml pipette Washing ear ball 70% alcohol 2% iodine 1% HCL (reagent)

PROCEDURE

PREPARING REAGENT:0.38ml diluent, 1% HCl was drawn into a small test tube

DRAWING BLOOD:20 μl of capillary blood was drawn from the tip of the index finger

DILUTING BLOOD20 μl of blood was poured into 0.38 ml of 1% HCL, mixing suitably. This is called a cell suspension.

FILLING HEMOCYTOMETERA drop of diluted blood was drawn and it was dropped to the edge below the coverslip. Immediately after that, the counting chamber was filled with cell suspension.

COUNTING CELLSThe hemocytometer contains 2 Neubauer counting chambers.

Each chamber contains:

*4 WBC counting squares

*Each contains 16 squares

100 RBC= 10 Platelets= 1 WBC

Chose 90° lines, count only the cells that are on those lines

(ex: L-shape)

apply it to all squares for maximum accuracy.

CALCULATING RESULT

Counting of WBC is done under a microscope in the 4 corner squares of 1mm² areas each. Each corner square is divided into 16 smaller squares of 1/10mm²(1/4 × 1/4) area each. The depth of the counting chamber is 1/10mm; hence the volume of the fluid in each smaller square is 1/16 × 1/10 or 1/160cumm. Therefore, the volume of each corner square is 1/160 × 16=1/10mm³. From the average number of WBC counted in 4 corner squares, number of WBC is calculated.

RESULTTotal cells counted in the four large squares = 37WBC = (37/4) × (20/0.1μl) = (37/4) × 20 × 10 × 106/L = (37/20) × 109/L = 1.85 × 109/L

DISCUSSION

Some precautionary measures were taken when the experiment was carried out.

The needle and finger must be cleaned and sterilized before the experiment.

The hemocytometer and coverslip must be kept clean before puncturing the finger.

No air gap/clot should appear in the pipette while sucking blood or diluting fluid.

Sucking of blood and diluting fluid must be carried out quickly to prevent clotting.

The diluting blood must be given some time to settle on the counting chamber before the calculation is done.

The white cell count (WBC) is the total number of leukocytes in a volume of blood, expressed as thousands/µl. As with the RBC, the WBC can be done by manual methods or by automated cell

counters. The WBC by any method is a count of nuclei or total nucleated cell count. If nucleated red blood cells (nRBC) are circulating in blood, they will be included in the nucleated cell count whether the count is done by manual methods or by automated analyzers. In such cases, the WBC represents the leukocyte count only after it has been corrected for the nucleated red cells (nRBCs). The number of nRBCs per 100 leukocytes is recorded during the differential leukocyte count. Then a correction is made as follows:

Corrected WBC = nucleated cell count x (100 ÷ [nRBC + 100])