HealthSeba

Smaller laboratories and academic institutions commonly perform manual leukocyte counting due to its affordability and educational value. Although this method requires careful technique, it provides reliable results when performed correctly.

Equipment Required

• WBC pipette

• WBC diluting fluid (Turk’s fluid)

• Improved Neubauer counting chamber

WBC Pipette

Identification

The WBC pipette features a wider bore than an RBC pipette. Inside the bulb, a white bead facilitates uniform mixing. Clear graduations appear at the 0.5, 1, and 11 marks, allowing precise dilution.

Dilution Principle

When blood reaches the 0.5 mark and diluting fluid fills up to the 11 mark, the final dilution becomes 1:20.

Diluting Fluid

Composition

• Glacial acetic acid

• Gentian violet

• Distilled water

Function

Acetic acid lyses red blood cells while preserving leukocytes. At the same time, gentian violet stains WBC nuclei, thereby enhancing visibility and contrast during microscopic examination.

Procedure

Dilution Step

Initially, draw blood up to the 0.5 mark of the WBC pipette. Next, add Turk’s fluid until the 11 mark is reached. Afterward, mix the contents thoroughly for one to two minutes to achieve uniform dilution.

Charging the Chamber

Before filling the chamber, discard the first few drops to eliminate uneven dilution. Subsequently, place the coverslip correctly on the Neubauer chamber. Fill the chamber carefully, ensuring the absence of air bubbles. Then allow the cells to settle for two to three minutes.

Counting Step

Leukocytes are counted in the four large corner squares of the chamber. Each square measures 1 mm², with a chamber depth of 0.1 mm.

Total volume counted:
4 × 1 × 0.1 = 0.4 mm³

Counting Rules

To maintain accuracy, follow standard counting rules consistently:

• Include cells touching the top and left boundary lines

• Exclude cells touching the bottom and right boundary lines

By applying these guidelines, observers minimize counting bias and improve reproducibility.

Calculation

Let N denote the number of cells counted.

Formula:

WBC = (N × Dilution) / Volume counted

WBC = (N × 20) / 0.4

WBC = N × 50 cells/mm³

Example

If 120 cells are counted:

120 × 50 = 6000 cells/mm³

Thus, the total leukocyte count equals 6000/mm³.

Bulk Dilution

This alternative approach replaces the WBC pipette with a measured blood volume, which simplifies handling and reduces technical errors.

Procedure

• Measure 0.02 ml of blood

• Add the blood to a calculated volume of diluting fluid

• Mix thoroughly for uniform distribution

• Load the Neubauer chamber using a Pasteur pipette

The final dilution remains 1:20.

Advantages

This method improves accuracy through precise volume measurement. Additionally, easier calibration minimizes dilution errors. Better mixing further enhances reliability. As a result, many laboratories prefer bulk dilution for routine work.