Prepare Nitution of 1:20 Sample for TLC. and the Procedures. 2) prepare Turks Diluting Fluid.

The standard dilution for Total Leukocyte Count (TLC) using a WBC pipette is approximately 1:20 (precisely 1:22).

• Sample Collection: Obtain a fresh capillary blood sample or use anticoagulated venous blood.
• Dilution: Perform the 1:20 (or 1:22) dilution as described above using a WBC pipette and Turk's diluting fluid.
• Mixing: Mix the diluted blood thoroughly in the pipette for 2-3 minutes.
• Discarding First Drops: Discard the first 2-3 drops from the pipette to ensure only well-mixed fluid is used.
• Charging the Hemocytometer: Carefully charge both sides of a clean Neubauer counting chamber by touching the tip of the pipette to the edge of the coverslip and allowing the fluid to flow in by capillary action.
• Settling: Place the charged hemocytometer in a moist chamber for 5-10 minutes to allow the cells to settle.
• Counting: Place the hemocytometer on a microscope stage. Using the $10\times$ objective, count the leukocytes in the four large corner squares (W squares) of the counting chamber.
• Calculation: Calculate the total leukocyte count using the formula: $$\text{WBC count} = \frac{\text{Number of cells counted} \times \text{Dilution factor}}{\text{Area counted} \times \text{Depth}}$$ For the four large corner squares, the area is $4 \text{ mm}^2$ and the depth is $0.1 \text{ mm}$. The dilution factor is 22. $$\text{WBC count} = \frac{\text{N} \times 22}{4 \text{ mm}^2 \times 0.1 \text{ mm}} = \frac{\text{N} \times 22}{0.4} = \text{N} \times 55$$ Where N is the total number of WBCs counted in the four large squares.

2) Prepare Turk's Diluting Fluid

Turk's diluting fluid is used to lyse red blood cells and stain the nuclei of white blood cells, making them easier to count.