Mammalian & Plant Physiology

Experiment 2

Principle

White cell count is an important component of the blood count. White cells can be counted either manually or automatically. Anticoagulated venous blood is added to a diluent at a specific volume. The diluent lyses the erythrocytes, but preserves leukocytes. The diluted blood is added to the hematocytometer chamber. The diluent used is 2% acetic acid +gentian violet.

Materials required

Neubar chamber, blood sample, cover slip, microscope, RBC Lysis solution

Procedure

1. Pipette 0.95 ml of the diluents in a 75x10 mm tube.

2. Add 0.05 ml (50 μl) of blood to the tube.

3. Tightly seal the tube and mix the contents for one min.

4. Fill the counting chamber by means of a capillary tube.

5. Count the cells in the four "white cell" corner squares.

Note:

Each one of the four corner squares "W" has an area of 1mm2. These large squares contain 16 smaller secondary squares, each with an area of 0.04 mm2

Calculation

Dilution: 50 μl blood/50= 1 volume of blood

1. 0.95 𝑚𝑙 (950 𝜇𝑙 𝑑𝑖𝑙𝑢𝑒𝑛𝑡)/50= 19

2. 1/19+1=1/20

3. Dilution factor = 20

Number of WBC in Whole Blood:

1. Count =No. of cells counted/volume× 𝑑𝑖𝑙 𝑓𝑎𝑐𝑡𝑜𝑟

2. =𝑁/0.1× dilution factor

3. = 𝑁 × 10 × 20 = 𝑁 × 200

Normal Range:

The normal range is: 4 – 10 × 10⁹/l

Result

The total WBC count in the given blood same is……………………………× 10⁹ WBC/litre of blood.

Conclusion

Hematology is the study of blood cell counting is one of the most basic and yet most reliable ways of evaluating blood for multiple conditions of disease

Reference Material

1. Riley, Watson, Sommer, Martin. How to Prepare & Interpret Peripheral Blood Smears

2. Complete blood count, MedLine Plus, NIH, USA.

3. Ichihashi et al. (1996) The Haematology Atlas, Nagoya University School of Medicine, Japan.

Questions

1. How many morphologically different cell types do you observe?

2. What is the ratio of number of RBCs compared to WBCs you observed on your slide?