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  Section: Biotechnology Methods » Cell Biology and Genetics
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Estimation of Number of Leucocytes (WBC) in Human Blood

Cell Biology and Genetics
  Cell Cycles
  Meiosis in Flower Buds of Allium Cepa-Acetocarmine Stain
  Meiosis in Grasshopper Testis (Poecilocerus Pictus)
  Mitosis in Onion Root Tip (Allium Cepa)
  Differential Staining of Blood
  Buccal Epithelial Smear and Barr Body
  Vital Staining of DNA and RNA in Paramecium
  Induction of Polyploidy
  Mounting of Genitalia in Drosophila Melanogaster
  Mounting of Genitalia in the Silk Moth Bombyx Mori
  Mounting of the Sex Comb in Drosophila Melanogaster
  Mounting of the Mouth Parts of the Mosquito
  Normal Human Karyotyping
  Black and White Film Development and Printing for Karyotype Analysis
  Study of Drumsticks in the Neutrophils of Females
  Study of the Malaria Parasite
  Vital Staining of DNA and RNA in Paramecium
  Sex-Linked Inheritance in Drosophila Melanogaster
  Preparation of Somatic Chromosomes from Rat Bone Marrow
  Chromosomal Aberrations
  Study of Phenocopy
  Study of Mendelian Traits
  Estimation of Number of Erythrocytes [RBC] in Human Blood
  Estimation of Number of Leucocytes (WBC) in Human Blood
  Culturing Techniques and Handling of Flies
  Life Cycle of the Mosquito (Culex Pipiens)
  Life Cycle of the Silkworm (Bombyx Mori)
  Vital Staining of Earthworm Ovary
  Culturing and Observation of Paramecium
  Culturing and Staining of E.coli (Gram’s Staining)
  Breeding Experiments in Drosophila Melanogaster
  Preparation of Salivary Gland Chromosomes
  Observation of Mutants in Drosophila Melanogaster
  ABO Blood Grouping and Rh Factor in Humans
  Determination of Blood Group and Rh Factor
  Demonstration of the Law of Independent Assortment
  Demonstration of Law of Segregation

In order to count the WBC, the lysing of RBC or erythrocytes should be done. For this 1.5% –2% acetic acid solution with a small quantity of crystal violet was used as diluting fluid. Acetic acid destroys the erythrocytes, leaving behind the leucocytes. Crystal violet was used as a stain. These stains stain the nucleolus of leucocytes. This enables the leucocytes to be easily identifiable, and to be counted. The number of leucocytes in a dilute fluid can be counted by using a neubauer counting chamber.

Reagents Required
  • EDTA: Ethylene diamine tetra acetic acid was used as anticoagulant.
  • WBC diluting solution: This was prepared by using 1.5 mL of 1.5%–2% acetic acid solution of crystal violet and 98.5 mL of distilled water.
  • Syringe
  • Needle
  • Alcohol
  • Cotton
  • Vials
  • WBC pipette
  • Neubauer counting chamber
  • Cover glass and microscope
Using a syringe, venous blood was drawn and poured into a vial containing anticoagulant (EDTA) and mixed well. EDTA acts by chelating and preserves the cellular elements. With the help of a WBC pipette, this well-mixed venous blood was drawn. Both the fluid and blood are mixed gently, avoiding bubbling. The tube is kept aside for 5 minutes without disturbing it. In the mean time, a cover slip is placed on the counting chamber at the right place. The fluid/blood mixture is shaken and transferred, using a fine-bore posture pipette, onto the counting chamber. This is called “charging the chamber”. While charging the chamber, care is taken not to overflow. Whenever there is an overflow, it is washed and dried and recharged again. After charging, the cells are allowed to settle down to the bottom of the chamber for 2 minutes. It is seen that the fluid does not get dried up. For counting, the under part of the chamber was cleaned and placed on the stage of the microscope using 10X or a low-power objective. The leucocytes are counted uniformly in the 4 larger corner squares and cells present on the outermost lines are counted on one side, and those present on the lines opposite are not counted.

Present determination of total WBC count shows that there are about 7100 WBC/cm3 of blood. This is well within the normal range (6000 to 8000 WBC/cm3). In some cases of parasitic infection, the total WBC count increases. However, in the case of leukemia, the number of WBC increases to more than 15,000 cells/cm3 of blood. Normally, it would be about 4000 to 7000 WBC/mm3 of blood. If the WBC count decreases, then that condition is called leukemia.

Number of cells in 1 mm3 of blood
Cells Counted × Dilution Factor × Chamber Depth
Area of chamber counted
Cells counted × 20 × 10
= Cells counted × 50.


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