This document provides instructions for performing a WBC count using a hemocytometer. WBCs are counted using a dilute solution of glacial acetic acid to lyse red blood cells while fixing WBC nuclei. Blood is mixed with the solution, incubated, and cells in the counting chamber are examined under a microscope to determine the number of WBCs per microliter. Normal WBC counts range from 4,000 to 11,000 cells/microliter. The results are interpreted as normal, leukocytosis, or leukopenia.
2. Aim.
Principle.
Materials & reagents.
Type of sample.
Method.
Result calculation.
Normal values.
Comment/ interpretation of results.
3. Aim:
1. For diagnosis of infections.
2. For antibiotics treatments.
Principle:
Blood is diluted with a diluent called Glacial
acetic acid which is a hypotonic solution, so
lyses the RBCs & Plts, leaving & fixing the
nucleus of WBCs which stained with the
nuclear stain.
4. Reagent:
2 % Glacial acetic acid (G.A.A), which is
composed of:
i. v/v: 2 ml of G.A.A + 98 ml D.W.
ii. Few drops of nuclear stain (e.g.: crystal violet,
methylene blue, gention …)
- When blood is added to the solution above, the
majority of solution is water “i.e. hypotonic
solution” so result in swelling of red blood cells,
expanding and finally rupture of them, also the
platelets too.
- While WBCs & nucleated red blood cells are
more resistant to rupture by water than mature
RBCs & Plts.
- This G.A.A is a simple fixative, that fix the
nucleus of WBCs, while the nuclear stain is
used to allow viewing of WBCs nucleus.
5. Sample:
- Usually EDTA anticoagulated venous blood
sample, or
- Free flow capillary blood sample.
Materials & reagents:
1. 2% G.A.A.
2. Small test tubes.
3. Micro pipette (20 µl) & 1 ml glass pipette.
4. Cotton.
5. Improved neubauer counting chamber.
6. Cover glass.
7. Capillary tube or Pasteur pipette.
8. Microscope.
6. Method:
1. In small test tube, put 0.38 ml of 2% G.A.A.
2. Add 0.02 ml of blood.
3. Apply well mixing.
4. Incubate for 3 – 5 mins.
5. Prepare the counting chamber:
Clean the counting chamber properly “free from debris, dust,
cracks”.
Fix the cover glass properly. “avoid finger print”.
6. Put a small drop from the mixture above using
capillary or Pasteur pipette.
Avoid formation of air bubbles.
Avoid over or under filling of counting area.
7. Wait for 2 – 3 min in moisted environment to allow
the cells to settle and avoid drying of the mixture.
8. By using L.microscope ×10 count the WBCs in the
four corners.
7. - Checking that the blood is on the mark.
- Removing the excess blood.
