Lab 4:
Hemocytometer
The hemocytometer is a device used to count cells. It was originally designed for
the counting of blood cells.
The Neubauer chamber is a thick crystal slide with the size of a glass slide. (30 x 70
mm and 4 mm thickness)
 It is constructed so that the distance between the bottom of the coverslip and
the surface of the counting area of the chamber is 0.1 mm.
 The surface of the chamber contains two square ruled areas separated by an
Hshaped moat.
 These two squares are identical, allowing the technologist to duplicate the cell
count.
 Each has a total area of 9 mm2 (1 mm on each side). These squares are
divided into nine primary squares with an area of 1mm2.
 The four corner primary squares are used when counting leukocytes. (If the
WBC is very low, you would count all 9 squares.) These 4 large corner
squares contain 16 smaller secondary squares, each with an area of 0.04 mm2.
All 25 secondary squares of the center primary square are used to count
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platelets, and each of these 25 squares is further divided into 16 smaller
tertiary squares.
 The boundary lines of the central primary square are either double or triple.
 When the boundary line is double, all the cells within the square and those
touching the innermost line are counted. If the boundary line is triple, all of
the cells within the squares and those touching the middle line inward are
counted.
 Hemacytometers and coverslips should meet the specifications of the
National Bureau of Standards and are so marked by the manufacturer. A
standardized coverslip should be used that has been ground to fit the specifics
of the hemacytometer, ensuring a uniform depth and therefore a constant
volume. A regular coverslip cannot be used.
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 The necessary elements to perform a cell count with Neubauer chamber are as
follows:
a) cellular dilution to measure
b) hemocytometer, or Neubabuer chamber
c) optical microscope
d) cover glass
e) pippette / micropippete with disposable tips.
f) Dilution buffer / PBS (if needed)
STEP 1. Sample preparation.
Depending on the type of sample, a preparation of a dilution with a suitable
concentration should be prepared for cell counting.
WBCs
 Specimen
EDTA-anticoagulated blood or capillary blood is preferred.
Reagents, supplies and equipment
1. White blood cells count diluting fluid which may be one of the following:
- Acetic acid 2% (v/v) in distilled water.
- HCL 1% (v/v) in distilled water.
- Turks' solution which is formed of:
A. Glacial acetic acid 3 ml
B. Crystal violet 1 ml
C. 100 ml distilled water.
2. Thoma white pipette.
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 Procedure
1. Draw the blood up to 0.5 mark in the thoma pipette.
2. Wipe the outside of the capillary pipette to remove excess blood that would
interfere with the dilution factor.
3. Holding the pipette almost vertical place into the fluid. Draw the diluting fluid
into the pipette slowly until the mixture reaches the 11 mark, while gently
rotating the pipette to ensure a proper amount of mixing.
4. Place the pipette in a horizontal position and firmly hold the index finger of
either hand over the opening in the tip of the pipette, detach the aspirator from
the other end of the pipette now the dilution of the blood is completed
5. Mix the sample for at least 3 minutes to facilitate hemolysis of RBCs.
6. Clean the hemacytometer and its coverslip with an alcohol pad and then dry with
a wipe.
7. Before filling the chamber, discard the first
four to five drops of the mixture on apiece of
gauze to expel the diluent from the stem.
8. Carefully charge hemacytometer with diluted blood by gently squeezing sides of
reservoir to expel contents until chamber is properly filled.
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9. Mount the hemacytometer on the microscope and lower its condenser.
RBCs
 Sample
Whole blood using EDTA or heparin as anticoagulant. Capillary blood may also be
used.
 Diluting fluid
One of the following solutions may be used:
1. Isotonic saline: 0.85%sodium chloride (NaCl) in distilled water.
2. Hayam’s solution
A. Sodium Sulphate 10 g.
B. Sodium Chloride 2 g.
C. Mercuric Chloride 0.25 g.
D. Distilled Water 100ml
3. Gower’s solution
A. Sodium Sulphate 12.5 g.
B. Glacial acetic acid 33.3 ml
C. Distilled water 100 ml.
4. Citrate-formalin solution
A. Tri-sodium Citrate
B. Formalin
 Equipments
(Pipettes) used one of the following:
A. Thoma pipette (RBCs)
B. Micropipette –20l is the desired volume.
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 Procedure
1. Dilution of the blood:
Micropipette (20) 1:201 dilution.
 Pipette 4.0ml of diluting fluid into a tube
 Pipette 20l of will mixed anticoagulated whole blood to the tube.
Thoma red count pipette.
 Draw the blood up to exactly the 0.5 mark and dilute to the 101 mark.
 Mix continuously for 2-3 minutes.
2. Load the cleaned hematocytometer.
3. Place the hematocytometer on the microscope stage, lower the condenser.
4. Focus with x10 objective lens on the large central square. This square is
ruled into 25 small squares, each of which is further divided into 16
smaller squares, of the 25 squares, only the four corner squares, and one
middle square are used to count RBCs.
5. Switch to 40 objective lens, and start counting in the five designated
squares.
STEP 2. Microscope set up and focus.
1. Place the Neubauer chamber on the microscope stage. If the microscope has a
fixing clamp, fix the Neubauer chamber.
2. Turn on the microscope light.
3. Focus the microscope until you can see a sharp image of the cells looking through
the eyepiece and adjusting the stage 4x, 10x, and 40 x.
4. Look for the first counting grid square where the cell count will start.
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Calculation
WBCs
for counting 4 squares :
Total WBC/mm3 = No. of cells x volume correction factor x dilution factor.
Total WBC/mm3 = No. of cells x (10/4) x 20.
 Correction for dilution:
The thoma pipette is 1:20
Dilution factor 20
 Correction of volume:
Volume of square: One large square is = 0.1 x 1 x 1= 0.1 μL.
Volume of 4 squares: Four large squares are 0.4 μL.
Volume correction factor: 1/0.4
RBCs
Total RBC Count = N x Dilution Factor x Volume Correction Factor
Where:
N = the total number of red cells counted in the counting chamber.
Dilution 1: 200
 Dilution Factor = 200
Counted Volume
 Each counted square has a volume of 0.2 X 0.2 X 0.1 = 0.004
 5 squares volume = 5 X 0.004 = 0.02 cumm
 Volume correction factor = 1/0.02 = 50
So,
Total RBC count = N X 200 X 50 = N X 10.000
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