Experiment 1 Hematology I: Methods of Blood Collection, Processing And Storage of Blood

EXPERIMENT 2
TOTAL RBC AND WBC
COUNTING

Structure
2.1 Introduction 2.4 Total WBC Counting

Expected Learning Outcomes Principle

2.2 Total RBC Counting Materials Required

Principle Procedure

Materials Required 2.5 Calculation of WBC count

Procedure 2.6 Result and Observations

2.3 Calculation of RBC count 2.7 Precautions

2.8 Terminal Questions

2.1 INTRODUCTION
You are aware that the primary function of red blood cells (RBCs) is to
transport oxygen to the body's cells, whereas white blood cells (WBCs) are
part of our immune system that protect us from infectious agents. In this
exercise. you will learn how to use a Hemocytometer to count total RBCs and
WBCs in a sample of a person’s blood. The Neubauer chamber will be used in
this exercise to count total RBC and WBC. The number of cells per cubic
millilitre of blood is the most commonly used unit of measurement for blood
cell counts.

Expected Learning Outcomes

After studying and performing this exercise, you should be able to:
 handle RBC pipette and WBC pipette;
 prick the finger and collect blood through capillary;
 charge the Neubauer chamber or hemocytometer;
 observe the blood cells in grid of Neubauer chamber under microscope;
and
 count and calculate total RBC and WBC present in the blood. 11
 BBCCL-116 Human Physiology: Lab

2.2 TOTAL RBC COUNT

2.2.1 Principle
The blood specimen contains large number of red blood cells. The counting of
such large numbers under the microscope is almost impossible. Therefore, the
RBC count is performed with the help of a hemocytometer. The methodology
involves the accurate dilution of blood specimen (200 times)with the RBC
diluting fluid for which preferably Hayem’s fluid is used as it is isotonic to
blood.

The total number of Red blood cells is then calculated by the number of blood
cells counted in Neubauer's chamber and multiplying by dilution factor.

2.2.2 Materials Required

1. Compound microscope

2. Hemocytometer (Neubauer’s Chamber and RBC pipette)

3. Capillary and lancet

4. RBC diluting fluid (preferably Hayem’s fluid)

5. Sterile gauze piece or Cotton and

How To Use a Compound
Light Microscope: Biology
6. Watchglass
Lab Tutorial
7. Coverslips
Watch YouTube video:

https://www.youtube.com/ General Feature of the Neubauer Chamber

watch?v=lo2aC_m2vyo
The Neubauer's chamber is a rectangular indentation in a thick glass slide(Fig.
2.1). With a grid of perpendicular lines, the rectangular chamber has a
precision volume chamber.

(a) (b)

Fig. 2.1: (a) Neubauer chamber (b) Empty grid of Neubauer chamber (100X)

Neuberger chamber has ruled area of total 9 square mm and the depth is 0.1
mm. When the coverslip is placed on the counting chamber, the space
between the cover glass and the base of grooved area measures 0.1 mm in
depth.
12
Experiment 2 Total RBC and WBC Counting

The counting of blood cells can be performed either in the central large square
or in the corner squares by observing them under the microscope (Fig. 2.2).

WBC Counting area: The four large squares located at the corners of
Neubauer’s Chamber are used for white blood cell count.

RBC Counting area: The red blood cells are counted in the 5 squares of the
Central square (divided into 25 squares, each of which is divided into 16
squares). These include four corner squares and one central square of the
Large square.

Fig. 2.2 : Grid area of Neubauer’s Chamber.

RBC pipette is used to suck the blood sample and mixing blood fluid with the
RBC diluting fluid. It has a round shape bulb which contains the Red bead to
mix the blood specimen and the diluting fluid (Fig. 2.3). On the top, a rubber
tube is attached to the pipette for sucking the blood specimen and diluting
fluid. It has two marking points 0.5,1 at the bottom and the top is marked
as101.
13
 BBCCL-116 Human Physiology: Lab

RBC diluting fluid

Fig. 2.3 : RBC Pipette.

Composition of RBC diluting fluid(Hayem’s Fluid): Sodium chloride (0.5g),

sodium sulphate (2.5g), mercuric chloride (0.25g) and distilled water (100mL).

2.2.3 Procedure
1. Assemble clean and dry equipment at the practical site.

2. Take adequate RBC diluting fluid or Hayem’s Fluid in a clean and dry
watch glass.

3. Prick the finger with the help of a lancet and let the second large drop
accumulate on the finger.

4. Immediately hold the RBC pipette horizontally and suck gently on the
mouthpiece to draw blood upto the 0.5 mark of RBC pipette stem. Wipe
out the pipette externally to avoid false results.
14
Experiment 2 Total RBC and WBC Counting

5. After that, fill the same pipette with the RBC diluting fluid up to the mark
101 using constant suction. Be careful to prevent entry of any air bubble.

6. Mix the blood and diluting fluid in the bulb of RBC pipette for 2-3minutes
by rotating the pipette (horizontally) between your palms.

7. The pipette is now ready for charging. Place it on a horizontal surface

until the cell count is performed.

Charging Neubauer’s chamber

8. Place the coverslip on the central grooved area of Neubauer’s chamber.

9. Now, hold the pipette carefully and mix the solution again. Discard the
first 1-2 drops of fluid from the pipette.

10. Place the tip of RBC pipette on the surface of chamber by touching the
edge of the coverslip making an angle of 45° approximately (Fig.2.4).

11. Allow the diluted blood to flow from the pipette to fill into the chamber by capillary action. Do not
overcharge the
chamber.

Fig. 2.4: Charging Neubauer’s Chamber.

12. After charging, allow cells settle down in the chamber for 3-5 min.

COUNTING THE RED BLOOD CELLS UNDER MICROSCOPE

1. Place the charged Neubauer’s chamber on the stage of microscope for

observation and adjust the microscope under low power (10X) by fine
adjustment. Check uniform distribution of cells in the chamber.

2. Focus the RBC squares (Central large square of Neubauer’s chamber)

under high power objective lens (X40).

3. Count the cells in five medium-sized corner RBC squares (Four corners
and one central medium-sized RBC square with 1/20 mm side and
containing a total of 80 small squares(16x5= 80)(Fig. 2.5).

4. Draw the count of RBC cells in your notebook.

15
BBCCL-116 Human Physiology: Lab

(a)

Red Cell dimension

Size: About 6-7 μm in diameter

Shape: Concave shape

Thickness- 2.0 μm

(b)

Fig. 2.5 : (a) Arrow indicates the direction of counting cells; (b) Pattern of
counting red cells in a medium RBC square.

2.3 CALCULATION
(i) Calculation of dilution factor

The final volume of fluid in bulb is 100 (101-1=100) which comprises0.5

volume of blood and 99.5 volume of diluting fluid. Hence, the dilution factor
is100/0.5 = 200.
16
Experiment 2 Total RBC and WBC Counting

(ii) Calculation of fluid in 5 RBC squares

Now, the volume of the fluid inside the chamber is the product of Area and
depth of the Neubauer’s chamber.

1. The central area is the 1 sq. mm which is divided into 25 parts so the
area is 25 squares = 1 sq. mm

2. Out of these 25 squares, the RBCs are counted in 5 squares. Thus, the
area of 5 small squares is 5/25 i.e. 1/5mm2 in length

3. Depth of chamber = 1/10 mm (1/10 =0.1 mm)

Hence, volume of fluid in 5 medium-sized squares

=1/5 mm2X 1/10mm = 1/50 mm3

(iii) Calculation of total RBC count

The formula for RBCs count is

= number of cells counted (N) × Dilution factor / Area x Depth

Where,

 Number of Red blood cells (N)= ?

 Dilution factor = 200

 Area of 5 small squares =5/25 i.e. 1/5sq. mm in length

 Depth of the chamber = 1/10 mm = 0.1 mm

Total RBCs/mm3 = N X 200 / (1/5 X 0.1) = N X 200 X 50

= N X 10,000 million/mm3

For example, suppose, N or number of RBCs in the five squares is 500,

then the total RBCs will be:

= 500 x 10, 000 = 5,000,000 (5.0 million)

Normal values of human Red Blood cells:

Men –4.8-5.5 million/mm3

Women –4.5-5 million/mm3

2.4 WBC COUNT

2.4.1 Principle
Principle of WBC count is the same as of the RBC count in the human blood.
The diluted blood suspension is placed in the Neubauer’s chamber and the
cells are counted under the microscope. Since the number of WBC in blood
sample is much lower than the RBCs, the dilution factor is much lesser (1: 20)
than that of RBC (1:200). 17
 BBCCL-116 Human Physiology: Lab

2.4.2 Material Required

1. Hemocytometer (WBC pipette (Fig. 2.6) and Neubauer chamber
2. Coverslips
3. Watch glass
4. Diluents
5. Microscope
6. WBC diluting fluid (Turk's fluid): It contains glacial acetic acid(2 mL),
Gentian violet stain (1 mL), NaCl (0.9 g) and distilled water (97 mL). The
glacial acetic acid causes destruction of red blood cells, the Gentian
violet stains the nuclei of leucocytes and distilled H2O acts as a solvent.

WBC diluting fluid

Fig. 2.6 : WBC pipette.

2.4.3 Procedure
Watch YouTube video on
1. Prick your finger and collect the blood from the puncture site with the
Haematology White Blood
Cell Count help of a WBC pipette. Fill the pipette exactly upto the 0.5 mark. The
https://www.youtube.com/ blood level should be maintained by holding the pipette in horizontal
watch?v=q6rfJQVSals position.

2. Take WBC diluting fluid from the watch glass exactly up to the mark 11
and maintain this level closing the pipette tip with the index finger.
18
Experiment 2 Total RBC and WBC Counting

3. By closing both ends of pipette, shake and mix content of bulb at right
angle. A rotation of bead inside pipette will mix the solution.

4. Keep the pipette in the horizontal position.

5. Discard first two drops of fluid present in stem as it does not contain
cells.

6. Load the sample on the chamber as described in the section 2.2 for RBC
counting.

7. Place the charged chamber on the stage of microscope and set it under
the low objective power lens for observation.

8. Count the cells in the WBC squares at four cornersof chamber.

9. Draw WBC square in the notebook to record your observation.

10. Calculate the number of cells counted.

2.5 CALCULATION OF WBC COUNT

Area of 4 WBC squares = 4 x 1 = 4mm3

Volume of 4 WBC square = 4 x 1/10 = 4/10 mm3

Dilution factor = 1:20 (20)

Let us consider WBC cells in 4/10 mm 3 volume of diluted blood = n

Therefore, cells in 1mm3 volume of diluted blood = n x 10/4cells in 1mm3

volume of undiluted blood

= nx10/4 x20 = n x 50 mm3

Normal reference range of WBC = 4500-11,000/mm3

2.6 RESULTS AND OBSERVATIONS

You can record your answers of the following questions in your practical
notebook while counting of blood cells.

 Are the number of RBC and WBC normal in range or less than normal
count?

 What are health consequences if blood cells (RBC &WBC) are less or
more than normal range?

Errors

About 20-30%errorscan occur possibly during RBC and WBC counting due to
pipetting errors, uncleaned microscope, chamber volume errors, and errors
due to volume of sample introduced into the chamber. 19
BBCCL-116 Human Physiology: Lab

2.7 PRECAUTIONS
1. The Neubauer's counting chamber, RBC pipette, WBC pipette, cover
slips and microscope should be cleaned and dust free.

2. Discard the first/two drops of the blood-diluting fluid mixture.

3. Draw blood exactly up to 0.5 marks in pipette and dilute accurately with
RBC diluting fluid /WBC fluid depending on the cell types to be counted.

4. Prevent the entry of air bubbles in the pipette bulb.

5. Gently mix the contents of the bulb using hand's palms.

6. Do not overcharge the Neubauer’s chamber.

7. Ensure that no air bubble enters in the chamber while charging.

8. Do not count cells twice.

2.8 TERMINAL QUESTIONS

1. Define the term "Hemocytometer."

2. Draw the grid cells of Neubauer chamber.

3. Distinguish RBC pipettes from WBC pipettes.

4. What is formula for calculating the number of RBCs and WBCs in blood?

5. Name the chemical components of Hayem's Fluid and Turk's Fluid.

Fill in the Blanks:

I. Blood cell counts are expressed as _________________________

II. Dilution factor for RBC count is_______________________

III. Dilution factor for WBC count is______________________

Acknowledgment of Figures

Fig. 2.1b
https://en.wikipedia.org/wiki/Hemocytometer#/media/File:Hemocytometer_r
id_100x.jpg

20