Blood Cell Count

(By Haemocytometer) - Academic Script

Course Name: Zoology

Second Year Under Graduates
U.G.C. Syllabus | Model - 1

Paper No. & Title: Z-202 B

Physiology

Topic No. & Title: Topic : C-4

Blood Cell Count
(By Haemocytometer)

 
 
 
 
 
 
 
Academic Script: -

RED BLOOD CELL COUNTING USING HAEMOCYTOMETER

1.RBC COUNTING - INTRODUCTION

• Blood is an important tissue of our body which contains cells like

red blood cells, white blood cells and platelets. Today, we are
going to perform an experiment wherein we learn to calculate their
number which is of great importance for clinical diagnosis of
various diseases. This experiment is divided in to two parts. The
first part we will learn to calculate the number of red blood cells
while in second part we will learn to calculate the number of white
blood cells present in blood by haemocytometer.
• We all have come across certain cases where the Medical
professionals ask us to take certain blood tests. A scientist or a lab
technician performs these tests. For this your blood is collected, it
is diluted and the number of blood cells in it are counted according
to the requirement of the medical professional.
• One of such test is blood cell count. The blood cell count gives us a
better idea about our general health status. For example when
your Red blood cell content is too low, you are having anemia.
Once you find out this anemic condition, it can be treated very
easily.
• Similarly during infection our immunological cells i.e. white blood
cells also increase. Hence, by performing the White blood cell
 count you can easily have an understanding about status of
infection.
• Similar is the case with platelet count. When a person is having
low platelet count, there are chances of blood coagulation
problems and hemorrhage.
• Hence it is very essential to perform blood cell count. In this
experiment we are going to learn about how to perform the blood
cell count, specifically of red blood cells.
• These tests are called as CBC or complete blood cell count. These
tests are performed to find out approximate number of red blood
cells in your blood.
• A scientist or lab technician performs this test. For this, the blood
is collected, it is diluted and the number of blood cells in it is
counted according to the requirement of the medical professional.
• Our blood contains 3 major types of cells;
1. Red blood cells (erythrocytes),
2. White blood cells (leukocytes),
3. Platelets (thrombocytes).
• Red blood cells perform the function of carrying oxygen and carbon
dioxide for respiration. White blood cells are our immunological
cells that fight infection. And platelets are the ones that are
responsible for blood clotting.
• Abnormally high or low counts may indicate the presence of
disease.
• Today we are going to find out the number of red blood cells in our
blood using equipment called as hemocytometer.
• Before going in to the detail of this experiment, let’s understand
what a hemocytometer is. This is a hemocytometer which is used
for finding out the blood cell count. This hemocytometer is made
up of two pipettes which are used for the diluting the blood.
Pipette which is used for Red blood cell counting is having red
colour bead in the bulb and a red colour knob, while the one used
for White blood cell counting is having white bead in the bulb and a
white colour knob. This makes it convenient for us to recognize the
Red blood cell and White blood cell dilution pipettes.
• This is the Red blood cell dilution pipette. We first fill up the blood
only up to this 0.5 mark. And then we dilute this blood using Red
blood cell dilution fluid up to this 101 mark.
• Hence, the dilution is 200 times. Because this is 0.5µL while the
entire volume is 101µL.
• This is the glass slide which is graduated for the counting of blood
cells and it is also called as Neubauer’s chamber.
• The larger squares are used for the calculation of white blood cells,
while smaller squares in the center are used for the calculation of
Red blood cells.
• Here in this slide you can see both smaller and larger squares.
• We count the number of Red blood cells in any 5 squares of the
total 25 squares which are observed in the field.
• Now let’s move on this experiment and learn how to calculate Red
blood cell count in your blood.
2.RBC COUNTING - EXPERIMENTAL PROTOCOL

Aim: Aim of this experiment is to calculate number of Red blood cells in

the blood. In this experiment we are going to take our own blood and
we will find out number of red blood cells present in a given unit of the
blood.

Principle: In this experiment we will take out our own blood, we will
dilute it 200 times and then in that diluted fluid we will calculate the
number of our Red blood cells. Then we will multiply it with 200 and
other multiplication factors to find out the actual number of Red blood
cells in one mL.
Before starting the actual experiment let’s gather the material which we
are going to need for this experiment.

Materials Required:
• Hemocytometer for the calculation of Red blood cells.
• Alcohol(Spirit) for disinfection
• Red blood cell dilution fluid for dilution of our blood.
• Cotton balls.
• Pricking needles.
• compound microscope.

Now let’s move on to this experiment and learn how to calculate Red
blood cells in our blood.
Method:
• First we will clean the glass slide. For this we will take cotton and
deep it in the alcohol (Spirit).
• Allow it to air dry, clean the cover slip.
• Take another cotton ball.
• Deep it in the alcohol and wipe your finger with alcohol.
• Allow this alcohol to air dry.
• take the pricking needle and puncture your finger
• Wipe off the first blood drop that is coming out.
• Now take Red blood cell dilution fluid immediately.
• Shake this vigorously for 2 min.
• Now, discard initial two drops of this dilution fluid.
• Take the drop on the hemocytometer.
• Now cover it with cover slip and mount it on the stage.
• First observe it under the low power and adjust it on the squares.
• The central squares in your observation field and focus them in the
central 25 squares. And count the number of red blood cells in any
5 squares.
• Once you have adjusted slide on low power, switch to high power.
• Now have an observation again.
• We can see 25 squares in this observation field.
• Out of these 25 squares, we will calculate the number of Red blood
cells only in 5 boxes.
• And we will sum up those numbers.
• Now count the number of Red blood cells in each square.
• Hence total we will be counting 16 X 5 = 80 squares.
• Calculate the total number of Red blood cells in these 80 squares.
 • Now calculate the total number of Red blood cells present in a unit
blood by given formula.

NBRC=N/NSq*Volume*Dilution = N/NSq*Area*Height*Dilution
NRBC=N/(80*(1/400)*(1/10)*(1/200)) = N/80*(400*10*200)=N*10000

3.RBC COUNTING - RESULTS & DISCUSSIONS

Hence from the formula which we have already understood, it can be

calculated that the number of Red blood cells in my blood is
686,000/µL. Hence, it would be approximately 686 billion/mL.
Hence from the formula which we have already understood we can
learn that the number of Red blood cells in given sample is 6.86
X106, i.e. almost 6.86 million cells/µL of blood. Hence form the given
formula it can be concluded that number of blood cells in the given
sample is 6.86 X 106 which falls under normal category hence the
person’s blood is having normal red blood cell count.

QUICK REVIEW
Now let’s have a quick review about what we have done in today’s
experiment.
In today’s experiment we had tried to learn the calculation of Red
blood cells in blood using haemocytometer. For this what we did was
• First we had pricked our finger
• Collected a drop of blood up to 0.5 mark
• Dilute it to 101 mark.
• Collect a drop of it on hemocytometer
 • Calculated the number of Red blood cells in any 5 squares.
• Multiplied it with dilution factors and obtain the number of Red
blood cells/mL.
• For our sample the number of Red blood cells was 6.86 X
106/µL.

Normal range:
– males: 4.3 – 6.2 X 106/ µL
– females: 3.2 – 5.5 X 106/ µL

Hence from our today’s experiment we learned how we can measure

the Red blood cell content of the given sample.

WHITE BLOOD CELL COUNTING USING A HEMACYTOMETER

4. WBC COUNTING - INTRODUCTION

In the first part of the experiment we had learned how we can calculate
the red blood cells in the given blood sample. Now let’s move on to the
second part i.e. white blood cells and how we can calculate the number
of white blood cells in the given blood sample.
• As we already know that our blood cells are of three types, i.e. Red
blood cells, white blood cells and platelets.
• Red blood cells are the ones which are performing the function of
carrying oxygen from the lungs to the tissues. And the white blood
cells are our immunological cells that are responsible for our
immunity. Our white blood cells are the ones that are fighting
infections in our body.
 • Normally the range of white blood cells in our blood is between 4
million to 11 million white blood cells/mL.
• Today we are going to perform an experiment to find out that what
the number of these white blood cells in our blood is. Increased or
decreased number of white blood cells in the given blood indicates
the disease. F.e. whenever you are having any infection of
bacterial or other origin the number of white blood cells is
increased to fight the infections in the body.
• White blood cells are also called as leucocytes. Whenever there is
an infection in the body and the number of white blood cells is
increased it is called as leucocytosis.
• However, contrary to this whenever the number of white blood
cells is falling down below average or the normal range, it is called
as leucocytopenia.
• This is the reason whenever we are having infection the medical
professionals are asking us to take the blood cell count
examination. And the increased or decreased number of white
blood cells indicate the presence of disease. Now as we all know
the importance of optimal number of white blood cells in our body
let’s move on to the experiment and learn how to find out the
number of white blood cells in the given blood sample.

5. WBC COUNTING - EXPERIMENTAL PROTOCOL

Before going in to the experimental detail let’s first understand the aim
of our experiment that what we want to do today.
Aim: The aim of this experiment is to find out the number of white
blood cells in the given blood sample and express it in the unit form so
that it is easy to interpret.

Principle: Now let’s first understand the principle behind this

experiment. In this experiment we will take out our own blood and we
will dilute it using a dilution fluid. This dilution fluid contains HCl as well
as a stain. HCl ruptures the non nucleated red blood cells and the stain
is staining the white blood cells. Because of this it is easy for us to
calculate only the white blood cells as the red blood cells are killed using
HCl.

Materials Required:
Now let’s gather the material which we are going to need in this
experiment. In this experiment we are going to need a
haemocytometer. And in the previous part of the experiment we had
already understood what a haemocytometer is. Apart from this we are
also going to need some pricking needles. We are going to need a white
blood cell dilution fluid. This white blood cell dilution fluid is prepared by
adding 1 mL of HCl to 100 mL distill water. And then we also added 5
mg crystal violet as a stain into this solution. We are also going to need
alcohol (spirit) for disinfection. And we are going to need some cotton
balls.
After this we are going to need a compound microscope for making the
observation.
Method:
• Now let’s start performing this experiment. For this first take a glass
slide and clean it with the alcohol.
• Also clean the cover slip using the alcohol (Spirit). After this allow the
alcohol to air dry.
• Then get ready to collect the blood from your finger. For this we will
prick our finger but before that we will have to disinfect it. For that
we will deep the cotton ball in the alcohol (Spirit) and clean our
finger. Once you have cleaned your finger, allow the alcohol (Spirit)
to air dry. Then take the pricking needle and puncture your finger.
After puncturing collect the blood up to 0.5 mark in the white blood
cell pipette.
• Once you have collected the blood immediately dilute this blood using
white blood cell dilution fluid up to 11 mark.
• Immediately after that shake the pipette well to allow the mixing of
two solutions.
• Now discard initial two drops of this fluid.
• Then put the drop of this solution on the haemocytometer and cover
it with cover slip and put it under the microscope for the observation.
• We will be making this observation at 10X power.
• Now calculate the number of white blood cells in the four squares
which are seen in this field.
• Now calculate the number of white blood cells in each square. And
sum up this number of white blood cells.
• After this you can calculate the number of white blood cells using the
given formula.
• WBCs per cu mm = Average number of chambers(4) WBCs counted x
dilution(20)/Volume(0.4)

6.WBC COUNTING - RESULTS & DISCUSSIONS

• These results are showing the number of white blood cells /µL.
• Hence we will be converting it to mL by multiplying it with 1000.

CONCLUSION:

Hence From the given formula it can be calculated that the number of
white blood cells in the given sample is about 7.9 million/mL, which
indicates the normal range of white blood cells in the given sample.

Hence in today’s experiment we learned how to calculate the number of

white blood cells in the given sample.

And we calculated the number of white blood cells in my blood is 7.9

million which falls under normal category.

Now let’s quickly summarize what we learned in today’s experiment.

Quick review:

In today’s experiment

1. we first pricked our finger,

2. we collected a drop of blood,
3. Collect the blood up to 0.5 mark
4. Diluted that blood 20 times using white blood cell dilution fluid.
5. Then we observed it under the haemocytometer.
6. we calculated the average number of white blood cells in the four
chambers and
7. We calculated the final number of white blood cells in the given mL
of blood using the formula which we already understood.