SCHOOL OF PHARMACY

BACHELOR OF PHARMACY (HONS)

COHORT 10

BPH 1024
Human Anatomy and Physiology

EXPERIMENT 3:
DETERMINATION OF WHITE BLOOD CELL
COUNT USING A HEMACYTOMETER

NAME : NUR HANNAH BINTI REIDZUAN

STUDENT ID : 2062221016
PROGRAM : BPH 10
DATE OF EXPERIMENT : 18TH OCTOBER 2022
NAME OF LECTURER : DR KRUPAVARAM B.
 EXPERIMENT 3: DETERMINATION OF WHITE BLOOD CELL COUNT USING A HEMACYTOMETER

TABLE OF CONTENTS

3.1 TITLE ............................................................................................................................ 3

3.2 OBJECTIVE ................................................................................................................. 3

3.3 INTRODUCTION ......................................................................................................... 3

3.4 REQUIREMENTS ........................................................................................................ 4

3.5 PROCEDURE ............................................................................................................... 4

3.6 RESULTS ...................................................................................................................... 6

3.6.1 The hemacytometers shown to count White Blood Cells present in several drops of

human blood. ..................................................................................................................... 6

3.6.2 Calculations of the White Blood Cells ....................................................................... 7

3.7 DISCUSSIONS .............................................................................................................. 9

3.8 CONCLUSION............................................................................................................ 10

3.9 REFERENCES ............................................................................................................ 11

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 EXPERIMENT 3: DETERMINATION OF WHITE BLOOD CELL COUNT USING A HEMACYTOMETER

3.1 TITLE
Determination of White Blood Cell Count Using a Hemacytometer.

3.2 OBJECTIVE
To determine the white blood cell count by using a hemacytometer.

3.3 INTRODUCTION

Figure 3.1 above shows the different type of white blood cells

A white blood cell is a particular sort of blood cell that is produced in the bone marrow
and present in both of blood and lymphatic tissue. The immune system of the body includes
white blood cells or known as leukocytes. They support the body's defences against illness and
infection. Granulocytes (neutrophils, eosinophils, and basophils), monocytes, and lymphocytes
are different types of white blood cells (T cells and B cells). A complete blood cell (CBC) test
often includes checking the blood's white blood cell count. It can be used to check for diseases
including leukaemia, allergies, inflammation, infection, and so forth. (Definition of White Blood
Cell - NCI Dictionary of Cancer Terms - NCI, n.d.)
White blood cells (WBC), or leukocytes, are involved in fighting infections and
clearing away dead cells. During infections, their numbers increase dramatically above the
normal range of 4,800 to 10,800 WBC/cubic mm. Finding white blood cells content above
10,800 WBC/cubic mm is suggestive of the infection, or other problems such as
leukaemia. Leukocytes are present without pigment and must be stained to be
counted. Therefore, the diluent contains crystal violet for this purpose are used. It also contains
acetic acid which fixes the white blood cells and causes the lysis of red blood cells, so they are
not visible.

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 EXPERIMENT 3: DETERMINATION OF WHITE BLOOD CELL COUNT USING A HEMACYTOMETER

3.4 REQUIREMENTS
➢ Hemacytometer
➢ 2 coverslips
➢ 1 WBC pipet with hose and mouthpiece
➢ WBC diluting fluid (Turk’s fluid)
➢ Autolet
➢ 2 lancets needless (for drawing blood)
➢ 2 platforms (for drawing blood)
➢ Microscope
➢ 70% of ethanol

3.5 PROCEDURE
All the equipment were set up on a desk to make sure to have everything at fingertips for the
procedure in experiment.
An autolet was prepared with a sterile lance and platform.
1. The side of the tip of a little-used finger was swabbed with 70% Ethanol. (but NOT
closely to the cuticle!)
2. The platform of the autolet was placing against the fingertip to lance it and the trigger
was pressed. Alternatively, a lancet was used with quick, firm jabbed to the side of the
pad of the finger. The first blood was wiped away.
3. By using dilution pipet with the WHITE mixer, the blood was drawn up to the 0.5
mark. Did not allow the blood to congeal in pipette! The next step was proceeded
immediately.
4. The pipet was filled to the mark of 11 with crystal violet diluents.
5. The mixture was shaken well while the tip sealed with a finger by rolling gently.
6. About 1-2 of pipet was emptied into waste container, a small amount of the diluted
blood was added to apply to the first and second chamber of the hemacytometer. It
should flow in to fill the chambers. Did not over fill. If the chamber was overfilled, the
overflow blood would be removed quickly with a paper towel.
7. Let the preparation sat for a minute for cells to settle.
8. The cells were examined under 100x, count the five fields indicated squares of blue-
stained white blood cells with a clicker (fields: top L & R, bottom L & R,

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 EXPERIMENT 3: DETERMINATION OF WHITE BLOOD CELL COUNT USING A HEMACYTOMETER

center). Included in the count all of cells touching left and bottom sides, the cells
touching top and right side were being ignored.
9. THE EQUIPMENT WERE CLEANED UP: The hemacytometers, pipettes and mouth
pieces were washed out thoroughly with soap and water. Then, the equipment would
rinse well and finish it with distilled H2O rinsing, replaced in case.

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 EXPERIMENT 3: DETERMINATION OF WHITE BLOOD CELL COUNT USING A HEMACYTOMETER

3.6 RESULTS
3.6.1 The hemacytometers shown to count White Blood Cells present in several drops of human
blood.

First chamber of the hemacytometer

1 0 0 2 1 1 2 3
1 0 1 3 2 0 2 2
3 1 2 4 1 1 2 2
2 2 0 1 0 3 0 2

0 0 2 2 1 1 3 2
2 0 0 2 3 0 2 2
6 0 3 0 2 2 1 1
3 3 4 1 3 0 1 0

Key;
RED BLOOD CELL

WHITE BLOOD CELL

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 EXPERIMENT 3: DETERMINATION OF WHITE BLOOD CELL COUNT USING A HEMACYTOMETER

Second chamber of the hemacytometer

0 1 1 2 0 0 1 2
6 1 2 2 1 0 3 2
2 2 0 3 3 2 1 5
0 0 1 0 1 2 1 0

2 3 1 0 1 2 0 1
3 1 2 2 2 1 2 1
2 3 0 1 1 3 2 2
1 3 3 0 6 1 1 1

Key;
RED BLOOD CELL

WHITE BLOOD CELL

3.6.2 Calculations of the White Blood Cells

Total counting from first chamber is 23 + 24 + 28 + 24 = 99 of White Blood Cells

Total counting from second chamber is 23 + 24 + 27 + 27 = 101 of White Blood Cells

In order to calculate the total number of WBCs per microliter of a sample, a general formula
used in which to convert the count into the number of cells/microliter;

𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠
𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 𝑝𝑒𝑟 𝑚𝑖𝑐𝑟𝑜𝑙𝑖𝑡𝑒𝑟 𝑣𝑜𝑙𝑢𝑚𝑒 =
𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑠𝑢𝑟𝑓𝑎𝑐𝑒 (𝑚𝑚2 ). 𝑐ℎ𝑎𝑚𝑏𝑒𝑟 𝑑𝑒𝑝𝑡ℎ (𝑚𝑚). 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛

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 EXPERIMENT 3: DETERMINATION OF WHITE BLOOD CELL COUNT USING A HEMACYTOMETER

Given the information is as below;

First chamber of the hemacytometer
Total White Blood Cells = 99 WBC from 64 small squares
So, N = 99
Area counted = 1 𝑚𝑚2 x 4 𝑚𝑚
= 4 𝑚𝑚2
Depth of Chamber = 1/10 𝑚𝑚
Dilution = 1:20
Hence, WBC/𝑚𝑚3 of whole blood is = N x 50
= 99 x 50
= 4950 WBC/𝑚𝑚3

Second chamber of the hemacytometer

Total White Blood Cells = 101 WBC from 64 small squares
So, N = 101
Area counted = 1 𝑚𝑚2 x 4 𝑚𝑚
= 4 𝑚𝑚2
Depth of Chamber = 1/10 𝑚𝑚
Dilution = 1:20
Hence, WBC/𝑚𝑚3 of whole blood is = N x 50
= 101 x 50
= 5050 WBC/𝑚𝑚3

(4950 + 5050 )
Average counting number of White Blood Cells =
2

= 5000 WBC/𝑚𝑚3

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 EXPERIMENT 3: DETERMINATION OF WHITE BLOOD CELL COUNT USING A HEMACYTOMETER

3.7 DISCUSSIONS

The main equipment to make this experiment successfully is by using a Neubauer’s

hemacytometer. This instrument is consisting of a special glass slide. The four corner squares
are divided into 16 equal-sized squares, and each has 1 square millimeter area. The central
square is ruled into 25 groups of 16 small squares, each group is separated by triple lines (DK
Biosolutions, 2019). The ruled surface area is 1/10 millimeter below the inner surface of the
cover glass placed over the middle platform. Thus, each smallest square of the corner has a
volume of 1/4 x 1/4 x 1/10 or 1/160 mm3. Whereas the smallest square in the center has a
volume of 1/20 x 1/20 x 1/10 = 1/4000 mm3 (DK Biosolutions, 2019). This central segment is
used for Red Blood Cells counting. However, in four big squares, leukocytes or White Blood
Cells is counted which are placed at the four corners of the erythrocyte counting chambers.
White Blood Cells diluting fluid or Turk’s fluid consists of Glacial Acetic Acid about 1.5 ml,
for the destruction and dissolution of Red Blood Cells and to fix White Blood Cells, as well as
this fluid is also contained Gentian violet solution about 1.5 ml of 1% solution to stain the
White Blood Cells and mix them well in order to see them clearly for counting in a light
compound microscope.

Fortunately, we can see almost all of the type of leukocytes including monocytes,
neutrophils, basophils and eosinophils. These white blood cells should be appeared to be white
colour due to lack of haemoglobin. However, we can see them in violet colour due to the adding
of Turk’s fluid. Based on the calculations from both chambers of hemacytometer above, the
total number of White Blood Cells of the first chamber is 4950 WBC/mm3 and meanwhile for
the second chamber is 5050 WBC/mm3. From these values, we can get the average total number
of counting of White Blood Cells drew up in this experiment is 5000 WBC/mm3. The average
value of using this method is called as experimentally. However, in theoretically, human
normal range number of White Blood Cells is between 4,500 to 11,000 WBC/microliter (4.5
to 11.0 × 109/L) (WBC Count - San Francisco, 2019). From this statement, we can deduce that
average value of counting in this experiment is acceptable because 5050 WBC/mm3 is within
the normal range number of white blood cells. A count less than 4,500 White Blood Cells per
microliter (4.5 × 109/L) that below normal is called as leukopenia meanwhile of white blood
cells that have more than normal is called as leukocytosis.

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 EXPERIMENT 3: DETERMINATION OF WHITE BLOOD CELL COUNT USING A HEMACYTOMETER

3.8 CONCLUSION

In conclusion, the purpose of counting the White Blood Cells by using hemacytometer
is to know how many White Blood Cells present in a particular human being. This counting
test is crucial as we can detect earlier or can get to know the diagnosis conditions of diseases
infection, allergic reaction, inflammation, blood cancer and more. These cells will circulate
through the bloodstream and tissues to respond to injury or illness by attach to it and attacking
any unknown organisms that enter through the body. (White Blood Cells: What Are They,
Normal Ranges, Role & Function, 2021). Nevertheless, the number of white blood cells is less
than number of red blood cells in our tissue, but it is really important for our immune system
to stay healthy and have a longer life span. In order to take care of our blood, we need to
practicing a good hygiene to prevent infection, taking vitamins to boost the immune system
and regularly do a medical check-up in clinic/hospital. (White Blood Cells: What Are They,
Normal Ranges, Role & Function, 2021)

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 EXPERIMENT 3: DETERMINATION OF WHITE BLOOD CELL COUNT USING A HEMACYTOMETER

3.9 REFERENCES

Definition of white blood cell - NCI Dictionary of Cancer Terms - NCI. (n.d.). National Cancer

Institute. Retrieved November 3, 2022, from

https://www.cancer.gov/publications/dictionaries/cancer-terms/def/white-blood-cell

DK Biosolutions. (2019, March 11). Total Leukocyte Count | TLC Blood Test Procedure | Total WBC

Count Experiment. YouTube. Retrieved November 3, 2022, from

https://www.youtube.com/watch?v=s-Zt9jCZyDQ

WBC count - San Francisco. (2019, January 29). UCSF Health. Retrieved November 3, 2022, from

https://www.ucsfhealth.org/en/medical-tests/wbc-count

White Blood Cells: What Are They, Normal Ranges, Role & Function. (2021, July 23). Cleveland

Clinic. Retrieved November 3, 2022, from https://my.clevelandclinic.org/health/body/21871-

white-blood-cells

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