manual cell count.pdf

The hemocytometer (or
hemocytometer) is a counting-
chamber device originally designed
and usually used for counting
blood cells. A hemocytometer. The
two semi-reflective rectangles are
the counting chambers.
manual cell count
1

A manual cell count of a blood specimen, also
known as a peripheral blood smear (PBS), is a
microscopic examination of blood stained on a
glass slide. It is used to:
1. Count the different types of white blood
cells (WBCs). This is called a differential
white blood cell count.
2. Examine the size and shape of red blood
cells (RBCs).
3. Look for abnormal cells, such as immature
blood cells or cancer cells.
manual cell count 2

A PBS is a more time-consuming
and labor-intensive test than
automated blood cell counts, but it
can provide more information
about the types and morphology of
blood cells. It is often used in
conjunction with automated blood
cell counts to investigate abnormal
results or to diagnose certain
conditions.
manual cell count 3

Here are the steps involved in performing a
manual cell count of a blood specimen:
1.Prepare a blood smear. A small drop of blood is
placed on a glass slide and spread into a thin
film. The slide is then stained with special dyes to
make the cells easier to see under a microscope.
2.Examine the slide under a microscope. A
trained technician will examine the slide under a
microscope to count the different types of white
blood cells and examine the size and shape of the
red blood cells.
3.Report the results. The technician will report the
results of the cell count to the doctor.
manual cell count 4

While still used in some cases,
manual cell counts are becoming
less common as automated blood
cell counters become more
sophisticated. However, they are
still an important tool for
diagnosing certain conditions.
manual cell count 5

a hemocytometer is the primary
tool used for manual cell counts in
blood specimens. It's essentially a
specialized microscope slide with a
precise, shallow chamber etched
with a grid. This grid allows for
accurate counting of cells
suspended in a known volume of
liquid.
Here's a closer look at
hemocytometers:
Hemocytometer microscope slide
manual cell count 6

manual cell count 8
Components:
•Counting chamber: This is the rectangular
indentation where the cell suspension is loaded. It
has a specific depth and volume (usually 10
microliters).
•Cover slip: This thin glass plate is placed over the
counting chamber to create a sealed chamber and
ensure even distribution of the cell suspension.
•Etched grid: The grid is typically divided into
several smaller squares, with specific areas
designated for counting different types of
cells. For example, the Neubauer improved
hemocytometer, a common type, has a large
central area with 16 squares further divided into
16 smaller squares each.
How it works:
1.Prepare the hemocytometer: Clean the
chamber and coverslip and ensure proper
adhesion.
2.Load the cell suspension: Carefully pipette a
known volume of the diluted cell suspension into
the counting chamber. Capillary action will draw
the liquid in.
3.Count the cells: Using a microscope with the
appropriate objective (usually 10x or
40x), systematically count the cells within the
designated grid areas according to the type of cell
being counted.
4.Calculate the cell concentration: Use the
counted number of cells, the grid area, and the
loaded volume to calculate the cell concentration
per unit volume (e.g., cells/mL).

Components:
•Counting chamber: This is the rectangular indentation where the cell
suspension is loaded. It has a specific depth and volume (usually 10
microliters).
•Cover slip: This thin glass plate is placed over the counting chamber to create
a sealed chamber and ensure even distribution of the cell suspension.
•Etched grid: The grid is typically divided into several smaller squares, with
specific areas designated for counting different types of cells. For example, the
Neubauer improved hemocytometer, a common type, has a large central area
with 16 squares further divided into 16 smaller squares each.
How it works:
1.Prepare the hemocytometer: Clean the chamber and coverslip and ensure
proper adhesion.
2.Load the cell suspension: Carefully pipette a known volume of the diluted
cell suspension into the counting chamber. Capillary action will draw the liquid
in.
3.Count the cells: Using a microscope with the appropriate objective (usually
10x or 40x), systematically count the cells within the designated grid areas
according to the type of cell being counted.
4.Calculate the cell concentration: Use the counted number of cells, the grid
area, and the loaded volume to calculate the cell concentration per unit
volume (e.g., cells/mL).
manual cell count 9

Advantages of using a
hemocytometer:
•Relatively inexpensive and
readily available.
•Provides detailed information on
cell morphology and types.
•Useful for low cell count samples.
Disadvantages of using a
hemocytometer:
•Time-consuming and labor-
intensive.
•Requires skill and experience for
accurate counting.
•More prone to errors compared
to automated methods.
manual cell count 10

Overall, hemocytometers remain
valuable tools for manual cell
counting in various applications,
including blood analysis, cell
culture experiments, and
environmental monitoring.

Using a Hemocytometer in Four Simple
Steps
1. Dilute Your Sample with Trypan blue
Trypan blue is a stain that allows you to
distinguish dead cells from living cells.
When mixed with your cell sample, any
dead cells will be stained blue by the dye,
meaning that you can count only those
cells that are living and viable.
You can dilute your sample with trypan
blue at any ratio, but a 1:1 ratio is the most
common. Whatever dilution you use, make
sure to note it down as you’ll need this for
your final calculation.

2. Loading the Hemocytometer
Before you get started, ensure that both the
hemocytometer and its coverslip are clean by
removing any dust particles with lens paper.
Coverslips used for mounting on hemocytometers
are specially made to be thicker than conventional
microscopy coverslips because they must be able
to overcome the surface tension of a drop of liquid.
Make sure you place the coverslip over the counting
surface before loading the cell suspension. Then
place the pipette tip with your sample into one of
the V-shaped wells, and gently expel the sample.
The area under the coverslip fills by capillary action.
Enough liquid should be introduced so that the
mirrored surface is just covered, usually around 10
µl, but don’t overfill the surface. You can load two
samples on one hemocytometer, one into each of
the two grids.

The loaded hemocytometer
is then placed on the
microscope stage and the
counting grid is brought into
focus at low power. Allow the
sample to settle for a couple
of minutes and avoid
moving the coverslip as it
might introduce air bubbles
and make counting difficult.

. Counting Cells in a Hemocytometer
The full grid on a hemocytometer contains
nine squares, each of which is 1 mm2 The
central counting area of the
hemocytometer contains 25 large squares
and each large square has 16 smaller
squares.
When counting cells that overlap an
exterior line or ruling, count only those cells
on the top or right-hand line of the large
square to avoid counting cells twice.
Suspensions should be dilute enough so
that the cells or other particles do not
overlap each other on the grid, and should
be uniformly distributed.

To perform the count,
determine the magnification
needed to recognize the
desired cell type and
systematically count the
cells in selected squares so
that the total count is
approximately 100 cells, a
minimum number of cells
needed for a statistically
significant count.

For large cells, you can simply count the
cells inside the four large corner squares
and the middle square For a dense
suspension of small cells, you may wish
to count the cells in the four outer and
middle squares of the central square or
make a more dilute suspension.
Remember if a cell overlaps a line, count
it as “in” if it overlaps the top or right-
hand line and “out” if it overlaps the
bottom or left-hand line

The area of the middle and each corner
square is 1 mm x 1 mm = 1 mm2. The depth
of each square is 0.1 mm. Hence, the final
volume of each square at that depth is
100 nl.

Calculating Cell Concentration
You can calculate your cell concentration
using the following formula:
Total cells/ml = (Total cells counted x
Dilution factor x 10,000 cells/ml)/
Number of squares counted
So, for example, if you diluted your sample
1:1 with Trypan blue (dilution factor is 2 in
this case), and you counted 325 cells in
the four corner squares plus the central
big square (number of squares counted is
5), then:
Total cells/ml = (325 cells x 2 x 10,000
cells/ml)/ 5 = 130 x 104 cells/ml

If you want to know how
many cells you have in your
original sample, just multiply
the cell concentration by the
total sample volume. For
example, if your original
sample volume is 5 ml, then:
Total cells in sample = 130 x
104 cells/ml x 5 ml = 650 x
104 cells

manual cell count.pdf

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manual cell count.pdf