Haemocytometer

Vinitha Unnikrishnan
D3
biotech
 Haemocytometer or
Counting chamber
The hemocytometer is a specimen slide which is used to
determine the concentration of cells in a liquid sample.
The hemocytometer was invented byLouis-Charles Malassez
It is frequently used to determine the concentration of blood
cells (hence the name hemo-) but also the concentration of
other cells in a sample.
The cover glass, which is placed on the sample, does not
simply float on the liquid, but is held in place at a specified
height (usually 0.1mm).
Additionally, a grid is etched into the glass of the
hemocytometer. This grid, an arrangement of squares of
different sizes, allows for an easy counting of cells.
This way it is possible to determine the number of cells in a
specified volume.
The two semi-
reflective
rectangles are
the counting
chambers.

Load a chamber
The parts of the hemocytometer (as
viewed from the side)
Appearance of the haemocytometer grid visualized under the
microscope.
Hemocytometer grid
RBC use 5 small squares in the center large
square
WBC , sperm cells, culture cells use 4 corner
large squares
 *** Hemacytometers are used when

1- automated cell counters and hematology

analyzers are unavailable
2- blood cell counts are extremely low
3- to get a cell count for body fluids (spinal fluid,
joint
fluid, semen counts, and other bodily fluids.
The most commonly used hemacytometer is the
Neubauer chamber.

It includes:
a) Neubauers slide
b) Cover slip
c) RBC pipette
d) WBC pipette
 NEUBAUERS SLIDE

It is the name given to a thick glass slide.

In the centre of the slide, there is an
H- shaped groove. On the two sides of
the central horizontal bar, there are
scales for counting the blood cells.
The depth of the scales is 1/10mm or
0.1mm. Each scale is 3mm wide and 3mm
long.
The whole scale is divided into 9 big squares.
Each square is 1mm
 PRINCIPLE OF
HAEMOCYTOMETER
The gridded area of the hemocytometer
consists of nine 1 x 1mm (1mm2) squares.
These are subdivided in 3 directions; 0.25 x
0.25mm (0.0625mm2), 0.25 x 0.20mm
(0.05mm2) and 0.20 x 0.20mm (0.04mm2).
The central square is further subdivided into
0.05 x 0.05mm (0.0025mm2) squares. The
raised edges of the hemocytometer hold the
cover slip 0.1mm off the marked grid,
giving each square a defined volume.
 Volume at 0.1mm
Dimensions Area
depth

1 x 1mm 1mm2 100nL

0.25 x 0.25mm 0.0625mm2 6.25 nL

0.25 x 0.20mm 0.05mm2 5nL

0.20 x 0.20mm 0.04mm2 4nL

0.05 x 0.05mm 0.0025mm2 0.25nL

 Setting up the
Haemocytometer:
1.Wet the raised glass rails with the tip of a
moistened finger
2.Carefully slide the cover slip over the raised glass rails
3.Draw up 10L and deliver into the gap between the cover slip and the countering chamber

4.Observed in microscope
 Equipment & Reagents

Haemocytometer plus a supply of

cover-slips
- 0.4% Trypan Blue stain (fresh &
filtered) in phosphate buffered saline
- Tally Counter
- Cell Suspension
- pipettes
- microscope
 procedure explaining how to
obtain a viable cell count from
a hemocytometer.
1.Preparing hemocytometer
If using a glass hemocytometer and coverslip, clean with
alcohol before use. Moisten the cover slip with water and
affix to the hemocytometer. The presence of Newton's
refraction rings under the cover slip indicates proper
adhesion.
Newton's Rings" which indicate that the cover slip has
adhered via suction to the haemocytometer. Newton's
refraction rings are seen as rainbow-like rings under the
cover-slip.

If using a disposable hemocytometer (for example,

INCYTO DHC-N01), simply remove from the packet before
use.
 2.Preparing cell suspension

Gently swirl the flask to ensure the cells are

evenly distributed.
Before the cells have a chance to settle, take
out 0.5 mL of cell suspension using a 5 mL
sterile pipette and place in an Eppendorf tube.
Take 100 L of cells into a new Eppendorf tube
and add 400 L 0.4% Trypan Blue (final
concentration 0.08%). Mix gently.
3.Counting

Using a pipette, take 100 L of TrypanBlue-treated cell suspension and

apply to the hemocytometer. If using a glass hemocytometer, very
gently fill both chambers underneath the coverslip, allowing the cell
suspension to be drawn out by capillary action. If using a disposable
hemocytometer, pipette the cell suspension into the well of the
counting chamber, allowing capillary action to draw it inside.
Using a microscope, focus on the grid lines of the hemocytometer with
a 10X objective.
Using a hand tally counter, count the live, unstained cells (live cells do
not take up Trypan Blue) in one set of 16 squares (Figure 1). When
counting, employ a system whereby cells are only counted when they
are set within a square or on the right-hand or bottom boundary line.
Following the same guidelines, dead cells stained with Trypan Blue can
also be counted for a viability estimate if required.
Move the hemocytometer to the next set of 16 corner squares and carry
on counting until all 4sets of 16 corners are counted.
 4.Viability

To calculate the number of viable

cells/mL:
Take the average cell count from each of the sets of 16 corner
squares.
Multiply by 10,000 (104).
Multiply by 5 to correct for the 1:5 dilution from the Trypan Blue
addition.

The final value is the number of viable cells/mL in the original cell
suspension.
Example:
If the cell counts for each of the 16 squares were 50, 40, 45, 52, the
average cell count would be:
(50 + 40 + 45 +52) 4 = 46.75
46.75 x 10,000 (104) = 467,500
467,500 x 5 = 2,337,500 live cells/mLin original cell suspension
 5.To calculate viability:

If both live and dead cell counts have been recorded for
each set of 16 corner squares, an estimate viability can be
calculated.
Add together the live and dead cell count to obtain a total
cell count.
Divide the live cell count by the total cell count to calculate
the percentage viability.

Example:
Live cell count: 2,337,500 cells/mL
Dead cell count: 50,000 cells/mL
2,337,500 + 50,000 = 2,387,500 cells
2,337,5002,387,500 = 97.9% viability
Close up view of a grid with cells
Counting system to ensure accuracy
and consistency
Advantages over hemacytometer cell counting:

Quick and simple takes 1 minute

No time consuming sample dilutions
No tedious counting at the
microscope
Accurate not affected by cell
clumping
Count multiple samples at once
Disadvantages of using this process:

Dead cells are not identified from the lives.

Small cells are difficult to locate and even
impossible to mention.
Precision is tough to achieve.
If the samples are not stained then it is
required to use a phase-contrast
microscope.
Motile cells must be immobilized before
counting.
 APPLICATIONS
1.To perform blood counts: blood is a fluid that
naturally carries cells throughout the human (or
animal) body. In turn, blood is a mix of different
types of cells that carry oxygen or fight infection,
among others. They are distinguishable to the
experienced eye by their shape and size. So, by
counting separately all the cell types visible in the
hemocytometer and calculating their concentration,
we can not only get the cell numbers in the whole
body, but also the percentage of each of them. This
is very valuable for doctors to know if youre within
the levels established for a healthy person.
2.To perform sperm counts: the
concentration of sperm in semen is
important in order to assess the
males fertility. For humans, values
above 15 million per milliliter are
normal. Because sperm cells are
moving cells, they need to be
immobilized prior to counting. There
are also special hemocytometers
that are used for sperm, due to the
cells smaller size: Makler or MTG
hemocytometers.
3.To process cells for culture: when
culturing cells in the lab, the medium
that contains the nutrients needs to
be renewed once in a while. Cells can
be counted as long as they have
been put in solution. This includes
adherent cells for cell culture, or
suspension cells if they originally
come from blood, but also bacteria
and yeast. A popular example is in
the preparation of yeast for the
fermentation of beer.
4.To process cells for downstream
analysis: accurate cell numbers are
needed in many tests for the
quantification of proteins or DNA (PCR,
flow cytometry), while some others
require high viabilities for them to be
valid.
5.To determine the size of a cell:
because the size of the
hemocytometers squares is known. In
a micrograph, the real cell size can be
inferred by scaling it to the width of a
hemocytometer square, which is known