an instrument for visual counting of the number of cells in a blood sample or other fluid under a microscope.

1. Haemocytometer
Vinitha Unnikrishnan
D3 biotech

2. 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 by Louis-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.

3. The two semi-
reflective
rectangles are the
counting
chambers.
Load a chamber

4. The parts of the hemocytometer (as viewed
from the side)

5. Appearance of the haemocytometer grid visualized under the
microscope.

6. Hemocytometer grid

7. RBC use 5 small squares in the center large square
WBC , sperm cells, culture cells use 4 corner large
squares

8. *** 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) Neubauer’s slide
b) Cover slip
c) RBC pipette
d) WBC pipette

9. NEUBAUER’S 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

10. PRINCIPLE OF HAEMOCYTOMETER
• The gridded area of the hemocytometer
consists of nine 1 x 1 mm (1 mm2) squares.
These are subdivided in 3 directions; 0.25 x
0.25 mm (0.0625 mm2), 0.25 x 0.20 mm
(0.05 mm2) and 0.20 x 0.20 mm (0.04 mm2).
The central square is further subdivided into
0.05 x 0.05 mm (0.0025 mm2) squares. The
raised edges of the hemocytometer hold the
cover slip 0.1 mm off the marked grid, giving
each square a defined volume.

12. Dimensions Area Volume at 0.1 mm depth
1 x 1 mm 1 mm2 100 nL
0.25 x 0.25 mm 0.0625 mm2 6.25 nL
0.25 x 0.20 mm 0.05 mm2 5 nL
0.20 x 0.20 mm 0.04 mm2 4 nL
0.05 x 0.05 mm 0.0025 mm2 0.25 nL

13. 1.Wet the raised glass rails with the tip of a moistened
finger
Setting up the Haemocytometer:

14. 2.Carefully slide the cover slip over the raised glass rails

15. 3.Draw up 10µL and deliver into the gap between the
cover slip and the countering chamber
4.Observed in microscope

16. 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

17. • 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.

18. 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 µL of cells into a new Eppendorf tube
and add 400 µL 0.4% Trypan Blue (final
concentration 0.08%). Mix gently.
•

19. 3.Counting
• Using a pipette, take 100 µL of Trypan Blue-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 4 sets of 16 corners are counted.

20. 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/mL in original cell suspension

21. 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
• 2,337,500 ÷2,387,500 = 97.9% viability

22. Close up view of a grid with cells

23. Counting system to ensure accuracy
and consistency

24. 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

25. 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.

26. 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 you’re
within the levels established for a healthy person.

27. 2.To perform sperm counts: the concentration
of sperm in semen is important in order to
assess the male’s 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.

28. 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.

29. 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 hemocytometer’s 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