Enumeration of Red Blood Cells using Haemocytometer.

1. ENUMERATION OF RED
BLOOD CELLS
VIT UNIVERSITY
16BBT0058
NIKITA SREENATH
BIT1006

2. RED BLOOD CELL COUNT
• A red blood cell count is typically ordered as
part of a complete blood count(CBC).
• RBCs circulate in the blood and carry oxygen
throughout the body.
• A red blood cell count is used to help diagnose
and monitor a number of diseases like
anaemia, kidney disease, etc.

4. HAEMOCYTOMETER
• Haemocytometer is a counting chamber
recommended for cell counting.
• The haemocytometer was invented by Louis-
Charles Malassez.
• It consists of a thick glass microscope
slide with a rectangular indentation that
creates a chamber.
• This chamber is engraved with a laser-
etched grid of perpendicular lines.

7. • The device is carefully crafted so that the area
bounded by the lines is known, and the depth of
the chamber is also known.
• The gridded area of the haemocytometer 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 haemocytometer hold the coverslip
0.1 mm off the marked grid, giving each square a
defined volume.

9. USING THE HAEMOCYTOMETER
• The special coverslip provided with the counting chamber is
properly positioned on the surface of the counting
chamber. When the two glass surfaces are in proper
contact Newton's rings can be observed.
• The cell suspension is applied to the edge of the coverslip
to be sucked into the void by capillary action which
completely fills the chamber with the sample.
• The number of cells in the chamber can be determined by
direct counting using a microscope, and visually
distinguishable cells can be differentially counted. The
number of cells in the chamber is used to calculate
the concentration or density of the cells in the mixture the
sample comes from.

13. CONCENTRATION CALCULATION
• We apply the formula for the calculation of the
concentration The number of cells will be the sum of all the
counted cells in all squares counted. The volume will be the
total volume of all the squares counted.
• Since the volume of 1 big square is: 0,1 cm x 0,1 cm = 0,01
cm2 of area counted. Since the depth of the chamber is
0.1mm, 0.1 mm = 0.01 cm, 0.01 cm*0.01 cm = 0.0001 cm2 =
0.0001ml = 0.1 µl
• In case a dilution was applied, the concentration obtained
should be converted to the original concentration before
the dilution. In this case, the concentration should be
divided by the dilution applied.

16. APPLICATIONS
• Blood counts: for patients with abnormal blood cells, where
automated counters don't perform well.
• Cell culture: when subculturing or recording cell
growth over time.
• Beer brewing: for the preparation of the yeast.
• Cell processing for downstream analysis: accurate cell
numbers are needed in many tests (PCR, flow cytometry),
while some others require high cell viability.
• Measurement of cell size: in a micrograph, the real cell size
can be inferred by scaling it to the width of a
haemocytometer square, which is known.

17. REFERENCES
• http://celeromics.com/en/resources/docs/Arti
cles/Cell-counting-Neubauer-chamber.pdf
• https://en.wikipedia.org/wiki/Hemocytometer

18. THANK YOU