The process of counting cell using Neubauer’s Slide

2.  What is hemocytometer?
 Uses of hemocytometer?
 Principle of cell counting using Neubauer's
slides
 Common sources of error during the process of
Hemocytometery

3.  Hemo: Blood
 Cyto: Cell
 Meter: Measurement/Counting
Thus , it is a device originally designed and
usually used for counting blood cells.

5.  Thick glass slides with two ruling areas on
center
 Ruling areas are separated by H-shaped
trough.
 Beyond trough, there are two raised arms to
support cover glass
 lining is coated by shiny metal or rhodium.
 Neubauer’s slide with a cover slip over it is
called Neubauer’s chamber.

8.  Each scale is 3mm wide and 3mm long.
 Depth of the chamber is 0.1mm.
 The whole scale is divided into 9 big
squares.
 Each square is 1mm long and 1mm wide.

11.  The four corner squares are further divided
into 16 smaller squares and are used for
WBC counting.
 Total 64 small squares are there which have
a dimension of
0.25mm x 0.25mm = 0.0625mm

12.  Central square is divided into 25 medium
sized square and are separated by triple
line
 The medium sized square are further
divided into 16 small square(tiny)
 The four corner and central square are
used for cell count

15.  Special cover glass with smooth surface
and even thickness
› Thickness= 0.3, 0.4, 0.5 mm
› Length= 16x22mm, 22x23mm

17.  Draw the cell suspension into pipette up to
100 μL
 Draw 100 μL of Trypan blue (to differentiate live and
dead cells)
 Load both chambers of the Neubauer's slide
by pipetting the suspension under the cover
slip.

19.  Now place the Neubauer’s chamber under
the microscope.
 Focus must be
› 4X to see the general formation of slides
› 10X for WBC counting
› 40X for RBC/ platelets counting

24.  Count the cells in
› 4 corner squares
› The center square

26.  Do not count cells
touching
› Bottom line
› Right line

27. count
Don’t
Count

29.  Count cells in the five selected chambers of
cell count.

31. 1. Average # of cell per square =
Total number of cells / Total squares
2. Dilution factor =
final volume/ volume of cell
3. Concentration of cells =
average # of cell / dilution factor

33.  Blood count:
› for patients with abnormal blood cells, where
automated counters don't perform well.
 Sperm count:

34.  Cell culture:
› when subculturing or recording cell growth over
time.

35.  Errors in this process are of two main types
› False high count
› False low count

36. › Uneven distribution of cell
 Improper mixing
 Error in pipetting
› Error in calculation
› Blood taken from area of hemo
concentration
› Yeast, dirt and leukocyte are
counted as RBC

37. › Blood diluted with tissue fluid
› Undue delay in counting of cell
› Clumping of cell(AIHA)
› Uneven distribution of cell
› Faulty technique of counting
› Improperly standardized counting chamber