Hemacytometer Cell Counting Procedure
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GENERAL PRINCIPLE :
The count of cells per volume unit is
quite useful as diagnostic tool.. The
only realistic alternative will be to
use a counting chamber in which the
cells are counted under the
microscope .
Depending on the kind of liquid to be
analysed (expected cell number), a dilution
and/or destruction of undesirable cells must
be done. A simple calculation taking in
accounting and it will give the number of
cells per
mm³
.
Ø Hemacytometer {the counting chamber}.Ø Cover glass.
Ø RBCs pipette with hose and Mouthpiece
{Thoma red count pipette}
.
Ø Lancet needless (for drawing blood).
Ø Platforms (for drawing blood).
Ø Microscope.
Ø 70% ethanol.
Ø Bottle ringer's solution(a clear diluent for
RBCs).
Ø Absorbent paper.
Set up all equipment on your desk so that you
are sure to have everything at
1
. Swab towards the side of the tip of a little-used
finger with 70% Ethanol.
2
. Lance by placing the platform of the auto let
against the fingertip and pressing the trigger.
3
. Using the dilution pipette with RED mixer
from hemacytometer kit, draw blood up to
the 0.5 mark (Do not allow air to be drawn
into the pipette or it will not draw the correct
volume of blood,Do not allow blood to
congeal in pipette Immediately proceed to the
next step:
4
. Continuing to hold the pipette as horizontal
as possible,draw
Ringer's solution
diluent
up to the 101 mark. (Dilution of 1 to 200.)
5
. Seal the tip with your finger and shake well
to mix.
6
. Empty ~1/2 of pipette into waste container
add a small amount of the diluted blood
to one chamber of the hemacytometer to just
fill the chamber of the hemacytometer.
(Do not over fill).
7
. Let the preparation sit for a minute (for cells to
settle).
8
. Center the grid at 100x,switch to 400x and count
and record the RBCs in each of five fields(each with
16 smallest squares) .Include in the count all cells
touching left and bottom sides,ignore cells
touching top and right sides.Calculate the RBCs/
mm
3
by adding the cells in the 5 groups and
multiplying by
10,000
(i.e., add four zeros).
RBCs/mm
3
.
9
. Wash out the pipette thoroughly with soap
and water, rinse well, finish with distilled H
2
O .
Routinely, blood is drawn to the 0.5 mark
and diluted to the 101 mark with RBC
diluting fluid. All the blood is washed
into the bulb of the pipette (which has a
volume of 100). Therefore, 0.5 volumes of
blood are contained in 100 volumes of
diluting fluid. The resulting dilution is
1:200.
N
RBCs/mm
3
=--------- x 400 x 200 x 10
80
N = numbers of RBCs counted in {80 smallsquares} .
80 = ( 5 x 16 ) small squares.
400 = ( 16 x 25 ) total squares.
200 = Dilution factor .
10 = depth of the chamber .
The hemacytometer cell counting method is a valuable diagnostic tool for determining the number of cells in a given volume of fluid. This process involves using a counting chamber under a microscope to calculate the cells per unit volume. By following a systematic approach, including proper dilution and preparation techniques, accurate cell counts can be obtained for diagnostic purposes. The detailed steps outlined in the procedure ensure precision and reliability in cell counting for various applications.
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Presentation Transcript
GENERAL PRINCIPLE : The count of cells per volume unit is quite useful as diagnostic tool.. The only realistic alternative will be to use a counting chamber in which the cells are counted under the microscope . GENERAL PRINCIPLE :
Depending on the kind of liquid to be analysed (expected cell number), a dilution and/or destruction of undesirable cells must be done. A simple calculation taking in accounting and it will give the number of cells per mm mm .
Hemacytometer {the counting chamber}. Cover glass. RBCs pipette with hose and Mouthpiece {Thoma red count pipette}. Lancet needless (for drawing blood). Platforms (for drawing blood). Microscope. 70% ethanol. Bottle ringer's solution(a clear diluent for RBCs). Absorbent paper.
Set up all equipment on your desk so that you are sure to have everything at 1 1. Swab towards the side of the tip of a little-used finger with 70% Ethanol. 2 2. Lance by placing the platform of the auto let against the fingertip and pressing the trigger. 3 3. Using the dilution pipette with RED mixer from hemacytometer kit, draw blood up to the 0.5 mark (Do not allow air to be drawn into the pipette or it will not draw the correct volume of blood,Do not allow blood to congeal in pipette Immediately proceed to the next step:
4 4. Continuing to hold the pipette as horizontal as possible,draw Ringer's up to the 101 mark. (Dilution of 1 to 200.) 5 5. Seal the tip with your finger and shake well to mix. 6 6. Empty ~1/2 of pipette into waste container add a small amount of the diluted blood to one chamber of the hemacytometer to just fill the chamber of the hemacytometer. (Do not over fill). Ringer's solution solution diluent
7 7. Let the preparation sit for a minute (for cells to settle). 8 8. Center the grid at 100x,switch to 400x and count and record the RBCs in each of five fields(each with 16 smallest squares) .Include in the count all cells touching left and bottom sides,ignore cells touching top and right sides.Calculate the RBCs/ mm3by adding the cells in the 5 groups and multiplying by 10,000 RBCs/mm3. 9 9. Wash out the pipette thoroughly with soap and water, rinse well, finish with distilled H2O . 10,000 (i.e., add four zeros). (i.e., add four zeros).
Routinely, blood is drawn to the 0.5 mark and diluted to the 101 mark with RBC diluting fluid. All the blood is washed into the bulb of the pipette (which has a volume of 100). Therefore, 0.5 volumes of blood are contained in 100 volumes of diluting fluid. The resulting dilution is 1:200.
N N RBCs/mm RBCs/mm3 --------- x 400 x 200 x 10 80 N = numbers of RBCs counted in {80 small squares 80 = ( 5 x 16 ) small squares. 400 = ( 16 x 25 ) total squares. 200 = Dilution factor . 10 = depth of the chamber 3 = =--------- x 400 x 200 x 10 80 N = squares} . 80 = ( 5 x 16 ) small squares. 400 = ( 16 x 25 ) total squares. 200 = Dilution factor . 10 = depth of the chamber . . numbers of RBCs counted in {80 small } .
