The object of the experiment is to determine the concentration of cells in a given sample.
A liquid sample containing immobilized cells when placed on the chamber, and is covered
with cover glass, capillary action completely fills the chamber with the sample. Looking
at the chamber through a microscope, the number of cells in it can be determined by counting.
Different kinds of cells can be counted separately as long as they are visually distinguishable. 0.4% Trypan blue stains.
Uniform cell suspension Fresh vial
Micropipette Pipette tips
Vial Rack. Arrange all materials inside LAF. PROCEDURE (Inside the LAF)
Set the micropipette scale to 10µl Insert a new pipette tip to the micropipette.
Take the vial containing uniform cell suspension, open it, and mix it two to three times with
the micropipette. Now take 10 µl of the suspension with the
micropipette. Take the fresh vial
Transfer the culture to the fresh vial with the micropipette.
Close the vial and keep the same in the vial rack.
Dispose the used pipette tip. Insert a new pipette tip to the micropipette.
Now take vial containing Trypan blue dye, open it and mix 2-3 times with the micropipette.
Now take 10 µl of Trypan blue solution. Now take the vial containing 10 µl cell suspension
and transfer the dye solution . With the micropipette mix the trypan blue-cell
suspension and keep the vials for 5- 15 min. Materials required for the Outside LAF
Haemocytometer plus a supply of cover-slip Spirit
Tissue paper Micropipette
Pipette tip box Glass beaker with water PROCEDURE:
Take the box containing hemocytometer, open it
Take the hemocytometer and place it on a white paper.
Take a tissue paper. Now take spirit.
Apply a small quantity on tissue paper Now with the tissue paper wipe the surface
of Hemocytometer. Take a micropipette.
Take the appropriate tip for the micropipette. Take a small quantity of water using the micropipette.
Place few drops on the surface of the Hemocytometer. Take the cover slip and clean it with tissue
paper sprayed with spirit. Now place the cover slip on the Hemocytometer.
Make sure that it does not Slide off. PROCEDURE:(Inside the LAF) Take the Hemocytometer and place it inside
LAF Set the scale of micropipette to 10 µl.
Insert a new micropipette tip to the micropipette. Now take the vial containing Trypan blue-
cell suspension and open it. Mix the suspension with micropipette.
Take 10 µl of the cell suspension . Now slowly transfer the cell suspension to
the Hemocytometer by carefully touching the edge of the cover slip with the pipette tip
and allowing the chamber to fill by capillary action. Do not overfill or under fill the
chambers. Now Observe under phase contrast microscope
at 10X View the cells under a microscope at 100x
magnification. Under the microscope, you should see a grid
of 9 squares. Focus the microscope on one of the 4 outer
squares in the grid. The square should contain 16 smaller squares.
Count all the cells in the four 1 mm corner squares.
Include cells on top and left touching middle line. Do not count cells touching middle line
at bottom and right. If there are too many or too few cells to
count, repeat the procedure either by concentrating or diluting the original suspension as appropriate.
For an accurate determination, the total number of cells overlying 1 mm2 should be between
15 and 50. Live cells appear colourless and bright (refractile)
under phase contrast. while the dead cells stain blue and are non-refractile.
Keep a separate count of viable and non-viable cells. Calculations %Cell Viability=Total Viable cells(Unstained)/Total
cells (Viable + Dead) X 100 Viable Cells/ml=Average viable cell count
per square x Dilution Factor x 104 Average viable cell count per square=Total
number of viable cells in 4 squares /4 Dilution Factor=Total Volume (Volume of
sample + Volume of diluting liquid) / Volume of sample
Total Viable cells/Sample=Viable Cells/ml x The original volume of fluid from which
the cell sample was removed Volume of media needed=Number of cells needed/Total
number of viable cells x 1000