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BACKGROUND
Hemocytometer is used exclusively for counting the number of cells in a liquid medium. So the objective of this method is to count the number of microorganisms by using hemocytometer.1
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REQUIREMENTS
Materials: Uniform cell suspension
Trypan blue stain
Equipments: Hemocytometer
Cover slips
Tally counter
Micropipettes
Microscope
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PROCEDURE
For getting the perfect cell count it is highly necessary to get a uniform cell suspension with single cells. The cell suspension is made by following trypsin neutralization protocol. Now transfer this cell suspension to conical centrifuge tube. Now thoroughly mix the solution by pipetting for 5-7 times. About 10 microliters of the suspension is required for charging one chamber of hemocytometer. Take another centrifuge tube and transfer 10 microlitres of 0.4% trypan blue to it. Now vortex the cell suspension and add 10 microlitres of cell suspension to trypan blue. Mix it properly by pipetting. Now keeping moistened ,fix the cover slip to hemocytometer. Now very carefully transfer the cell suspension-trypan blue to a single chamber through the age of the cover slip by pipette, so that the suspension reaches all chambers by capillary action. Now observe the cell under microscope at a magnification of 100x.There will be grid of 9 squares. Keep a focus on one of the four outer squares of the grid. There will be 16 smaller squares. Then make a count of the cells in the four 1mm corner squares. If there are many cells or very less cells then the procedure must be repeated for getting perfect results. Keen observation should be made on viable and non-viable cells. If the number of non-viable cells exceeds 25% then culture is not properly maintained.
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CONCLUSION
This is an easy and reliable method for counting microorganisms. The results obtained are more accurate than other methods of counting cells.
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REFERENCES
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KS Louis, AC Siegel – Mammalian Cell. Viability, Springer, 2011.
