IISc UG Programmer: Bio 102
Semester II (2025)
Cell Biology: Lab 2
Haemocytometer: A tool to make cell counting easy
Finding out the number of cells in a given volume of a sample is useful for many reasons. A
common example is urine analysis, where a pathologist checks the numbers of cells (e.g. red
and white blood cells, puss and epithelial cells, yeast or bacterial cells) in a certain volume of
your urine to see if they fall within a normal range. Microbiologists count cells/ microbes to
understand their growth patterns. For a molecular biologist or a biochemist, knowing the
number of cells helps in determining concentrations or volumes of chemicals used for certain
experiments. Sometimes, it becomes important to gauge the proportion of dead to viable cells
in a sample.
While several techniques are available to estimate the cell density, haemocytometer is one of
the most commonly used devices. It was invented by Louis-Charles Malassez and was
originally used to count blood cells.
The haemocytometer is divided into nine large squares. Each square has a surface area of
1mm2 and the depth of the chamber is 0.1mm. Based on the numbers in these squares, the
subsequent cell concentration per ml can be determined.
There are many stains that help to distinguish between viable and non-viable cells. Live cells
have intact membranes, enabling them to eliminate certain stains like Trypan blue (or
Erythrosine B) and appear colourless. On the other hand, dead cells, with their damaged
membranes take up the stain and appear blue in colour.
Materials Required: Haemocytometer, Cover slips, 70% Ethanol, Uniform cell suspension
( HeLa ), phosphate buffered saline (PBS), Micropipette and tips, Compound microscope.
Procedure:
A. Preparing haemocytometer
Clean the mirror-like polished surface of the haemocytometer and the cover slip
carefully with tissue paper and ethanol.
Place the cover slip on the counting surface carefully.
B. Preparation of Cell suspension & Counting cells
1. Grow cells to 70-80 % confluence in a T25 cell culture flask
2. Remove the growth medium completely
3. Wash/ rinse the flask with warm PBS and then remove PBS completely
4. Add warm 1 ml of trypsin/EDTA into the flask and make sure that trypsin/EDTA
solution covers the cell layer and spread evenly in the flask
5. Transfer the flask to an incubator (set at 370 C) and incubate for 2 minutes
6. Take out the flask from the incubator and tap the side of the flask, and examine the
flask under a microscope for cell lifting. If necessary, return the cells to the incubator
for an additional 2 minutes, with occasional tapping, until lifting is complete
7. Quickly quench the Trypsin reaction by adding 2 ml Complete Cell Culture Medium
(Bovine fetal serum which is a component of cell culture medium has a strong trypsin
inhibitor)
8. Transfer 3 ml of cell suspension to a 50 ml conical tube and take 50 ul of cell
suspension by using a 200 ul pipette and place in a hemocytometer for counting
9. Count the cells from four corners and determine cells per ml (average count x 104)
10. Seed 0.1 x cells/ml in PBS in a well of cell culture 6 well plate
3. Counting cells
Take 100ul of cell suspension into a 1.5 ml microfuge tube and add 100ul of trypan
blue solution. Mix the cell suspension with trpan blue by pipetting up & down gently
With the help of a pipette, transfer 10µl of the cell suspension to a chamber on
the haemocytometer.
Allow the chamber to fill by capillary action. Ensure that the chamber is neither over-
filled nor under-filled.
Place the haemocytometer on the microscope stage and focus on the counting grid.
Count all the cells in the 1mm corner square. Repeat the same for other 3 corner
squares. Dead cells take up trypan blue and appear blue. On the other hand, live cells
exclude trypan blue and appear normal. Register the live and dead cell count
separately. If cells are touching the 4 perimeter sides of a corner square, only count
cells on 2 sides, either the 2 outer sides or 2 inner sides as shown below
If the cells are too few (<10) or too many (>100) repeat the protocol by either concentrating
or diluting the original cell suspension with PBS
4. Calculations:
Viable Cell count = No. of live cells counted x Dilution factor x 104
No. of large corner squares counted
Non-Viable Cell count = No. of dead cells counted x Dilution factor x 104
No. of large corner squares counted
Cell Viability = No. of live cells x 100
Total No. of cells
Precautions:
Observe the grids first under 10X before loading the sample.
Clean to hemocytometer and coverslip (else dust particles will hinder counts)
Make a uniform cell suspension. Mix the cell suspension before taking 10-15 L
Ensure there are no clumps of cells
Don’t incubate the cells in trpan blue for long. If incubated long, live cells also
take up trypan blue
Follow a counting convention/ pattern of counting to minimise errors.
