Counting cells using a haemocytometer

Kate Andrews
Lorraine Bruce
Cells and Proteins (Advanced Higher)
(f) Aseptic technique and cell culture
Use of inoculum, explants or cells. Use of:
haemocytometers to estimate total cell
counts; vital staining to estimate viable cell
counts. Complex media containing growth
factors from serum for animal cell lines.
Lifetime of primary cell lines and cancer
cell lines in culture. Use of growth
regulators in plant tissue culture.

Sterilisation of containers, equipment and

materials. Disinfection of working area.
Culture bacterial, yeast and algal cells
using aseptic technique.
Use a haemocytometer to make an
estimate of cell count.
Yeast cells x100
Saccharomyces cerevisiae as seen using an electron microscope ~ x 500,000

Image: Society for General

Microbiology
Blood counts: for patients with abnormal blood cells, where automated counters
don't perform well.

Sperm counts

Cell culture: when subculturing or recording cell population growth over time.

Beer brewing: for the preparation of the yeast cultures.

Cell processing for downstream analysis: accurate cell numbers are needed in
many tests (PCR, flow cytometry) while some others require high cell viability.

Measurement of cell size: in a micrograph, the real cell size can be inferred by
scaling it to the width of a haemocytometer square, which is known.
 www.sserc.org.uk

Home / Biology Home / Resources / Microbiological Techniques /

Enumerating micro-organisms / Counting cells using a haemocytometer
See SSERC website:

http://www.sserc.org.uk/index.php/biol
ogy-2/biology-resources/microbiological-
techniques265/enumerating-micro-
organisms141
C-Chip Haemocytometer

•Cheaper!

•Disposable

•No need for coverslips!

•Virtually non-breakable!

•Reusable (within limitations)

Image: www.invitrogen.com

Image :
https://www.labtech.com/disposable-haemocytometers
https://www.labtech.com/disposable-haemocytometers
 1mm

0.2mm

The grid has 25 squares, we will sample 5 squares

C-Chip grid view x40
C-Chip grid view x100
 Boundary
lines

Top left square x400

Bottom right square x400
Loading the haemocytometer
Yeast cells in grid x100
Yeast cells in one square of the grid x400
Systematic counting....
What should be counted?
 Volume in sample area (5 squares)

Length of side of grid = 1 mm

Area of grid = 1 mm2

Depth between coverslip

and slide = 0.1 mm

Volume under grid = 1 mm2 x 0.1 mm

(25 squares)
= 0.1 mm3

Volume under 5 squares = 0.1 / 5 mm3

= 0.02 mm3
Estimating cell concentration (in 5 squares)

Number of cells in sample = n

Number of cells in 1 mm3 = n x 50 (because 0.02 mm3 is 1/50 of 1 mm3)

Number of cells in 1 cm3 = (n x 50) x 1000

If the concentration of cells is very low, all the cells in the 25 square grid
could be counted.
Volume in grid (25 squares)

Length of side of grid = 1 mm

Area of grid = 1 mm2

Depth between coverslip

and slide = 0.1 mm

Volume under grid = 1 mm2 x 0.1 mm

(25 squares)
= 0.1 mm3
  For counting large cells, some protocols
advocate counting the cells in the ‘big’ squares...

 Some protocols suggest averaging the number of cells counted prior to

doing the calculation of cell concentration
 In some situations cell viability is also taken into consideration –

 Trypan blue

 Methylene blue

Yeast cells (x 400) stained with

methylene blue

Live cells actively pump out the dye. Image: http://braukaiser.com

Non-viable cells are dark blue.
 Cell counting apps iPhone HemocyTap
£1.49

https://itunes.apple.com/us/app/hemocytap/id617767138?mt=8&ign-mpt=uo%3D4
 Counting cells
National 4
Cell Biology Properties include rapid growth,
5. Properties of diverse use of food sources
microorganisms and use in
industries
Life on Earth Nitrogen in fresh water
4. Fertiliser design and impact increases algal growth, blocking
of fertilisers out light.
Investigate the effects of
fertilisers e.g. algal blooms
 Counting cells
National 5
Cell Biology Appropriate growth media include
3. Producing new cells various nutrient broths and agars
Carry out numeracy activities
based on cell growth
graphs/curves
Life on Earth Fertilisers supply nitrates to
2c. Nitrogen in ecosystems increase yield
5. Impact on the environment Fertilisers can leach into fresh
water, increasing algal blooms.
This leads to a reduction in
oxygen levels
 Counting cells
Higher Biology
Metabolism and survival
3. Metabolism in
microorganisms –
microorganisms to include
archaea, bacteria and some
species of eukaryota
(a)Environmental control of
metabolism
Phases of growth and doubling or
generation time of exponential
growth and changes in culture
conditions
Interpretation of exponential
growth on normal and semi-
logarithmic scales.