Tissue Culture

LAB # 9

TISSUE CULTURE
CELL COUNTING
T.A . Hanadi Qashqari
 Aim

The hemocytometer or haemocytometer is a

device originally designed for the counting of
blood cells.

It is now also used to count other types of cells as

well as other microscopic particles.
 What is cell counting?
Quantitation in cell culture is required for
experimental analyses.

Although we can estimates the growth of a

culture from its appearance under microscope,
proper quantitative experiments are difficult to
analyze and reproduce unless the cells are counted
before and after each experiment.

Cell counting is a general name for various

methods for the quantification of cells in molecular
biology & medicine.

By counting the cells in a known volume of

 Why to count?
Certain experiments require an exact number
of cells to be used.

A lot of kits for molecular biology have the

amount of reagents for a certain number of cells

Experiments of proliferation and survival

(usually toxicity studies)
With addition of dyes alive and dead cells can be
distinguished and counted

White blood cells count

Sperm count
 What is
hemocytometer?
It is a microscope slide
that is especially designed
for cell counting by
determining the number of
cells per unit volume.

Invented by Louis-
Charles Malassez.
 Hemocytometer is
consist of

This device has 2 separate cell counting areas,

each containing 0.1 cubic mm of sample when
covered by a cover slip (1 x 1 x 0.1).

1 mm
0.1
mm 1 mm
 A special cover slip mounts on two ground
glass bars to form the top of the two chambers.

Cover slip has to lay exactly in the middle and

cover both chambers.
 Counting grid
Fine rulings on the
floor of each chamber
provide grids to help
count the number of cells
in suspensions.

There are 9 big

squares on each side of
hemocytometer.

Cells are counted in a

big square.

You should count

 Grid area: 9 squares.

5 squares are usually

used for cell counting.

One central square,

consist of 25 smaller
squares.

4 outer squares, each

consist of 16 smaller
 Hemocytometer
grid
1 mm
0.1
mm 1 mm
Volume = length x width x
height
Red Square V = 1mm x 1mm x
0.1mm = 0.1mm3
Grid area:
This gives the number of cells Red square= 1.0000
per 0.1L mm2
Multiplying by 10 gives the # of Green square = 0.0625
cells per 1L mm2
Multiplying by10000 gives the #
of cells per 1mL
Yellow square = 0.040
Total number of cells per ml mm2
multiply the total number of Blue square = 0.0025
 How to count

Establish a rule to avoid counting cells twice or

not at all

Example:
Count only cells that lie on the top and left hand
lines of each box the ones on the lower line and right
hand line will be counted with next box when you get
to them.
 Trypan blue
A stain which will only enter across the
membranes of dead/nonviable cells, while live cell
membrane is impermeable to trypan
blue.
Cells must be counted within 3-5 min because
the number of blue staining cells increases with time
after addition of the dye.
Trypan blue is carcinogenic, so be careful while
use!
 Two methods for counting
Method A:
Counting the 4 outer
squares.

Cell concentration /ml=

Total cell count in 4 squares/
4 squares x dilution factor x
104

Example: If one counted

450 cells in 4 squares after
diluting an aliquot of the cell
suspension
450/4 x 10 x1:10
104 =1125 x 104 =
cell
1.125 concentration/
x 107 = 11,250,000 mL =
 Cont ..
Method B:
Counting 5 squares in the
center square.
Cell concentration/ml=
Total cell count in 5 squares
x 5 squares x dilution factor
x 104 .

Example: If one counted

45 cells in 5 squares after
diluting an aliquot of the cell
suspension 1:10
45x cell
concentration/
5 x 10 x 104 = 2250 xml
104==
2.250x 107 = 22,500,000
Cont
 How to perform the procedure?
1) materials

q 0.4 % Trypan blue (0.4 g trypan blue disolve in

100 ml P.B.S).
Hemocytometer
Cover slip
70% ethanol
Pipetteman and pipette tips
Cells
Trypsin
10% F.B.S Media
q PBS
q Microscope
q Hand-held counter
 2) Preparation of cells

Aspirate media.
Add 2 mL PBS and aspirate.
Add 2 mL trypsin.
Leave in 37O C incubator for 2 min.
Add at least 2 mL media (to stop trypsin
reaction).
Mix well and trasfer to a 15 mL centrifuge
tube.
 3) Loading the
sample
Clean the hemocytometer and the cover slip with
70% ethanol.
Put the cover slip on the hemocytometer.
Add 100 L cell solution in a small tube.
Add 100 L Trypan blue.
Mix gently (avoid bubbles).
Take out 100L by Pipette.
Place the tip of the pipette in the V-shaped groove
to load the
sample into the chamber (10 L ).
Let sample settle for 2 min so that the cells stop
drifting around the chamber.
Not to allow the sample to settle too long or it will
dry out.
 4) How to
count?
Observe your sample at the lowest magnification
under the microscope (10x).
Focus on the grid lines of chamber.
Count the number of cells in the chamber.
Calculate the dilution factor (DF) = Final volume/
initial volume
Derive the concentration of the sample as follows:

Cell concentration (cells/mL) = total cells counted x (DF) x

(104)
number of squares
Total # of cells in sample = Concentration (cells/ml) x sample
volume
 Cell viability
Cell viability is a measure how many of your cells
survived your cell culture technique.
Rely on the alteration in membrane integrity as
determined by the uptake of trypan blue by dead
cells, thereby giving a direct measure of cell
viability.
Determine the percentage of viable cells as follow:
% Cell viability = number of living cells
x 100
total number of cells counted (live + dead)

Example: number of living cells is 54, number of

dead cells is 8, calculate cell viability?
Answer: % viability = 54/62 x 100 =87.09%
 What if the cells
concentration are very
low?
We can always adjust the concentration we
need in the appropriate volume of media.
Procedure:
Re-suspended cells as follow:
Transfer the cell suspension to a sterile
centrifuge tube and centrifuge for 10 minutes
at 800 g.
Carefully remove the supernatant without
disturbing the cell pellet.
Add the desired volume of fresh medium gently
and slowly pipette up and down 2 to 3 times to
resuspend the cell pellet.
Automated trypan blue method for
optimal cell viability determination
Th
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