Grade 9 2022-2023

Cell Biology

USING BIOLOGICAL
STAINS TO SHOW OSMOSIS
IN CELLS OF AN ONION

A PROJECT IN THE LAB

Cells are so tiny that a microscope is
needed to see them.

Often it is necessary to use the High

Power (HP) objective lens (x400).

Cells are usually transparent and

sometimes a stain has to be added to
the cells, which reacts with chemicals
inside the cells, changing their colour,
and making organelles and structures
visible.
 Part 1 - WHICH STAIN IS BEST?

Not all stains work well in all tissues,

so our first decision is to decide which
stain is best to use, either methylene
blue or iodine. For the investigation
into osmosis, we specifically want to
see the central vacuoles of the onion
cells, so we are evaluating the stains
for that purpose.
Collect the following materials and
equipment and take them to your
work-place:

Materials
● piece of red onion leaf

Equipment
● Microscope
● Plastic pipette
● Slides
● Coverslips
● needle

Chemicals
● Methylene blue stain
● Iodine stain
● Distilled water
● 5% solution NaCl (Sodium chloride
- salt)
 PROCEDURE
What you do, to prepare 3 slides for comparison:
1. Carefully read and follow these instructions:
● Collect a small piece of onion and identify which
is the inside of the leaf
● With a knife or sharp point, lift up a piece of the
inner layer of the leaf. (This is called the inner
epidermis). This layer is just one cell thick.
● Cut a square of the epidermis, about 2mm x 2mm
– much smaller than you think!
● Lay this square onto the middle of a clean slide.
Check that the layer of cells do not roll over.
● Add 2 or 3 drops of distilled water to the
epidermis.
● Put a cover slip on top. Make sure that you do
this properly. Remember how? You do not want
bubbles to appear.
● Examine the slide with Low Power and then
Medium Power. Maybe, carefully, go to High
Power.

2. You can use stains to make the inside of a cell

more visible. The best two stains are iodine and
methylene blue.

3. Prepare another piece of onion ‘skin’ and add two

drops of methylene blue instead of water. Leave
the stain for 3 minutes before putting on the cover
slip. Examine the cells with the microscope.

4. Repeat this a third time, using iodine stain.

[Show your slides to John 5 points]

Evaluating the results
● How will you decide which stain works
best? Remember, it is the vacuoles
and the tonoplast membrane around
them which we want to see. Use a 5
point scale like the one below:

1 2 3 4 5
vacuoles still cells show up
difficult to see very clearly

● Put the results into this results table:

STAIN USED SCORE - 1 to 5

Water
Iodine
Methylene Blue

[2 points]
When you compare two different variables in an
investigation, you must make sure that your
investigation is a FAIR TEST. Here you were
comparing the two stains, iodine and methylene
blue. Was your investigation a fair test? If you
answer “Yes!” then write why. If you answer “No!”
then write why not.

Was the investigation a FAIR TEST? YES or NO.

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Explain why you have answered “Yes!” or “No!”

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[3 points]
Part 2 - OSMOSIS – a form of diffusion
THE CONTEXT
Membranes have an important function in all cells, to
control what passes in and out , through the membranes.
Not all chemicals and particles can pass through all
membranes. There are 3 main ways in which substances
can pass through membranes:

1. If there is a concentration gradient, with a higher

concentration of the substance on one side than the
other, then this substance may be able to simply flow
through the membrane, with the membrane unable to
control the flow. This is called DIFFUSION. Very few
substances can pass through a membrane in this way.

2. Some chemicals which have a concentration gradient

but cannot simply diffuse across a membrane may be
helped through the membrane by special proteins. This
is like a flip-flop system! No energy is used by the cell.
It is called FACILITATED DIFFUSION.

3. Sometimes a cell needs to move chemicals or

substances where there is no concentration gradient.
Energy is required in this case to actively bring
substances across the membrane, through special
protein channels. This is called ACTIVE TRANSPORT.

4. Many cells have the capacity to engulf liquids and solid

substances, using their membranes. Think about those
phage bacteria which are able to engulf viruses. This is
called BULK TRANSPORT.
Membranes in cells are highly complex and organised.
Their role is to control what passes into or out of a cell or
its vacuole or its organelles, all of which are surrounded
by membranes.
And then we have OSMOSIS!
Osmosis is actually very simple to understand
but students get frightened by osmosis!
Osmosis is the movement of only water from a
solution on one side of a membrane to the
other side. If there is more water on one side
than the other, then water will diffuse across
the membrane. How can there be more water
on one side than on another? We need to know
about SOLUTIONS.

Google Slides:
https://docs.google.com/presentation/d/1yrV
mwOYTwGahTEGTNhrthe5isLEYddVhDkLOrp8f
LgE/edit?usp=sharing
YouTube video:
https://www.youtube.com/watch?v=L-osEc07vMs

Cornell notes on solutions, membrane transport

and osmosis. One Grade in Idukay [10 points]
Investigation into OSMOSIS
1. Prepare a slide of a piece of red onion epidermis, exactly as you
did previously, using the stain that gave you the best results.
Place a cover slip on the slide.
2. Use a microscope to check that you can properly see the cells.
3. Carefully remove the slide from the microscope and place it on a
flat surface.
4. Use a pipette to place 2 drops of 5% NaCl solution along one side
of the cover slip – in contact with the liquid beneath the cover slip
but not on the coverslip.
5. Now place a piece of paper towel along the other side of the
coverslip. This will draw the 5% NaCl solution through the slide, so
that the onion cells are flooded by salt solution.

6. Carefully replace the slide on the microscope and examine the

cells. What do you see?
7. Describe what you see, below, and make a drawing of a small
group of cells on the next page.

Describe what you see:

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[2 points]
Labelled Drawing

[3 points]
 8. Remove the slide from the microscope.
9. Lift off the piece of paper towel and dry the slide on both sides
of the coverslip. (Be careful not to move the coverslip and the
onion cells.)
10. Now use the pipette to place drops of distilled water along one
side of the coverslip and draw the distilled water through the
piece of onion with a paper towel, as you did before.
11. Describe what you see and make a drawing of a small group of
cells in the spaces below.

Describe what you see:

……………………………………………………………………………………………………….................................

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[2 points]

Labelled Drawing

[3 points]
Conclusion – the hard part!
If this investigation has worked out properly for you, you should have
been able to observe OSMOSIS in action. Write a conclusion – a
summary of your observations – using the words OSMOSIS, TURGOR
PRESSURE and PLASMOLYSIS to describe what you have seen in this
investigation.

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[5 points]
Total 25 points