Preparing specimens for observation under the microscope
 slides, cover slips, stains
 slowing down moving specimens
 permanent slides
 oil immersion lenses
Microscope slides
Microscope slides are thin, flat pieces of glass on which your specimen is placed or mounted prior
to observation under the microscope.
Size is usually about 75 mm x 25 mm (3 inches x 1 inch).
Thickness is usually 1.0 to 1.2 mm, but sometimes thinner slides (0.8 mm) are used specifically
for high power and oil immersion lenses.
Other types of slide that you may use are:


Cavity slides
Also called ‘well’ slides.
They are used mostly to
observe cultures of
microorganisms under Low
Power.
They can be used with or
without a cover slip
These slides
have a well or
cavity
Side view
showing well
Graticle slides
These are also called graduated slides
and have a minute scale (ruler) or a grid
pattern. They are used for direct
measurement or for counting cells as in
a blood test.
Photo of actual Graticle slide is shown (left).
This slide is well used and has been scratched!
Cover slips
Cover slips (also called cover glasses) are much smaller and thinner pieces of glass than slides.
They are used on top of the specimen in a ‘wet mount’.
Size is usually about 18 to 20 mm square. Smaller ones are available.
Thickness can vary depending on the intended use:
 Number 2 cover slips are thicker and less prone to breakage. They are about 0.20 mm thick
and are used with normal objective lenses. These are the cover slips you will probably use.
 Number 1 cover slips are about 0.15 mm thick and are used with oil immersions lenses.
These are thinner and allow the lens to get closer to the specimen.
Reasons for using cover slips for ‘wet mounts’ include:
1. They provide a flat surface above the specimen for easier focussing. Note that water
droplets have a convex (curved) surface which can distort the image.
2. They protect the objective lenses from moisture and stains that are used to mount the
specimen.
3. They help to flatten the specimen. Reducing its thickness also reduces the amount of
focussing that needs to be done when observing the specimen.
4. They prevent the specimen from drying out. This is especially important when using a selfilluminated microscope (one with a built in light) as heat from the light soon dries out the
specimen on the slide.
For this reason it is good practice to turn the lamp off when the microscope is not in use.
How to lower a cover slip
This method avoids getting air bubbles in your wet mount. Often some air bubbles are unavoidable
but their presence should be minimised because they can impede the view.
Slide is held at 45o and the top edge is
lowered gently. You can use a pencil or
forceps to support the edge of the cover slip
Specimen in water droplet or stain
AIR
As cover slip touches liquid it
spreads. As the cover slip is
lowered further all the air is
pushed out
Final slide without air
bubbles
Cleanliness
When handling slides and cover slips your hands should be clean and dry. Finger prints on the
cover slip in particular can impede your view.
Mounting:
This term refers to the preparation of a specimen on a slide before it is placed on the microscope
stage ready for focussing and observation.
Types of mount
1.
Dry mount
This is the simplest mount. The specimen is placed on the slide without and water or stain.
A cover slip may be used but it is not essential. It will keep the specimen flat, prevent it being
blown off the slide and protect the microscopes objective lenses.
Items that can be dry mounted include:

feathers and hair

insect wings

pollen
2.
Wet mount
In a wet mount the specimen is placed in a drop of water or stain and a cover slip is always used.
When preparing a wet mount the following procedure is used

ensure the slide and cover slip are clean

with the slide on a horizontal surface place one drop of liquid in the middle of the slide.
This liquid may be water, stain solution, blood or a culture of micro-organisms

place the specimen into the drop of liquid

if the specimen is large and dry on top it may be necessary to place another drop of liquid
on top of it

lower the cover slip as described on the previous page
What liquid should be used when mounting cells?
When cells are subjected to solutions of different concentrations water can move in or out of the
cells by osmosis. This means that the cell can swell (become more turgid) or shrink (become
flaccid or plasmolysed).
When mounting cells in fluid there are four options:
1. use a stain solution. If the specimen requires staining then this is the only option.
2. use water from the natural habitat of the organism i.e. the water the cells have been
growing or living in. This is the best option for algae and ciliates. Use pond water for
freshwater organisms and use sea water for marine specimens.
3. use a specially prepared isotonic solution eg. Ringers solution or 0.9% salt (NaCl)
solution (saline). This avoids any osmotic movement in or out of the cells.
4. tap water is used for specimens that are not affected by osmosis eg. dry specimens like
pollen.
Too much liquid?
If there is too much liquid the cover slip will float and move easily as you carry the prepared slide.
It is easy to remove some of the liquid with a piece of paper towel or other absorbent material.
Touch the liquid at the edge of the liquid with the paper and hold it there until enough has been
drawn into the paper by capillary action.
Small piece of torn absorbent
paper
Liquid is absorbed by the paper
Thick specimens, the raised cover slip method.
Sometimes you want to mount a thick piece of tissue and the cover slip rides on it like a mini
seesaw. This means that the surface of the cover slip will not be horizontal which does not help
focussing. This issue can be overcome by using some small pieces of broken cover slip as
supports. This is an alternative to a cavity slide.
Thicker than normal specimen
mounted in liquid
Small broken pieces of cover
slip used to bridge cover slip
Adding a stain to a wet mount after it has been prepared
This technique can be used to replace water with a stain or it can be used to observe plasmolysis
(in which case, a 10% salt solution is used instead of the stain).
Slide with specimen mounted in
water
Place a drop of stain (or NaCl
solution) on the edge of the cover
slip
Using a piece of absorbent paper
draw the stain or salt solution under
the cover slip until the specimen
has been surrounded
Stains
Stains are pigments used to prepare microscope specimens to assist their visualisation.
Stains are used for a number of reasons:

Stains can enhance contrast between different parts of the specimen. This is especially
important if some or all parts of the specimen are transparent and are thus impossible to
see without staining.
Staining increases visualisation of the specimen and allows you to see more than in if the
specimen is unstained.

Stains can be used to identify certain substances or compounds within the specimen.
This involves using a class-specific dye. Examples include:
o Iodine which stains starch, blue-black
o Aceto-orcein is used to stain chromosomes in cells undergoing mitosis

Stains can be used to help identify cells (with particular properties). Example: in blood
samples, Wrights stain is used to identify white blood cells (which have nuclei).
Demonstration of the effect of a stain
To show the effect of a stain on two specimens on the same slide, use two specimens and two
cover slips. One specimen is stained while the other is left unstained. This allows you to change
from a stained to an unstained specimen quickly so that you can easily see if staining has a
desirable effect.
Onion skin cells and
iodine stain make a
good demonstration
of the value of using
a stain
Unstained specimen
mounted in water
Unstained onion cells
Specimen mounted
with stain
Stained onion cells
Note: Nuclei are now visible
In the school laboratory, there are two commonly used stains. Both have relatively low toxicity
but should still be handled with care. These two stains are:
1. Iodine (also called Lugol’s iodine solution)
Used primarily as a general stain for plant cells. It will colour many plant cell components
increasing visualisation.
Iodine also stains starch (remember only plant cells contain starch – usually in small
granules within the cytoplasm).
2. Methylene blue
Used primarily as a stain for animal cells. It is absorbed in to the cell and helps visualisation
of nuclei and other cell organelles within the cytoplasm. It is also useful for staining
bacteria.
There are a number of stains used in a large range of biological applications. To make matters
more complicated stains are often combined.
In a school laboratory other useful stains include:

Aceto-orcein used to stain chromosomes in preparations designed to show cells
undergoing mitosis

Carmine is used to show glycogen (red) in animal cells

Congo red shows up food vacuoles where it changes from red to blue as a result of acidic
conditions inside the vacuole

Crystal Violet stains cell walls purple (and is used to ‘Gram’ stain bacteria)

Wrights stain is a combination of methylene blue and other stains. It is used to show up
white blood cells
Caution:
 Stains are used because they ‘stain’. In some cases, spillages and stained clothing are
impossible to clean up. The stain is irreversible.
 Some stains are toxic. Others have adverse effects especially those chemicals that attach
to chromosomes; these potentially could cause mutations.
Experimentation with stains
There are common pigments that you could experiment with. Try mounting specimens in any of the
following:
 Food colouring. These stains are non-toxic.
 Inks from computer printers. It is usually possible to obtain small amounts of stain from
used printer cartridges.
Slowing down rapidly moving protozoa
One of the most frustrating microscope activities is trying to observe fast moving protozoa. The
issue is compounded by the fact that the image observed under the microscope is inverted. It is
useful to slow these organisms down so that they can be observed in detail before they disappear
from the field-of-view.
There are 3 techniques that can be used to do this:
1. ‘Pinned-down by the cover slip’ method
This method is successful only if the protozoa are relatively large. The protozoa are held in
place between the slide and the cover slip.
There are three ways of achieving this:
a. Use only a small drop of liquid when preparing the slide. Gentle pressure may be
used to squash the drop of liquid out so that it forms a very thin film
b. Prepare the slide as normal but well ahead of time. Leave it to stand so that much of
the water evaporates from under the cover slip
c. Prepare the slide as normal and then use a piece of absorbent paper to remove
some of the liquid by touching it at the edge of the cover slip
2. Using lens tissue
Rip up a small section of lens
cleaning tissue so that it is
slightly smaller than a cover
slip.
The method is as follows:
i. place this in the centre of
the slide
ii. add a drop of your protozoa
culture
iii. add the cover slip as
normal. The protozoa
become trapped within the
fibres of the tissue and are
more easily observed
i.
ii.
iii.
3. Using gels
There are 2 gels that can be used in this method
a. Methyl cellulose (10% i.e. 10 g in 90 ml water)
b. Gelatine (2 – 3 % solution, needs to be heated so the gelatine will dissolve and
then allowed to cool)
The method is as follows:
i. using a dropper make a
circular ring of the gel
solution in the centre of the
slide
i.
ii.
add a drop of the culture
containing the protozoa into
the middle of this ring so
that the gel surrounds it
iii. use a needle or toothpick to
mix the culture and the gel.
The idea is that the
viscosity (thickness) of the
gel will slow down the
protozoa
iv. add a cover slip as normal
and observe
ii.
iii.
iv.
Temporary slides
In the school laboratory, it is usual to mount specimens for immediate observation. These
temporary mounts have many advantages:





students learn the skills involved in mounting their own specimens
they are quick to prepare
there are usually few artefacts that are the result of complex preparations eg dehydration
and shrinkage
the specimens can be observed living and moving
colours are often more natural because natural pigments have not deteriorated
There are also disadvantages:


living organisms move and are difficult to focus on
the moisture in the slide preparation evaporates, often as you observe the specimen
Permanent slides
It is possible to make more permanent slides that can be stored. When doing so there are two
main considerations:


the specimen must be fixed (killed) and preserved so it does not decay. The simplest way
of doing this is to soak the specimen in alcohol or an alcohol/acetic acid mixture
the specimen must be sealed so that it does not dehydrate. One way of doing this is the
seal the cover slip with nail varnish. However you must allow the varnish to dry very
thoroughly before putting the slide on the microscope stage as it will be impossible to
remove from an objective lens
Permanent slides can also be purchased. These slides have been professionally prepared,
stained and mounted. Of particular use are slides that show:


cross sections of plant parts, roots, stems, leaves etc.
longitudinal sections of root tips showing cells undergoing mitosis (these are particularly
recommended)
Oil Immersion lenses
These are specialised objective lenses that give high magnification and excellent resolution.
Most school microscopes do not have Oil immersion (OI) objectives but the following is included for
those that do. Generally, they have a magnification of 100X and when used in conjunction with a
10X eyepiece give a total magnification of 1000X.
Because the lens needs to be close to the specimen, thinner cover slips are used (0.16 mm
instead of 0.20 mm). OI lenses can also be used for bacterial and blood smears when no cover slip
is used.
The space between the cover slip (or specimen) and the OI objective lens is filled with special oil
that has a refractive index similar to the glass of the lens allowing a greater magnification.
OI
x100
Oil immersion objective
Immersion oil
Thin cover slip (0.16 mm thick)
Specimen
Microscope slide
To use an OI objective the procedure is:
 focus the specimen at high power as usual
 close the diaphragm as much as possible to concentrate the light on the centre of the slide
 swing the HP objective out of the way
 add a drop of special ‘Immersion oil’ to the centre of the slide (immediately above the
specimen)
 swing the OI objective into place
 if necessary lower the OI lens into the oil, watching carefully from the side (this will depend
on if the OI lens is parfocal or not). OI objective are usually spring-loaded to prevent
damage if the objective makes contact with the cover slip. When doing this operation watch
from the side.
 focus with the OI objective upwards (moving the objective lens away from the slide). Focus
slowly as it is easy to pass through the focal point quickly, without realising it.
 Once focus is established close the diaphragm and adjust the condenser for best image.
 When finished, rack the OI lens upwards, remove the slide and clean the lens with lens
tissue to remove oil. If needed you can also use solvents supplied with the oil to further
clean the OI lens.
Because most school microscopes do not have OI lenses none of the photographs in the resource
were taken with an OI Lens.