Preparation of Slides to be

observed under microscope

The microscopic specimen has to be prepared for
studying under bright field microscopy. It should
be prepared in such a manner that the
• cells and other components in the specimen are
preserved in their original state.
• specimen should be thin, flat and transparent so
that light can pass through it.
• components which have been differentially
coloured (stained) can be clearly distinguished.

Objects or specimen or section to be studied is

initially mounted on a slide.
 Slides
A microscopic slide is a rectangular (corners are at
90o) piece made of either soda lime glass or
borosilicate glass or plastic.

Variations of Slides
• Slides may contain surface depressions for liquid
solutions, a variation known as concave
microscopic slides or depression slides. This type
of slide may have dual concavity for immediate
comparisons of samples.
• Slides may have a positive electrostatic charge or
surface modifications allowing adherence of
certain cells and tissues which cannot adhere
directly on a plain glass slide. Such slides are
called electrostatic charged slides.
• Surface of a slide may be treated with certain
biological reagents to make it water-proof or
resistant to certain chemicals in order to reduce
chances of cross-contamination.
• Slides may have an etched grid system to help
geographical plotting or to monitor a particular
area of interest.
• Slides may be made of transparent mica as it is
less prone to dust and scratching.
 Cover Slips

Cover slips are thin and transparent usually

square/rectangular/circular borosilicate or
silicate glass pieces and hold the sample at
one place protecting it from any damage or
contamination. Cover slips also protect the
objective lens of the microscopes by
preventing any direct contact with the sample
or mountant or stain and are removable for
reuse, if required.
 Slide Preparation Techniques

Slide preparation depends on the type of sample

and type of staining to be used to study it. The
various methods include dry mount, wet
mount, smear, squash, whole mount or
sectioning followed by staining, wherever
required (staining may be done before
preparation depending on the sample and/or
the technique).
Dry Mount
It involves placing a thinly sliced section on the slide
and a cover slip over it for further study (If the
specimen is thin and transparent than thin slicing
may not be required).

Wet Mount
It involves suspension of specimen or sample in
fluids such as water or glycerine and is used for
aquatic samples and live observations. Methyl
cellulose or albumin or cotton fibres may be used
for live observations depending on the type of
specimen.
Smear
Smear slides are prepared for those cells which are
suspended in some fluid such as culture of
microorganisms, blood etc or for cells which are
scraped or aspirated or brushed from some
surface or within an organ.
Squash
Squash is prepared for soft samples in which the
sample is intentionally squashed or crushed on
the slide for study or further processing. Mostly
the sample is stained before preparing a squash.
Whole Mount
It includes mounting a small and/or thin organism
or structure by placing directly on a slide (e.g. a
membrane which can be stretched on the slide or
a small thin unicellular / multicellular organism)
using the wet mount preparation.
Sectioning
For sectioning, the sample is supported in a way
allowing it to be cut into very thin slices and then
adhered to slides and stained. Sectioning is done
using an instrument called microtome or
ultramicrotome depending on the desired
thickness of the slices.
 Processing of the Slide

Temporary Mounts
The sample can be mounted in water or stain
(obstructs observation if the colour is dark) or
glycerine or a mixture of water and glycerine
immediately after staining. This type of slide
processing doesn’t require the steps of
dehydration and cannot be preserved for
further viewing and is known as temporary.
Permanent Mounts
The sample is mounted in a water-insoluble
mounting medium and requires to be dehydrated
before mounting. This type of slide can be
preserved for years for further viewing and is
known as permanent.
Samples are usually adhered on the slide using any
(desired) method of fixation. Depending on the
method of fixation and the slide preparation
technique, the sample may be rehydrated before
staining. Depending on the stain used for staining
the sample/slide may be dehydrated before
mounting. The Downgrading/Rehydration and
Upgrading/Dehydration is carried out with series
of ethyl alcohol.
 Some Examples of Mountants

• Euparal
• Canada Balsam
• Eukitt and other resin-based media
• Clear nail polish
• DPX (Di -N-Butyl Phthalate in Xylene)
Precautions:
• Always use clean glassware.
• Label the slides with suitable marker (glass marker) to identify the
specimen and staining later (avoid using markers which can later dissolve
in alcohol series).
• Choice of fixation, staining and type of slide preparation should be
according to the desired component to be studied.
• Set up coplin jars with solutions before you start and label each jar clearly
and keep them covered at all times.
• All coplin jars contain grooves for holding slides. Make sure to use one slot
for one slide to avoid any sticking of slides to each other.
• Drain slides on absorbent tissue between each solution.
• Always use the solutions afresh, especially the alcohol series for hydration
and dehydration.
• Always use separate alcohol series for hydration and dehydration.
• The final dehydration before applying the cover slip is critical. If any water
is present, xylene will cloud and the stain will diffuse or fade.