1

Making and staining a wet mount

Refer to the Practical Manual Introduction Sections F.1 and F.2

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Preparation of a wet mount

A wet mount is a temporary preparation of material for light

microscope that can be made quickly and that may be stained with water-

based stains.

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Equipment

Water bottle

Dissecting needles

Stain

Tweezers

Slides

Cover slips

Absorbent paper

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Precautions Make sure that the microscope slides

and cover slips are clean Slides can be easily cleaned with

water, then alcohol and then flamed or wiped with a tissue

Cover slips are very fragile and can be treated in the same manner but with great care

5

Precautions Always handle the glass slides and

cover slips by their edges Always place the glass slide and

cover slip on a piece of absorbent paper towel

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Step 1 Using a dropper bottle or a pipette add one to two

drops of distilled water to the centre of the slide Stain may be added with or instead of water at this stage

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Attention An ‘e’ has been used in the next step

of this demonstration as it is easy to see in photographs, and you need a slide using an ‘e’ from an OHT for the first practical

Specimens are usually used Specimens viewed with a

transmission light microscope must be very thin, to allow the light to pass through them

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Step 2 Add the specimen to this drop Another drop of water may be added at

this point if necessary

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Step 3 The specimen is then covered with a

thin cover slip to exclude air and dust, and to protect the high power microscope objectives

This is done by holding the cover slip by the edges, and then placing it on the slide at an angle of about 45° so that the angle includes the specimen and the drop of water (or mounting medium)

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Step 3 – continued Gently and slowly lower the cover

slip using a dissecting needle If this is done with care and done

very slowly there should be few, if any, air bubbles trapped on the specimen or under the cover slip

Air bubbles will be seen under the microscope as perfect circles with distinct dark edges

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Drawing of lowering cover slip

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Start – Step 3

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Slowly lower – Step 3

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Lowered – Step 3

Air bubble

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Problems: if an air bubble occurs

Using extra water to irrigate slide Add a drop of water to the side of the cover

slip on the side away from the bubble Then apply absorbent paper to the edge of

the cover slip near the bubble Water will be drawn across the slide and

often the bubble moves with it Raise the cover slip, add more water

and then lower it again Tapping the cover slip very gently

may release the bubble

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Lowered with no air bubble

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Finishing slide Hopefully the specimen is near the

centre of the cover slip and the cover slip is close to the centre of the slide

Carefully remove any excess water from the top and bottom of the slide

There should be NO water on the top of the cover slip

When you are happy with the slide then proceed to Step 4

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Step 4 Look at the specimen under low

power and if there are too many air bubbles in the specimen then mount another specimen

If specimens begin to dry out then add a drop of water to the edge of the cover slip and draw it under the cover slip, using the same method as staining a mounted specimen (see next section).

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Different mounting medium

If the specimen is to be viewed for more than 10 minutes then it can be mounted in glycerine = mounting medium

Hydramount is a fairly permanent water based mounting medium

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Staining orirrigating a wet mount

Stains are chemicals that dye parts of cells. A great variety of stains are available and are used to reveal and

identify various structures

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Drawing for staining

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Staining while on microscope

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Step 1 Place the slide on a small piece of paper

towel

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Step 2 Add a drop of stain (or water) to the slide

at the edge of the cover slip as shown

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Stain drawn across slide On the opposite side of the cover slip place a

small piece of tissue or filter paper. This will draw the stain from one side of the cover slip to the other and so through the specimen

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Stain drawn across slide (1)

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Stain drawn across slide (2)

Absorbent paper towel

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Stain drawn across slide (3)

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Stain drawn across slide (4)

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Stain drawn across slide (5)

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Blot excess from right-hand side

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Blot excess from left-hand side

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Finished slide

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Other uses This technique can be used to:

Change the concentration of the solution under the microscope

Increase intensity of the stain by adding more stain

Decrease the intensity of the stain by adding more water

Add water to a specimen that is drying out

See the effect that different solution concentrations have on cells etc.

35

Common problems

Water on top of cover slip

Air bubble

Excess stain or water on top of

slide beside cover slip and/or bottom

of slide

Specimen badly positioned

36

Slide dried out