1 Making and staining a wet mount Refer to the Practical Manual Introduction Sections F.1 and F.2

2 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.

3 Equipment Water bottle Dissecting needles Stain Tweezers Slides Cover slips Absorbent paper

4 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

6 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

7 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

8 Step 2 Add the specimen to this drop Another drop of water may be added at this point if necessary

9 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)

10 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

11 Drawing of lowering cover slip

12 Start – Step 3

13 Slowly lower – Step 3

14 Lowered – Step 3 Air bubble

15 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

16 Lowered with no air bubble

17 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

18 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).

19 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

20 Staining or irrigating 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

21 Drawing for staining

22 Staining while on microscope

23 Step 1 Place the slide on a small piece of paper towel

24 Step 2 Add a drop of stain (or water) to the slide at the edge of the cover slip as shown

25 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

26 Stain drawn across slide (1)

27 Stain drawn across slide (2) Absorbent paper towel

28 Stain drawn across slide (3)

29 Stain drawn across slide (4)

30 Stain drawn across slide (5)

31 Blot excess from right-hand side

32 Blot excess from left-hand side

33 Finished slide

34 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