SLIDE PREPARATION

Wet mount slide preparation

Wet mounts are used for living samples, transparent liquids, and aquatic samples. A wet
mount is like a sandwich. The bottom layer is the slide. Next is the liquid sample. A small
square of clear glass or plastic (a coverslip) is placed on top of the liquid to minimize
evaporation and protect the microscope lens from exposure to the sample.

To prepare a wet mount using a flat slide or a depression slide:

1. Place a drop of fluid in the middle of the slide (e.g., water, glycerine, immersion oil, or a
liquid
Sample).
2. If viewing a sample not already in the liquid, use tweezers to position the specimen
within the drop.
3. Place one side of a coverslip at an angle so that its edge touches the slide and the outer
edge of the liquid.
4. Slowly lower the coverslip, avoiding air bubbles.

Note :( Most problems with air bubbles come from not applying the coverslip at an angle,
not touching the liquid drop, or from using a viscous (thick) liquid. If the liquid drop is
too large, the coverslip will float on the slide, making it hard to focus on the subject using
a microscope.)

Dry mount slide preparation

Dry mount slides can consist of a sample placed on a slide or else a sample covered with
a cover slip. For a low power microscope, such as a dissection scope, the size of the
object isn't critical, since its surface will be examined. For a compound microscope, the
sample needs to be very thin and as flat as possible. Aim for one cell thickness to a few
cells. It may be necessary to use a knife or razor blade to shave a section of sample.

To prepare a dry mount slide

1. Place the slide on a flat surface.

2. Use tweezers or a forceps to place the sample on the slide.

 3. Place the coverslip on top of the sample. In some cases, it's okay to view the sample
without a coverslip, as long as care is taken not to bump the sample into the
microscope lens. If the sample is soft, a "squash slide" may be made by gently
pressing down on the coverslip.

If the sample won't stay on the slide, it may be secured by painting the slide with clear
nail polish immediately before adding the specimen. This also makes the slide semi-
permanent. Usually slides can be rinsed and reused, but using nail polish means the slides
must be cleaned with polish remover before reuse.

How to Stain Slides

There are many methods of staining slides. Stains make it easier to see details that might
otherwise be invisible.

Simple stains include iodine, crystal violet, or methylene blue. These solutions may be
used to increase contrast in wet or dry mounts. To use one of these stains:

1. Prepare a wet mount or dry mount with a coverslip.

2. Add a small drop of stain to an edge of the coverslip.

3. Place the edge of a tissue or paper towel on the opposite edge of the coverslip.
Capillary action will pull the dye across the slide to stain the specimen.
 Human cheek cells (squamous cell) Wet Slide Preparation

Materials:
 Glass microscope slides
 Plastic cover slips
 Paper towels or tissue
 Methylene Blue solution
Note: (0.5% to 1% (mix approximately 1 part
stock solution with 4 parts of water))
 Plastic pipette or dropper
 Sterile, individually packed cotton swabs

Methods
1. Take a clean cotton swab and gently scrape the inner portion of your cheek.

2. Smear the cotton swab on the centre of the microscope slide for 2 to 3 seconds.

3. Add a drop of methylene blue solution on the smeared specimen and place a coverslip on
top.

Note: Concentrated methylene blue is toxic if ingested. Wear gloves and do NOT allow
children to handle methylene blue solution or have access to the bottle of solution.

4. Remove any excess solution by allowing a paper towel to touch one side of the coverslip.

5. Place the slide on the microscope, with 4 x or 10 x objective in position and find the cells.

Note: Methylene blue stains negatively charged molecules in the cell, including DNA
and RNA. This dye is toxic when ingested and it causes irritation when in contact
with the skin and eyes.

The cells seen are squamous epithelial cells from the outer epithelial layer of the
mouth. The small blue dots are bacteria from our teeth and mouth.
 Preparing a Dry Mount (Bread Molds)

Materials:
 Bread mold
 Glass slide
 Cover slip
 Tweezers

Methods:
1. Select a clean slide. Hold a slide up to a light source and look through to make sure
it’s free from smudges and dirt.

2. Inspect the specimen to determine if it needs to be sliced. The sample specimen

needs to be translucent (or semi-transparent) to transparent in order for light to pass
through.

3. Slice off a thin piece of the sample specimen. Use a razor blade to cut your
specimen material into a thin, translucent slice.

4. Place the sample specimen on the slide. Use a pair of forceps to pick up the thin
slice of your sample specimen.

5. Set a cover slip over the sample specimen. The cover slip prevents the sample
specimen from tumbling off the slide.

Note: always wear your laboratory gown, protective goggles, gloves and mask. Be
sure to read instructions of laboratory activities properly.
References:
↑ http://www.microscopemaster.com/microscope-slides.html
↑ https://learning-center.homesciencetools.com/article/microscope-slide-making-
ideas/
↑ https://learning-center.homesciencetools.com/article/microscope-slide-making-
ideas/
↑ https://www.carolina.com/teacher-resources/Interactive/make-microscope-slides-
for-science-fair-projects/tr10768.tr
↑ https://www.carolina.com/teacher-resources/Interactive/make-microscope-slides-
for-science-fair-projects/tr10768.tr
↑ https://www.carolina.com/teacher-resources/Interactive/make-microscope-slides-
for-science-fair-projects/tr10768.tr
↑ https://www.carolina.com/teacher-resources/Interactive/make-microscope-slides-
for-science-fair-projects/tr10768.tr
↑ https://learning-center.homesciencetools.com/article/microscope-slide-making-
ideas/
↑ http://utahscience.oremjr.alpine.k12.ut.us/sciber00/7th/cells/sciber/slidepre.htm
↑ https://www.youtube.com/watch?v=YU0DYrU_0FM&feature=youtu.be&t=1m40s
↑ https://www.youtube.com/watch?v=YU0DYrU_0FM&feature=youtu.be&t=1m51s
↑ http://utahscience.oremjr.alpine.k12.ut.us/sciber00/7th/cells/sciber/slidepre.htm
↑ https://www.youtube.com/watch?v=YU0DYrU_0FM&feature=youtu.be&t=1m54s
↑ https://www.youtube.com/watch?v=YU0DYrU_0FM&feature=youtu.be&t=2m11s
↑ http://utahscience.oremjr.alpine.k12.ut.us/sciber00/7th/cells/sciber/slidepre.htm
http://www.marietta.edu/~biol/introlab/Onion%20root%20mitosis.pdf
http://staff.jccc.net/pdecell/celldivision/oniontip.html
http://www.wisc-online.com/objects/ViewObject.aspx?ID=BIO204
 Onion Root Tips (mitosis)

Materials required:

 Onion plant with root

 Feulgen stain
 1 N HCl
 Scissors
 Forceps
 Razor blade
 Pasture pipette
 1.5 ml microfuge tubes
 Dissection probe with wooden
back
 Microscopic slides and cover slips
 Water bath
 Light Microscope

Procedure:

1. Take the onion plant with newly sprouted roots and cut two root tips using
scissors and transfer them into a plastic microfuge tube.
2. Fill 2/3 of the tube with 1N HCl using a dropper.
3. Place the tube in a 60°C water bath and incubate the tube for 12- 15 minutes.
4. Remove the tube from the water bath after the incubation.
5. Discard the HCl from the tube using a Pasture pipette to the running tap water.
6. Add some drops of distilled water into the tube and rinse the root. Then remove
the water from the microfuge tube using the Pasture pipette. (Rinse the roots at
least three times).
7. After the washing step add 2-3 drops of Feulgen stain into the tube with root tips
and incubate the roots for 12-15 minutes. (During the incubation, the very tip of
the root will begin to turn red as the DNA stains the numerous small actively
dividing cells at the time).
8. After the incubation remove the stain using a Pasture pipette.
9. Again rinse the root tips with distilled water. (Rinse the roots at least three
times).
10. Transfer a root from the tube to the centre of the microscopic slide and add a
drop of water over it.
11. Take a razor blade and cut most of the unstained part of the root.
12. Cover the root tip with a cover slip and then carefully push down on the cover
slide with the wooden end of a dissecting probe. (Push hard, but do not twist or
push the cover slide sideways). The root tip should spread out to a diameter of
about 0.5- 1cm.
13. Observe it under a compound microscope in 10x objective. Scan and narrow
down to a region containing dividing cells and switch to 40x for a better view.

An Alternative Procedure

1. Cut the tip 5 to 8 mm from the tip of the freshly sprouted root. Discard the rest
of the root.
2. Place the cut tip on a clean microscope slide.
3. Add 2-3 drops of acetocarmine stain to the slide.
4. Warm the slide gently over the alcohol lamp for about one minute. (Do not allow
the slide to get hot to the touch; you don't want to cook either your fingers or
the root. Do not let the root dry out).
5. Cover the slide with a cover slip or lens paper.
6. Squash the slide with your thumb using a firm and even pressure. (Avoid
squashing with such force that the cover slip breaks or slides).
7. Observe it under a compound microscope in 10x objective. Scan and narrow
down to a region containing dividing cells and switch to 40x for a better view.