how to prepare a slide for a light microscope
How to Prepare Microscope Slides ?
1 Wet Mount Slides
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:
Place a drop of fluid in the middle of the slide (e.g., water, glycerin, immersion oil, or a liquid sample).
If viewing a sample not already in the liquid, use tweezers to position the specimen within the drop.
Place one side of a coverslip at an angle so that its edge touches the slide and the outer edge of the drop.
Slowly lower the coverslip, avoiding air bubbles. 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.
Some living organisms move too quickly to be observed in a wet mount. One solution is to add a drop of a commercial preparation called “Proto Slow”. A drop of the solution is added to the liquid drop before applying the coverslip.
how to prepare a slide for a light microscope
Some organisms (e.g., Paramecium) need more space than what forms between a coverslip and flat slide. Adding a couple of strands of cotton from a tissue or swab or else adding tiny bits of broken cover slip will add space and “corral” the organisms.
As the liquid evaporates from the edges of the slide, living samples may die. One way to retard evaporation is to use a toothpick to coat the edges of the cover slip with a thin rim of petroleum jelly before dropping the coverslip over the sample. Press gently on the coverslip to remove air bubbles and seal the slide.
how to prepare a slide for a light microscope
2 Dry Mount Slides
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.
Place the slide on a flat surface.
Use tweezers or a forceps to place the sample on the slide.
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 semipermanent. Usually slides can be rinsed and reused, but using nail polish means the slides must be cleaned with polish remover before re-use.
3 How to Make a Blood Smear Slide
Some liquids are either to deeply colored or too thick to view using the wet mount technique. Blood and semen are prepared as smears. Evenly smearing the sample across the slide makes it possible to distinguish individual cells. While making a smear isn’t complicated, getting an even layer takes practice.
Place a small drop of a liquid sample onto the slide.
Take a second clean slide. Hold it at an angle to the first slide. Use the edge of this slide to touch the drop. Capillary action will draw the liquid into a line where the flat edge of the second slide touches the first slide. Evenly draw the second slide across the surface of the first slide, creating a smear. Its not necessary to apply pressure.
At this point, either allow the slide to dry so that it can be stained or else place a coverslip on top of the smear.
4 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:
Prepare a wet mount or dry mount with a coverslip.
Add a small drop of stain to an edge of the coverslip.
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.
5 Common Objects to Examine With a Microscope
Many common foods and objects make fascinating subjects for slides. Wet mount slides are best for food. Dry mount slides are good for dry chemicals. Examples of appropriate subjects include:
Table salt
Epsom Salt
Dishwashing detergent powder
Sugar
Alum
Mold from bread or fruit
Thin slices of fruits or vegetables
Human or pet hair
Pond water
Garden soil (as a wet mount)
Yogurt
Dust
how to prepare a slide for a light microscope
How ot Slide Mount Instructions?
Before you start building your slides, make sure you have everything you will need, including slides, cover slips, droppers or pipets and any chemicals or stains you plan to use.
You will be using two main types of slides, 1) the common flat glass slide, and 2) the depression or well slides. Well slides have a small well, or indentation, in the center to hold a drop of water or liquid substance. They are more expensive and usually used without a cover slip.
Standard slides can be either plastic or glass and are 1 x 3 inches (25 x 75 mm) in size and 1 to 1.2 mm thick.
Wet slides will use a cover slip or cover glass, a very thin square piece of glass (or plastic) that is placed over the sample drop. Without the cover in place, surface tension would cause the droplet to bunch up in a dome. The cover breaks this tension, flattening the sample and allowing very close inspection with minimal focusing. The cover also serves to protect the objective lens from interfering with the sample drop.
MOUNTS
There are four common ways to mount a microscope slide as described below:
Dry Mount
In a dry mount, the specimen is placed directly on the slide. A cover slip may be used to keep the specimen in place and to help protect the objective lens. Dry mounts are suitable for specimens such as samples of pollen, hair, feathers or plant materials.
Wet Mount
In a wet mount, a drop of water is used to suspend the specimen between the slide and cover slip. Place a sample on the slide. Using a pipette, place a drop of water on the specimen. Then place on edge of the cover slip over the sample and carefully lower the cover slip into place using a toothpick or equivalent. This method will help prevent air bubbles from being trapped under the cover slip.Your objective is to have sufficient water to fill the space between cover slip and slide. If there is too much water, the cover slip will slide around. Take a piece of paper towel and hold it close to one edge of the cover slip. This will draw out some water. If too dry, add a drop of water beside the cover slip. Practice this until you get used to it.
Wet mounts are suitable for studying water-bound organisms such as paramecium or bodily fluids such as saliva, blood and urine.
Section Mount
In a section mount, an extremely thin cross-section of a specimen is used. Using a microtome, cut a thin slice of your selected specimen such as an onion, and carefully set it on your slide. Then follow the instructions for a dry or wet mount. A stain can often be applied directly to the specimen before covering with a cover slip.
Section mounts are suitable for useful for a wide variety of samples such as fruit, vegetables and other solids that can be cut into small slices.
Smear
A smear is made by carefully smearing a thin layer of the specimen across a slide and then applying a cover slip. Typically, a smear should be allowed to air dry before applying a stain.
STAINS
Stains are used to help identify different types of cells using light microscopes. They give the image more contrast and allow cells to be classified according to their shape (morphology). By using a variety of different stains, you can selectively stain different areas such as a cell wall, nucleus, or the entire cell. Stains can also help differentiate between living or dead cells.
Stains tend to be grouped as neutral, acidic or basic, depending upon their chemical makeup and will attract or repel different organisms accordingly. For example, scientists and health professionals use Methylene Blue, a slightly alkaline stain, to reveal the presence of deoxyribonucleic acid, more commonly known as DNA.
Stain Types
Iodine is one of the more commonly available stains and is used to identify starch in a variety of samples. It will stain carbohydrates in plants and animal specimens brown or blue-black. Glycogen will show as red.
Methylene Blue is an alkaline stain useful in identifying acidic cell nuclei and DNA in animal, bacteria or blood samples. It’s also useful in aquariums to prevent the spread of fungal infections in fish. See more details >
Eosin Y is an acidic stain which stains pink for alkaline cells (cytoplasm, for example). It colors red for blood cells, cytoplasm and cell membranes. Eosin’s most important medical uses are in blood and bone-marrow testing, including the PAP smear. See more details >
Gram’s Stain is one of the most frequently used processes in identifying bacteria – used daily in hospitals. It is a primary test that quickly and cost effectively divides bacteria into one of two types: Gram positive or Gram negative. See more details >
STAINING STEPS
Prepare a wet mount slide.
Collect a drop of stain with an eye dropper or pipette.
Put a drop of stain on an outer edge of your cover slide.
Place a piece of napkin or paper towel against the opposite side of your cover slip, right up against the edge. This will help draw the stain under the cover and across the specimen.
You may need to add another drop to ensure complete coverage.
The slide is now ready for viewing.
