52 PART III Sample Preparation in Light Microscopy
✔✔Samples must be thin and flat, otherwise you will have too many out-of-
focus regions.
✔✔To distinguish different structures, a nice colorization helps.
Samples for transmitted light brightfield are normally put on glass slides. The
main methods of placing samples onto microscope slides are wet mount, dry
mount or sectioning, smear, squash and staining.
Dry Mounts
A dry mount is the most basic technique: simply put the complete sample or a
thinly sliced section of your sample on the center of the slide and place a coverslip
over the sample. Dry mounts are ideal for observing hair, feathers, airborne particles
such as pollens and dust as well as dead matter such as insect and aphid legs
or antennae. Opaque specimens require very fine slices for adequate illumination.
Since they are used for primarily inorganic and dead matter, dry mounts can
theoretically last indefinitely.
Wet Mounts
Used for aquatic samples, living organisms and natural observations, wet mounts
suspend specimens in fluids such as water, brine, glycerol and immersion oil.
A wet mount requires a liquid, tweezers, pipette and paper towels for preparation.
Although wet mounts can be used to prepare a significantly wide range of
microscope slides, they provide a transitory window as the liquid will dehydrate
and living specimens will die. Organisms such as protozoa may only live 30 minutes
under a wet mount slide. In addition, protozoa like Paramecium may be too
large and/or move too quickly under the wet mount (see Figure 8.1).
Smear Slides
In smears, the sample is spread thinly across one slide’s surface using a second
slide
as the spreader. The resultant smear is dried, sometimes additionally fixed
and stained. In medicine, samples of bodily fluids such as blood, cerebrospinal
fluid or semen are smeared. The angle of the smearing slide determines the length
of the smear; a steeper angle creates a shorter smear. For samples like blood, begin
by backing the smearing slide into the sample and then push across the slide,
pulling the blood in the opposite direction to create a smooth layer (Figure 8.2).