Preparation of Wet Mount Slide
Wet mount preparations are especially valuable for demonstrating motility in microorganisms. Fresh cultures must be used for maximum motility. No stain is employed since most stains kill the organisms (except vital stains). Therefore focusing is more difficult (see suggestions for focusing). If your microscope has dark field capabilities, this is ideal illumination for this purpose (use the “D” position on the sub-stage condenser dial). Alternatively, a small disc of dark paper may be placed in the center of the condenser to approximate dark field optics.
Motility needs to be distinguished from Brownian motion which is due to molecular bombardment. Brownian motion occurs in all microscopic bodies suspended in water and appears as a random shimmying-shaking. Motility will be in the form of cork-screw spiraling, movement in a given direction, or tumbling in place.
Wet mount preparations are also useful for giving clear images of fresh specimens under the microscope. Features which may be particulate, such as spores of fungi and ferns, and pollen grains may be best observed using this technique.
FOR CULTURES: Place 15 – 20 uL of the culture in the middle of the slide. (Here is a larger view of this image.)
FOR COLONIES: Place a small drop of dH2 O in the center of a slide. [For greater volume of sample, or for hanging drop preparations, use a depression slide for this preparation.]
CULTURES: The sample of liquid culture is place on the slide.
COLONIES: Sterilely transfer a tiny portion of a single colony to the drop with a loop and suspend (be certain to allow the loop to cool before picking up specimen). For solid specimens or dry spores, transfer a small portion of the specimen with a scalpel.
Lower a clean cover slip over the drop as though it were hinged at one side.
Finished preparation. Note that it is essentially transparent, making focusing difficult. (Ignore the handle of a drawer which appears in the background. It plays no role here…)
First focus with the 4x objective on the edge of the coverslip. It is easier to find and focus on than the nearly transparent suspension.
Find a bubble in the liquid suspension, and adjust the fine focus on the edge of the bubble.
Switch to the 10x objective, repeat the careful focusing.
Switch to the 40x objective, repeat the careful focusing. You should be able to discern bacteria at this power (magnification = 400x).
Apply oil and examine with the 100x oil immersion lens, again using the edge of the bubble as a focusing point. At 1000x, maximize the depth of field by narrowing the iris diaphragm, and adjust the focus so that most bacteria are in focus. (Because of the depth of the water, not all bacteria will be in focus at a given point.) Illustrate the types of motility you observe.
Clean up the slide with alcohol first (because it had live bacteria on it), followed by soap and water. Discard the cover slip.