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Laboratory Exercise 7
Wet mount
Expectations of this lab:
 Learn that this is one of the quick ways to see what a microbe looks like and also
is it is motile.
 At the end of this lab be able to accurately adjust the microscope and view a wet
mount.
 Learn that the approximate bacterial cell size and shape can be determined with a
wet mount.
 Understand the limitations of bright-field microscopy and the advantage of phase
contrast microscopy.
 Understand that the phase-contrast microscope can be utilized for quick
observations of microbes without staining. e.g., wet mounts, endospore stains. In
a wet mount the cells will appear dark against a light background and with an
endospore stain the endospores will be refractile amidst dark cells.
Introduction
Making a wet mount is a quick and easy way to see the size and shape of a microbe using
a bright-field or a phase contrast microscope. With a wet mount it is easy to characterize
the size and shape of a bacterium. However, a transparent microbe is difficult to see in a
transparent background when you use a bright-field microscope. The diaphragm of the
compound microscope has to be adjusted properly according to the specimen to gain
good contrast between the cells and the background in order to clearly see the size and
shape of organisms. The method of simple staining discussed in the next chapter is an
easier way to see the bacteria more clearly.
Brownian movement occurs when water molecules bombard the cells and shake them
giving the impression of locomotion. The truly motile bacteria will be easy to spot and
you may see tumbling and change of direction in them as well. Positive chemotaxis is
when bacteria move towards a positive stimulus (e.g., nutrients), and negative
chemotaxis is when bacteria move away from a negative stimulus (e.g., bacterial toxins).
The hanging drop slide method is another common way of looking at wet mounts for
motility. In the hanging drop procedure Vaseline is applied to the four edges of a
coverslip, a droplet is placed in the middle of the coverslip, and a special glass slide with
a depression in the middle is lowered onto the coverslip. Next, the slide is turned upside
down. This slide will now have the droplet hanging into the concavity of the slide. Under
oil immersion the motility will be visible. This method requires special thick glass
depression slides. This lab will cover the common way of making a wet mount using a
glass slide and a coverslip.
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Materials:
Clean glass slides
Distilled water bottle
Sharpie marker
Lens paper
Immersion oil
Compound light microscope
Splash/windex
Inoculating loop
Micro incinerator
Gloves
Tryptic soy broth culture of Staphylococcus epidermidis
Tryptic soy broth culture of Proteus mirabilis or Proteus vulgaris
Procedure:
1. Obtain a clean glass slide. Using the sharpie marker, draw two ovals. Label as shown
below.
Proteus Staphylococcus
2. Mix the broth culture and aseptically transfer a loopful to the middle of the oval.
3. Take a glass coverslip and drop the coverslip at an angle over the droplet.
4. Repeat steps 1-2 for the second microbe.
5. Observe the smears under a bright-field microscope 100x objective lens using oil
immersion. Adjust the diaphragm to give proper lighting. Low lighting is preferable
for wet mounts.
6. Record your results in the note book. Record the colors, cell shape, and arrangement.
7. Clean the work area and dispose of the used stain in the proper location.
8. Clean the 100x objective lens of your microscope with clean lens paper and splash,
lower the stage, wrap the cord, and return the microscope to the cabinet.
Data/Results:
Maintain detailed notes of your results in your lab notebook. Clearly labelled drawings
denoting the cell shape, arrangement, and colors are recommended.
Common problems and tips:
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Use a fresh culture, especially if you are looking for motility. An old culture will have
dead cells and motility will not be apparent.
Mix the culture well before making a slide.
It is better to do one slide for each organism if you are working with unknowns. Look
for motility on a freshly made wet mount. Leaving the slides too long under the
microscope is not recommended. The droplet can dry out and not show motility.
This is not a suitable method for checking motility on pathogens.
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