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It's a typical clinic day. A patient has noticed some itching, or maybe an unpleasant vaginal odor.
During her exam, the clinician will check vaginal pH, examine any discharge that's
present, and collect a sample. Then, it's on to the microscope.
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This is where you'll gather more specific information about what's causing those vaginal symptoms.
We'll show you how to prepare and examine vaginal wet preps and how to do a whiff test.
The results, combined with the patient's vaginal pH test, will aid in the diagnosis.
Under the microscope, you'll be looking for trichomonads, yeast, and the clue cells
associated with bacterial vaginosis. We'll show you how to recognize them.
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First, the microscope itself: This is a compound light microscope. It has several objective
lenses on a rotating mount. For our purpose, one of these has to be a 10x low power objective,
and one has to be a 40x for greater magnification. This flat part, under the objectives, is the
stage. Under the stage is the condenser. Below that, at the base of the microscope, is the
light source. There are two knobs that control focus; one for coarse adjustment and one for
fine adjustment. And these are the oculars, or eyepieces.
We'll come back to the microscope in a minute, but first, let's look at how to prepare wet mount slides.
The complete vaginal wet mount involves both a saline prep and a potassium hydroxide, or
KOH, prep. When the vaginal sample was collected, the swab was placed in a test tube with approximately
half a milliliter of saline. So, for the saline prep, you only have to take a drop of the
suspension and place it on a slide. Add a coverslip, being careful to avoid trapping
air bubbles. Your saline slide is ready. Place a second drop of the vaginal sample
on another slide and add one drop of 10 percent KOH. Sniff the preparation immediately, using
your hand to waft any odor toward your nose. This is the whiff test. Note if there's a
fishy or amine odor. Then add a coverslip, avoiding air bubbles. Keep in mind that you
must work quickly to prepare and examine the wet mounts. That's because trichomonads may
lose their characteristic motility within 15 to 20 minutes.
Before we move on now, though, let's look at the cast of characters you may discover.
These are normal squamous epithelial cells found in the vagina. They're large, flat cells
with a small nucleus and a large area of cytoplasm. Note that there is some granularity in the
cytoplasm.
Polymorphonuclear leukocytes are known as Polys, or PMNs. They may also be called white
blood cells, or WBCs. These are small round cells. Several lobes of the nucleus are visible
within the surrounding cell cytoplasm. Finding many PMNs may indicate infection.
Trichomonads are pear-shaped protozoa which move by means of flagella. Trichomonads are
similar in size to PMNs and are identified by their characteristic jerking movement.
The actual flagella may be too thin and too rapidly-moving to be seen.
A clue cell is a squamous epithelial cell coated with enough small bacteria that at
least 75 percent of the cell's border is obliterated. It may look as if someone has spread glue
over the cell and pressed it in sand. Clue cells are associated with bacterial vaginosis,
a condition in which the normal microbial flora of the vagina is disrupted.
Yeast may be found in two forms. Pseudohyphae are the long, tubular, branching forms. Budding
yeast are paired yeast cells that resemble a shoe print. The larger part is the sole
and the smaller bud is the heel of the shoe.
The saline prep will allow you to see epithelial cells, PMNs, trichomonads, and clue cells.
You can also see yeast in saline, but sometimes it's hidden by epithelial cells or by PMNs.
Red blood cells, sperm, and bacteria can also be seen.
The KOH prep will only help you detect yeast. Epithelial cells, PMNs, trichomonads, and
clue cells will be broken apart, or lysed. Lysing the other cells makes it much easier
to see yeast. A positive whiff test, defined by a fishy odor, may indicate either trichomoniasis
or bacterial vaginosis. KOH volatizes amines which are associated with both these conditions.
When that happens, the fishy odor is easier to detect.
Now that you know what to look for, let's get back to the microscope. Put your slide
on the stage, and rotate the 10x objective into place. Turn on the light. Bring the sample
into focus using the coarse adjustment knob. Next, you'll need to find the best contrast,
which is important for seeing unstained slides clearly. You can lower the condenser away
from the stage until you achieve good contrast. Or, if you're using a microscope without a
moveable condenser,you can adjust the diaphragm lever to allow more or less light through.
Cells should appear clearly delineated against the bright background. Then, rotate the 40x
objective into place and use only the fine adjustment knob to focus. You may need to
increase the illumination.
Got all that? Here's the sequence again: Put your slide on the stage. Rotate the 10x objective
into place. Turn the light on. Use the coarse adjustment knob to bring the sample into focus.
Adjust the condenser and/or diaphragm lever for maximum contrast. Rotate the 40x objective
into place. Raise the light level if necessary. Use the fine adjustment knob to bring the
sample into focus. Readjust the condenser and/or diaphragm lever if necessary. Begin
reading. One other important point is that the objective
lenses must be free of oil and dirt. Use lens cleaning solution and lens paper to clean
each lens at the beginning or end of the day—or, of course, any time they become dirty.
The length of time you'll need to read the slides thoroughly depends on your level of
experience. A good rule of thumb is to spend at least three minutes reading each slide.
First, use the 40x objective to examine the saline slide. If you don't see trichomonads
or yeast pseudohyphae, move back to the10x objective and reexamine the slide with a wider
view. Then, if you see trichomonads or pseudohyphae, switch back to 40x for confirmation. Next,
use the same procedure on the KOH slide, to look for yeast. During your examination of
both saline and KOH slides, move from one side of the coverslip to the other in a Z
pattern.
And now, let's look at some patient samples. We'll start with the 40x objective. This saline
prep shows a cluster, or sheet,of normal squamous epithelial cells. Each of them clearly shows
a single nucleus. There's not a lot of granularity to the cells. As we focus up and down, the
edges of the cells are well defined. Not all squamous epithelial cells will be perfectly
flat. Some cells can be folded, or rolled-looking.Here you can see normal epithelial cells and red
blood cells. The red blood cells have no interior detail. We can also see PMNs here, which are
approximately the size of the nucleus in the epithelial cells, and a little bigger than
the red blood cells. Inside a PMN you can see several lobes of the nucleus.
In this field, we also see bacteria, which are most likely lactobaccilli. These epithelial
cells are surrounded by PMNs. Many PMNs can indicate infection. Another saline slide shows
a trichomonad moving in a jerky, circular motion. It's causing the PMN and the epithelial
cell near it to move as well. The trichomonad is about the same size as the PMN, but the
trichomonad does not show an internal structure the way the PMN does. We also see a sharp-edged
artifact. An artifact can be a fiber, glass chip, or other foreign object on the slide.
More trichomonads. Again, these are large, pear- or oval-shaped organisms, moving independently
of the flowing liquid. The flagella are a blur of motion.There may be only a few trichomonads
on the entire slide, so a thorough examination of the slide is essential. Here, the trichomonads
are becoming sluggish, making them harder to identify. That's why it's important to
examine the slide quickly, because once trichomonads stop moving, they can't be recognized.
This saline slide shows a normal squamous epithelial cell on the right, with a fold.On
the left, we see a clue cell, which looks as if it's been pressed in sand. The nucleus
is still visible, but as we focus up and down, we can see that there are so many bacteria
attached to the cell that they appear to spill off the edges. Moving on to another field,
we see two definite clue cells.
And here's a slide that does NOT show clue cells. This is an epithelial cell with some
bacteria adhering to it, but not enough to call it a clue cell. Most of the edges are
well defined, not obscured by bacteria. To the right is a partial cell.
These ARE two clue cells, surrounded by normal epithelial cells. The epithelial cell at lower
right is suspicious, but should not be considered a clue cell.
These dark circles are artifacts.
This is a budding yeast floating in. Note that the yeast bud is at least as large as
the nucleus of a squamous epithelial cell. The two parts of the little shoe print are
always connected. Here, again, is a yeast bud floating by a clue cell. The bacteria
make the edges of the clue cell appear to glitter. There are also lots of bacteria floating
in the background, which are much smaller than the yeast bud. These pseudohyphae are
also visible in this saline prep. However, a thick cluster of epithelial cells, such
as this one, could hide pseudohyphae, so we always prepare a KOH slide as well.
This KOH prep reveals pseudohyphae and budding yeast cells, with lysed epithelial cells in
the background. Once again, these are pseudohyphae and budding yeast on a bed of lysed epithelial
cells. The pseudohyphae look tubular, and they appear to branch. When we adjust the
focus, we see more pseudohyphae on the lower plane, surrounded by more buds. It's important
to focus up and down, because cells and organisms may be in different planes of focus. And that's
obvious here, where budding yeast show up in different focal planes. In the upper left
of this slide we see an artifact.
These are normal squamous epithelial cells seen through the 10x objective. It's especially
important to use 10x when trichomonads and pseudohyphae are not evenly distributed throughout
the slide. Here, we're able to see several trichomonads, recognizable by their characteristic
motion. Now that we've spotted them, we can move them to the center of the field, switch
to 40x, and confirm identification.
Returning to 10x, this saline prep shows pseudohyphae and epithelial cells. There may be yeast buds
floating by, but they are too small to identify here. Switching to 40x, we're able to confirm
identification. Sometimes, you may notice air bubbles on a slide. They look like this.
KOH preps can be examined in the same way, starting with 10x and moving to 40x for confirmation.In
this case, a slide seen through 10x seems to show pseudohyphae. At 40x, however,it turns
out to be an artifact.
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For diagnosis, the clinician will consider these test results: In trichomonas vaginitis,
the saline prep shows motile trichomonads. No trichomonads will be seen on the KOH prep.
The whiff test may reveal a fishy odor. The pH will generally be greater than 4.5.
In bacterial vaginosis, the saline prep may reveal clue cells. To be considered positive
for clue cells, the slide should show at least one clue cell in each of ten fields. Or, one
out of five epithelial cells should be clue cells. The KOH prep will not show clue cells.
The whiff test usually has a fishy odor. The pH is generally greater than 4.5.
In yeast vaginitis, the saline prep may have budding yeast and/or pseudohyphae. The KOH
prep will show those budding yeast and pseudohyphae more clearly. The whiff test will not have
a fishy odor, and the pH will generally be less than 4.5. Of course, a patient may have
more than one cause of vaginal symptoms. You may see a combination of microorganisms and
cells, indicating multiple infections. This patient, for example, has both trichomoniasis
and yeast. But now, treatment can begin.
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