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Examination of Vaginal Wet Preps

August 18, 2019

music 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. music This is where you’ll gather more specific
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. music 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. music 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. music

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