Evaluation of Wet Mount and KOH Preparations

Phase 1 Pharmacokinetic Trial of Two Intravaginal Rings (IVRs) Containing Different Dose Strengths of

Vicriviroc (MK-4176) and MK-2048 MTN-028 Study Specific Training

Wet Mount for Clue Cells and Trichomonas

• Remove the swab from the tube with saline and dot a liberal amount onto a glass slide

• Place a coverslip over the specimen• Scan the slide on 100X and 400X

– Positive for clue cells: >20% – Negative for clue cells: <20% – Positive for Trichomonas: any motile trichomonads seen– Positive for yeast: any budding yeast and/or pseudohyphae

seen

KOH preparation for Yeast

• Place a coverslip over the KOH preparation after smelling for amine odor

• Scan the slide at 100X and 400X – Positive for yeast: any budding yeast and/or

pseudohyphae– Negative for yeast: no yeast cells seen on slide

Gram stain of vaginal flora

Normal flora BV

Wet Mount Evaluation

• Minimum of 5 fields should be evaluated.• Ask yourself: What is your first impression?• Assess the epithelial cells and background

bacteria present.Shape: Symmetrical rods or pleomorphic coccobacillary?Numbers: Fewer or many?

Normal flora: No clue cells

400X1000X

BV: clue cells and WBC

400X1000X

BV: Clue cells

400X1000X

Wet Mount Evaluation: Clue Cells• The following tips should be utilized for determining clue

cells:

1. Count the number of distinguishable epithelial cells in your field of view.

2. To determine if any of the epithelial cells are clue cells, it is important to study ONLY THE BORDERS OF THE CELL.Note: Normal variation in cell membranes can result in a “grainy” appearance of the cell and can mimic bacterial adhesion.

3. To determine the percentage of clue cells in your field:a. Count the number of clue cells and divide that number by

the total number of distinguishable epithelial cells.

Normal Cells Clue Cells

400X 400X

Normal Cells Clue Cells

400X 400X

Clue Cells

400X

Normal Cells Normal Cells and yeast

400X 400X

Wet Mount Evaluation: Yeast

• Determining Yeast in Wet Mounts or KOH preps• In order for yeast/pseudohyphae not to be mistaken

for amorphous material, nuclei or artifacts there must be “budding”.

“Bud”

Pseudohyphae with “buds”

Pseudohyphae

Budding yeast

400X

Budding yeast

400X

Pseudohyphae and budding yeast

Budding yeast

KOH 400X KOH 400X

Pseudohyphae and budding yeast

Pseudohyphae, budding yeast and

amorphous material

KOH 400X KOH 400X

Pseudohyphae, budding yeast and

amorphous material

Budding yeast

KOH 400X KOH 400X

Amorphous material Amorphous material

KOH 400X KOH 400X

Amorphous material Amorphous material

KOH 400X KOH 400X

Pseudohyphae, budding yeast and

amorphous material

Pseudohyphae and budding yeast

KOH 400X KOH 400X

Wet Mounts: Other Common Morphotypes

Trichomonas vaginalis Neutrophils

Sperm Red blood cells

Epithelial Count = 8

Clue Cells = 3(38%)

BV MorphotypeDominatedFlora

Clue Cell NegativeEpithelial Count = 4

Epithelial Count = 6

Clue Cell Negative

Mixed Flora

Epithelial Count = 11

Clue Cell = 3(27%)

Budding yeast

Artifact

Pseudohyphae

“Bud”

artifact

RBCs

WBC covered with bacteria

Clue cell

Microstructures of leaves called stellate hairs found in a vaginal KOH prep

Know Your Microscope

Adjusting the Condenser for Contrast of Cells

Move the condenser up and down

Adjustments for Optimal Illumination

Adjust condenser aperture diaphragm

Centers the condenser

Adjust light source diaphragm

Care and Cleaning of Microscope

• Cover the scope when not in use• Use water or mild cleaning solutions for the body of

the scope• Clean the lenses with optics cleaning paper, Kimwipes,

or a cotton cloth (do not use facial tissue)• Use an optics cleaning solution to remove oily or

greasy dirt (fingerprints, immersion oil)• Check the alignment of the condenser with “Kohler

illumination”