evaluation of wet mount and koh preparations phase 1 pharmacokinetic trial of two intravaginal rings...
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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”
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