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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 1000X 400X BV: clue cells and WBC 1000X 400X BV: Clue cells 1000X 400X 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 400X Clue Cells 400X Normal Cells 400X Clue Cells 400X Clue Cells 400X Normal Cells 400X Normal Cells and yeast 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” Budding yeast Pseudohyphae 400X Budding yeast 400X Pseudohyphae and budding yeast Budding yeast KOH 400X KOH 400X Pseudohyphae and budding yeast KOH 400X Pseudohyphae, budding yeast and amorphous material KOH 400X Pseudohyphae, budding yeast and amorphous material KOH 400X Budding yeast KOH 400X Amorphous material KOH 400X Amorphous material KOH 400X Amorphous material KOH 400X Amorphous material KOH 400X Pseudohyphae, budding yeast and amorphous material KOH 400X Pseudohyphae and budding yeast KOH 400X Wet Mounts: Other Common Morphotypes Trichomonas vaginalis Sperm Neutrophils Red blood cells Epithelial Count = 8 Clue Cells = 3 (38%) BV Morphotype Dominated Flora Clue Cell Negative Epithelial Count = 4 Mixed Flora Epithelial Count = 6 Clue Cell Negative Epithelial Count = 11 Clue Cell = 3 (27%) Budding yeast Artifact “Bud” Pseudohyphae Clue cell RBCs artifact WBC covered with bacteria 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 Centers the condenser Adjust condenser aperture diaphragm 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”