1. 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

2. 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

3. 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

4. Gram stain of vaginal flora Normal flora BV

5. 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?

6. Normal flora: No clue cells 400X1000X

7. BV: clue cells and WBC 400X1000X

8. BV: Clue cells 400X1000X

9. 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.

10. Normal Cells Clue Cells 400X

11. Normal Cells Clue Cells 400X

12. Clue Cells 400X

13. Normal CellsNormal Cells and yeast 400X

14. 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”

15. Pseudohyphae Budding yeast 400X

16. Budding yeast 400X

17. Pseudohyphae and budding yeast Budding yeast KOH 400X

18. Pseudohyphae and budding yeast Pseudohyphae, budding yeast and amorphous material KOH 400X

19. Pseudohyphae, budding yeast and amorphous material Budding yeast KOH 400X

20. Amorphous material KOH 400X

21. Amorphous material KOH 400X

22. Pseudohyphae, budding yeast and amorphous material Pseudohyphae and budding yeast KOH 400X

23. Wet Mounts: Other Common Morphotypes Trichomonas vaginalisNeutrophils SpermRed blood cells

24. Epithelial Count = 8 Clue Cells = 3 (38%) BV Morphotype Dominated Flora

25. Clue Cell Negative Epithelial Count = 4

26. Epithelial Count = 6 Clue Cell Negative Mixed Flora

27. Epithelial Count = 11 Clue Cell = 3 (27%)

28. Budding yeast

29. Artifact Pseudohyphae “Bud”

30. artifact RBCs WBC covered with bacteria Clue cell

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

32. Know Your Microscope

33. Adjusting the Condenser for Contrast of Cells Move the condenser up and down

34. Adjustments for Optimal Illumination Adjust condenser aperture diaphragm Centers the condenser Adjust light source diaphragm

35. 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”