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100X objective. Examine the saline portion for trichomonads and clue cells under the 400X objective.
- For dry specimens of skin scrapings or biopsies place a drop of KOH over the specimen already on the
slide, cover with a coverslip (probably not practical with biopsy), and observe under microscope as above. - What to Look For:
Candida (Yeast) and Fungi (tinea): KOH causes the epithelial cells to swell and break open which will
allow a clearer view of any filamentous or branching yeast or fungi.
Clue Cells: Vaginal epithelial cells to which bacteria are attached, obscuring the cell border. The edges
of vaginal epithelial cells are normally sharp and clear. The attached bacteria make the edges of the cell
appear fuzzy on low power. Small bacteria will be visible on higher power.
Trichomonas: Motile flagellated protozoa. They look like sperm with balloon-shaped heads
and are about the size of a normal epithelial cell.
What Not To Do:
Do not add too much KOH or saline allowing the fluids to mix or run off the slide.
Do not forget to perform the whiff test.
Lab Procedure: Gram Stain
18D Skills and Training ManualWhen: You have a culture with one or more organisms growing in it. Prepare and stain a smear so
that gram-negative organisms appear pink to red and gram-positive organisms appear blue to purple with
100% accuracy.
What You Need: A sample for testing, a Bunsen burner, Gram stain reagents, an inoculating loop, an
inoculating needle, glass slides, water, a staining rack, a diamond point pen or lead pencil, needle, syringe,
disposable transfer pipets, a timer, and a logbook.
What To Do:
- Label a slide with a lead pencil or diamond point pen.
- Prepare the smear.
a. From a liquid specimen or medium, (if applicable).
(1) Place 1 drop in the center of a labeled slide using a sterile pipet, syringe, or loop.
(2) Spread inoculum to about the size of a dime.
(3) Allow the smear to completely air-dry.
b. From a colony on an agar plate (if applicable):
(1) Place a small drop of water in the center of the labeled slide.
(2) Touch the top center of the colony with a sterile inoculating needle and transfer a small amount
of bacteria to the drop of water.
(3) Mix the bacteria and water with the needle and spread it to about the size of a dime.
(4) Allow the smear to completely air-dry.
c. From a swab (if applicable):
(1) Roll the swab across the center of a dry labeled slide.
(2) Allow the smear to completely air-dry. - Use either of the following methods to fix the smear:
a. Methanol.
(1) Flood the smear with methanol and allow it to stand for 1 minute.
(2) Tilt the slide to drain off excess methanol and allow it to completely air-dry.
b. Heat. Gently heat the smear by passing it through the burner flame two or three times. - Stain the slide.
a. Flood the slide with crystal violet for 1 minute.
b. Rinse the smear with tap water.
c. Flood the slide with Gram’s iodine for 1 minute.