1. Inspect external genitalia, vagina, and cervix
2. Collect vaginal secretions
3. Collect cervical secretions
4. Measure the vaginal pH

Inspect external genitalia, vagina, and cervix

Specimens for vaginal secretion examination are collected as part of the pelvic examination. First examine the external genitalia and perineum, looking for vulvar edema, erythema, or excoriations that may be due to candidiasis or trichomoniasis. Candida satellite lesions are frequently found on the vulva or inner thighs. Next insert the speculum. Click here to review speculum placement.

Examine the vaginal mucosa and secretions. Carefully inspecting the mucosa and amount, color, viscosity, and odor of vaginal secretions can be quite helpful in securing a diagnosis. Click here for the characteristics of vaginal discharge in common conditions. Vaginal dryness and atrophy are seen in atrophic vaginitis. The vaginal mucosa is moist and pink in the healthy state or with bacterial vaginosis. Erythema and edema are often present with candidiasis and trichomoniasis or other inflammatory state.

Next examine the cervix, noting any discharge that may be coming from the os. Normal cervical discharge is clear in the early phase of the menstrual cycle, turning thicker and scant later in the cycle. If a yellow discharge is present, this is always abnormal and indicates cervicitis (Morse, Eschenbach). If a large amount of vaginal secretions is present, wipe the cervix using a large cotton swab to better visualize the cervical os and any evident discharge. Examine the appearance of the cervix itself, looking for erythema or petechiae, which may be seen in approximately 10% of cases of Trichomoniasis (Eschenbach , Dunkelberg). If any atypical appearance is noted, a Papanicolaou (Pap) smear should be done.

Collect vaginal secretions
Rub a small cotton-tipped applicator along the vaginal walls and fornices. Insert the swab into a small test tube in which approximately 1 ml of saline solution has been placed. Set this aside for later microscopic analysis.

Another sample using a second swab may be obtained if Gram stain or culture is indicated. For Gram stain, the swab may be placed in transport media and sent directly to the laboratory or rolled along a glass slide and allowed to air dry. There is no need to heat fix the glass slide because the vaginal smear is stable at room temperature for several weeks (Sweet). In the laboratory, the slide will be heat fixed and Gram stained. If cultures of vaginal secretions are desired (looking for candidiasis or trichomoniasis), a standard culturette swab should be used. Simply swab the vaginal walls and fornices with a culture swab, and place in culterette media for transport to the laboratory (Sweet). Make sure to label with the patient's name, culture source, and any additional information the laboratory might find helpful. Do not hesitate to call the lab with any pertinent clinical information.

Collect cervical secretions
Next, sample cervical secretions if indicated by symptoms or examination findings (such as purulent cervical discharge). Whether a Gram stain, culture, or molecular probe assay is done, the collection technique is the same. Separate swabs should be used for each. First remove ectocervical secretions using a large swab. Place a small-tipped swab in the endocervical canal. (If you are doing a molecular probe assay, use the swab provided in the sampling kit). Be sure that the cotton tip is completely in the os. Gently twirl slowly in a clockwise direction for 10 to 15 seconds. Carefully extract the specimen, avoiding contact with the vaginal mucosa (Sweet, PACE2.) To better assess color, inspect the secretions on the swab tip against a white background.

• Molecular probe assays Molecular probe assays for Chlamydia and gonorrhea have become very popular because of their accuracy and ease of specimen handling. Simply place the swab with the cervical secretions on it into the vial of transport media provided with the molecular probe kit. Apply a label, and send it to the laboratory for processing. No special environment or temperature is required, and the specimen remains stable for 7 days after collection. Both Chlamydia and gonorrhea assays may be done from one specimen (Pace).
  
• Gram stain
  If a Gram stain of cervical secretions is desired, either roll the swab on a glass slide or place it in transport media to be sent to the laboratory. See the section on interpreting results for the typical appearance of gonorrhea on the Gram stain.
  
• Cultures
  Although many clinics are turning to alternative methods of diagnosing gonorrhea and Chlamydia, cultures are still considered the gold standard (Morse, Phillips). If a standard culture technique is to be used, the swab should be immediately streaked onto Neisseria-selective media (chocolate agar or Thayer Martin media) and incubated at 35° to 36.5° C in a CO₂-enriched atmosphere. Special transport containers (e.g., candle jars) should be used to maintain the proper environment until transported to the laboratory incubator (Sweet).
  
  The standard laboratory technique for Chlamydia culture uses cycloheximide-treated McCoy cells stained with fluorescein-labeled monoclonal antibodies (Morse). Again, proper collection of the specimen and handling are essential for high diagnostic yield. A swab with a shaft made of inert material (i.e., plastic rather than wood) is used. Individual manufacturer directions for sample collection materials should be followed with transport media specific for the Chlamydia culture used. Chlamydia trachomatis in clinical specimens has limited viability at room temperature; place specimens on ice and transport immediately (Morse, Weinstock).

Measure the vaginal pH

Remove the vaginal speculum and note any exudate or secretions. Apply a strip of pH paper to the pooled secretions in the withdrawn speculum or directly to the vaginal wall using a cotton swab. Make sure that the pH paper is directly on the vaginal secretions and not sampling water or lubricant (which may be alkaline). Cervical secretions are also more alkaline than normal vaginal discharge and should be avoided as well. Narrow-range pH paper (4.0 to 5.5) is easier to read than broad-range paper (4.5 to 7.5), but either may be used. Compare the color of the test tape used on secretions to the color guide on the pH tape dispenser to determine the pH of the secretions.

The vaginal pH can be helpful in the differential diagnosis of vaginitis:

• pH 4.0 - 4.5: Normal flora, Vulvovaginal Candidiasis
• pH >4.5: Bacterial vaginosis
• pH 5 - 6.0: Trichomoniasis
• pH >6.0: Atrophic vaginitis

The remainder of the evaluation is done in the office laboratory. Click here to jump to the section on office testing of vaginal secretions.