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Wet Mount Examinations

Reprint from Focus On, 1998, Issue 3

The direct examination of a clinical specimen is the most rapid, cost-effective diagnostic aid in the laboratory. Microscopic examination of a sample is used to identify the motility and morphology of microorganisms, fungal elements, protozoan trophozoites, and helminth eggs and larvae. The visual examination assists the laboratory and the clinician in quickly identifying the causative agent of infection so treatment may begin. Proper sample collection is important in diagnosing infection. Samples may be treated with potassium hydroxide (KOH) to enhance the recovery of fungi or stained with iodine to enhance the recovery of trophozoite nuclei and vacuoles. Wet mount samples may be obtained from various clinical sites and sources. This article should answer your questions about specimen collection, common causes of infection, and the enhancements used in examining the specimens.

Vaginal Wet Mounts

Vaginal wet mounts aid in the diagnosis of vaginitis, which is caused by a variety of organisms. Patients with vaginitis may present with vaginal discomfort, itching, discharge, dysuria, or a foul odor. Proper diagnosis and treatment of vaginitis can prevent the onset of a more serious infection. The examination of vaginal secretions lends to the recovery of organisms causing infection and allows for more accurate treatment regimens.

The vaginal area requires a balance between normal and abnormal bacteria. Normal vaginal bacteria primarily consists of Lactobacillus acidophilus appearing as gram positive rods. Lactobacillus produces lactic acid which deters the growth of opportunistic pathogens by keeping the vaginal environment acidic with a pH between 3.8-4.2. Other bacterial species and small numbers of yeast may normally be present in the vaginal environment. Any disturbances in this natural balance, such as taking antibiotics for other bacterial infections, may cause an over-abundance of the other organisms leading to vaginitis.

Proper specimen collection and handling of vaginal samples is crucial for accurate results. The vaginal vault and walls should be swabbed using one or two swabs. If any fluid has pooled in other areas, these areas should be swabbed as well. The swabs should then be placed in a tube containing 0.5 ml saline and examined within two hours of collection. The sample should remain at room temperature.

After obtaining the sample, it is equally important to properly prepare the slide for examination. The direct, unstained slide should be prepared as follows:

  1. Vigorously mix the swab(s) in and out of the saline making sure to collect all the material adhering to the side of the tube.
  2. Remove the swab from the saline and depress onto a clean, dry microscope slide expressing a small amount of fluid.
  3. Coverslip the sample and examine under a microscope.

The KOH slide may be prepared by adding a drop of KOH to the sample after following the directions as noted above. The saline slide should be examined first to allow the KOH to properly digest other cellular elements in the sample such as epithelial and blood cells.

The pH and the amine ("whiff") test often are performed before the microscopic examination. The clinician tests the pH at the time of sample collection. A pH greater than 4.5 may indicate bacterial vaginosis (BV) or trichomoniasis. The amine test is performed prior to cover slipping the KOH preparation. A "fishy" or amine odor is characteristic in the presence of Trichomonas vaginalis and Gardnerella vaginalis.

Begin the microscopic review of the slide by examining the saline preparation. The examination should begin using the 10x objective noting cellular distribution and obvious cellular and fungal elements. The 40x objective is used to identify the presence of white and red blood cells, quantity and type of bacteria present, clue cells, motile Trichomonas, yeasts, and fungal hyphae. The KOH slide should be examined with the 10x objective for any yeast and hyphae present and the 40x objective is used to distinguish smaller budding yeasts and hyphae.

Common causes of bacterial vaginitis include Trichomonas vaginalis, Gardnerella vaginalis, and Candida albicans. All of these infections can be diagnosed by direct examination. The absence or presence of white blood cells and normal vaginal flora are clues that can be directly related to the causative agent of infection. The amine test and vaginal pH also can help to determine the cause of infection.

Trichomoniasis is caused by Trichomonas vaginalis. This single-celled parasite is transmitted sexually. Commonly, the parasite is motile with its flagella whipping back and forth. However, it may not be motile and can easily be missed or confused with white blood cells. The infection is associated with large numbers of white cells with a positive amine test and the absence of normal vaginal flora.

Gardnerella vaginalis is often characterized as non-specific vaginitis. Microscopically it is characterized by a lack of normal vaginal flora and a predominance of many small coccobacilli. The small bacteria adhere to the surface of the epithelial cells creating a speckled appearance. These speckled cells are called "clue cells" and their presence is considered diagnostic for Gardnerella vaginalis induced vaginitis. Gardnerella infections do not provoke a large white blood cell response and have a positive amine test.

Yeast infections, commonly caused by Candida albicans, are easily identified upon direct examination. As expected, the direct examination will yield budding yeasts and hyphae. The use of KOH enhances the recovery of these fungal elements. Yeasts provoke a large white blood cell response with a negative amine test. Normal vaginal flora will be present.

Laboratories should establish their own criteria for diagnosing vaginitis. In most cases, the direct examination of vaginal secretions in a symptomatic patient will result in the proper diagnosis of the infection and allow for prompt treatment. Use of vaginal lubricants, douches, tampons, contraceptive jelly, and medications may interfere with the examination. Patients should avoid using these items before sample collection. In some cases, the Gram stain may be used to aid in the diagnosis of these patients.

Skin Scrapings

Skin scraping examinations are used to identify fungal infections of the skin, mouth and nails. Fungus lives in the stratum corneum layer of the skin. KOH is used to digest this layer of skin so that the fungal growth with its visible hyphae and spores is exposed. Skin scraping examinations are indicated in areas of broken hair or baldness, presence of scaly lesions on the skin, crumbling or scaly nails or nail beds, and white plaque in the mouth.

The proper collection of a skin scraping sample is crucial to the recovery of the fungus. In obtaining and preparing the sample:

  1. Clean the infected area with 70% alcohol. Scrape the infected area using a scalpel, skin curette, tongue blade, or other blunt object. Infected nails may require a portion of the infected nail to be clipped.
  2. Place the sample on a microscopic slide and coverslip.
  3. Apply gentle heat to enhance the digestive capabilities of the KOH. If 40% dimethyl sulfoxide (DMSO-KOH) is used, heating the sample is unnecessary.
  4. Let the slide sit for 10 minutes to allow for KOH digestion.
  5. Examine the slide using the 10x objective. All suspicious areas should be examined using the 40x objective.

Keep the light intensity of the microscope low. If the light intensity is too high, the fungal elements may not be seen.

Fungal infections are commonly caused by Tinea species, Trichophyton rubrum, and Candida albicans. These fungi infect various parts of the body and have varying characteristics.

The Tinea species infect different parts of the body and their names reflect their common sites of infection. Tinea versicolor infects the skin and is characterized by clustered, rectangular spores and long, branched hyphae. Short, small spores or hyphae parallel to the hair shaft are characteristic of Tinea capitis. Tinea corporis causes what is commonly referred to as ringworm. Hyphae will always be present upon examination. Athlete's foot, caused by Tinea pedis, does not require a KOH preparation to be diagnosed as few diseases mimic this infection. Onychomycosis, caused by Tinea ungunium, infects the nails and nail beds and is the hardest fungal infection to treat.

Trichophyton rubrum is a fungal infection commonly seen in adults. It causes a dry, inflamed rash on the hands, feet, nails, and bathing suit area. The KOH smear is often negative and may require a fungal culture to diagnosis.

Oral candidiasis (thrush) is commonly caused by Candida albicans and is most often seen in infants and small children. It is characterized by red, moist lesions and pustules in and around the mouth. The KOH examination will reveal filaments of budding spores and oval yeast bodies.

Fecal Suspensions

The direct examination of a fresh stool sample is of great importance when diagnosing a parasitic infection. Examination of a fecal suspension is indicated when a patient has experienced several days of watery diarrhea or diarrhea containing blood or mucous. The sample should be collected in a clean dry container devoid of urine or water. A series of three samples should be obtained for examination. Do not place the sample in the refrigerator. Fecal material received with preservative is not suitable for direct examination because trophozoite motility would be nonexistent.

The protocol for handling stool samples may be different depending on the consistency of the samples. Protozoan trophozoites and cysts normally are found in loose, watery stools. Helminths eggs and larvae are commonly found in formed stools. The detection and motility of a trophozoite can be made from direct examination of a fresh stool sample. Watery stools may not require the use of saline to prepare the slide. Fecal suspensions from a formed stool specimen may be prepared by mixing a small amount of stool with a drop of saline.

Direct examination of fecal material is beneficial to assess the worm burden of a patient, provide a quick diagnosis in a heavily infected sample, and to detect motile protozoa. Prepare the slide and examine it under the 10x objective. One-third of the slide should be reviewed for suspicious organisms. Any suspicious organism should be more closely examined under the 40x objective. The smear may be treated with a drop of iodine. Iodine will cease protozoan motility and enhance the nuclei and vacuoles in the organism.

Sperm Examination

The presence or absence of sperm may be determined from vaginal or semen samples. In vaginal samples, this procedure may be indicated in the examination of a post-coital or sexual assault incident. Direct examination of a semen sample is indicated in a post-vasectomy sample to measure the efficacy of the surgical sterilization procedure. For either clinical sample, the direct examination is performed using the direct, unstained slide preparation steps noted under the vaginal wet mount section.

Though often an under-used tool in the clinical laboratory, wet mount examinations provide a quick and efficient way in which to diagnose vaginitis, fungal skin infections, parasitic infections, and the effectiveness of a sterilization procedure. Direct examination should be performed before more invasive procedures are used to make a diagnosis. Costing less than $1 per examination, wet mounts provide a cost-effective way to make a rapid diagnosis. In some cases, when the direct wet mount preparation may not be the best means to obtain a diagnosis, the laboratory may employ staining methods, rapid antigen detection methods, or culture techniques to identify the causative agent of infection.

References:

Fischer PM, Addision LA, Curtis P, Mitchell JM. The Office Laboratory. Appleton-Century-Crofts: East Norwalk, CN, 1984.

Mass, D. "Ensuring Correct Diagnosis in Testing for Vaginitis." Medical Office Report; Volume XI (3): 2-3.

Murry, PR et al. Manual of Clinical Microbiology. 6th ed. Washington, DC: ASM; 1995.

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