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Program Operations Guidelines for STD Prevention
Medical and Laboratory Services

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Appendix ML-C

Commonly Used Stat Tests-Useful Tips

GRAM STAIN FOR MICROORGANISMS

Test Principles

The Gram stain is the most commonly used stain in bacteriology. It is classified as a differential stain and serves to distinguish the gram-positive from the gram-negative bacteria. The original Gram stain technique has been modified a number of times, and the usual recommended procedure is the Hucker modification.

Although the Gram stain is among the least complicated and least time-consuming of all microbiological tests, the information that may be obtained from a properly stained smear of a specimen from a client is one of the most valuable aids to the clinician and the laboratorian. A properly performed stain can provide important diagnostic information concerning the type of organisms present, and the therapy to initiate while waiting for other test results. In the stat STD laboratory setting, the Gram stain is used to aid in the diagnosis of gonorrhea, candidal vulvovaginitis, and bacterial vaginosis, and in the assessment of urethritis, cervicitis, proctitis, and other infections characterized by infected discharges. Both the numbers of polymorphonuclear leukocytes (PMNs) and microbial flora present can be assessed (Stamm, 1988).

Specimen Collection

Male urethral smear

Patient should not urinate prior to specimen collection. Insert a small swab into the urethra.

Cervical smear

Wipe the cervix before collecting the specimen to reduce the amount of vaginal bacteria and cells in the smear.

Rectal smear

Use an anoscope to collect the specimen and sample areas containing pus.

Smear Preparation

To prepare a direct smear from a patient, roll swab with patient's specimen on a clean glass slide, making a thin spread; do not smear (leukocytes may be disrupted) or prepare a thin smear from a culture in a drop of water on the slide. Air dry the smear and fix to the glass by rapidly passing the slide through a Bunsen burner flame two or three times. The slide should be slightly warm to the skin on the back of the hand. Do not use swab from a DNA probe or Pap smear for a Gram stain.

Staining Schedule

1. Stain smears with crystal violet ammonium oxalate.
2. Wash in tap water.
3. Apply Gram's iodine solution.
4. Wash in tap water.
5. Decolorize with 95% ethyl alcohol until washes are no longer blue
6. Wash and shake off excess water.
7. Apply counterstain of safranin.
8. Wash in tap water and blot dry.

Examination of Slide and Interpretation of

Results

1. Scan the stained smear with the 10X objective to locate the best area for viewing.
2. Examine the smear microscopically with the oil immersion objective.
3. Gram-positive organisms appear purple and gram-negative organisms appear red. Search for organisms and count PMNs. Cells and mucus should stain pink. Yeast stain purple. Bacteria are characterized as gram-positive (purple) or gram-negative (pink) and as cocci (round), bacilli (rod shaped), or coccobacilli (in between rods and cocci).
4. Control slides of representative gram-positive and gram-negative organisms should be examined each time Gram stains are performed.

Note: If using commercial kits or reagents, follow manufacturer's instructions in the product insert.

Sources of Error

1. Scrubbing, not rolling, the swab across the slide may destroy cellular morphology.
2. Failure to heat-fix the slide may cause material to wash off during staining.
3. Overheating the slide may cause artifacts to be stained and cells to be distorted.
4. Use of Gram's Iodine solution beyond expiration date (shelf life of reagent at room temperature is approximately 90 days).
5. Over-decolorizing the slide may cause gram-positive organisms to appear gram-negative.
6. Under-decolorizing the slide may cause gram-negative organisms to appear gram-positive.
7. Reagents contaminated with microorganisms may give erroneous results.

 

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