Demonstration of Treponema pallidum Using Dark-Field Microscopy

Treponema pallidum is a thin, delicate, tightly wound spirochaete that cannot be seen in Gram-stained smears. So, dark-field microscopy is used to demonstrate the presence of motile Treponema pallidum in lesions or aspirates in early-stage (primary or secondary) syphilis. Serous fluid from genital chancre or skin lesion must be examined immediately (within 20 min) to observe motile treponemes.

Dark-field microscopy has a sensitivity of approx 80% in primary syphilis but the sensitivity declines as the infection progress and also if the patient has already been treated with topical antibiotics. The sensitivity of this test largely depends on proper sample collection (i.e obtaining serous exudates from lesions while avoiding blood or pus) followed by placement of collected specimens on a slide and examination within 20 minutes of collection.

As this method requires a special microscope and a trained microscopist close to the site of specimen collection, it is not done in routine clinical practice. 

Materials

1. Dark-field microscope with parfocal 10, 40 to 45, and 100 oil immersion objectives, 10 oculars, dark-field immersion condenser (single or double deflecting), and a 6.0- to 6.5-V high-intensity lamp with variable transformer for regulating light intensity
2. Microscope Slides, 1 by 3 in.
3. Coverslip, 22 by 22 mm
4. Immersion oil, nondrying

Caution: T. pallidum spirochaetes are highly infectious.

Specimen Collection

To detect motile Treponema pallidum spirochaete, collect serous fluid from lesion prior to antimicrobial therapy.

1. Wearing protective rubber gloves, clean the surface of the lesion (chancre) using a swab moistened with physiological saline, and blot dry.
2. Gently remove any crusts (scab), and discard.
3. Abrade superficially until slight bleeding occurs, using a needle, scalpel blade, or broken glass slide. Irrigate with sterile saline and wipe away the first few drops of blood, etc.
4. Gently squeeze the lesion to obtain serous fluid. Collect a drop on a cover glass and invert it on a microscope slide.
5. Apply gentle pressure at lesion base, touching clear exudate in ulcer base with a glass slide.
6. If no exudate is present, add a drop of saline to the lesion or insert a needle and syringe at the lesion base, aspirate, and then draw a drop of saline into the needle. Express the material onto a slide and place a coverslip immediately.
7. Examine the slide by dark-field microscopy within 20 min of collection.

Microscope Examination Procedure

• Search the entire specimen with a high dry objective for spiral or helical organisms.
• Center suspicious objects, and examine them under oil immersion objective.
• Upon completion of the examination, discard slide into a container of appropriate disinfectant.

Interpretation

• T. pallidum organisms appear as delicate, corkscrew-shaped, rigid, uniform, tightly wound, deep spirals; coil appearance is maintained even while organisms are actively motile.
• Observe for rotational motility around longitudinal base; backward and forward movement; flexion, bending, or twisting from side to side; and snapping motion.
• Spirochetes are 6 to 14 lm long, which is slightly longer than the diameter of an erythrocyte.

Reporting Results

1. When organisms are seen that have characteristic morphology, shape, and motility of T. pallidum, report “Treponemas resembling T. pallidum observed.”
2. When no treponemas are observed, report “No treponemas resembling T. pallidum observed.”

References and further readings

• Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology. https://doi.org/10.1128/9781555818814
• District Laboratory Manual in Tropical Countries, Part 2; Cambridge University Press