Darkfield Microscopy

St. Louis STD/HIV Prevention Training Ctr.

Washington University School of Medicine
St. Louis, Missouri

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 Overview of syphilis
Infection with Treponema pallidum
Spirochetal organism
Primary infection: genital ulcers
Secondary infection: rash
Tertiary infection: brain, cardiovascular
system, skin, internal organs

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 Diagnosis of syphilis
Clinical evaluation of ulcers, rash
Darkfield exam of lesion exudate
Serologic testing
- non-treponemal tests (RPR, VDRL)
- treponemal tests (FTA-ABS, TP-PA)
Less widely available: DFA testing

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 Role of darkfield examination

Extremely specific for T. pallidum

Test of choice for moist ulcers
Offers immediate diagnosis
Opportunity for immediate treatment

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Primary syphilis-chancre

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Primary syphilis - chancre

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Primary syphilis - chancre

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Primary syphilis - chancre

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Primary syphilis - chancre

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Primary syphilis - perianal chancre

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 Secondary syphilis
Rash,
plantar
syphilids

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Secondary syphilis
Patchy alopecia
of secondary
syphilis

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 Who should be tested ?
Rule: EVERY genital lesion in
sexually active patients should be
examined by direct darkfield
microscopy
Possible exception: recurrent genital
herpes in known HSV+ patients

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 Specimen Collection / Preparation

Always observe universal precautions!

Rapid transport from patient to
microscope
Patience in viewing and interpreting
slide

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Darkfield microscopy
Uses oblique
illumination to
enhance contrast
Direct light is blocked
by a stop in substage
condenser
Light from oblique
angles forms bright
image on dark
background

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 Moist lesions
Remove scab, crust, exudate
Squeeze base of lesion to produce
tissue fluid on lesion surface
Apply glass slide directly to exudate, or
collect fluid with sterile loop
Alternative method: wash lesion with
sterile saline, transfer fluid to slide

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 Dry skin lesions
Gently remove superficial skin with
scalpel, needle tip, or mechanical
abrasion
Squeeze lesion base to collect tissue
fluid
Avoid contamination of specimen with
blood

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 Cervical/vaginal mucosal lesions
Use speculum to better visualize lesion
Remove cervical or vaginal discharge
or exudate
Compress lesion to produce serous
fluid accumulation at base of lesion
If necessary, use Kelly clamp to
compress

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Examination of lesion material
Place slide on darkfield microscope
stage
Apply oil to light source (not the slide)
Dim external room lighting
Scan slide slowly for characteristic
corkscrew-appearing organisms
Minimum 10-minute review of slide

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Darkfield view of T. pallidum

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Human treponemes
Organism Location Size Motility

T. pallidum Skin, mucosa ~10um, very thin, Slow with slow/rapid

tight coils (~10 coils) corkscrew, soft
bending in middle
T. refringens Normal genital flora 5um, thick, loose Rapid with rapid
coils (2-3 coils) rotation and marked
bending
T. phagedereis Normal genital flora 10um, thick, loose Slow, jerky, twists or
coils (10-12 coils) moves side to side

T. denticola Normal oral flora ~10um, very thin, Slow with slow/rapid
tight coils (6-8 coils) rotation, soft
bending

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Typical bended appearance of T.
pallidum

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Do NOT perform darkfield on:
Oropharyngeal lesions
Lip
Tongue
Tonsil
Cheek
Normal mouth flora may resemble syphilis
under the microscope (nonpathogenic
treponemes)

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Treponema

Left: darkfield view of T. pallidum

Above: brightfield view of T. denticola,
an agent of periodontal disease

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Spirochetes on darkfield microscopy

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Chancre of tongue

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Confounders of darkfield exam
Use of topical antibiotics by patient may
kill some organisms
Lack of sufficient serous exudate on
lesion surface
Insufficient time spent scanning slide

All of these factors can lead to a

false-negative interpretation
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Confirmatory testing for syphilis
Stat serologic test at time of clinical
exam
Careful evaluation of patient sexual
exposure history and lesion
characteristics
When in doubt, treat patient empirically
for syphilis, even in the face of
negative test results

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