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Dark Field Microscopy

Theory of darkfield microscopy

First, the stop blocks the center of the beam of light that would
otherwise fill the objective lens.

Second, only the light which is scattered by the specimen and

enters the objective lens is seen. Therefore, the best viewing
result requires increasing the light intensity as much as possible:
by setting the light intensity adjustment at maximum, by opening
the field diaphragm, by opening the condenser aperture, and by
removing any color or other filters. The correct microscope slides
also should be used; they should be 1mm thick.

The standard brightfield microscope may be equipped for darkfield

examination by replacing thebrightfield, or Abbe, condenser with either
a double-or single-reflecting darkfield condenser.Illumination for
darkfield microscopy is obtained when light rays strike the object in the
field at an oblique angle so that no direct light rays enter the microscope
objective, only the rays reflected from the object.
Darkfield microscopy relies on a different illumination system. Rather
than illuminating the sample with a filled cone of light, the condenser
is designed to form a hollow cone of light. The light at the apex
of the cone is focused at the plane of the specimen; as this light
moves past the specimen plane it spreads again into a hollow
cone. The objective lens sits in the dark hollow of this cone; although
the light travels around and past the objective lens, no rays enter it .
The entire field appears dark when there is no sample on the microscope
stage; thus the name darkfield microscopy. When a sample is on the
stage, the light at the apex of the cone strikes it. The image is
scattered by the sample and
captured in the objective lens .The
image appears bright against the dark
background. This situation can be
compared to the glittery appearance
dust particles in a dark room
illuminated by strong shafts of light
coming in through a side window. The
dust particles are very small, but are
easily seen when they scatter the light
rays. This is the working principle of
darkfield microscopy and explains how
image of low contrast material is
created: an object will be seen against a dark background if it
scatters light which is captured with the proper device such as
an objective lens.
Darkfield microscopy reduces the amount of light entering the lens
system of a microscope in two ways.

The illumination needs to be aligned and adjusted to achieve the best

Before making the darkfield modification, align the light beam in

the center of the field of view according to the manufacturers

To facilitate focusing the substage condenser and the objective

lens, use a slide filled with samples that are easy to find;
instructions for making a cheek cell slide follow.

Focus the sample slide at low magnification (10X) in the

brightfield mode.

Insert the darkfield stop without changing the focus.

Make sure that the maximum amount of light is available. Rack

up the condenser to its highest position with the condenser focus

Look at the sample and slowly lower the condenser until the
sample is visible against a dark background and in sharpest

Finally, adjust the view of the image with the fine focus knob.

Limitation of darkfield microscopy

The advantage of darkfield microscopy also becomes its disadvantage:
not only the specimen, but dust and other particles scatter the light and
are easily observed. More care in sample preparation needs to be
exercised in darkfield application. Glass slides need to be thoroughly
cleaned of extraneous dust and dirt. It may be necessary to filter sample
media (agar, water, saline) to exclude confusing contaminants. Sample
materials need to be spread thinly; too much material on the slide
creates many overlapping layers and edges making it difficult to
interpret structures.
To create the image, this technique relies on scattered light from
specimens. Color is lacking or minimal; this can be disappointing to the
viewer. The actual size of specimens is also impacted; the width of
objects becomes exaggerated.
Result Interpretation

The demonstration of treponemes with characteristic

morphology and motility for T. pallidum constitutes a positive
diagnosis of syphilis in primary,secondary, or early congenital
stages, whatever the outcome of serologic testing.
False-positive darkfield tests may occur with oral specimens;
therefore, such positive specimens must beconfirmed by
direct fluorescent antibody tests specific for identification of T.
When patients with untreated primary syphilis are positive by
darkfield microscopy but are serologically nonreactive, they
usually become serologically reactive within several days to
several weeks. In other stages, the patient should be
seroreactive; if not, the darkfield interpretation and
serologicresults should be analyzed to decide whether test
results may be false-positives or false-negatives, respectively.

Every genital lesion should be considered syphilitic until proven

otherwise. Extragenital lesions characterized by indolence,
induration, and regional lymphadenopathy should be regarded as
possibly syphilitic. Failure to find the organism does not exclude a
diagnosis of syphilis.
Negative results may be reported for the following reasons:
1. The number of organisms was insufficient for detection.
2. The patient has received antitreponemal drugs, topically or
3. The lesion is "fading" or approaching natural resolution or
4. The lesion is one of late syphilis.
5. The lesion is not syphilitic.
When the darkfield examination is negative in patients suspected of
having primary syphilis, repeated examination (on as many as 3
consecutive days) or aspiration of enlarged regional lymph nodes may
be indicated. Theoretically, serologic tests for syphilis should be
repeated at 1 week, 1 month, and 3 months. If nonreactive serologic
results are obtained for longer than 3 months inuntreated patients,
syphilis may be excluded as the cause of such lesions. Among
patientssuspected of having syphilis in other stages, a negative darkfield
examination and nonreactiveserologic tests suggest that syphilis is
extremely unlikely, and follow-up tests are unnecessary .


Specimen Collection for Syphilis


To avoid accidental infection when collecting specimens,

observe universal precautions
Before collecting specimens, always make sure that the
darkfield microscope is in good working order.

1. The ideal specimen for darkfield examination is a serous fluid that
is rich in T. pallidum but that contains few blood cells (treponemes
may be obscured if many cells are present).
2. Consider every genital lesion in sexually active patients as syphilis
until subjected to a darkfield examination and proven otherwise.
Other lesions on the skin or mucous membranes should also be
examined when syphilis is suspected.
3. Darkfield examination of oral lesions is not recommended. All
positive darkfield tests with mouth specimens must be confirmed
by a direct fluorescent antibody test. The indigenous flora of the
oral cavity frequently contain a spiral organism, T. denticola, which is
indistinguishable from T. pallidum.
4. If topical antimicrobial therapy has been applied to a syphilitic lesion,
it may not be possible to demonstrate motile T. pallidum, even if
several specimens are examined. In this instance, an aspirated
sample from an enlarged regional lymph node may be used
for diagnosis.
1. Lesions



Remove any scab or crust covering the lesion.

Secondary infection exudate, if any, should be removed
with a gauze sponge.
If necessary, compress the base of the lesion or apply a
suction cup to the lesion to promote the accumulation of
tissue fluid on the ulcer surface.
Apply a glass slide to the oozing lesion or use a sterile
bacteriological loop totransfer the fluid from the lesion to
the glass slide.
Place a cover glass on the specimen and flatten or depress
it evenly on the slide,using the blunt end of an applicator

stick to remove air bubbles.


Examine the slide immediately.

To prevent drying, place additional slides with specimens in
a moist chamber such as a large plastic petri dish
containing a moistened paper towel.
Note: The slide preparations should not contain a large volume of
fluid (largevolumes cause a rapid liquid flow across the field), nor
should the preparation be so thin that it begins to dry before an
adequate examination can be made.
2. Dry papulosquamous lesions of the skin
a. Gently remove the superficial layer of skin with a scalpel,
needle tip, or mechanical abrader.
b. Try not to cause bleeding. If very little serous fluid appears,
compress the lesion.
c. Touch the corner of the surface of a microscope slide to the
fluid or use a sterile bacteriological loop to transfer the
material to the slide.
d. Material can also be collected by injecting a small drop of
sterile saline into the baseof the lesion and aspirating the
fluid with a small-gauge needle and syringe.
3. Cervical/vaginal lesions
a. With visualization by a bivalve speculum, remove any
cervical or vaginal discharge.
b. Obtain serous exudate with a sterile bacteriological loop.
c. Serous exudate, if necessary, can be produced from the
lesion by compressing itwith a Kelly clamp.
d. Prepare slides as described in no. 1.
4. Mucous patches
a. Using a sterile bacteriological loop, collect some of the
mucous material and place iton a clean glass slide.
b. Place a cover glass on the specimen and examine
5 . Lymph node
a. Disinfect the skin over the node by swabbing it with iodine
and alcohol or anothersuitable agent.
b. Rinse a sterile 20-gauge needle and a 2-ml syringe with
sterile physiological saline.
c. Allow 0.2 ml or less of the saline to remain in the syringe.
d. Hold the node firmly and insert the needle well into the
node. The ability tomanipulate the node freely with the

needle tip is a good indication that the capsuleof the node

has been pierced.
e. Inject the sterile physiological saline into the node.
f. Macerate the tissue by gently manipulating the needle in
various directions.
g. Aspirate as much fluid as possible.
h. Discharge the aspirated material onto slides for immediate

Reference :


blockers, lithium, salicylates, or corticosteroids
Lack of male circumcision
Multiple sex partners, lifetime or current
Nonrecognition of ulcers in prodrome stage
Serodiscordant sex partners (i.e., one partner with herpes simplex
virus and one without)
Unprotected sexual contact
Unprotected skin-to-skin contact with ulcers

Laboratory evaluation of an initial genital ulcer outbreak should
include culture or polymerase chain reaction testing for HSV
infection, HSV type-specific serology, serologic testing for
syphilis, and culture forH. ducreyi in settings with a high
prevalence of chancroid.

HSV Infection

reaction testing is 96 to 100 percent sensitive and 97 to

98 percent specific (positive likelihood ratio = 49,
negative likelihood ratio = 0.02), much more sensitive
than culture.

Diagnosis Evaluation of Genital

The diagnosis of genital ulcer disease is based on the presence of one or
more mucocutaneous ulcers involving the genitalia, perineum, or
anus.5 Diagnosing the specific cause of genital ulcers is based on history,
physical examination, and laboratory findings.


Risk Factors for Genital Ulcers

History of inflammatory disease (e.g., psoriasis) and exposure to
trauma or medications such as nonsteroidal anti-inflammatory drugs,

Darkfield microscopy and direct fluorescent antibody tests

of exudate or tissue material are the definitive methods
for diagnosing primary syphilis.


Risk factors for infectious causes of genital ulcers are similar to those for
STIs, whereas risk factors for noninfectious causes vary

For the diagnosis of HSV infection, polymerase chain

a presumptive diagnosis of syphilis can be made with a

serologic nontreponemal test (i.e., Venereal Disease
Research Laboratories or rapid plasma reagin).

But, because of possible false-positive results, positive

nontreponemal test results should be confirmed with
serologic treponemal testing (i.e., fluorescent treponemal
or T.
pallidum passive
agglutination). Nontreponemal titers typically decline and
may become nonreactive after treatment, but most
positive treponemal test results tend to remain
persistently active.

Reference :



Although a definitive diagnosis of chancroid requires

identification of H. ducreyi, testing with special culture
media is less than 80 percent sensitive and polymerase
chain reaction testing for H. ducreyi is not available in the
United States.

A presumptive diagnosis is possible with a painful genital

ulcer, regional lymphadenopathy, no evidence
pallidum infection, and negative HSV test results.

Even with appropriate laboratory testing, no pathogen is identified in up

to 25 percent of patients with genital ulcers. Biopsy is rarely needed to
diagnose the cause of genital ulcers, but it may be considered if an ulcer
persists after treatment.

If primary syphilis is treated, up to 25 percent of patients

may have nonreactive treponemal results in two to three
years; however, treponemal titers are not recommended
to evaluate response to treatment

professionals may collect and send rectal specimens to

state health departments for referral to the Centers for
Disease Control and Prevention for testing and validating
diagnostic methods for lymphogranuloma venereum.

of T.

lymphogranuloma venereum

To diagnose lymphogranuloma venereum, genital swabs

for C.
trachomatisserotypes L1, L2, and L3 by culture, direct
immunofluorescence, or nucleic acid amplification. Nucleic
acid amplification tests for lymphogranuloma venereum
are not approved by the U.S. Food and Drug
Administration for rectal specimens. Health care


The Penis
Inspect the penis, including:
- The skin
- The prepuce (foreskin). If it is present, retract it or ask the patient
to retract it. This step is essential for the detection of many
chancres and carcinomas. Smegma, a cheesy, whitish material,
may accumulate normally under the foreskin.
- The glans. Look for any ulcers, scars, nodules, or signs of
o Check the skin around the base of the penis for
excoriations or inflammation.
o Look for nits or lice at the bases of the pubic hairs.
o Note the location of the urethral meatus.
o Compress the glans gently between your index finger
above and your thumb below. This maneuver should open
the urethral meatus and allow you to inspect it for
discharge. Normally there is none.

If the patient has reported a discharge but you do not see

any, ask him to strip, or milk, the shaft of the penis from
its base to the glans. Alternatively, do it yourself. This
maneuver may bring some discharge out of the urethral
meatus for appropriate examination. Have a glass slide
and culture materials ready.

- Palpate any abnormality of the penis, noting any tenderness or
- Palpate the shaft of the penis between your thumb and first two
fingers, noting any induration. Palpation of the shaft may be
omitted in a young, asymptomatic male patient.
- If you retract the foreskin, replace it before proceeding on to
examine the scrotum.
The Scrotum and Its Contents
Inspect the scrotum, including:
_ The skin. Lift up the scrotum so that you can see its posterior surface.
_ The scrotal contours. Note any swelling, lumps, or veins.

- Palpate each testis and epididymis between your thumb and first
two fingers.
o Note size, shape, consistency, and tenderness; feel for
any nodules.
o Pressure on the testis normally produces a deep visceral
- Palpate each spermatic cord, including the vas deferens,
between your thumb and fingers from the epididymis to the
superficial inguinal ring.
o Note any nodules or swellings.
o Swelling in the scrotum other than the testicles can be
evaluated by transillumination. After darkening the room,
shine the beam of a strong flashlight from behind the
scrotum through the mass. Look for transmission of the
light as a red glow.
Reference :
- Bates Guide to Physical Examination and History Taking