A diagnosis of syphilis is confirmed by using darkfield microscopy
to demonstrate Treponema pallidum in material from suspected lesions
or regional lymph nodes (Creighton, 1990). A positive darkfield result is an
almost certain diagnosis of primary, secondary, or early congenital syphilis.
In primary syphilis, the darkfield examination may provide a means by which
to identify the etiologic agent of syphilis and diagnose the disease before
antibodies to T. pallidum can be detected.
Proper equipment and adequately trained personnel are required
to demonstrate the presence of T. pallidum in lesion material by darkfield
microscopy. The examination of several slides may be required.
Principles of Darkfield Microscopy
The standard brightfield microscope can be equipped for darkfield examination
by replacing the brightfield condenser with a darkfield condenser. Illumination
for darkfield microscopy is obtained when light rays strike the object in
the field at such an oblique angle that no direct rays enter the microscope
objective, only the rays reflected from the object. Therefore, the object
appears self-luminous against a dark background, hence the term darkfield.
When a fluid containing particles, including bacteria or treponemes, is placed
on a slide, the oblique rays are reflected from the surfaces upward into
the barrel of the microscope; these particles appear brightly illuminated
against a black background.
Specimen Collection
Lesions in general
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 to transfer the fluid from the lesion to the glass slide.
Three specimens should be collected from each lesion.
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.
The slide preparations should not contain a large volume of fluid
(large volumes cause a rapid liquid flow across the field), nor should the
preparation be so thin that it begins to dry before and adequate examination
can be made.
Microscope adjustment should always be completed and the microscope
should be in satisfactory working condition BEFORE collecting the patient's
specimen for examination.
Procedure for adjustment of the microscope
Place a blank slide on the stage and raise the substage containing
the darkfield condenser to its maximum height. The top of the darkfield condenser
should be slightly below the level of the stage but as close to the glass slide
as possible without pushing it up. Remove blank slide.
Turn on the variable transformer to produce maximum light
intensity.
Lower the substage slightly and place immersion oil on the
top of the condenser.
Place the slide with specimen (gingival scraping) on the stage
and use the mechanical slide carrier to center the specimen over the condenser.
Slowly raise the substage until there is a complete oil contact
between the top of the condenser and the bottom of the slide.
Rotate the nosepiece to center the low power objective over
the specimen.
Bring the specimen into focus by using the coarse adjustment
knob.
At this point, center the light in the field by rotating the
two centering screws located at the base of the darkfield condenser.
Focus the darkfield condenser by slightly raising or lowering
the substage until you observe the smallest diameter of the circular area of
intense light.
Rotate the nosepiece until the high-dry objective is in place
over the specimen.
Bring the specimen into focus by using the fine adjustment
knob only.
If a satisfactory image is obtained, place a SMALL drop of
immersion oil on the cover glass.
Rotate the nosepiece until the oil-immersion objective is
in place over the specimen and is in contact with the oil on the cover glass.
If the oil-immersion objective is equipped with an iris diaphragm, close the
diaphragm to reduce the numerical aperture below that of the darkfield condenser.
A funnel stop will serve the same purpose in an oil-immersion objective without
an iris diaphragm.
Bring the specimen into focus by using the fine adjustment
knob only. The light intensity from the illuminator may be decreased or increased
slightly to give the best contrast.
Examination of the client's specimen for T. pallidum
Place the slide to be examined (client's specimen) on an adjusted
darkfield microscope. Since minor adjustments may be required, it may be necessary
to repeat steps already described.
Search the entire specimen methodically with the high-dry
objective for spiral organisms that have the characteristic morphology and
motility of T. pallidum. (T. pallidum is a thin, delicate,
spiral organism, with 6- 20 (average 10) rigid, tightly wound coils, capable
of extreme bending which occurs in the middle and is stiffly executed, snapping
back to its original form, like the bending of a coil spring. The average organism
is slightly longer than the diameter of a red blood cell. Coil appearance is
maintained despite active motility of the organism. It may spin rapidly about
the longitudinal axis (like a corkscrew) without any forward or backward movement,
move slowly forward and backward without obvious change in direction of rotation
or pitch of coils, or the organism may move slowly, threading its way corkscrew-fashion
in viscous material. A springlike rigidity is constant, and T. pallidum does
not move rapidly from place to place with a serpentine motion. Any coarsely
wound spiral organism exhibiting great flexion and rapid movement from place
to place is NOT T. pallidum. Search carefully, systematically, and
exhaustively before making a negative report. A typical systematic scheme for
searching the specimen adequately is to start in the upper left corner of the
cover glass area, traverse to the right edge of the cover glass, drop down
slightly and traverse to the left; continue to search in this pattern, until
the entire cover glass area has been searched.
At least 10 minutes should be spent on each of the three specimens
collected before a negative report is rendered. The additional slides may be
placed in a moist chamber (moistened paper towels placed in a Petri plate,
for example) until examined.
Safety precautions should be followed, including gloves for
the microscopist and proper biohazard discard containers for the disposal of
slides.
Reporting and Interpretation of Results
Report
Results
Darkfield
positive
Organisms that have the
characteristic morphology and motility of T. pallidum
Darkfield
negative (inconclusive)
No treponemal organisms or spiral organisms; no organisms
with characteristic morphology and motility of T. pallidum
Darkfield
unsatisfactory
No T. pallidum found, but specimen has too many refractile
elements (blood cells, air bubbles, tissue fragments), or specimen is
drying
Every genital lesion should be considered syphilitic until proven
otherwise. Extragenital lesions characterized by indolence (causing little
or no pain), induration (firm or hard), and regional lymphadenopathy should
be regarded as probably syphilitic. Failure to find the organism does not exclude
a diagnosis of syphilis.
Negative results may mean that
The number of organisms was insufficient for detection.
The patient has received antitreponemal drugs locally or systemically.
The lesion is "fading" or approaching natural resolution
or disappearance.
The lesion is one of late syphilis.
The lesion is not syphilitic.
Sources of Error
Preparation Errors
If the specimen contains too many blood cells, air bubbles,
or tissue fragments, these refractile elements can obscure the presence of T.
pallidum.
If the microscope slides are not of the proper thickness,
or if slides and cover glasses are dirty or scratched, it will be difficult
to obtain a good darkfield.
If there is excessive fluid in the specimen or too little
fluid, the examination will be difficult.
Microscopy Errors
If immersion oil is not placed between the condenser and slide,
no light will reach the specimen.
If the darkfield condenser is not properly centered or focused
the illumination will not be optimum.
If immersion oil is on the lens of the low-power or high-power
objectives, the resulting view will be hazy.
Interpretation Errors
If one is unfamiliar with the morphology and motility characteristics
of T. pallidum, a false-positive or false- negative report could be
issued.
If one mistakes nonspecific spiral organisms or objects, tissue
debris, fibrin strands, and other extraneous objects for treponemes, a false-positive
report could be issued.
If one sees occasional erratic movement of T. pallidum or
no movement at all, too much time may have elapsed between making the slide
and examining it.
Program Operations Guidelines for STD Prevention
