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Lab
Diagnostic Testing: Acanthamoebae
Acanthamoebae
keratitis is a serious and devastating ocular problem. Rapid confirmation
of the presence of Acanthamoebae in a patient specimen is essential.
The laboratory diagnosis of Acanthamoeba is made through culture
and examination of cornea smears on glass slides by giemsa.
Specimen
Materials
Procedure
Interpretation
Quality
Control
Specimen
Specimens for Acanthamoebae cultures include corneal scrapings or
biopsies. Other possible and highly recommended specimens include
the patient's contact lenses and contact lens solutions.
Materials
Non-nutrient agar plates (1.5% noble agar)
0.9% sterile non-preserved saline
Sterile test tubes
30oC incubator with humidity
Procedure
A. Non-nutrient agar plates should be warmed to room temperature
prior to inoculation.
B. Inoculation
of specimen:
- Corneal scrapings
are inoculated directly onto the agar plate. Corneal material
is also placed on a slide for direct microscopic examination for
Acanthamoebae cysts and trophozoites using the Giemsa stain.
- Biopsy material
is ground first before inoculation onto the agar plate.
To culture a contact lens case, thoroughly rub a swab inside the
chambers of the case and then inoculate the agar plate. Contact
solutions, saline, and water samples are inoculated onto the agar
plate by placing one drop of each sample on a separate agar plate.
- NOTE: Specimens
in addition to cornea should be inoculated onto a DIFFERENT plate
than the cornea. Due to the swarming nature of the acanthamoebae
growth, it would be difficult to determine where the growth was
coming from.
- Transport
swabs: Rub the swab onto the agar plate making sure to roll it
around on the surface. Cut the swab tip off and place onto surface
of media.
C. After specimen
inoculation, the agar plate is overlayed with a thick suspension
of Enterobacter aerogenes or E. coli in sterile saline. For transport
swab tips, place an extra amount of bacterial suspension (approximately
100ul) directly onto the swab tip.
D. The plates
are incubated in a 30°C incubator and examined every day up
to seven days for the presence of Acanthamoebae trophozoites or
cysts.
Interpretation
The non-nutrient agar plates are examined for fronts of trophozoites
that appear grossly as a distinct circular arch from the point of
agar inoculation. Initially all trophozoites are present but as
the circle of the front becomes larger, cysts are present between
the front and point of inoculation. Trophozoites appear as Acanthopodia
that are irregularly shaped with a prominent nucleus. These nuclei
may appear to blink when observed under the adverse conditions of
light and heat. Cysts appear as smaller, circular, and polygonal
with a double wall. On smear, trophozoites and cysts can be observed
in the corneal tissue.
Quality
Control
Each time a culture is set-up for the isolation of Acanthamoebae,
the lot of non-nutrient agar is also checked for growth of stock
Acanthamoebae. A negative control, one without stock Acanthamobae,
is also set up.
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