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IMMUNODIAGNOSTICS
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Limulus Tests is a sensitive
method for detection of bacterial endotoxins. It
depends on the in vitro gelation of Limulus amebocyte lysate (LAL), prepared from the circulating blood (amebocytes) of the horseshoe
crab, by the endotoxin or related compound.
Hemagglutination Inhibition: Certain organisms can
agglutinate red blood cells. If you have antibodies to impede that
agglutination, then you have a positive test result (antibody is present).
Direct Fluorescent Antibody: Fluoro tagged antibody checks for presence of antigen
directly.
Indirect Fluorescent Antibody: First allow
antigen-antibody reaction, then use a second Fluoro
tagged antibody (anti-antibody) to identify presence of the first antibody.
Complement Fixation: Usually avoided;
expensive.
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SUSCEPTIBILITY TESTS:
1.
KIRBY-BAUER
METHOD of Antibiotic Susceptibility Testing: Testing is done
using the Disk-Diffusion Method
PROCEDURE: Grow confluent bacterial
colony on plate.Add antibiotic disc in center.
Measure
the Zone of Inhibition (diameter) to determine bacterial susceptibility.
RESULTS: The larger the zone of inhibition, the more susceptible are the bacteria.
Resistant:
No zone of inhibition is found.
Intermediate:
Intermediate zone of inhibition.
Susceptible:
Larger zone of inhibition.
An
interpretive table or computer, along with known concentrations of antibiotic,
must be used to interpret results.
2. E-TEST: ANAEROBE Susceptibility Test.
Strict Anaerobes must be cultures under special conditions. Instead of a disk,
the antibiotics are contained in capillary disks.
A
novel version of the disk/agar diffusion method employs a quantitative
diffusion gradient, or epsilometer (E-test), and uses
an absorbent strip with a known gradient of antibiotic concentrations along its
length. When the strip is placed on the surface of an agar plate seeded with a
bacterial strain to be tested, antibiotic diffuses into the medium, and
bacterial growth is inhibited. The MIC is estimated as the lowest concentration
that inhibits visible growth.
3.
SPIRAL GRADIENT
ENDPOINT TEST: Another susceptibility test
Line several isolates (can be from different patients) up on a
single plate in wheel-spoke fashion.
Growth
inhibition at each line can then be observed and interpreted by computer.
4.
SERUM
BACTERICIDAL TEST: Susceptibility test for patients that are
extremely sick. Take serum sample and test amount of antibody actually in
patient's serum. Then plate that antibody out onto culture and verify that it
actually does kill the bugs at its peak and trough concentrations in the
patient's serum.
A
1:8 dilution of serum inhibiting growth should be indicative that antibiotics
are effective enough to kill bacteria in vivo.
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