General Microbiology

·         IMMUNODIAGNOSTICS

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.

·         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.

·         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.