A Microtiter Plate Method to Assess the Minimum Inhibitory Concentration of an Antibiotic

Take microtiter plates containing a serially diluted aminoglycoside antibiotic. One of the plates contains NTBC, a synthetic drug.

Introduce Pseudomonas aeruginosa, a gram-negative bacteria, and incubate.

The bacteria produce pyomelanin — a protective extracellular pigment — in the NTBC-untreated wells, while the drug prevents the production of pyomelanin.

The cationic antibiotic binds to anionic lipopolysaccharides, or LPS, on the bacterial outer membrane, displacing the cationic bridges and destabilizing the membrane.

Upon entering the bacteria, the antibiotic binds to the aminoacyl site, or A site, in the 30S ribosomal subunit.

The bound antibiotic prevents the aminoacyl-tRNA binding and ribosomal movement across mRNA, interfering with protein synthesis and inhibiting bacterial growth.

Post-incubation, measure the culture's optical density to assess bacterial growth.

The lowest antibiotic concentration that prevents bacterial growth is the minimum inhibitory concentration or MIC.

Similar MIC values for NTBC-treated and NTBC-untreated wells indicate no impact of NTBC on the antibiotic sensitivity of bacteria.