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

Abstract

Source: Ketelboeter, L. M., et al. Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress. J. Vis. Exp. (2015).

This video demonstrates a microtiter plate-based method for determining the Minimum Inhibitory Concentration (MIC) of test antibiotics against bacteria. Upon adding a suspension of the bacterial pathogen Pseudomonas aeruginosa to a microtiter plate containing serial dilutions of a test antibiotic, and an inhibitor of the production of pyomelanin by the bacteria, the optical density of the wells is measured to compute the minimum inhibitory concentration of the antibiotic, and the impact of the pyomelanin production inhibitor the antibiotic sensitivity of the bacteria.

Protocol

1. Antibiotic Minimum Inhibitory Concentration (MIC) Assay in 96-well Plates Set up overnight cultures of the strains to be tested in LB (Luria-Bertani) with and without NTBC (2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione). NOTE: This protocol is described using the representative level of 300 µM NTBC. Add 300 µM NTBC to 2 ml LB. Add an equivalent volume of vehicle (Dimethyl sulfoxide, DMSO) to 2 ml LB for the no NTBC condition….

Representative Results

Figure 1: Schematic of antibiotic MIC assay 96-well plate setup. (A) 100 µl of 2x antibiotics of the highest starting concentration are in row A. Rows B through H are filled with 50 µl of either LB + NTBC or LB + DMSO without antibiotics. (B) Two-fold serial dilutions are performed in rows A through G, resulting in 50 µl of diluted…

Materials

2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)	Sigma-Aldrich	SML0269-50mg	Also called nitisinone. Soluble in DMSO.
H2O2	Sigma-Aldrich	216763-100ML	30 wt. % in H2O. Stabilized.
Gentamicin	Gold Bio	G-400-100	Soluble in H2O. Filter sterilize.
Kanamycin	Fisher Scientific	BP906-5	Soluble in H2O. Filter sterilize.
Tobramycin	Sigma-Aldrich	T4014-100MG	Soluble in H2O. Filter sterilize.