Microscopy for Rapid Antibiotic Susceptibility Test Using Membrane Fluorescence Staining and Spectral Intensity Ratio

A method of determining a minimum inhibitory concentration (MIC) of one or more bacteria in a sample comprising:

• preparing a plurality of bacterial suspensions in a plurality of receptacles;
• adding different amounts of an antimicrobial agent to two or more of the plurality of bacterial suspensions, thereby creating a plurality of suspensions comprising a combination of bacteria and an antimicrobial agents;
• incubating the plurality of suspensions comprising a combination of bacteria and an antimicrobial agent at a suitable temperature for a suitable period of time to produce a plurality of incubated suspensions comprising a combination of bacteria and an antimicrobial agent;
• adding a single membrane-associated dye to the plurality of incubated suspensions;
• illuminating the incubated suspensions comprising the dye with a light at a one or more excitation wavelength for the dye;
• obtaining microscopic images of the incubated suspensions comprising the dye at two emission wavelengths of the dye;
• determining, with at least one processor, an intensity of emitted light at the two emission wavelengths of the dye for individual bacterial cells in each image;
• determining, with the at least one processor, an MIC for bacteria in the sample based upon the relative intensity of emitted light at the two emission wavelengths of the dye for individual bacterial cells in each image.