Real-Time Digital Bright Field Technology for Rapid Antibiotic Susceptibility Testing.

Optical scanning through bacterial samples and image-based analysis may provide a robust method for bacterial identification, fast estimation of growth rates and their modulation due to the presence of antimicrobial agents. Here, we describe an automated digital, time-lapse, bright field imaging system (oCelloScope, BioSense Solutions ApS, Farum, Denmark) for rapid and higher throughput antibiotic susceptibility testing (AST) of up to 96 bacteria-antibiotic combinations at a time. The imaging system consists of a digital camera, an illumination unit and a lens where the optical axis is tilted 6.25° relative to the horizontal plane of the stage. Such tilting grants more freedom of operation at both high and low concentrations of microorganisms. When considering a bacterial suspension in a microwell, the oCelloScope acquires a sequence of 6.25°-tilted images to form an image Z-stack. The stack contains the best-focus image, as well as the adjacent out-of-focus images (which contain progressively more out-of-focus bacteria, the further the distance from the best-focus position). The acquisition process is repeated over time, so that the time-lapse sequence of best-focus images is used to generate a video. The setting of the experiment, image analysis and generation of time-lapse videos can be performed through a dedicated software (UniExplorer, BioSense Solutions ApS). The acquired images can be processed for online and offline quantification of several morphological parameters, microbial growth, and inhibition over time.