Development of a micromanipulation method for single cell isolation of prokaryotes and its application in food safety

For nearly a century, conventional microbiological methods have been standard practice for detecting and identifying pathogens in food. Nevertheless, the microbiological safety of food has improved and various rapid methods have been developed to overcome the limitations of conventional methods. Alternative methods are expected to detect low cell numbers, since the presence in food of even a single cell of a pathogenic organism may be infectious. With respect to low population levels, the performance of a detection method is assessed by producing serial dilutions of a pure bacterial suspension to inoculate representative food matrices with highly diluted bacterial cells (fewer than 10 CFU/ml). The accuracy of data obtained by multiple dilution techniques is not certain and does not exclude some colonies arising from clumps of cells. Micromanipulation techniques to capture and isolate single cells from environmental samples were introduced more than 40 years ago. The main limitation of the current micromanipulation technique is still the low recovery rate for the growth of a single cell in culture medium. In this study, we describe a new single cell isolation method and demonstrate that it can be used successfully to grow various types of microorganism from picked individual cells. Tests with Gram-positive and Gram-negative organisms, including cocci, rods, aerobes, anaerobes, yeasts and molds showed growth recovery rates from 60% to 100% after micromanipulation. We also highlight the use of our method to evaluate and challenge the detection limits of standard detection methods in food samples contaminated by a single cell of Salmonella enterica.