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

Date Published: May 31, 2018

Publisher: Public Library of Science

Author(s): Marisa Hohnadel, Myriam Maumy, Renaud Chollet, Jeffrey Chalmers.

http://doi.org/10.1371/journal.pone.0198208

Abstract

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.

Partial Text

Diseases caused by foodborne pathogens have long been a serious threat to public health and food safety and remain a major concern to society. According to reports from the Centers for Disease Control and Prevention (CDC), approximately 48 million people in the United States become ill, 128,000 people are hospitalized, and 3000 people die from foodborne diseases each year [1, 2, 3].

Since the introduction of micromanipulation techniques for the isolation of single cells from environmental samples 40 years ago, several attempts have been made to improve the micromanipulation of single microbial cells [9, 11, 13]. Each time, suggestions were based on the state of the art at that time. With further improvements in microscopy and the development of hydraulic systems such as CellTram, the capillary can be positioned quickly and precisely, which increases the efficiency of micromanipulation of such small cells as microorganisms.

 

Source:

http://doi.org/10.1371/journal.pone.0198208

 

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