DETECTION OF VIABLE BUT NONCULTURABLE CELLS OF LISTERIA MONOCYTOGENES WITH THE USE OF DIRECT EPIFLUORESCENT FILTER TECHNIQUE

作者：Olszewska Magdalena A^*; Panfil Kuncewicz Helena; Laniewska Trokenheim Lucja

来源：Journal of Food Safety, 2015, 35(1): 86-90.

DOI：10.1111/jfs.12130

Direct epifluorescent filter technique was studied as a method to enumerate Listeria monocytogenes in artificially contaminated cheese. Occurrences of viable but nonculturable (VBNC) cells of L.monocytogenes prompted us to investigate the viability of this pathogen in hard cheese during refrigerated storage under different packaging conditions. This study compared the results of two enumeration methods: epifluorescent microscopic counting and plating. The microscopic enumeration was based on fluorescent staining with carboxyfluorescein diacetate (CFDA) for tracing cells that display metabolic activity. In this study, the L.monocytogenes persisted well throughout a prolonged storage of cheese, regardless of the packaging conditions. Based on a discrepancy between CFDA and plate count results, a dormant fraction of VBNC cells was distinguished, thus greater quantities of VBNC cells were estimated along with storage time. We conclude that direct epifluorescent filter technique may be an attractive approach for assessing bacterial viability, especially for pathogens with the ability to enter a dormant state.
Practical ApplicationsListeria monocytogenes is a foodborne pathogen with a remarkable long-term ability to persist in a variety of ready-to-eat foods. The plating method for enumeration of L.monocytogenes in food is well established, but it is time consuming and relies only on cell reproduction, so that injured and nonculturable cells have no chance to be counted. As a solution, we describe a rapid method for the enumeration of L.monocytogenes in cheese using the direct epifluorescent filter technique. This method is based on fluorescent staining of cells with a metabolic activity indicator and cell counting with the help of epifluorescence microscopy. The method presented in this study allows the rapid enumeration of both active and nonculturable cells of L.monocytogenes and could be used by predictive microbiologists for precise estimation of the safety of dairy products with respect to pathogenic bacteria.

• 出版日期2015-2