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Petrova, Vera K.

Nutritional and Food Sciences

2004

M.S.

In foods, Listeria can become injured when subjected to physical and chemical stresses. Injured Listeria have the ability to repair and regain their pathogenicity, thus detection of injured Listeria in foods is consequential. Traditional methods for detecting and identifying injured Listeria have proven to be inadequate in precision, speed and accuracy. We investigated the use of flow cytometry (FCM) for assessment of dead, viable and injured Listeria monocytogenes cells after their exposure to heat and acid stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI) and oxonol (DiBAC4 (3))were used to analyze esterase activity, membrane integrity, and membrane potential as indicators of bacterial viability. Untreated and heat killed (75Cʻ for 30 min) L. monocytogenes F5069 cells were used as control populations. Different levels of heat and acid injury were achieved by subjecting the cells to heat at 56ʻC for 1, 3, 5, 7, 10, 13, 15, 20,25,30,35 minutes, and to acid in TPB adjusted to pH 3.0 for 2,5, 10 and 17 minutes. The results of single staining demonstrate a clear discrimination between viable intact, depolarized and permeabilized subpopulations of Listeria. A strong inverse correlation between the cFDA and DiBAC4 (3) or PI labeling was observed: cells that absorbed cFDA absorbed neither DiBAC4(3) nor PI. A direct and high correlation between DiBAC4 (3) and PI was discovered: the percentage of cells that appeared PI-stained was similar to the percentage of DiBAC4 (3)-stained cells. The percentages of PI and DiBAC4(3)-stained cells were highly correlated and inversely related to the percentage viability as obtained by plate counts. The percentage of cFDA-positive cells exhibited the same trend as plate counts; however, a lower estimation of viability for both heat and acid treatments was revealed. During double staining with PI and cFDA, three subpopulations were identified based on their uptake of the dyes: PI-positive, PI and cFDA-positive and cFDA-positive, which represent dead, injured and viable cells, respectively. The study demonstrates that the use of differential dyes and molecular probes affords a powerful and sensitive scientific tool for assessment of relative injury and physiological heterogeneity of Listeria populations, which may have applications for assessment of fate of Listeria in high risk foods.