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Applied and Environmental Microbiology, May 2001, p. 2240-2247, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2240-2247.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Morphological and Physiological Characterization of Listeria monocytogenes Subjected to High Hydrostatic Pressure

M. Ritz,1 J. L. Tholozan,2 M. Federighi,1 and M. F. Pilet1,3,*

Unité Mixte de Recherche, 1014 INRA/ENVN d'Hygiène des Aliments, Ecole Nationale Vétérinaire de Nantes, F-44307 Nantes,1 Laboratoire de Microbiologie Alimentaire et Industrielle, ENITIAA, F-44322 Nantes,3 and Laboratoire du Génie des Procédés et Technologies Alimentaires, INRA, F-59651 Villeneuve d'Ascq,2 France

Received 28 July 2000/Accepted 16 January 2001

High hydrostatic pressure is a new food preservation technology known for its capacity to inactivate spoilage and pathogenic microorganisms. That inactivation is usually assessed by the number of colonies growing on solid media after treatment. Under normal conditions the method does not permit recovery of damaged cells and may underestimate the number of cells that will remain viable and grow after a few days in high-pressure-processed foodstuffs. This study investigated the damage inflicted on Listeria monocytogenes cells treated by high pressure for 10 min at 400 MPa in pH 5.6 citrate buffer. Under these conditions, no cell growth occurred after 48 h on plate count agar. Scanning electron microscopy, light scattering by flow cytometry, and cell volume measurements were compared to evaluate the morphological changes in cells after pressurization. All these methods revealed that cellular morphology was not really affected. Esterase activity, as assessed either by enzymatic activity assays or by carboxy fluorescein diacetate fluorescence monitored by flow cytometry, was dramatically lowered, but not totally obliterated, under the effects of treatment. The measurement of propidium iodide uptake followed by flow cytometry demonstrated that membrane integrity was preserved in a small part of the population, although the membrane potential measured by analytical methods or evaluated by oxonol uptake was reduced from -86 to -5 mV. These results showed that such combined methods as fluorescent dyes monitored by flow cytometry and physiological activity measurements provide valuable indications of cellular viability.

* Corresponding author. Mailing address: Unité Mixte de Recherche, 1014 INRA/ENVN d'Hygiène des Aliments, Ecole Nationale Vétérinaire de Nantes, Route de Gachet, BP 40706, F-44307 Nantes, France. Phone: 33 0 2 40 68 78 11. Fax: 33 0 2 40 68 77 62. E-mail: pilet{at}vet-nantes.fr.

Applied and Environmental Microbiology, May 2001, p. 2240-2247, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2240-2247.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.