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Applied and Environmental Microbiology, April 2009, p. 2132-2138, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.01796-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Stress- and Growth Rate-Related Differences between Plate Count and Real-Time PCR Data during Growth of Listeria monocytogenes{triangledown} ,{dagger}

Franziska Reichert-Schwillinsky,1 Carmen Pin,2 Monika Dzieciol,1 Martin Wagner,1 and Ingeborg Hein1*

Institute for Milk Hygiene, Milk Technology, and Food Safety, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria,1 Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom2

Received 4 August 2008/ Accepted 21 January 2009

To assess the overestimation of bacterial cell counts in real-time PCR in relation to stress and growth phase, four different strains of L. monocytogenes were exposed to combinations of osmotic stress (0.5 to 8% [vol/vol] NaCl) and acid stress (pH 5 to 7) in a culture model at a growth temperature of 10°C or were grown under optimal conditions. Growth curves obtained from real-time PCR, optical density, and viable count data were compared. As expected, optical density data revealed entirely different growth curves. Good to moderate growth conditions yielded good correlation of real-time PCR data and plate count data (r2 = 0.96 and 0.99) with similar cell counts. When growth conditions became worse, the numbers of CFU decreased during the stationary phase, whereas real-time-PCR-derived bacterial cell equivalents differed in this regard; the correlation worsened (r2 = 0.84). However, fitted growth curves revealed that maximum growth rates calculated from real-time PCR data were not significantly different from those derived from plate count data. The overestimation of bacterial cell counts by real-time PCR observed in the stationary phase under higher-stress conditions might be explained by the accumulation of viable but nonculturable bacteria or dead bacteria and extracellular DNA. Considering these results, real-time PCR data collected from naturally contaminated samples should be viewed with caution.

* Corresponding author. Mailing address: Institute for Milk Hygiene, Milk Technology, and Food Safety, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria. Phone: 43 1 25077 3507. Fax: 43 1 25077 3590. E-mail: ingeborg.hein{at}vu-wien.ac.at

{triangledown} Published ahead of print on 30 January 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, April 2009, p. 2132-2138, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.01796-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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