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erate and virulent phages, demonstrating its influence on plaque size and definition in all cases, but mainly on the virulent phages studied (YAB, Ib3 and BYM). No significant effect on enumeration was observed. A calcium ion requirement for proliferation and plaque formation for LAB bacteriophages has been demonstrated in several phage-cell systems. According to Sechaud et al. (13), Ca2+ (or Mg2+) ions not only stabilize the coiled DNA inside the phage capsid and greatly improve the adsorption rate but also regulate the penetration efficiency of phage DNA into bacterial cells. After adsorption (possibly at the DNA injection step), the divalent cations could act as counter-ions during translocation of the phage DNA across the cellular membrane (5) or be involved in DNA stabilization following the injection step. The conventional method of double-layer plaque titration uses Ca2+ in the support media (agarized). This study demonstrated that calcium addition to soft agar increased the availability of this cation for the bacteriophage. Significant differences in phage particle counts, of at least one log order, were found for all phages assayed. The data obtained for L. delbrueckii (temperate and virulent) phages studied in this work indicate the need to modify methodology for phage counting to include calcium. In addition, the preparation of the inoculum used was important to define the methodology. The strains cultured overnight in broth and RSM were diluted in broth up to a carefully controlled OD560nm value. This methodology implemented in our laboratory allowed us to increase counts of phage Cb1/204 and also to obtain more visible plaques. In contrast, for Cb1/342 phage, the MRS overnight inoculum was more suitable. On the basis of our results, each phage/host strain system should be considered individually when performing detection and enumeration of bacteriophages. Even if all these modifications are implemented in other laboratories, at present there is no information about changes of the conventional methodology (17). ACKNOWLEDGMENTS This work was supported by the Universidad Nacional del Litoral (Santa Fe, Argentina) (C.A.I.+D. Nº 37–200, Resolución C.S 119/06, 2006–2008) and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) (Project PIP N° 5321, 20062007). REFERENCES 1. Brüssow, H., M. Frémont, A. Bruttin, J. Sidoti, A. Constable, and V. Fryder. 1994. Detection and classification of Streptococcus thermophilus bacteriophages isolated from industrial milk fermentation. Appl. Environ. Microbiol. 60(12):4537– 4543. 2. Bruttin, A., F. Desiere, N. D’Amico, J-P Guérin, J. Sidoti, B. Huni, S. Luccini, and H. Brüsow. 1997. Molecular ecology of Streptococcus thermophilus bacteriophage infections in cheese factory. Appl. Environ. Microbiol. 63(8):3144–3150. 3. Capra, M., A. Quiberoni, and J. Reinheimer. 2005. Phages of Lactobacillus casei⁄paracasei: response to environmental factors and interaction with collection and commercial strains. J. Appl. Microbiol. 100:334–342. 4. Javis, A. W. 1989. Bacteriophages of lactic acid bacteria. J. Dairy Sci. 72:3406–3428. 5. Josephsen, J., and H. Neve. 1998. Bacteriophages and lactic acid bacteria, p. 385–436. In S. Salminen and A. von Wright (eds.), lactic acid bacteria. Microbiology and Functional Aspects, Marcel Dekker, Inc., New York. 6. Lillehaug, D. 1997. An improved plaque assay for poor plaqueproducing temperate lactococcal bacteriophages. J. Appl. Microbiol. 83:85–90. 7. Moineau, S. 1999. Applications of phage resistance in lactic acid bacteria, p. 377–382. In W. Konings, O. Kuipers and J. H. J. Huis in ‘t Veld (eds.), Proceedings of the sixth symposium on lactic acid bacteria: genetics, metabolism and applications, Kluwer Academic,Veldhoven. 8. Neve, H. 1996. Bacteriophage, p. 157–190. In T. M. Cogan, and J-P Accolas (eds.), Dairy starter cultures,VCH Publishers, New York. 9. Neviani, E., D. Carminati, and G. Giraffa. 1992. Selection of some bacteriophage- and lysozymeresistant variants of Lactobacillus helveticus CNRZ 892. J. Dairy Sci. 75:905–913. 10. Quiberoni, A., D. Guglielmotti, and J. Reinheimer. 2004. Inactivation of Lactobacillus delbrueckii bacteriophages by heat and biocides. Int. J. Food Microbiol. 84:51–62. 11. Quiberoni, A., and J. Reinheimer. 1998. Physicochemical characterization of phage adsorption to Lactobacillus helveticus ATCC 15807 cells. J. Appl. Microbiol. 85:762–768. 12. R e i n h e i m e r, J . , A . B i n e t t i , A. Quiberoni, N. Bailo, A. Rubiolo, and G. Giraffa. 1997. Natural milk cultures for the production of Argentinian cheeses. J. Food Prot. 60:59–63. 13. Séchaud, L., P-J Cluzel, M. Rousseau, A. Baumgartner, and J-P Accolas. 1988. Bacteriophages of lactobacilli. Biochimie 70:401–410. 14. Suárez, V., S. Moineau, J. Reinheimer, and A. Quiberoni. 2008b. Argentinean Lactococcus lactis bacteriophages: genetic characterization and adsorption studies. J. Appl. Microbiol. 104:371–379. 15. Suárez, V., A. Quiberoni, A. Binetti, and J. Reinheimer. 2002. Thermophilic lactic acid bacteria phages isolated from Argentinian dairy industries. J. Food Prot. 65:1597–1604. 16. Suárez, V., M. Zago, A. Quiberoni, D. Carminati, G. Giraffa, and J. Reinheimer. 2008a. Lysogeny in Lactobacillus delbruekii strains and characterization of two temperate prolate-headed bacteriophages. J. Appl. Micrbiol. 105:1402–1411. 17. Svensson, V., and A. Christiansson. 1991. Methods for phage monitoring, FIL-IDF Bull. 263:29–39. 18. Watanabe, K., M. Shirabe,T. Fukusaki, Y. Kakita, Y. Nakashima, and F. Miake. 1993. Electron microscope studies on the host cell energy requirement for injection of PL-1 phage DNA into Lactobacillus casei. Curr. Microbiol. 26:293–298. JANUARY 2010 | FOOD PROTECTION TRENDS
Food Protection Trends - January 2010

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