Cronobacter Support
06-03-2009, 05:13 PM
1st International Conference on Cronobacter Poster Abstract 51
Validation of D-value determination methodology to be used to screen environmental and clinical Enterobacter sakazakii.
The use of D-values as a means to evaluate the thermal resistance of bacteria is widespread in the food industry, but the methods used to determine them may vary resulting in different published D-values for the same bacterial strains in similar media. The aim of this study was to determine whether the use of a thin walled glass tube could be used to calculate D-values of Staphylococcus aureus and to validate the method by comparison with peer reviewed values in published literature. Bacterial cultures of S. aureus were grown in Tryptone Soya Broth (TSB) at 37°C for 18-24 h, inoculated to a level of 107 cfu/ml in infant formula milk and was added as 1 ml aliquots into a series of thin walled (ca 0.15mm) glass tubes (9.6 cm long, internal diameter 0.6 mm), and sealed with a rubber bung. The inoculated tubes were immersed into a temperature controlled water bath. Initial experiments were carried out at 55, 60 and 65ºC. Duplicate tubes were prepared for each time point, and a minimum of three independent experiments were performed. Serial dilution was carried out in Ringers (Oxoid) solution and microbial enumeration done using spread plate technique on TSA. The method was also used to determine D-values of Enterobacter sakazakii strain NCTC8155 at 56 and 60ºC where the effect of overlaying with VRBGA (Oxoid) to inhibit Bacillus spore outgrowth was also investigated. Using the thin walled glass tubes, the D-values for S. aureus were calculated as, 2.3, 1.5 and 0.1 min for 55, 60 and 65°C respectively. These values fall well within the range of published values for this microbe in reconstituted milk products. D-values of E. sakazakii NCTC8155 at 56°C was 8.1 min (which falls between 5.1 and 10.5 min previously reported), and at 60°C was 0.98 min (compared to 1.1min reported in literature). There was no difference in E. sakazakii counts obtained by direct plating on TSA or on TSA overlaid with VRBGA, however the latter method eliminated the risk of Bacillus outgrowth, particularly when testing at temperatures less than 60°C. The use of the thin walled tubes gave more rapid heat-up and cool down times than conventional TDK tubes. D-values obtained using these tubes are similar to published values. This method is suitable for the screening of clinical and environmental E. sakazakii strains.
Jenna M. Warby, Niall Mullane, Carmel McNamee, F. Byrne, Ciara Walsh, Séamus Fanning, J. Lyng and Amalia Scannell
College of Life Sciences,School of Agriculture, Food Science & Veterinary Medicine, UCD Agriculture and Food Science Centre, UCD, Dublin, Belfield, Dublin 4, Ireland.
Validation of D-value determination methodology to be used to screen environmental and clinical Enterobacter sakazakii.
The use of D-values as a means to evaluate the thermal resistance of bacteria is widespread in the food industry, but the methods used to determine them may vary resulting in different published D-values for the same bacterial strains in similar media. The aim of this study was to determine whether the use of a thin walled glass tube could be used to calculate D-values of Staphylococcus aureus and to validate the method by comparison with peer reviewed values in published literature. Bacterial cultures of S. aureus were grown in Tryptone Soya Broth (TSB) at 37°C for 18-24 h, inoculated to a level of 107 cfu/ml in infant formula milk and was added as 1 ml aliquots into a series of thin walled (ca 0.15mm) glass tubes (9.6 cm long, internal diameter 0.6 mm), and sealed with a rubber bung. The inoculated tubes were immersed into a temperature controlled water bath. Initial experiments were carried out at 55, 60 and 65ºC. Duplicate tubes were prepared for each time point, and a minimum of three independent experiments were performed. Serial dilution was carried out in Ringers (Oxoid) solution and microbial enumeration done using spread plate technique on TSA. The method was also used to determine D-values of Enterobacter sakazakii strain NCTC8155 at 56 and 60ºC where the effect of overlaying with VRBGA (Oxoid) to inhibit Bacillus spore outgrowth was also investigated. Using the thin walled glass tubes, the D-values for S. aureus were calculated as, 2.3, 1.5 and 0.1 min for 55, 60 and 65°C respectively. These values fall well within the range of published values for this microbe in reconstituted milk products. D-values of E. sakazakii NCTC8155 at 56°C was 8.1 min (which falls between 5.1 and 10.5 min previously reported), and at 60°C was 0.98 min (compared to 1.1min reported in literature). There was no difference in E. sakazakii counts obtained by direct plating on TSA or on TSA overlaid with VRBGA, however the latter method eliminated the risk of Bacillus outgrowth, particularly when testing at temperatures less than 60°C. The use of the thin walled tubes gave more rapid heat-up and cool down times than conventional TDK tubes. D-values obtained using these tubes are similar to published values. This method is suitable for the screening of clinical and environmental E. sakazakii strains.
Jenna M. Warby, Niall Mullane, Carmel McNamee, F. Byrne, Ciara Walsh, Séamus Fanning, J. Lyng and Amalia Scannell
College of Life Sciences,School of Agriculture, Food Science & Veterinary Medicine, UCD Agriculture and Food Science Centre, UCD, Dublin, Belfield, Dublin 4, Ireland.