Comparison of plate counting with flow cytometry, using four different fluorescent dye techniques, for the enumeration of Bacillus cereus in milk.

This study aimed to compare the performance of flow cytometry methods with plate counting for the enumeration of bacteria, using Bacillus cereus as a model organism. It was found that the cFDA-propidium iodide, CellROX™ Green-propidium iodide, and DiOC2 dye techniques had similar accuracy to plate counting, while the SYTO 24-propidium iodide dye technique was not as accurate. The four dye techniques had comparable precision to plate counting, with the CellROX™ Green-propidium iodide dye having the greatest precision. The consistency of the position and shape of the cell clusters on the flow cytometry plots, and the extent of separation of the cell from background clusters, was greatest with the DiOC2 and CellROX™ Green-propidium iodide dyes. Furthermore, the DiOC2 and CellROX™ Green-propidium iodide dyes performed well, even when a sample was measured containing reconstituted whole milk powder at a 10-1 dilution, without the use of sample preparation to specifically remove the milk constituents prior to measurement. Given gating of only one cell cluster was required to be managed with the DiOC2 dye, to determine the viable number of cells, it was found that the DiOC2 dye had the greatest ease-of-use. Overall, results indicated that the DiOC2 dye is an ideal candidate for the enumeration of viable bacteria in dairy samples on a high-throughput, routine basis.

Preconditioning with cations increases the attachment of Anoxybacillus flavithermus and Geobacillus species to stainless steel.

Preconditioning of Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 with cations prior to attachment often significantly increased (P ≤ 0.05) the number of viable cells that attached to stainless steel (by up to 1.5 log CFU/cm(2)) compared with unconditioned bacteria. It is proposed that the transition of A. flavithermus and Geobacillus spp. from milk formulations to stainless steel product contact surfaces in milk powder manufacturing plants is mediated predominantly by bacterial physiological factors (e.g., surface-exposed adhesins) rather than the concentrations of cations in milk formulations surrounding bacteria.

Influence of cations on growth of thermophilic Geobacillus spp. and Anoxybacillus flavithermus in planktonic culture.

Free ions of Na(+), K(+), Ca(2+), and Mg(2+) influenced the optical density of planktonic cultures of thermophilic bacilli. Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 (milk powder manufacturing plant isolates) and A. flavithermus DSM 2641 and G. thermoleovorans DSM 5366 were studied. Ca(2+) and Mg(2+) were associated with increases in optical density more so than Na(+) and K(+). Overall, it appeared that Ca(2+) and/or Mg(2+) was required for the production of protein in thermophilic bacilli, as shown by results obtained with A. flavithermus E16, which was selected for further study.

Recovery of Cronobacter sakazakii from environmental surface swabbing materials using a 5-h enrichment procedure.

This study aimed to reduce the time taken to detect low numbers of Cronobacter sakazakii inoculated onto environmental swabs (100, 10 or 1 cfu per swab) using a simple plating procedure for application in a dairy testing laboratory. Three types of environmental swabs (Biolab FlexiSwab, gauze swabs and the Whatman SwabCheck Polywipe sponge) were inoculated with either Cron. sakazakii in single culture or Cron. sakazakii together with Citrobacter freundii. A 5-h enrichment procedure of swabs in Cronobacter enrichment broth at 37 degrees C prior to plating was then compared with no enrichment or 24-h enrichment. The 5-h enrichment procedure was as efficient at detecting Cron. sakazakii on environmental swabs at low cell concentrations (100 cfu per swab), and in pure culture or in competition with other coliforms (Citrobacter), as pre-enrichment for 24 h. This protocol was also successful in detecting 10 cfu per swab 80% of the time. The results also indicated that the type of swab selected for use in environmental safety programmes is influential on the outcome, with the FlexiSwab and gauze swabs being the most efficient swabbing materials evaluated in this study.