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

• Published in: Journal of microbiological methods 2024-08, Vol.223, p.106978, Article 106978
• Main Authors: Somerton, Ben T., Morgan, Brooke L.
• Format: Article
• Language: English
• Subjects: Bacillus cereus; Bacteria; CellROX™ green; DiOC2; dried milk; Flow cytometry; fluorescent dyes; iodides; Milk quality; Propidium iodide; whole milk
• ISSN: 0167-7012; 1872-8359; 1872-8359
• DOI: 10.1016/j.mimet.2024.106978

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. •DiOC2 and CellROX™ Green-propidium iodide dyes were found to have similar accuracy and precision compared to plate counting, and high ease-of-use, for the enumeration of B. cereus in milk by flow cytometry.•DiOC2 and CellROX™ Green-propidium iodide dyes had a consistent position and shape of cell clusters on flow cytometry plots when comparing from sample-to-sample. In addition, a high degree of separation of cell clusters from background, non-bacterial clusters, was found, even without the use of sample preparation to remove milk constituents prior to measurement.•Results indicated that DiOC2 would be an ideal candidate when using flow cytometry for the routine enumeration of viable bacteria in dairy samples.