A novel image-based method for simultaneous counting of Lactobacillus and Saccharomyces in mixed culture fermentation

Abstract Mixed microorganism cultures are prevalent in the food industry. From sour beer to sourdough bread, mixed culture popularity has been growing steadily. A variety of microbiological mixtures have been used in these unique fermenting processes to create distinctive flavor profiles and potential health benefits. Although mixed cultures may seem ubiquitous, they are typically not well characterized, which may be due to the lack of user-friendly and simple measurement tools. Image-based cytometry systems have been employed to count bacteria or yeast cells directly and rapidly in plastic chambers, as opposed to traditional cell counting methods that can take days to grow and count. In this work, we aim to develop a novel image cytometry method to distinguish and enumerate mixed cultures of yeast and bacteria in beer products. We demonstrate that the proposed image cytometry method can be used to rapidly distinguish and simultaneously count the bacteria and yeast cells mixed cultures. The Cellometer X2 from Nexcelom was used to automate the counting of Lactobacillus plantarum and Saccharomyces cerevisiae in a mixed culture using fluorescent dyes and size exclusion image analysis algorithm. The proposed image cytometry method was tested with a series of experiments. (1) Yeast and bacteria monoculture titration, (2) mixed culture with various ratios, and (3) monitoring a Berliner Weisse mixed culture fermentation. All experiments were validated by comparing directly to manual counting of yeast and bacteria colony formation on agar plates. The two counting methods were highly comparable with ANOVA analysis showing p-value > 0.05 for all trials. The image cytometry method was the most accurate for yeast concentrations between 105-107 cells/mL and 106-108 cells/mL for lactic acid bacteria. Overall, the novel image cytometry method was able to distinguish and count mixed cultures consistently and accurately, which may provide better characterization and assessment of mixed culture brewing applications that may produce more consistent and higher quality products.