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. 2023 Feb 27;23(1):48.
doi: 10.1186/s12866-023-02792-2.

Insights into the enumeration of mixtures of probiotic bacteria by flow cytometry

Harry Tracey #  1 , Niall Coates #  2 , Eleri Hulme  1 , Daniel John  1 , Daryn Robert Michael  1 , Susan Frances Plummer  1
Affiliations

Insights into the enumeration of mixtures of probiotic bacteria by flow cytometry

Harry Tracey et al. BMC Microbiol. .

Abstract

The use of flow cytometry to enumerate microorganisms is gaining traction over the traditional plate count technique on the basis of superior accuracy, precision and time-to-result. Here, we assessed the suitability of live/dead flow cytometry for the enumeration of mixed populations of probiotic bacteria (L. acidophilus, L. paracasei, L. plantarum, L. salivarius, B. lactis and B. bifidum) whilst comparing outcomes with plate counting. Using a novel gating strategy designed specifically for the enumeration of mixed populations, the application of flow cytometry resulted in the detection of higher numbers of viable bacteria with a greater level of repeatability than plate counting (RSD of 6.82 and 13.14% respectively). Across all multi-species blends tested, viable cell input was more accurately recovered by flow cytometry (101.8 ± 6.95%) than plate counts (81.37 ± 16.03%). However, when certain probiotic mixtures contained preparations with high numbers of non-viable cells in their total population, flow cytometry had the potential for overestimation of the viable population. Nevertheless, the comparative plate counts of these mixtures were low and variable, thus supporting the use of flow cytometry for the enumeration of viable bacteria in mixed populations.

Keywords: Flow cytometry; Multistrain; Plate count; Probiotic.

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Conflict of interest statement

All authors are/were employees of Cultech Ltd.

Figures

Fig. 1
Fig. 1
Quantification of bacterial numbers present in probiotic preparations. Bacterial numbers present in 3 batches of A CUL21/60, B CUL20/34, C CUL08, D CUL66N or E CUL61 were determined by PC (CFU/g) and FC (AFU/g and n-AFU/g). Data represent the mean ± SD of 10 experimental replicates per batch. Values of p were determined using the Student’s paired t-test where *p < 0.05, **p < 0.01 and ***p < 0.001. Abbreviations: PC, plate count; FC, flow cytometry; CFU, colony forming unit; AFU, active fluorescent unit; n-AFU non-active active fluorescent unit; SD, standard deviation
Fig. 2
Fig. 2
Specific and general flow cytometric gating strategies. Representative flow cytometric multi-parameter dot plots (forward (FSC-H)/side (SSC-H) scatter and green (SYTO24-H)/red (PI-H) fluorescence) for each organism overlaid with A the specific gates or B a general gating strategy
Fig. 3
Fig. 3
General gating strategy for probiotic blends. Representative flow cytometric multi-parameter dot plots (forward (FSC-H)/side (SSC-H) scatter and green (SYTO24-H)/red (PI-H) fluorescence) for each of the blends overlaid with the general gating strategy
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