Application of microplate scale fluorochrome staining assay for assessment of viability and stability of probiotic Bifidobacterium sp.

Probiotic cultures encounter harsh conditions during production and in the GI-tract. There is a need for rapid and reliable methods predicting survival and activity of the probiotic cultures in various applications. In this study we describe a fluorescence staining assay for the determination of viability of Bifidobacterium cells in microplate scale. LIVE/DEAD BacLight Bacterial Viability Kit (SYTO9 and propidium iodide) was utilized for viability testing of fresh and freeze-dried Bifidobacterium cells and the fluorescence intensity was detected by a microplate fluorometer. Validation of the microplate scale assay was performed by comparing results to colony forming units, fluorescence microscopy and spectroscopy. Fresh and freeze-dried B. animalis cultures treated in acidic conditions (pH 2.5 and pH 3.0 alone and in combination with pepsin) or stored at different temperatures were used to study the applicability of the microplate assay for viability assessment of stressed cells. To reveal changes in membrane functions during acid treatment, DIBAC4 (a potentiometric fluorochrome) was additionally used for the analysis of the acid treated cells. In general, the results obtained with the microplate assay were comparable with plate count analysis and fluorescence microscopy. Microplate assay with viability stains gave an estimate of the viability of the probiotic preparations (detection level 106 cfu and the assay was applicable also for acid-treated cells. In the acid tolerance test, B. animalis was sensitive to pH 2.5, although pepsin had clearly protective effect in acidic conditions. Potentiometric measurements with DiBAC4 fluorochrome indicate that acidic conditions caused hyperpolarization of the cell membrane in B. animalis cells. Benefits from the fluorochromic viability assays are that changes in cell state in probiotic preparations can be estimated earlier compared to the results obtained with traditional cultivation methods