Probiotic Interference of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 with the Opportunistic Fungal Pathogen Candida albicans
Figure 4
Viability assays of C. albicans cells following coculture with bacteria and exposure to lactic acid. (a) Microscopic viability assay: C. albicans cells were stained with the FUN 1 cell stain (Invitrogen) for viability after coculture with L. rhamnosus GR-1 (Ca + Lb-GR-1). A C. albicans culture without lactobacilli served as control (Ca). Cell images were obtained by confocal laser scanning microscopy. Results for the vital staining procedure are depicted above the images. White arrows in the zoomed insert image on the left indicate CIVS formation in metabolically active cells of the control culture. The presence of lactobacilli increases the number of metabolically inactive and dead cells (right image). (b) Fluorometric assay of C. albicans cells viability following 16 hr incubation in MRS, MRS (HCl) pH 4.5, and MRS (lactic acid) pH 4.5. Cell viability was measured by monitoring the conversion of FUN 1 to red fluorescent intravacuolar structures in a microplate assay. The red/green fluorescence ratio increases in the presence of viable, metabolically active cells, particularly within the first 30 min of the assay. Metabolically inactive cells show no increase of the red/green ratio. For assay details see Section 2. The figure shows the results of a representative experiment. Incubation of C. albicans cells in MRS at pH 4.5 in the presence of lactic acid appears to reduce cell viability much more than incubation in MRS that was adjusted to pH 4.5 using HCl. (c) Example of a fluorometric FUN 1 cell viability assay of C. albicans following coculture with L. rhamnosus GR-1, L. reuteri RC-14, L. johnsonii, or S. aureus. A C. albicans culture without bacteria served as control (Ca + MRS). GR-1 and RC-14 consistently caused the strongest reduction in C. albicans viability. All fluorometric experiments were conducted in triplicate. Error bars indicate standard deviations.
