The use of flow cytometry to accurately ascertain total and viable counts of Lactobacillus rhamnosus in chocolate

The goals of this study were to evaluate the precision and accuracy of flow cytometry (FC) methodologies in the evaluation of populations of probiotic bacteria (Lactobacillus rhamnosus R0011) in two commercial dried forms, and ascertain the challenges in enumerating them in a chocolate matrix. FC analyses of total (FCT) and viable (FCV) counts in liquid or dried cultures were almost two times more precise (reproducible) than traditional direct microscopic counts (DCM) or colony forming units (CFU). With FC, it was possible to ascertain low levels of dead cells (FCD) in fresh cultures, which is not possible with traditional CFU and DMC methodologies. There was no interference of chocolate solids on FC counts of probiotics when inoculation was above 107 bacteria per g. Addition of probiotics in chocolate at 40 °C resulted in a 37% loss in viable cells. Blending of the probiotic powder into chocolate was not uniform which raised a concern that the precision of viable counts could suffer. FCT data can serve to identify the correct inoculation level of a sample, and viable counts (FCV or CFU) can subsequently be better interpreted.