Development of a Fluorescence-Based Method to Estimate Bacterial Density in In Vitro Fermentation Studies
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Zamorano: Escuela Agrícola Panamericana
Human gut microbiota composition, structure, and density varies among individuals mainly due to age, eating habits, gender, and other factors. Although human microbial biodiversity varies between individuals with respect to species composition, variable fecal bacterial load may introduce an artifact during in vitro fermentations, resulting in variable fermentation outcomes in terms of metabolite production and composition and structure of the microbiota. Multiple methods have been developed for the estimation of bacterial density; however, these tend to require considerable time to obtain results and/or expensive equipment, making their use difficult for measurement of bacterial density in inoculum before in vitro experiments. Here, we describe a convenient and cost-effective method to estimate bacterial density from diluted stool by staining bacteria with a fluorophore and measuring its fluorescence intensity. For our preliminary test, Escherichia coli cells were stained with SYBR Gold, SYBR Green, and DAPI to identify the fluorophore with the highest fluorescence intensity. SYBR Gold was selected as the best fluorophore and the method was optimized by maintaining the sample wash step and fluorophore concentration. Fluorescence intensity measurements were collected on a microplate reader to assess staining efficiency and cell numbers were estimated using a hemacytometer. Finally, the method was used to estimate the bacterial density in diluted feces obtained from two donors to later use this method to test the effect of normalizing the fecal bacterial load before in vitro fermentation.
bacterial density, fluorophore, normalization, microbiota