An Indirect Fluorescence Microscopy Method to Assess Vaginal Lactobacillus Concentrations

Lactobacillus species are the main colonizers of the vaginal microbiota in healthy women. Their absolute quantification by culture-based methods is limited due to their fastidious growth. Flow cytometry can quantify the bacterial concentration of these bacteria but requires the acquisition of expensive equipment. More affordable non-culturable methods, such as fluorescence microscopy, are hampered by the small size of the bacteria. Herein, we developed an indirect fluorescence microscopy method to determine vaginal lactobacilli concentration by determining the correlation between surface area bacterial measurement and initial concentration of an easily cultivable bacterium (Escherichia coli) and applying it to lactobacilli fluorescence microscopy counts. In addition, vaginal lactobacilli were quantified by colony-forming units and flow cytometry in order to compare these results with the indirect method results. The colony-forming-unit values were lower than the results obtained from the other two techniques, while flow cytometry and fluorescence microscopy results agreed. Thus, our developed method was able to accurately quantify vaginal lactobacilli.