A common feature among Gram-negative bacteria is the production of outer membrane vesicles (OMVs). Once regarded as cell debris or microscopy artifacts OMVs are being increasingly associated with disease status and as bacterial virulence factors. Quantitative and qualitative analysis of OMVs is severely complicated by their small size (10-300nm in diameter) which requires high resolution methods, such as electron microscopy for visualisation. However, enumeration of OMVs via SEM/TEM is very time consuming and a straightforward and rapid method of counting OMVs is required. An accurate method of counting OMVs would allow comparative studies analysing the molecular composition of OMVs produced by different bacteria and under different growth conditions, and immunological evaluation.
Using an Apogee A50-Micro Flow Cytometer we initially optimised a protocol for counting bacterial OMVs produced by periodontal pathogen P.gingivalis. Several protein, lipid and DNA fluorescent dyes were found to be unsuitable for labelling OMVs as they severely altered the OMV structure as determined by forward/Small Angle Light Scatter (F/S-ALS). The lipid Fluorescent Dye, PKH-26, was found not to alter OMV F/S-ALS and was used to specifically label the lipid layer of the OMVs and facilitate enumeration of vesicles/μL/preparation. Vesicles counts were compared to bacterial counts from the culture in which they were produced to calculate vesicle to bacteria ratios. The optimised OMV counting protocol was then validated by counting OMV preparations from a variety of Gram-negative oral bacteria T.denticola; T.forsythia and the Gram-negative ESKAPE pathogens; Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter aerogenes.