Use of Silica Microspheres Having Refractive Index Similar to Bacteria for Conversion of Flow Cytometric Forward Light Scatter into Biovolume

This research describes an alternative approach for the rapid conversion of flow cytometric Forward Angle Light Scattering (FALS) into bacterial biovolume with the aim to develop a rapid and direct method to quantify active bacterial biomass in activated sludge and wastewater for detailed mass balances. The Rayleigh–Gans theory was considered for explaining the main parameters affecting FALS intensity: sensitivity analysis of the model was carried out, taking into account the parameters characteristic of bacterial cells and characteristics of the flow cytometer. For particles with size in the typical range of bacteria, the FALS intensity is affected mainly by volume and refractive index of bacterial cells and is approximately independent of the shape of the cells. The proposed conversion from FALS intensity into bacterial biovolume is based on a calibration curve determined by using silica microspheres having relative refractive index as far as possible similar to that of bacteria. The approach was validated for two different flow cytometers (the first equipped with an arc lamp and the second with a laser) by comparing the biovolume distribution obtained from FALS conversion with the biovolume measured conventionally under epifluorescence microscopy. The specific case of bacteria taken from wastewater treatment plants (influent wastewater, effluent wastewater and activated sludge) was addressed. Compared to the time-consuming conventional microscopic approach, the application of FALS for sizing bacterial biovolume could be a very promising tool being completed in few minutes, simultaneously to the enumeration of bacteria during the flow cytometric analysis and allowing a rapid and direct determination of active bacterial biomass in activated sludge and wastewater (for mass balances).