**3. Fluorescence-activated cell sorting**

Flow cytometry separates cells based on their intrinsic physical and chemical characteristics, integrating electronics, fluidics and optics. The sample, from which the cells of interest are to be sorted, is passed through a flow cell. The sheath fluid escorts the cells down the channel, where they encounter a laser beam. Light beams of specific frequencies and wavelength are emitted. Detectors measure the forward scatter (FSC) and the side scatter (SSC) based on cell size and granularity. FSC and SSC are unique for every particle. A combination of the two can differentiate among cell types within a cohort of cells. This way, the qualitative and quantitative data of a particular kind of cell can be assessed.

Fluorescence-activated cell sorting is an application of flow cytometry. The cells of interest are fluorescently tagged and sorted by the machine. Here the GFP-tagged fluorescent *E. mundtii* is isolated from a mixture containing insect gut homogenate with other bacteria. The solution is delivered to the flow channel and carried by the sheath fluid. The pressure from the compressor, which is adjustable, forces the solution through a laser beam using hydrodynamic focusing. Then monochromatic beams of high intensity interrogate cells one at a time. Depending on the excitation wavelength of the fluorophore, the laser wavelength is chosen. The scatters are then recorded. The forward scatter (FSC), which refers to light that is refracted by the cell and continues in the same direction, tells us about the size of the cell. In contrast, the side scatter (SSC), which refers to light that is refracted by the cells and travels at right angles to the excitation axis, tells us about the fluorescence and granularity of the cells. The more granular a cell, the more scattered light it produces. Furthermore, each cell enclosed in a droplet is assigned a charge, depending on the extent of the cell's deflection [90]. After passing through an electrical field, the cells are deflected to the collection tubes and the uncharged droplets are directed to the waste. The detector system consists of a set of photo multiplier tubes that have specific filters to select for certain wavelengths of the beam and are set at the excitation range to view GFP.

Once the larvae are fed with the fluorescent *E. mundtii*, the number of larvae that survive can be determined and eventually recovered for further studies. The *E. mundtii* cells are sorted and their transcriptomes can be studied. This technology has made it possible to focus on a single cell or cells of interest, to study their function or their physiological state.
