Fluorescence Flow Cytometry (FFC) for K

In Fluorescence Flow Cytometry (FFC), bacteria are first mixed with fluorescent stains. The stained sample is then injected into the flow cytometer, where the cells are arranged into a single-file. As each bacterium passes through a laser beam one at a time, it scatters light and emits fluorescence from the bound stains. A typical approach for bacterial vitality is to use SYTO-9 as a total stain and Propidium Iodide (PI) as a dead stain. PI can only penetrate cells with damaged membranes. This will result in red-staining of the bacteria while PI-negative cells with intact membranes will remain green.

Illustration of staining principle for fluorescence flow cytometry (FFC). Propidium iodide (PI) can only penetrate cells with compromised membranes, while the total stain, SYTO-9, can freely diffuse through the membrane.

Advantages of FFC for K

Flow cytometry can rapidly count thousands of cells per second, giving highly precise estimates of cell concentrations. This method uses direct counting, eliminating problems associated with size changes and calibration factors, unlike optical methods.

The fluorescent staining principle makes it possible to distinguish between cells and debris by selective gating. It is possible to use the method to acquire information on live and dead subpopulations. The total cell concentration stays relatively constant once the plateau in stationary phase is reached and therefore FFC allows for a wide sampling window, unlike the plate count method.

FFC comes with 96-well formats which enables walk-away, high-throughput automation.

Disadvantages of FFC for K

The main disadvantages of FFC is that the instruments and reagents are costly. Furthermore skilled operators are required and it is not trivial to optimize staining and gating for different bacteria.

The sample workup is relatively involved with dilution, staining and filtering. This is time-consuming and may introduce variability.

Practical recommendations

FFC provides accurate cell concentration estimates and can detect individual species, showcasing its versatility. Despite its power, the high cost of equipment, need for skilled operators, and extensive method development have hindered its widespread adoption.