3 Ways Flow Cytometry Can Be Used To Research Bacteria

The global bacterial biomass has been estimated to be 5×1030, which is significantly higher than plants and animals. We are intimately dependent on bacteria for processing waste, producing vitamin B12, fixing nitrogen and so much more.  While some bacteria are known pathogens, most are not.  These organisms live in all environments from the soil to hot springs to deep thermal vents.  

Bacteria, on the whole, are typically 0.5 to 5 µm in size, just at the edge of the detection of our flow cytometers.  However, flow cytometry has become an essential tool for microbiologists to characterize bacteria as well as isolate them.  Table 1, taken from Current Protocols (Robinson, 2004 11.1.1-11.1.4) shows a rough comparison between bacteria, yeast, and larger eukaryotes.  

The article goes on to make the following estimate of fluorescence signal. Assuming an average eukaryote has a signal of 100,000 MFI, the average bacteria would have a surface signal of 181-727 MFI, and an intracellular signal of 30-300 MFI. So it is important that the probes that are used are as bright as possible. 

This blog highlights the power of flow cytometry focusing on activity assays, bacterial susceptibility testing, and cell sorting. These techniques are just the tip of the iceberg when it comes to studying bacteria using flow cytometry. 

Assays: Viability And Metabolic Activity Of Bacteria.

Traditionally, bacterial viability is measured using plate growth techniques. These are not always ideal, especially for slow-growing or hard to culture bacteria. This is where fluorescence is useful, and one of the tools for this is the Live/Dead BacLight kit from ThermoFisher. 

This kit contains a cell-permeant dye (Syto 9) and a cell impermeant dye (PI).  Labeling cells with these two dyes allow for the detection of live vs dead cells, as shown from this data taken from the ThermoFisher website. 

Figure 1: Use of the Live/Dead BacLight Kit. Dead cells show a lower green fluorescence than the live cells. Data from ThermoFisher.

By adding a bead to this kit, it is possible to enumerate the live cells at the same time. The advantage of this assay is that it works with both gram-positive (S. aureus, left) and gram-negative bacteria (E. coli, right).

If metabolic activity is important, the use of a dye to measure that parameter is also available, as shown in the figure below.

Figure 2: Measuring viability and metabolic activity of bacteria. Data from ThermoFisher.

One of the common assays for bacterial flow is viability. Our example uses the Thermo-Fisher Live/Dead Bac Light Kit, pictured [in Figure 1].

As you can see, this kit uses two dyes: Syto 9, a cell-permeant nuclear dye, and propidium iodide (PI), a cell-impermeant dye. When you plot Syto 9 fluorescence vs. that of PI—using S. aureus per this example—you can discriminate accurately between live cells and dead ones.

In fact, using flow cytometry to characterize bacteria is out of this world! Leys and coworkers (2009) used the bacteria Cupriavidus metallidurans CH34 to assess its survival in space. Samples were sent to the International Space Station, with controls grown on Earth. The cells were tested for growth on plates as well as a variety of metabolic characteristics that are summarized in this table from that publication.

Flow cytometry is an excellent tool for measurements of bacterial metabolic characteristics and can provide a great deal of information in a relatively short period of time.   

Flow Cytometry And Susceptibility Testing.

Tuberculosis, caused by the bacteria Mycobacterium tuberculosis, has been estimated to cause about 3 million deaths per year, with 100 million new cases diagnosed. M. tuberculosis is also a slow-growing bacteria, which impacts the ability to rapidly test new compounds for treating this disease. A typical experiment takes 2 to 3 weeks before results are known.

Norden and coworker (1995) turned to flow cytometry as a possible solution, using the fluorescent dye fluorescein diacetate (FDA), which is colorless until it is hydrolyzed to fluorescein by cellular processes. As shown in the figure below, viable M. tuberculosis (left) were able to hydrolyze the FDA and a clear fluorescent signal was detected. Nonviable M. tuberculosis (right), on the other hand, did not fluoresce.  

Figure 3:  Measuring the metabolic activity of M. tuberculosis using FDA.

This allowed the researchers to measure the effect of increasing concentration of different drugs on the viability of M. tuberculosis. These results were obtained in 24 hours, saving weeks of time using traditional methods and allowing for the more rapid screening of multiple compounds. 

Figure 4:  Effects of increasing doses of drugs on M. tuberculosis viability. 

The ability to rapidly test the effect of different compounds on the growth of bacteria using flow cytometry saves time and money.  

Sorting Bacteria

Isolation of bacteria can be a tedious process, especially if the bacteria are not amenable to culture in the laboratory. Additionally, rather than the suite of antigens used for phenotyping, bacteria are characterized by their 16S ribosomal RNA (rRNA) sequences. There is extensive literature on different probes to characterize different bacterial species.

To address this issue Batni and coworkers (2019) developed a methodology to introduce these probes into living cells without a permeabilization step. Their live FISH procedure is summarized below. 

Having developed this method, the authors set out to test this with samples from Baltic seawater. Here, they used a probe for alphaproteobacteria labeled with 6-carboxyfluorescein. Their sorting strategy is shown below. 

Batani et al. applied this methodology to sorting Baltic Sea water (shown in Figure 5) for alphaproteobacteria. The Control group “ALF968_6-FAM + PI,” shown in sub-figure “E,” was labeled with an alphaproteobacteria probe and propidium iodide. Positive cells were identified and sorted into a 96-well plate, and researchers were then able to identify positive sorts based upon turbidity of the culture—something the other controls were not able to do.

This demonstrated that Batani et al.’s technique is applicable to isolating new bacteria based upon their ribosomal sequence. Suffice it to say, this opens up some really exciting new avenues of possibility.

Concluding Remarks

To summarize, flow cytometry can be applied to bacterial populations, even though they are sized below normal limits of detection. Viability and metabolic activity are two common assays that can be applied to bacteria—and you can count bacteria without having to culture them on a plate.

Flow cytometry can speed up susceptibility testing, as demonstrated by the Mycobacterium tuberculosis studies. And of course, bacteria can be sorted by flow. Most interesting of all is that a new bacteria-sorting technique based on RNI identification has been identified and validated. So, if you’re looking to characterize different populations, this may be a great tool for you to explore.

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