Adding flow cytometry to your platform repertoire for microbial research can elucidate aspects unseen by traditional bulk observations. Various industries use flow cytometry to investigate microbial biology, such as biomedical sciences, food safety, and genetics. Flow cytometry can help clinicians detect bacterial infections and determine susceptibility to antibiotics, public health agencies use flow cytometry to detect pathogenic bacteria in water or food, and flow cytometry can also be useful for genetic studies when utilizing bacteria as a vehicle. Conventional methods used to examine bacteria are based on observations of either single-cell morphology or colony characteristics when grown on agar. However, highly sensitive flow cytometers provide tools for detecting and analyzing microbes independent of their cultivability, as not all microbes will grow equally on agar. Here we will highlight some of the advantages of using flow cytometry for microbial research as well as overview some experimental tips and tricks to jumpstart your research.
Benefits of using flow cytometry for microbial research
Single cell analysis
Flow cytometry is a technique that allows one to analyze bacteria rapidly and individually, while simultaneously offering quantitative analysis of microbial heterogeneity. Traditional methods of single-cell bacterial analysis are limited in the capacity to analyze individual cells while time-consuming microscopy limits the total number of cells that can be analyzed. On the other hand, flow cytometry is capable of analyzing thousands of cells per second, improving statistics, and allowing the quantification of unique and rare cell types.
Absolute cell counting
Using flow cytometry for bacterial cell counting offers a significant advantage in both speed and accuracy over traditional counting methods such as by microscopy, cell mass determination, or optical density readings. A flow cytometer can obtain cell counts of microbes either by direct volumetric counting or with the addition of counting beads. Also, by coupling cell counts with additional measurements, such as viability or size, cell counts of specific cell subpopulations can be easily determined.
Multiparameter analysis
One advantage of using a flow cytometer for bacterial characterization is the capability to perform multiparameter analysis. Light scattering characteristics can reveal the size (FSC) and complexity (SSC) of bacteria, however, additional information can be obtained by concurrent analysis with specific fluorescent stains such as DNA content, cellular viability, and membrane potential. These additional parameters can provide valuable insights into cellular health, growth, as well the capability to distinguish different species of bacteria in a mixed culture.
Tips and tricks for analyzing bacteria with a flow cytometer
Detecting bacteria on a flow cytometer
Proper PMT Voltage Settings/Range
Flow cytometers are capable of analyzing a wide variety of particles ranging from large cultured cell lines to microbes smaller than 1 µm. Many instruments cannot simultaneously collect a broad range of signal, therefore optimization of photomultiplier tube (PMT) voltage settings must be done to ensure proper collection of sample, especially of small particles such as bacteria. Some new flow cytometers, such as the NovoCyte®, have a wide range for signal detection, eliminating the need to make complicated PMT voltage adjustments, traditionally reserved for experienced individuals. Using a flow cytometer with this wide dynamic range of signal detection simplifies the experimental setup and ensures sufficient PMT settings for a broad range of assays.
Correct Threshold Setting
Figure 1. Detection of bacteria in natural waters with NovoCyte® Flow Cytometer
Fresh natural water was filtered through a 300 mesh sieve, diluted with deionized water (filtered through 0.1μm membrane) to desired concentration. A 100x SYBR® Green I dye was added to the sample, incubated at 37˚C for 13 min and acquired on the NovoCyte flow cytometer. Bacteria was differentiated from background by Green versus Red plot. Absolute counts were obtained automatically in each sample.
To ensure signal collection is specific, a threshold is used to eliminate background noise. The threshold is an electronic hurdle that establishes a criteria whether events are recorded or not. It is important to properly determine the threshold because anything that does not meet this criterion will not be displayed or saved for analysis. Forward or side scatter are generally used as the thresholding parameter, since no additional reagents are needed. However, bacteria may pose an issue because of their small size and make it difficult to distinguish them from background or debris. If this is the case, it is recommended to set a threshold using a fluorescence signal (i.e. fluorescent triggering) to specifically identify bacteria and ensure separation from the debris. Bacterial detection by fluorescence commonly includes a dye that binds to nucleic acids such as SYBR Green I, Sytox Green I, etc. Fluorescent triggering with SYBR Green to detect bacterial contamination in water is shown in Figure 1. This plot demonstrates sufficient discrimination of bacteria from both background caused by electronic noise and inorganic particles. In conclusion, using proper threshold settings, you can ensure quality data acquisition of your samples.
Obtaining bacterial cell counts on a flow cytometer
Flow cytometry can also be used to obtain accurate, absolute cell counts, or concentration of cells in the entire population or cell subpopulations. Currently, most flow cytometers depend on counting microbeads as reference particles to calculate cell counts, however, some flow cytometers feature direct volumetric cell counting without the need for counting beads. Either method produces precise and accurate absolute cell counts, but direct volumetric counting provides cell counts without the need for additional reagents or increased experimental complexity. Counting beads have a defined concentration, which allows acquisition of a certain number of bead events, and uses that number to calculate the cell concentration in the sample. Counting beads come in a variety of sizes, with and without fluorescence. If you plan on using fluorescence triggering, it is important to also use counting beads with a similar fluorescence. For direct volumetric counts, the cell concentration is automatically calculated by the flow cytometer without any additional steps, generally using a very precise syringe pump. Overall, both cell counting methods result in absolute counts that are fast and easy to achieve.
Multiparameter analysis of bacteria
Although light scattering alone can provide valuable information, the addition of multiparametric analysis using specific fluorescent dyes or markers offers further insights regarding viability, metabolic activity, cellular stress responses, and cell cycle analysis. However, there are specific features that are important to keep in mind when using fluorescence to analyze bacteria. The fluorescent reagents needed for cell analysis may be different for bacteria than mammalian cells, or the concentrations used might need to be altered. The size, mass, nucleic acid, and protein content of bacteria are approximately 1/1000 the magnitude of mammalian cells, thus the signal may be affected. Bacteria also tend to behave differently from eukaryotes in their interaction with reagents used in cytometry; this can be caused by differing uptake and efflux of reagents, which are affected by the structure of the cell wall and the presence of pores/pumps that may not be analogous to those in eukaryotes. One example of this is that the outer membrane of gram-negative bacteria excludes more lipophilic and hydrophilic molecules, commonly used to measure membrane potential. Therefore, it is important to empirically determine the correct concentration of reagents needed for staining in bacteria and ensure that it can be used for microbial analysis.
Conclusion
Including flow cytometry for microbial research can enhance experimental results, offering the capability to perform single-cell multiparametric analysis as well as absolute cell counts. Using the tips offered in this note will help prevent many of the pitfalls that occur in microbial analysis on the flow cytometer.
Lauren Jachimowicz is application development scientist at ACEA Biosciences.
Images: ACEA Biosciences
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