How small can you go? Flow cytometry of bacteria and viruses

Flow cytometers are traditionally designed for measuring particles, like beads and cells. These tend to fall in the small micron size range. Looking at the relative size of different targets of biological interest, it is clear the most common targets for flow cytometry (cells) are comparatively large (figure 1).

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Figure 1:  Relative size of different biological targets of interest. Image modified from Bioninja.   

In the visible spectrum, where most of the excitation light sources reside, it is clear the cells are larger than the light. This is important as one of the characteristics that we typically measure is the amount of light that the targets scatter. This we term as either forward or side scatter, based on the position of the detector relative to the excitation light source. Forward scatter is actually the Mie scatter, first described by the German physicist Gustav Mie. Mie scatter is  the light scattered by a spherical particle that is larger than the wavelength of the light illuminating the particle. A third component impacting Mie scatter is the refractive index of the particle and media. If you are interested in exploring the impact that the wavelength and refractive index have on the Mie scatter, this calculator can be very useful.  

Even though scatter is less useful for these smaller particles, it is interesting to note that flow has been used for years to measure them. In 1979, Hercher and coworkers described the construction of a purpose built instrument capable of measuring viruses, and were able to identify different test viruses by their scatter profiles alone. A review article by McSharry titled ‘Uses of Flow Cytometry in Virology’, that was published in 1994 summarized much of the work in this field, and the possibilities have continued to expand over the years.  

Understand your instrument

If you want  to be successful in any of these applications, the first thing that you need to understand is how sensitive your instrument is and how to get the best performance out of the system. We are fortunate that a lot of work has been done in this area because of the importance of extracellular vesicles and microparticles. In this paper by Tang et al (2017), the authors do a good job of discussing some of the work that you need to do in order to ensure that you can successfully measure smaller particles. Some of these tests include determining an appropriate threshold, how detector voltage and laser power affects the ability to resolve particles and best dilution factors to use. Take figure 2, which is taken from this paper showing one such study.  What is nice is that at the bottom there is a summary of the data looking at counts and aborts, so that it is possible to identify the best combination of voltage and laser power. 

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Figure 2:  Effect of laser power and detector voltage on the ability to measure small particles.  

Once you have your system characterized, the fun can begin. 

Speeding up assays 

Rabies virus infected cells are detected by PCR, ELISA or electron microscopy. However, flow cytometry offers a way to potentially detect the infection of cells sooner than these techniques. Bordignon and coworkers (2002) developed an intracellular flow cytometry based assay to identify cells that were infected with the rabies virus. The literature suggested that the cells infected with rabies virus could be detected 72 hours after infection. Using the flow based assay, these researchers were able to detect virus infected cells within 12 hours post infection.  

Norden and coworkers (1995) took a similar approach to measure Mycobacterium tuberculosis by flow cytometry. In this case, they used the fact that the bacteria can hydrolyze fluorescein diacetate. The work set out to find a faster way to determine the susceptibility of M. tuberculosis to different antimicrobial agents. The standard methods took 4 to 12 days for results, and required the cells to be infected. However, the flow based assay was much faster and reduced this time to 24 hours in a cell free manner.  

Sorting bacteria and viruses

Cell sorting is an incredibly powerful tool to isolate cells. Bacteria are responsible for many processes from food production, to sewage treatment to nitrogen fixation. But many of these bacteria have never been cultured. This is where cell sorting can become highly useful, especially in marine biology. Zehr and coworkers (2008) used cell sorting to study a recently discovered single-cell nitrogen fixing cyanobacteria. This work was able to isolate small cells ( <1𝝻m in diameter) and discovered that these cells lacked the components of photosystem II, and were able to fix nitrogen during daylight hours. Since nitrogen is an element that limits plant growth, the discovery and characterization of these cells is important to better understand the overall nitrogen budget in the oceans. 

Viruses are also amenable to cell sorting, as shown in the work of Bonar and Tilton (2017). Using fluorescently labeled HIV-1 viruses, they were able to isolate  infectious HIV particles. They even performed an experiment where they mixed two different particles. One was labeled with GFP and the other with an orange emitting protein. As a proof of concept, they mixed these two viruses together and identified three populations, (top right), single positives and a double positive population. When this double positive population was sorted and reanalyzed, as expected, it was composed of the two different viral particles binding to each other as shown in the bottom panel labeled 3. 

Figure 3:  Results of viral sorting a mixture of HIV-1 labeled cells.  Taken from Bonar and Tilton (2017).

Another result from this paper demonstrated the sensitivity of flow based assays. Detection of the HIV particles was determined to be under ≤80 particles per ml. This is over 1000 times lower than the p24 ELISA and close to the sensitivity of PCR-based assays. Bonar and Tilton (2017)

Concluding Remarks

Flow cytometry is for more than just characterization and isolation of cells. Proper care and instrument characterization, enables flow cytometry to measure small particles as bacteria and viruses. Using this technique, it is possible to develop assays to speed up discovery, identify previously unknown organisms. The sensitivity of these assays as good or better than many standard assays. Perhaps it is time to consider adding flow cytometry to your experimental repertoire, if you have not already.

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Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

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