Combining Flow Cytometry With Plant Science, Microorganisms, And The Environment

My first introduction to flow cytometry was talking to a professor who’d brought one on a research cruise to study phytoplankton. It was only later that I was introduced to the marvelous world that’s been my career for over 20 years.  

In that time, I’ve had the opportunity to work with researchers in many different areas, exposing me to a wide variety of cell types and more important assays. What continues to amaze me is the number of different parameters we can measure, not just the number of fluorochromes, but the information we can extract from samples – animal, vegetable (but not quite mineral). Everything from beer and wine making to flowering plants to cancer and immunology can be measured using flow cytometry, as long as you know what question you want to ask and understand how you can answer it with flow cytometry. It’s this power of flow cytometry that keeps the field interesting for me, and while phenotyping and sorting of mammalian cells may be the bread and butter of many facilities around the world, it’s nice to take a look at all the possibilities out there.

Plant Science And Flow Cytometry

The ploidy level of a given plant can have profound impacts on the progeny. Many plants can undergo polyploidization, which causes dramatic effects on the observed phenotypes and the physiology of the plant. Plant breeding programs can depend on knowing the ploidy level of the cultivars being studied. Imagine if there was an easy way to determine this for the plant breeder. Now, I don’t remember where I was first introduced to this method but I remember who introduced me to this technique (Thanks  Dr. David Galbraith). He demonstrated an amazing, simple, and efficient way to extract nuclei from plant tissue and analyze it using flow cytometry. There’s a great protocol here and I have borrowed the figure below from this source. Briefly, you chop up the tissue in the chopping buffer, filter the sample, and stain with a nuclear dye. It’s that simple. 

Figure 1:  Determining plant ploidy by flow cytometry.

Why is this important? There are around 360,000 species of angiosperms but only about 2,500 are crop plants. As we lose genetic diversity, our food supply becomes more susceptible to diseases and pests.  Since we don’t know what we don’t know, finding a way to start characterizing these critical plants is important. Coupling flow cytometry methods to identify the ploidy level of different species with techniques like NGS sequencing, who knows what we will find.  

Another area where flow cytometry can impact plant sciences is the ability to isolate plant protoplasts, which are plant cells that have had the cell wall removed, allowing for many different manipulations that are difficult or impossible on intact plant cells. These fragile cells offer their own unique challenges in flow cytometry, but as was illustrated in this article, using flow cytometric analysis on these cells is yielding much fruit (pardon the pun, or not).

Microorganism Flow Cytometry

I once worked on a project where the researchers were interested in isolating fecal bacteria from newborns to understand how their microbiomes changed during development. It was a challenge several levels below the normal mouse and human cells we worked with, from sample preparation to isolation and sorting of cells. Not to mention the sterilization of the instrument after the sorts to prevent contamination of the next sample. I’m happy to report that we were able to provide the researcher with their bacteria, and clean the system before the next sort.  

Bacterial flow requires you to delve into the details of the instrument performance, and optimize those conditions for smaller cells, which we’ve talked about here. Some of the newer instruments are looking for solutions for this issue, including the use of PMTs for detection and different lasers to measure scatter.  

Two of the most common measurements of bacteria by flow cytometry include viability measures and cell counting. However, with the use of fluorescent proteins and other markers, it’s possible to look at the membrane potential and expression of given genes of interest. Bacterial flow has also been performed on the International space station

Bacterial flow is also important in food science, as shown in this article on using flow cytometry to study the microorganisms in wine.  If you’re in a wine producing area, maybe a trip to the local winery to see if you can offer your expert services might be in order?  Not to be left out, beer brewing has also been impacted by flow cytometry as shown in this article from the Journal of the Institute of Brewing.

Flow cytometry has also been quite useful in the rapid susceptibility testing of bacteria to drugs. In this article, the authors developed a method to test the resistance of Klebsiella pneumoniae to carbapenem.  Their assay showed excellent correlation to the gold standard and could be completed in some hours, whereas the gold standard took 24 hours.

Environmental Flow Cytometry

Flow cytometry of environmental samples provides its own unique sets of challenges. Take, for example this paper where the authors used flow cytometry to aid in the isolation and cultivation of filamentous bacteria from the soil (figure 2).

Figure 2:  Flow sorting to assist isolation of soil microorganisms.

Another use of flow cytometry has been to detect the presence of Cryptosporidium oocytes in water. Cryptosporidium can cause cyrptosporidiosis, which is especially rough on immunocompromised patients.  

In marine sciences, flow cytometry continues to show its power. In this paper, the authors explored the changes in phytoplankton as a function of depth.

Concluding Remarks

When confronted with a new challenge, flow cytometry may be the go-to tool that you should consider.  With the ability to analyze thousands of cells in a short time period, we’re able to build a complete picture of what is occurring in the populations of interest. Add the power of cell sorting, and we can isolate these cells for many downstream applications, from cell culture to NGS analysis.

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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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