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.

To learn more about important control measures for your flow cytometry lab, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

Join Expert Cytometry's Mastery Class

ABOUT 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.

Tim Bushnell, PhD

Similar Articles

The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry

The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry

By: Tim Bushnell, PhD

What photon from yonder fluorochrome breaks?  It is … umm… hmmm. Let me see. Excitation off a 561 nm laser, with an emission maximum of 692 nm. I’m sure if Shakespeare was a flow cytometrist, he might have written that very scene. But the play is lost in time. However, since the protagonist had difficulty determining what fluorochrome was emitting photons, let’s consider how this could be figured out. In my opinion, one of the handiest flow cytometry tools is the spectral viewer. This tool helps visualize the excitation and emission profile of different fluorochromes, as well as allowing you…

Fickle Markers: Solutions For Antibody Binding Specificity Challenges

Fickle Markers: Solutions For Antibody Binding Specificity Challenges

By: Tim Bushnell, PhD

Reproducibility has been an ongoing, and important, concept in the sciences for years.  In the area of biomedical research, the alarm was sounded by several papers published in the early 2010’s.  Authors like Begley and Ellis, Prinz and coworkers, and Vasilevsky and colleagues, among others reported an alarming trend in the reproducibility of pre-clinical data.  These reports indicated between 50% to almost 90% of published pre-clinical data were not reproducible.  This was further highlighted in the article by Freedman and coworkers, who tried to identify and quantify the different sources of error that could be causing this crisis.  Figure 1,…

5 Common Flow Cytometry Questions, Answered

5 Common Flow Cytometry Questions, Answered

By: Tim Bushnell, PhD

I want to thank all of you who send us your questions about flow cytometry, so I thought I would dip into the old email bag and answer a few of the common ones here.  If your question isn’t answered this time, look for it to be answered in a future blog post.  Of course, if you want us to cover a specific topic, drop us a line.  1. How Fast Can I Go? This is  a common question. The allure of the ‘hi’ button is hard to resist.  The faster you go, the sooner you are finished with data…

Common Numbers-Based Questions I Get As A Flow Cytometry Core Manager And How To Answer Them

Common Numbers-Based Questions I Get As A Flow Cytometry Core Manager And How To Answer Them

By: Tim Bushnell, PhD

Numbers are all around us.  My personal favorite is ≅1.618 aka ɸ aka ‘the golden ratio’.  It’s found throughout history, where it has influenced architects and artists. We see it in nature, in plants, and it is used in movies to frame shots. It can be approximated by the Fibonacci sequence (another math favorite of mine). However, I have not worked out how to apply this to flow cytometry.  That doesn’t mean numbers aren’t important in flow cytometry. They are central to everything we do, and in this blog, I’m going to flit around numbers-based questions that I have received…

3 Must-Have High-Dimensional Flow Cytometry Controls

3 Must-Have High-Dimensional Flow Cytometry Controls

By: Tim Bushnell, PhD

Developments such as the recent upgrade to the Cytobank analysis platform and the creation of new packages such as Immunocluster are reducing the computational expertise needed to work with high-dimensional flow cytometry datasets. Whether you are a researcher in academia, industry, or government, you may want to take advantage of the reduced barrier to entry to apply high-dimensional flow cytometry in your work. However, you’ll need the right experimental design to access the new transformative insights available through these approaches and avoid wasting the considerable time and money required for performing them. As with all experiments, a good design begins…

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

By: Tim Bushnell, PhD

Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting.  FLUOROCHROMES Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached…

Flow Cytometry Year in Review: Key Changes To Know

Flow Cytometry Year in Review: Key Changes To Know

By: Meerambika Mishra

Here we are, at the end of an eventful year 2021. But with the promise of a new year 2022 to come. It has been a long year, filled with ups and downs. It is always good to reflect on the past year as we move to the future.  In Memoriam Sir Isaac Newton wrote “If I have seen further, it is by standing upon the shoulders of giants.” In the past year, we have lost some giants of our field including Zbigniew Darzynkiwicz, who contributed much in the areas of cell cycle analysis and apoptosis. Howard Shapiro, known for…

What Star Trek Taught Me About Flow Cytometry

What Star Trek Taught Me About Flow Cytometry

By: Tim Bushnell, PhD

It is no secret that I am a very big fan of the Star Trek franchise. There are many good episodes and lessons explored in the 813+ episodes, 12 movies (and counting). Don’t worry, this blog is not going to review all 813, or even 5 of them. Instead, some of the lessons I have taken away from the show that have applicability to science and flow cytometry.  “Darmok and Jalad at Tanagra.”  (ST:TNG season 5, episode 2) This is probably one of my favorite episodes, which involves Picard and an alien trying to establish a common ground and learn…

5 Flow Cytometry Strategies That Sun Tzu Taught Me

5 Flow Cytometry Strategies That Sun Tzu Taught Me

By: Tim Bushnell, PhD

Sun Tzu was a Chinese general and philosopher. His most famous writing is ‘The Art of War’, and has been studied by generals and CEOs, to glean ideas and strategies to help their missions. I was recently rereading this work and thought to myself if any of Sun Tzu’s lessons could apply to flow cytometry.  So I have identified 5 points that I think lend themselves to thinking about flow cytometry.  “Quickness is the essence of the war.” In flow cytometry, speed is of the essence. The longer the cells are out of their natural environment, the less happy they…

Top Industry Career eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.