Scientists have long known that bacteria come in many shapes and sizes, but understanding what those differences mean has remained a major challenge, especially for species that can't be grown in the lab. Now, a new study led by Nina Wale , an Assistant Professor in MSU's Department of Microbiology, Genetics, & Immunology, introduces a groundbreaking method that could change how researchers study bacterial diversity.
The research, published in mSphere , focuses on a tiny, unculturable pathogen called Pasteuria ramosa, which infects water-dwelling crustaceans known as Daphnia. These bacteria are pleiomorphic, meaning they can take on multiple shapes during their life cycle. Until now, scientists had to rely on fluorescent labels — custom-made tags that require detailed knowledge of a bacterium's biology — to sort and study these different forms. But for most bacteria, especially those that live in soil, water, or inside animals, that kind of information simply doesn't exist.
Wale's team found a way around this problem. Using imaging flow cytometry, they developed a label-free technique that identifies bacteria based on how they scatter light and naturally fluoresce. These "light signatures" allow researchers to sort different bacterial shapes without needing to tag them first.
Wale likens the different bacterial shapes, known as morphologies, to members of a football team. Although they all belong to the same team, each has a distinct role: some are coaches, some are players, and they each behave differently.
"To understand what each morphology does, we need to separate it from the other morphologies so we can understand what sorts of genes or proteins they express," said Wale. "Per the analogy, we need to get the coaches and players on their own so we can biologically 'interview' each group about what it does. It's kind of like we've invented a procedure to identify, for the first time, football players vs. coaches based on their uniforms; now we don't have to go up to them and give them a badge saying 'player' and 'coach' in order to tell them apart."
The method is not only accurate, yielding samples that are over 90% pure, but also opens the door to studying bacteria that were previously off-limits. Researchers can now investigate how different shapes contribute to bacterial behavior, such as causing disease, growing, or spreading to new hosts even if they can't grow in a dish or haven't been studied before.
This foundational work could have wide-reaching implications. It may help scientists understand how bacteria evolve and cooperate and even lead to new ways of counting or culturing hard-to-study microbes.
Daniel Vocelle, then Assistant Director of MSU's Flow Cytometry Core, is lead author on the paper. He notes that this new technique has helped launch broader interest in imaging cytometry.
"This method really highlights where the field of flow cytometry is heading, particularly the shift toward autofluorescent phenotyping which means using cells' natural fluorescence to identify them," said Vocelle. "It also showcases the advantages of imaging cytometry and how it can refine cell populations, enhance discovery, and rapidly identify rare events."
Next, Wale hopes to use the technique to explore how Pasteuria ramosa manipulates its Daphnia hosts, sometimes making them grow abnormally large or turn bright orange before dying. By separating and analyzing each bacterial shape, her team aims to uncover the genetic and chemical strategies behind these dramatic effects.
"The fact that there's a relationship between form and function is familiar to everybody: butterflies' wings allow them to fly, hummingbirds' beaks allow them to drink nectar. But we've only been able to study the role of bacterial shape in the small fraction of bacteria that can be grown in the lab, and even those bacteria don't always exhibit their full range of shapes inside of a dish," said Wale. "Our new methodology will allow scientists to explore the relationship between form and function across the bacterial tree of life and in the environments where they live naturally".
