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To study organisms at the genetic level, we need their DNA. Which means that we need to be able to wade through all the bits and pieces lying within their tiny bodies to pick out something even tinier—something we can’t just dig out with a shovel. So how does James manage to get the precious DNA from Legendrea loyezae and the other ciliates he’s interested in studying?

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We here at the Journey to the Microcosmos headquarters have been very excited about this ciliate, which is called Legendrea loyezae. James, our master of microscopes, found it in a lake, which made him just one of five people in human history who had ever found this organism. In our last episode, we talked about how his discoveries managed to reconcile the contradictions that had gone unexplained in the observations previous scientists had made.

What we didn’t talk about was the fact that James and his collaborators, Dr. Demetra Andreou and Professor Genoveva F. Esteban, were the first people to gather genetic data about the Legendrea genus, thanks in part to their little Legendrea loyezae friends.

Now when we say “genetic data,” we could be referring to a lot of different things, in part because genes are such an extensive, varied landscape that humans are only in the early days of being able to describe. Genes contain the instructions that tell our cells how to make the proteins that carry out our lives as they drive chemical reactions or produce key structures or pass along information. And those instructions are written in the language of DNA, a double helix whose components spell out individual bits of a message, like letters building up to a word that builds up to a manual for life.

To make that language comprehensible to us, scientists use the letters A, T, G, and C to represent the chemical bases that link together to form that code. But within every living organism, whether it is us or the organisms we’re watching, that code is a living, churning, evolving force that enshrines our identity in relation to so many others. In our journey through the microcosmos, we have often seen the way genetic material and the use of phylogenetic techniques reveal new ways to see an organism’s identity or to upend a set of relationships we thought existed in the microcosmos, like when scientists realized that the organisms they had gathered together in the taxonomic category of “amoeba” were not really so closely related after all.

The idea is simple: let’s compare organisms and see if we can figure out—based on their similarities and differences—how closely related they are. We can—and have—done this with microscopes and extensive descriptions of what different microbes look and act like. But like we said, genetics has often upended what we have gleamed just through observation.

But to study organisms at the genetic level, we need their DNA. Which means that we need to be able to wade through all the bits and pieces lying within their tiny bodies to pick out something even tinier—something we can’t just dig out with a shovel. So how?

How does James manage to get the precious DNA from Legendrea loyezae and other ciliates he is interested in studying? He starts by grabbing the whole organism, separating it from the crowd in a manner similar to what you can see him doing here with some other microbes. There they are, just chilling, until James appears with a tool called a micropipette that draws them right up.

After taking the ciliate he is interested in, it’s time for James to give it a little bath to make sure that it is clean and removed any organism that might cloud up the results. He starts by squirting the organism into a clean drop of water and letting it swim around for a while. Then, grabbing a new, clean micropipette, James repeats the process again.

And again, and again, and again. Each time, using a new micropipette to drop the organism into a fresh drop of water. The goal is to get the ciliate very, very clean.

And that means James sometimes has to leave them swimming for as long as 30 minutes in that drop of water. And that’s not just to scrub their outsides. The insides need to be empty of any ciliates they might have eaten, that are now taking up residence inside of a food vacuole.

Yes, even a master of microscopes has to wait around a bit for a ciliate to poop sometimes. When he’s done and the ciliates are cleaned up, James freezes them in a tiny tube, getting them ready for the next step: isolating the DNA. The process is simple: he takes the tube out of the freezer and lets it thaw out.

And then he puts it back into the freezer and lets it ice up again. Just like the washing, this is a repetitive process. Over and over again, the organism is frozen, then thawed, frozen, then thawed.

Each time, the cell changes a little bit. Ice pierces through the membrane, and the insides of the cell begin to disintegrate. Buried in the cellular soup that remains is the organism’s DNA.

Now separating that DNA requires a technique that you have probably heard a lot about in the past few years: PCR, which stands for polymerase chain reaction. The goal of PCR is to take a small amount of DNA and then make many, many more copies of it. During the pandemic, we relied on PCR to find fragments of viral RNA in our mucus, and then to amplify it so that those small quantities of genetic material could be detectable.

But PCR is so important that even describing it as a tool to save lives during a pandemic feels like we are underselling it. It is one of the tools whose premise is so simple that it can be used and adapted for any number of applications. Since its discovery in 1985, PCR has been fundamental to the work of scientists who are doing everything from hunting down proteins to engineering new technologies to creating new medical therapies.

For today though, PCR’s main use to us is to gather small segments of the Legendrea loyezae’s DNA that can be used to find where it belongs taxonomically. And as with the previous steps that James had to undertake, there is a lot of repetition. The ciliate that was cleaned over and over again, and then frozen and thawed over and over again will now have its contents heated and cooled in a mixture that will tear apart the strands of DNA and find the parts that James wants to amplify, over and over again.

With each repetition, the number of DNA fragments goes up exponentially so that in the end, James is holding a tube full of them, ready to be sent off so that a machine can determine its sequence of As, Ts, Cs, and Gs for scientists to study further. With his prized DNA, James was able to find a family for the Legendrea loyezae: the family Spathidiidae, Order Haptorida. He was even able to find a sibling: Epispathidium papilliferum.

They even have similar protruding structures, making their family resemblance apparent beyond the genetic similarities. This is a relationship that the early observers of Legendrea loyezae might not have been able to infer. And even if they did, the tools to verify them didn’t exist.

Each time we visit the microcosmos, it’s with knowledge and tools that we did not have before. That is why it matters to discover again what someone found once before, to dig through old texts and familiar ponds. Because buried deep within, there is always something unknown remaining to be found.

Thank you for coming on this journey with us as we explore the unseen world that surrounds us. And thank you again to Private Internet Access for sponsoring today’s episode. PIA provides a secure, reliable VPN connection.

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