It is impossible to know just how many microbial species there are in the world.

One recent estimate put the number somewhere around 1 trillion species. And the vast majority of those organisms still have yet to be discovered.

But even with our relatively limited knowledge of the microcosmos, being able to identify microbes when we can is important, from helping doctors diagnose their patients to guiding scientists through their observations. And sometimes the goals are a little less urgent but still meaningful. Like for James, our Master of Microscopes who collects these organisms and tends to them, and for the rest of us on the Journey to the Microcosmos team who want to share what we've learned.

We love showing off these organisms, and the more specific we can get when we identify them, the more we can tell you about their lives. But even if we humans have only discovered a small fraction of all the microbes, there's still a lot that we have found and named, so figuring out what exactly is in our samples can be difficult. It’s like playing the board game Guess Who?, but first you have to solve a maze to find the right board.

And even when you do find it, you realize you are playing with the largest Guess Who? board ever, and still, you can’t be sure it even holds the answer. As we'll talk about later, scientists have devised a number of molecular methods that make this task less daunting. But we do things the old-fashioned way: we just look at the organisms.

We collect our samples from nature, peering in to an assembly of microscopic animals and single-celled creatures, looking for clues in their morphology that will hopefully reveal what they are. So come along with us. Let’s take you through some of the questions that help guide us through this process.

The best place to start is to ask yourself, in the broadest sense, what you are looking at. Is it multicellular? Is it a unicellular eukaryote?

A prokaryote? We'll start with the multicellular organisms, in part because they're generally a little easier to identify. There are fewer of them in total, and their bodies just have more going on, and that means that there’s more clues for us to find.

Let's take the tardigrade, as an example, but pretend that you don't know what tardigrades look like. You went down to your favorite pond, gathered some water, and now you're looking at what you collected through the microscope. On the other side is this delightful, bumbling bit of life.

You see some stubby legs waving around, a pair of eyes, and maybe some strange things moving around inside its body. All those details are helpful. Legs are a great clue, as are eyes.

And inside, the moving things you're watching seem a bit like organs. If you weren't sure that you were looking at a multicellular organism before, you probably are now. And beyond what you see, there’s also what you know, you know that you got this little guy from a pond, meaning a freshwater environment.

That means you can rule out organisms that only live in marine environments. So what now? You might be able to get help from a freshwater biology book, but if you're a hobbyist, those books might not be worth the cost.

Luckily, as old-fashioned as our methodology is, we have a resource that the microbe hunters of yore did not: the good ol’ internet, a source not just of cat pictures and arguments, but also of the many microorganisms that scientists and science enthusiasts like yourself have studied and documented. Sometimes, all you need to do is just a simple search for something like "freshwater microscopic animal" and look through images until you find something that matches what you're looking at. Now, of course, "freshwater microscopic animal" can bring up a whole range of species, like rotifers, hydras, and gastrotrichs.

But each of those animals is very different from the others, which can help narrow down what your unidentified microbial organism is and is not. If you find this process overwhelming at first, because you are trying to narrow down the vastness of the microcosmos into just one species, that’s okay! Every time you do this, you will get better at it.

It takes years of practice and experience to figure out how to use all of the information available to you and pick out the most useful details. And like a baby tardigrade waving its legs for the first time, we all have to start somewhere. With single-celled eukaryotes, the process starts to get more difficult.

You won't see the complex structures and systems that distinguish multicellular organisms, but you will see organelles that can help clarify what you're looking at. For example, one major clue can be picking out which organisms have those beating hairs called cilia versus which ones have more whip-like hair called flagella. And if you see the green color of chloroplasts, you know that you need to find an organism capable of photosynthesis.

For example, what if you see the tell-tale trumpet morphology in your collection? “Aha,” you might say, “that is a Stentor!” And you would be correct, but which Stentor? While Stentors have varying numbers of micronuclei, these are hard to see without staining. The macronucleus, however, is visible and can be helpful.

For Stentor coeruleus, the macronucleus resembles beads strung together across the cell which is called a moniliform nucleus. Stentor polymorphous also has a moniliform nucleus, but it’s easier to distinguish from the Stentor coeruleus because of the endosymbiotic algae that turn it green. Stentor igneus, by contrast, is smaller, and has pink pigments, and contains an oval macronucleus.

Stentors are a relatively easier genus to pick apart because there are only about 20 species of Stentors to decide between. Other genera can have hundreds of species, and the differences might be more subtle. That’s why sometimes you’ll see our organisms labeled by the most narrow description we feel comfortable giving, whether that’s identifying an organism solely by its genus, or even going more broad and just calling it a “ciliate.” These are cases where we just don’t know enough...yet.

For those of you who remember our mystery, real time video, it took James a year to be sure of what organisms he was watching. And until he identified the mystery organism, we didn’t know what was happening in that video. And of course it’s even more challenging to identify bacteria.

We can use morphology to parse out cyanobacteria and sulfur bacteria. But with most of the bacteria in our samples, it's just impossible to tell without genetic sequencing, a technique that is not in our toolkit at the moment. And for us, that's okay.

But there are cases where it's important to dig further, and scientists have developed many molecular techniques to identify the microbes in their samples. There are stains to emphasize certain identifying structures, and biochemical tests to reveal the presence of specific enzymes. Also, of course, there’s gene sequencing and other more advanced chemical methods to probe what species a sample contains.

For James, who does the work of identifying our microbes without these molecular tests, what he's able to name comes from years of studying and memorizing the drawings and pictures he’s found in various books and keys, as well an ongoing habit of reading papers every day. It's more a way of life, a habit borne out of science, an art, a labor of love. Thank you for coming on this journey with us as we explore the unseen world that surrounds us.

And all of these people on the screen right now, these are our patrons on Patreon. We only have one Patreon tier, it’s $8 a month. And with it, you get your name featured in the end of our video here, a weekly hi-resolution image of one of our micro friends, and an extended video featuring uncut footage with no narration from me, just Andrew Huang’s wonderful music.

And if you want to join up and help us out, you can do that at patreon.com/journeytomicro. If you want to see more from our Master of Microscopes James, check out Jam & Germs on Instagram. And if you want to see more from us, just go to youtube.com/microcosmos.