How to Find Tardigrades (Water Bears) in Your Own Backyard

Tardigrades, also known as Water Bears or Space Bears, are incredible members of the microcosmos that can be found just about anywhere in the world. These eight-legged invertebrates are only 0.1-1mm long, yet they are certainly the world’s toughest animals!

enhanced-buzz-wide-4651-1402568065-6 05aeec0af88f914315ffd113893524d6

Just how tough are they? They have been shown to survive temperatures as low as -272°C and as high as 151°C (Rahm, 1923, 1924, 1926), pressures 6 times greater than those at the bottom of the deepest ocean trench on Earth(Seki & Toyoshima, 1998), over 1000 times the human lethal dose of heavy ion and gamma radiation (Horikawa et al. 2006), and more. In fact, tardigrades have even survived for weeks in space (Jönsson et al. 2008)!

DCIM148GOPRO

Typically considered aquatic animals, these small creatures require a film of water around their bodies to permit locomotion and gas exchange (Kinchin, 1994).

anigif_enhanced-21357-1402484030-9

Fortunately for the tardigrade, if there isn’t enough water, or environmental conditions become unfavorable, they can enter a state of “cryptobiosis” – a reversible suspension of metabolism (Keilin, 1959) – from which they can be revived decades later (Tsujimoto et al. 2016) when favorable conditions return. With the aid of some helpful, protective proteins, they release much of their body-water content, shrink into a “tun,” and remain in the same spot for as long as they need to.

enhanced-buzz-wide-2551-1402568258-6

This cryptobiotic adaptation has allowed many species of tardigrades to thrive on land in mosses, colonies of lichen, and liverworts, where periodic drying and wetting is common (Ramazzotti & Maucci, 1983). These habitats also contain plenty of food for both herbivorous and carnivorous tardigrades: algae, cyanobacteria, rotifers, nematodes, and other tardigrades.

enhanced-buzz-wide-22691-1402568163-6 enhanced-buzz-wide-11843-1402570940-6

In this post, I hope to share a step-by-step guide to finding tardigrades in your own backyard. Because these amazing creatures comprise an entire phylum of animals (phylum Tardigrada), of which only about 1000 species have been described, there is even a chance that you’ll find tardigrades from an entirely new species, never before seen!

Note: This guide also works great for finding nematodes and rotifers, which are almost always found occupying the same habitats as the tardigrades.

***

Each morning on my way to work, I pass a short, wooden fence that is home to many colonies of lichen.

IMG_3227

IMG_3229

Every time I’ve seen it, I have wondered whether these lichen hold tardigrades in their leafy folds.

IMG_3232

IMG_3233

IMG_3234

I thought this would be the perfect place to demonstrate how to look for tardigrades.

Here are the materials you will need:

  •                Paper bags
  •                Razor blade or pocket knife (optional)
  •                Pen or Marker
  •                GPS-enabled device (optional) – for coordinates
  •                Bag or backpack (optional) – to hold samples
  •                Paper towels (optional)
  •                2 oz plastic cup (any small cup or container will do, but I prefer this size)
  •                Pipette or eye-dropper (optional)
  •                Spring water (tardigrades prefer this, but any water should be fine for short-term use)
  •                Foldscope

When I’ve found the lichen that I would like to collect, I take out a paper bag, open it up, and then take out my razor blade.

IMG_3235

I position the paper bag below the lichen, then begin to scrape the lichen into the bag using the razor blade. Keep the blade nearly flat, and try to lift the lichen off of the surface rather than crushing it up. The bag should catch any small pieces that happen to break off.

IMG_3236

IMG_3237

IMG_3238

Pick up any large chunks and place them in the bag.

IMG_3239

IMG_3240

I will usually scrape the entirety of a small colony, but I always remember to respect the lichen, and take only what I will use. Some lichen colonies grow only a few mm per year, and can be many decades old.

IMG_3242

The lichen from these two small colonies should be enough for a couple samples that I can use many times over.

IMG_3243

I close the bag, and fold it such that I have space to write down information about my sample.

IMG_3244

I now take out my marker and write down my initials, the date on which the sample was collected, and the sample number (not shown here).

IMG_3245

I next write a description of the location that I took the sample from. For instance, my sample was taken from a wooden fence at the Governor’s Corner dormitories at Stanford University in California.

IMG_3246

If you have access to a GPS-enabled device, it is very useful to write down the coordinates of the location from which you collected the sample. By doing this, you can always return to the spot if you forget where it is, or even tell other people exactly where to find the original location of your sample.

I use a compass application on my smartphone to determine the coordinates of my sample locations.

IMG_3247

IMG_3248

Last, I write down a description of the sample itself. If I knew more about lichen taxonomy, I would make a guess at the genus and/or species, but I usually settle for a description of the color and/or texture.

IMG_3251

The same process also works for mosses on trees, rocks, dirt, etc. There was a tree nearby with a lot of moss at its base, so I decided to collect samples there as well.

IMG_3252

IMG_3253

First I took a sample of the moss that grew on the tree itself.

IMG_3254

I scraped chunks of the moss from the tree using my razor blade and placed them in a new paper bag.

IMG_3256

With this particular sample, I found it easier to pull off the moss with my hands.

IMG_3259

IMG_3257

After placing this moss in the paper bag, I next moved to the moss growing on the dirt around the tree.

IMG_3255

Again, I’m no expert at identifying moss taxa, but from my untrained perspective this looked like Bryum argentum, which has been reported to house a species of tardigrade called Milnesium tardigradum. The silver tips and conical shape of the moss led me to this guess.

IMG_3260

I used the razor blade to separate this moss from the ground, taking as little dirt as possible. Although tardigrades could be in the soil, it is much more likely that they are within the pockets of the moss, and too much dirt in the sample will make it difficult to search through later on.

IMG_3261

Once the chunks were free from the dirt, I collected them with my hands and put them in the paper bag that already contained moss from the tree.

IMG_3262

I labeled the bag as I did for the lichen, writing my name, the date, sample number, location description, coordinates, and sample description. I stored this in the plastic bag with my lichen sample, and headed back to the lab.

IMG_3264

Once I arrived at the lab, I found a drawer which could serve as a cool, dry place that I could store my samples in.

Please note that you do not need access to a laboratory or any laboratory equipment for finding tardigrades.

IMG_3265

If you won’t be using your samples right away, store them in a cool, dry place like this drawer until you need them. This will allow them to dry out slowly before use.

IMG_3267

Since I planned to use my samples right away, I didn’t actually bother storing them.

When tardigrades are taken from dry moss and lichen, they are most likely in their cryptobiotic “tun” state. This means that you will have to “rehydrate” the sample, soaking it in water for at least 4-8 hours before you can search it for tardigrades. Once the tardigrades are introduced to water, they will begin to enter their “active” state, leaving the pockets of the moss/lichen that they originally inhabited and swimming out into the water to find food. From this water, the tardigrades can be collected and imaged. I usually start the rehydration process the day before I intend to search for tardigrades in my samples.

First, I lay out a paper towel on a flat surface for each sample I plan to rehydrate. This isn’t absolutely necessary, but helps in the case of spills or wet containers.

IMG_3270

I set out my sample bags on the corner of each paper towel so that I know which sample I’m handling at all times.

IMG_3271

I place a 2 oz plastic cup on each paper towel that I will use for rehydrating the samples. You can use any small container that you have access to.

IMG_3272

I only use these particular plastic cups because my samples tend to fit well within them, and I don’t have to use too much water to completely soak the samples (more water means more to search through).

IMG_3273

Next I set a pipette on each paper towel for future use in transferring water after rehydration. I use glass Pasteur pipettes with a rubber bulb, but any pipette or eye-dropper should work fine. If you don’t have access to these items, you could also try pouring water after rehydration, using a straw, etc.

IMG_3275

IMG_3276

IMG_3277

Once everything is in place, open up your sample bags, and remove their contents.

IMG_3278

IMG_3279

Place your collected sample face-down within the plastic cups. This helps to better soak the parts of the moss and lichen that are likely to house tardigrades.

IMG_3280

Repeat this with all other samples you plan to use.

IMG_3281

IMG_3282

If the cup becomes too full to allow everything to soak properly within a small volume of water, grab another cup and transfer some of your sample, dividing the contents so that they will fit.

IMG_3283

IMG_3284

I now take a bottle of spring water and pour out about 20 ml for each sample. It’s fine to do this without measuring the volume of water that you’re using. Just use as much as it takes to comfortably soak your samples.

IMG_3287

IMG_3288

IMG_3289

I pour the water into each sample cup.

IMG_3290

To make sure that my samples are sufficiently soaked, I use a pipette to submerge them in the water. Again, you don’t need a pipette. You can do this with a stick, your finger, etc.

IMG_3291

IMG_3293

All that’s left to do is wait. As I mentioned, I usually let these soak overnight, but I’ve had success finding some active tardigrades in as little as 4 hours after soaking.

IMG_3294

When I return to my samples the next day, I usually find that some water has evaporated. This is fine, so long as there is still some water remaining in the cup. If all of it evaporates, your tardigrades will return to their “tun” state, and you will need to repeat the rehydration process.

IMG_3341

Sometimes moss samples will also absorb the water that they were soaked in. When this happens, I squeeze my moss like a sponge to release its water into the cup, then discard the moss in a separate container. The tardigrades will be happily swimming in the water that you squeezed out, so you don’t have to worry too much about them lingering around in the moss that you set aside.

IMG_3342

I now prepare my Foldscope slides, placing a cover slip on only one side in preparation for a water sample wet mount.

IMG_3343

IMG_3344

I take my pipette and attach the rubber bulb, then collect some of the water from my soaked samples.

IMG_3345

I like to take water from the edges of the cups, since I’ve found that tardigrades often crawl among the small pieces of debris that settle there.

IMG_3347

IMG_3350

I place a drop of the water on the sticky side of the slide, then place another cover slip on top of the slide to encapsulate my sample. I usually use my fingernail to press down the edges of the coverslips and ensure that no water escapes, creating a sealed pocket of water on the slide.

IMG_3355

The slides are now ready for viewing with the Foldscope!

IMG_3226

I use this flat, movable lamp as a uniform source of transmitted light (no light module), similar to the methods in Manu’s recent post on focus-locking and ambient light.

IMG_3356

I do a quick scan around the sample and look for tardigrades. If you don’t find them right away, don’t worry. Just put a new drop of water from your samples on the slide and continue your search.

IMG_3357

I found the first tardigrade of many after searching through five drops of water from the lichen sample taken off of the wooden fence. You can use my images and videos as a reference for what you should be looking for.

IMG_3358

Take a look at what I found!

I used Manu’s angular illumination technique to alter the contrast of my images and get a some different views of the tardigrades.

Lastly, I achieved what I call an “aquarium” view of the tardigrades by sealing off a large droplet of water near the edge of a wet mount slide, and then viewing it using angular illumination. As you’ll see, it looks like the tardigrade is in a tiny aquarium!

I hope you found this guide helpful, and I wish you the happiest of times searching for these amazing creatures around the globe.

I’m very excited to hear your answers to the following questions: 

What do the tardigrades in your area look like? What did you find them on? What other creatures were in the water with them? 

For more info on tardigrade biology and working with tardigrades, please see the following resource:

 http://tardigraderesearch.blogspot.com/

 

15 Comments Add yours

  1. MaxCoyle says:

    Wow, this is incredibly detailed and helpful!! Amazing videos too. Thank you for sharing your methods so openly

    1. Stanford Tardigrade Project says:

      Thank you, Max! In my opinion, all science should strive to be open and easy to understand.

  2. Saad Bhamla says:

    @Ricky – what a detailed post. I think this sets the bar for the detail a post should have, including step by step images so that others can follow.

    And the videos are superb – Fantastic job Ricky.

    Saad

    1. Stanford Tardigrade Project says:

      Thanks very much!!

  3. Mckanstryprep says:

    Super video!

    Our students have started this project since last semester! We have encountered rotifers and different types of specimens.. no tardigrade as of yet! Showed our students your post today, they are determined as ever to make this find aswell!

    Thanks for the inspiration!!

    McKanstry Prep. Staff

    1. Stanford Tardigrade Project says:

      Wow! Thanks so much! It’s very humbling to hear of the next generation of scientists being inspired by what we do. I wish you and your students the best of luck! Let me know if you have any questions along the way.

  4. Manu Prakash says:

    What an inspiring post @Ricky and team. After reading this; I wanted to leave anything I was doing and go and collect samples. You have inspired so many people by just a single post. Keep going.

    Now; lots of fun questions come to mind watching your videos. The gait patterns and how a tardigrades struggles with a dust particle reminds me of “little prince” stuck on a tiny island.

    Also; I am curious about the cuticle markings; it’s things like that don’t show up in an SEM. Is that typical to have cuticle markings. Are they species specific.

    Let’s talk soon about building “slide making” materials that are compatible to sequencing/barcoding.

    Absolutely inspiring post – keep going.

    Cheers
    Manu

    1. Stanford Tardigrade Project says:

      Thank you very much, Manu! I love the little (tardigrade) prince analogy. The pigmentation is specific to species, and can be similar across a genus. I have found some very large tardigrades with four red “racing stripes” running down their backs. Perhaps they are a new species. I’m excited to talk about sequencing whenever you are available!

  5. Matthew Rossi says:

    Oh man, this is a great tutorial! Time to go find some lichen and moss and get hunting. Out of curiosity, what kind of desk lamp are you using for your illumination? I’m leaning more and more that way, myself, and yours looks like it has most of the things I’d like for my personal workflow.

    1. Stanford Tardigrade Project says:

      Thanks so much, Matthew! The desk lamp I use is a Humanscale Element Disc lamp. I’ll paste a link to it here:
      http://www.humanscale.com/products/product.cfm?group=elementdisc

  6. newyorklenny says:

    This blog post is clearer and easier to follow/reproduce than 99.99% of research papers. I know this is an incredible amount of work and want to thank the author(s) for taking the time to open science in this manner.

    I bet the author is an amazing teacher.

    Thank you!

    Lenny

    1. Stanford Tardigrade Project says:

      Thanks for the kind words, Lenny! I’m incredibly happy to see people of all ages following this work with ease, since a lack of descriptive protocols made tardigrade research quite a daunting task for my team when we became interested in tardigrades. I really appreciate your belief in my ability to teach, as I am only an undergraduate going into my junior year here at Stanford, and I assure you this won’t be my only post!

      Thanks again,
      Ricky

  7. laksiyer says:

    What a fantastic post. Just the kind of detail one needs. I would love to join the hunt to find new tardigrade species. If you have any reference for what features to look for in particular, it would help. Also, any tips for culturing them would be great too. I have tried milk powder in the past but I dont remember being really successful. Would be great to see a full cycle.

    1. Stanford Tardigrade Project says:

      Thanks very much! I would certainly love to have people all over the world join the hunt for tardigrades! For simple dichotomous key made for tardigrades, check out https://sun.iwu.edu/~tardisdp/keypage1.html

      It’s not the most detailed tool for taxonomy, but it can be very helpful down to the genus level. From there you can search online for some more detailed identification for the species within that genus.

      I’ll look to make a post for identification and culturing in the near future. I culture my tardigrades in small plates atop a thin pad of agarose. I provide volvic water for them to swim in, and I feed them algae (Chlorella vulgaris). I transfer them to new plates and food every 3-6 days.

  8. marcosbruno says:

    Amazing job! Thank you for posting the whole process! Loved the way you easily explained how to do it by ourselves. Gonna look for Tardigrades where I live!

Leave a Reply