Step 1: Collect Substrate
A) Tardigrades are found in moss and lichen. Moss/lichen on trees is better than moss/lichen on the ground or rock surfaces. Gray lichen is better than yellow/green lichen. If you're collecting moss, look for leafy-looking, julaceous moss.
B) Scrape moss or lichen off a surface with a sharp edge (knife, razor, etc.) into a Ziploc bag or paper envelope. Try to get about a tablespoon of moss or lichen per sample. Avoid getting large amounts of dirt or bark in your sample.
C) Clearly label the bag or envelope with all necessary information (location, date, etc.) Substrate (especially if dry) can be stored for as long as necessary.
Step 2: Make Tardigrade Tea
A) Complete submerge substrate in deionized water by putting lichen in a petri dish and filling with water.
B) Let soak for at least 20 minutes.
C) When ready to search substrate, strain water through cheesecloth to filter out largest bits of substrate. You can strain water directly into another petri dish in preparation for searching your Tardigrade Tea using the microscope. Keep the cheesecloth/substrate bundle handy - if you don't find any organisms it helps to re-strain it or sprinkle some larger chunks into your Tardigrade Tea, as the organisms are often clinging to the pieces of moss or lichen.
Step 3: Scan for Life Forms
A) Search for life in the petri dish using a dissecting microscope. All of these organisms may be transparent. In the micro-world, it’s easier to detect movement than it is to spot a specific shape. Start by letting the substrate bits settle. Then watch for movement. Once you see movement, zoom in and focus to find out what it is.
B) Move petri dish around manually to ensure adequate observation of entire sample. To standardize your data, plan on limiting yourself to looking at each sample for 15 minutes.
C) Every time to discover an organism, record it and remove it using a pipette set to 30 ul. Removal is important so that you avoid counting the same organisms more than once. You can keep using the same pipette tip, but organisms (especially nematodes) can get stuck to the inside. To prevent this, pump water up and down several times when releasing the organism into the new container, to make sure it’s dislodged.
You will likely see:
o Tardigrades: recognizable by their legs, which will be constantly moving if tardigrade is alive. Slightly smaller than a grain of sand.
o Nematodes: recognizable due to their worm-like or snake-like body. Come in a wide variety of sizes.
o Rotifers: recognizable by their “squishy” way of moving. Body stretches and contracts. No legs; they swim rapidly using a propeller-like apparatus near their mouths. (If something is zooming around, it’s a rotifer, not a tardigrade!) Generally smaller in size than tardigrades.
Step 4: Put your Tardigrades to the Test
A) If you or another group is conducting an experiment that involves survival rates, put each organism you find into a microcentrifuge tube. Your pipette tip should be holding only 30 ul of water (plus a tardigrade) so the microcentrifuge tube should contain only a small drop of water. Only put one individual per microcentrifuge tube.
B) Label the tube clearly on both the top and side so you know exactly which sample it came from and what kind of organism (tardigrade, rotifer, or nematode) it contains.
C) Depending on your experiment, you can induce cryptobiosis through either:
Dehydration - let the water evaporate (leave the microcentrifuge tube with its cap open until it appears dry, then close the cap and find a place to store it)
Freezing - close cap and put microcentrifuge tube in the freezer