Find and keep tardigrades
Tardigrades live in almost every damp patch of moss on a wall or tree, and the lab species will live for years in a dish of spring water with a little green alga. We gathered the field methods, the lab protocols and every culture recipe we have read into one place, with the numbers and a source for each.
Finding them
The easiest place is moss. Scrape a small cushion from a wall, roof, tree bark or dead wood, and put each sample in its own labelled paper bag, so it dries slowly and the animals have time to curl into their dry "tun" state. Dry samples keep for years at room temperature, and better in a freezer. Lichens, liverworts, leaf litter, soil and pond sediment hold them too; tardigrades are patchy even within one cushion, so several small samples beat one big one (Degma et al. 2018).
- Wake them up. Soak the moss in water in the fridge for about a day, even if the sample is years old (Degma et al. 2018).
- Shake them out. Tear the moss into pieces and shake each in water, then pour it through a sieve series, or through a 1 mm mesh to hold back the debris, let the rest settle for 30 minutes and pour off most of the water.
- Look. Spread the residue in a Petri dish and search under a dissecting microscope at 25–80×, with light from above or a dark background; a grid ruled on the dish base helps. Pick animals and eggs with a fine pipette or a small wire loop.
Where to look first. Thin moss in full sun, on walls, roofs and rocks, holds the most. After a soaking rain, fully exposed moss is dry again in 1–2 days, half-shaded moss in 4–5 and shaded forest moss in 15–20, so the animals in sunny moss spend most of their lives as tuns. Moss by busy roads is poor. Carry samples in paper envelopes rather than plastic bags, which trap moisture and grow mould, and note the date, place, altitude and what the moss grew on (Ramazzotti & Maucci 1983, pp. 100–101, 124–125).
Soil. A tube with an 11.3 mm bore takes a core of exactly 1 cm², so its depth in centimetres is its volume in millilitres. Counting is slow: searching one 5 cm³ core took four days (Ramazzotti & Maucci 1983, pp. 125, 128).
Marine species live between sand grains and on barnacles and algae; freshwater samples have to stay wet until you extract them.
The lab tardigrade
Almost every lab working on Hypsibius exemplaris keeps one clonal strain. Robert McNuff isolated it on 13 November 1987 from a single female in rotting leaves at the bottom of a pond at Darcy Lever, Bolton, Lancashire (53°33′32″ N, 2°23′48″ W), and sold it through his company Sciento as catalogue number Z151; papers before 2018 call it H. dujardini (McNuff 2007; Gąsiorek et al. 2018; Goldstein 2018). All the animals are female and lay eggs without mating.
Keeping a culture
Two ways work, and both use the same food, the green alga Chlorococcum, which the animals pierce and suck empty.
| Small dish | Flask | |
|---|---|---|
| Container | 35–60 mm Petri dish, vented or glass. Coat plastic with algae for a few hours first: the animals stick to bare plastic | 250 mL Erlenmeyer flask sealed with cling film or Parafilm against mites and fungal spores |
| Water | About 2–15 mL bottled spring water | 150 mL Chalkley's medium with soil extract (recipe below) |
| Food | 0.5–1 mL Chlorococcum culture | 3–5 mL concentrated Chlorococcum |
| Start with | About 1 mL of tardigrades, or eggs in their moults | |
| Split | Every ~3 weeks | Every 4–6 weeks, when most of the algae is grazed |
| Where | Cool and shaded, 10–20 °C, normal daylight or dark | |
| Source | McNuff 2018; Coke et al. 2026 | McNuff 2007 |
- Never tap water. Chlorine and chloramine harm them; use bottled spring water (McNuff 2018; Heikes & Goldstein 2018).
- Temperature. They survive from freezing to 30 °C, but lay most eggs at 10–18 °C (McNuff 2007).
- Eggs. A female lays her eggs into the old cuticle as she moults, usually 3–12 at a time and up to 36; they hatch in 4–4.5 days, and the young lay their own eggs about 13–14 days later (Gabriel et al. 2007; Gąsiorek et al. 2018).
- Reading the gut. Right after a meal the gut is green; it turns brown, then very dark just before the animal defecates. A clear, narrow gut means it hasn't fed. Adults can go about five weeks without food, living on their storage cells (Ramazzotti & Maucci 1983, pp. 47, 56).
- Seeing inside. Sealed in a drop of water under a coverslip, an animal runs out of oxygen within a day or two and lies stretched out and transparent, ideal for looking at its organs; Doyère used this in 1840. Eyespots vanish in mounting media, so note them on live animals (Ramazzotti & Maucci 1983, pp. 81, 127).
- Parking a culture. Concentrate an old culture and keep it in the fridge at 2–4 °C: the animals go quiet and survive for months (McNuff 2007).
- Freezing. Add glycerol to 15% and cool slowly to −80 °C in a closed polystyrene box; about half the animals revived after a year and restarted the culture (Goldstein lab protocol).
Growing the food
Grow Chlorococcum in 150 mL of Bold's basal medium with soil extract in a 250 mL flask: autoclave, leave for a day, add less than 2 mL of algae, and keep it under a fluorescent tube (14 h light, 10 h dark) at 15–25 °C. After 4–6 weeks the cells have settled on the bottom, ready to pipette off, and they stay good food for months (McNuff 2007).
| Chalkley's medium with soil extract | Per litre of distilled water |
|---|---|
| NaCl | 5 mL of a 2.0 g / 100 mL stock |
| KCl | 5 mL of a 0.08 g / 100 mL stock |
| CaCl2 | 5 mL of a 0.12 g / 100 mL stock |
| Soil extract | 20 mL: 1 part air-dried rich soil to 2 parts water, autoclaved, settled for a few days, the clear liquid poured off |
Other species you can keep
Lab cultures exist for a handful of other species. Most sit on a thin layer of agar in a dish with a film of mineral water; what changes is the food (Altiero et al. 2018; Degma et al. 2018).
| Species | Dish | Food |
|---|---|---|
| Ramazzottius varieornatus | 35 or 90 mm dish, 1.5% agar in Milli-Q or Volvic water | Chlorella vulgaris, about 1:100 in Volvic |
| Milnesium | 35 mm dish, 2% agar in a KCl–CaCl2–MgSO4 salt solution, under water | Rotifers and nematodes for adults; algae for the young |
| Paramacrobiotus fairbanksi | 15 mm dishes, 1.2% agar with a few drops of mineral water | Nematodes |
| Thulinius stephaniae | Freshwater culture, 15–25 °C | Algae; 2–12 eggs laid into each moult |
| Diphascon, Isohypsibius, Acutuncus, Dactylobiotus | Agar-lined dishes | Algae |
| Hypsibius, Mesobiotus | Agar-lined dishes | Rotifers and algae |
Culturing was mostly a failure until the 1960s: adults lived for months in bell jars, but the young died. The first lasting cultures used Chlorella grown as a carpet on the dish bottom (Baumann 1961), and one parthenogenetic Hypsibius culture ran for more than six years on green algae in dilute soil extract (Ammermann), a forerunner of today's lab strain. Armoured tardigrades (Echiniscidae) had never been cultured by 1983 (Ramazzotti & Maucci 1983, pp. 135–138).
Egg development runs from 4 to more than 100 days across species, depending on temperature; females lay from 1 to 44 eggs at a time. Some species lay "resting" eggs that only hatch after drying and rewetting, like seeds (Altiero et al. 2018).
Where they live, from the papers
Sources
- McNuff R. 2007. Hypsibius dujardini collection notes and culture protocol. Goldstein lab, UNC (tardigrades.bio.unc.edu).
- McNuff R. 2018. Laboratory culture of Hypsibius exemplaris. Cold Spring Harbor Protocols.
- Degma P, Guidetti R et al. 2018. Collection and extraction methods. In Schill RO (ed.), Water Bears: The Biology of Tardigrades, Springer.
- Altiero T, Suzuki AC, Rebecchi L. 2018. Reproduction, development and life cycles. In Schill RO (ed.), Water Bears, Springer.
- Gąsiorek P, Stec D, Morek W, Michalczyk Ł. 2018. An integrative redescription of Hypsibius dujardini, with the description of H. exemplaris. Zootaxa 4415: 45–75.
- Goldstein B. 2018. The emergence of the tardigrade Hypsibius exemplaris as a model system. Cold Spring Harbor Protocols.
- Gabriel WN et al. 2007. The tardigrade Hypsibius dujardini, a new model for studying the evolution of development. Dev Biol 312: 545–559.
- Heikes KL, Goldstein B. 2018. Live imaging of tardigrade embryonic development by differential interference contrast microscopy. Cold Spring Harbor Protocols.
- Coke AN et al. 2026. Modified meiosis in the tardigrade Hypsibius exemplaris maintains heterozygosity across the genome.
- Ramazzotti G, Maucci W. 1983. Il Phylum Tardigrada, 3rd ed. Memorie dell'Istituto Italiano di Idrobiologia 41; English translation by C. W. Beasley (1995). Older work it summarises (Marcus, Baumann, Ammermann) is named in the text.
- Goldstein lab. Tardigrade cryopreservation protocol (tardigrades.bio.unc.edu).
How this is built
- Data
- Robert McNuff's culture protocols (2007, 2018) and the Goldstein lab's protocols; the collecting and life-cycle chapters of Water Bears (Degma et al. 2018; Altiero et al. 2018); Ramazzotti & Maucci's monograph (1983); and the papers on the lab strain (Gabriel et al. 2007; Gąsiorek et al. 2018). The habitat figures come from our figure library of openly licensed papers.
- Built with
- We read these sources into our knowledge graph as claims with their pages, then wrote this page from them by hand, with a source for every step and number.
figures.pypicks the habitat figures from the library when the site is built. - Checked
- Not yet tested against data: the steps and numbers are as the sources give them.
- Limits
- Most numbers are for the lab species, Hypsibius exemplaris. The recipes for other species come from two review chapters.
- More
- How the whole project is built