Laboratory Culture of Hypsibius exemplaris

Culture of Chlorococcum

• 1. Add 150 mL of Bold's basal medium plus soil extract to each of several 250-mL Erlenmeyer flasks, and stopper the flasks with nonabsorbent cotton wool and aluminum foil. Autoclave for 20 min at 15 psi, and allow the flasks to stand for 24 h.

  • Each flask, when harvested, will provide adequate algal food for sustaining ∼200 tardigrades over 4–6 wk.

• 2. After 24 h, inoculate each flask with <2 mL of a preexisting Chlorococcum culture.

• 3. Cover the flasks as described in Step 1, and leave them at room temperature (or between 15°C and 25°C). Illuminate under fluorescent lighting (with 40-W tubes) either continuously or controlled to give a photoperiod of 14 h of light and 10 h of darkness each day.

  • Agitating the flasks is not essential. The cultures will be ready to feed to the tardigrades after 4–6 wk. Chlorococcum cells are nonmotile and settle the bottom of the flasks, where concentrated cells can be collected by pipetting and used for standard culture (Steps 4–7) or small-scale culture (Steps 8–10) of H. exemplaris.

  • Chlorococcum cultures will perish if frozen; always keep them at room temperature. The cultures can be maintained indefinitely by subculturing as described in Steps 1–3 as necessary.

Standard Culture of Hypsibius exemplaris

• 4. Pour 150 mL of Chalkley's medium plus soil extract into a sterile 250-mL Erlenmeyer flask.

• 5. Add ∼3–5 mL of concentrated Chlorococcum cells to the flask, and inoculate with 1 mL of tardigrades or embryos. (Chlorococcum cells are nonmotile and can be pipetted from the bottom of the Chlorococcum flasks.) Seal the flask with clingfilm or Parafilm.

  • The seal prevents contamination from fungal spores and mites.

• 6. Place the flask in a cool (10°C–20°C), shaded location, with continuous darkness or with a photoperiod of 14 h of light and 10 h of darkness each day.

• 7. Subculture every 4–6 wk as necessary by repeating Steps 4–6.

  • Optimal subculturing intervals are subject to temperature, algal food inoculum, and density of tardigrades. Cultures generally peak between 4 to 6 wk, after most of the algae have been grazed over. Microscopical observation of cultures for abundant tardigrades or the presence of eggs will reveal the best time for setting up and inoculating fresh culture media.

  • Old cultures can be kept in reserve if necessary by concentrating and storing in the refrigerator at 4°C, where the tardigrades will survive for many months (or years) in an inactive or lethargic state.

Small-Scale Culture of Hypsibius exemplaris

• Small-scale cultures consisting of hundreds of tardigrades per dish can be maintained indefinitely in commercial bottled spring water in glass (Gabriel et al. 2007) or plastic Petri plates. Tardigrades stick to plastic, but this can be avoided by precoating the plates with algae.

• 8. Pour 15 mL of spring water and 1 mL of Chlorococcum algal culture into a 6-cm-diameter Petri plate. If the Petri plate is plastic, incubate for several hours to overnight at room temperature before proceeding to Step 9.

• 9. Add ∼1 mL of tardigrade inocula into the Petri plate, cover with the Petri dish lid, and incubate in a shaded location at room temperature.

• 10. Change algae and water every 10 d to 2 wk as follows.

  • i. Gently pipette the algae and tardigrades up and down to loosen clumps.

  • ii. Allow the tardigrades to settle, and then pour out most of the water.

  • iii. Add ∼7 mL of spring water to the dish.

  • iv. Repeat Steps 10.i–10.iii four or five times.

  • v. After the last water change, add 1 mL of algal culture.