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Tardigrade Lab Protocols

Preserving & Isolating:

Aquatic Samples (Must be processed within 12 hours of collection)

  1. Rinse through 1mm sieve, collecting runoff in Erlenmeyer flask
  2. Retrieve large material on sieve, bottle and agitate aggressively, rewash, collecting runoff
  3. Allow to settle, then decant to 30 ml
  4. Add 90 ml boiling 95% ETOH to bring to approximately 70% ETOH, decant to 30 ml

Below, a water bear is shown with its head under a fragment of Lumbriculus sp. Read more about water bears in the Spring, 2005 ATBI Quarterly Newsletter.

Photo by Paul G. Davison.

Preserving & Isolating:

Terrestrial Samples (May be stored up to 6 months in paper bags in lab, if >6 months store at –70 o C)

  1. Soak sample in beaker in tap water at least 3 hrs (24 hrs for anoxybiotic specimens). Leaving overnight works well. Add enough water to just cover sample
  2. Tease apart mosses, then blend for two short bursts in small food processor (or lab homogenizer with mini-container) to break up sample
  3. Pour through 1mm sieve. Wash thoroughly, collecting water in large Erlenmeyer flask.
  4. Place moss sample in jar with additional tap water and tight fitting lid, agitate aggressively, again wash on sieve. (This process loosens eggs.)
  5. Let runoff settle 3-5 min, decant to 30 ml.
  6. Add 90 ml boiling 95% ETOH, let settle again, decant to 30 ml

 Centrifuge Technique: All Samples

  1. Place 4 ml Ludox AM TM in 15 ml centrifuge tubes. Slowly trickle a few drops of 70% ETOH on surface of Ludox. Carefully add 5-8 ml of sediment-ETOH sample on top of Ludox in each tube.
  2. Centrifuge 1 min @ 1000 rpm. (Sediment settles into Ludox, specimens remain at ETOH/Ludox interface).
  3. With pipette, draw off supernatant just down to sediment, carefully avoiding sediment.
  4. Store in 50 ml screw cap vials with conical bottom. Specimens concentrate for easy retrieval.

Note: Thoroughly wash all equipment between samples to avoid cross contamination. Backwash sieves.

Permanent Mounting

  1. Transfer all specimens and debris from bottom of storage vial and place in small petri dish
  2. Clean coverslip and slide
  3. Place small drop of Hoyer’s solution* on center of slide
  4. Use 15-20X dissecting scope with transmitted light to find animals and eggs.
  5. Transfer specimens from petri dish Hoyer’s drop with small Irwin loop.
  6. Place tardigrade in medium and position either laterally or ventral side up.
  7. Cover with 18 mm circular coverslip with forceps or needle (press gently to spread specimen)
  8. Circle tardigrade with permanent marker to aid in locating
  9. Label and let dry lying flat for 1 week.
  10. After fully dry (cover slip doesn’t move), ring with epoxy paint

Note: We have been mounting one specimen per slide, making 50 slides/sample when that many specimens are available. 100 slides/sample would be preferable if time and resources allowed.

*Hoyer’s Solution

  1. 30 g extremely fine pulverized gum arabic are dissolved in 50 ml distilled water
  2. (dissolve slowly, this takes time)
  3. 20 ml glycerin are added
  4. 15 g chloral hydrate dissolved in resultant solution
  5. 1g of crystalline iodine and 1 g of potassium iodide (for light staining).
  6. Mix with magnetic stirring bar overnight or over weekend to dissolve.
  7. Filter (use cotton wool or millepore filter or Buchner funnel; vacuum pump speeds it up)

Note: Too much chloral hydrate can clear specimens to total transparency. Also note, chloral hydrate is a controlled substance and very difficult to obtain.

Direct Observation of Live specimens: All Samples
Temporary Wet Mounts

  1. Skip the alcohol, preservation stage.
  2. You may also skip the Ludox stage, although centrifuging makes matters much simpler even for live specimens.
  3. Pipette small amount of sediment into small petri dish, add enough water to cover. Don’t add too much sediment to make finding specimens difficult, nor too little yielding low numbers of specimens.
  4. Transfer all specimens and debris from bottom of storage vial and place in small petri dish
  5. Clean coverslip and slide
  6. Use 15-20X dissecting scope with transmitted light to find animals and eggs.
  7. Transfer specimens from petri dish w/ pipette, adding small drop of water and specimen to slide.
  8. Place tardigrade in medium and position either laterally or ventral side up.
  9. Cover with 18 mm circular coverslip with forceps or needle to avoid air bubbles
  10. Ring with vaspar (1/2 paraffin, ½ Vaseline melted) using pipette tip
  11. Wet mounts should allow observation for several hours
  12. An alternative method is to ring cover slip with silicone grease. This preparation doesn’t last as long as vaspar, but the silicone allows oxygen exchange and some organisms may remain active longer.