Artemia (Brine Shrimp) Cyst Decapsulation Protocol

Created 2015.03.28
Last modified 2021.10.19

Note – this protocol is based generally on the protocol published by the Zebrafish International Resource Center (ZIRC).  It has been modified to accommodate smaller decapsulation batches and to make measuring and material handling a bit easier.  The original protocol, as well as many other useful protocols can be found at https://zebrafish.org/wiki/protocols/start

Materials

  • 50 grams dried artemia cysts. This equates to approximately about 75ml of cysts, or 5 tbsp
  • 500ml reagent-grade 5% sodium hypochlorite or germicidal bleach (5-6.25%)
  • At least 2-4l purified water – distilled or reverse osmosis filtered
  • At least 340g rock or non-iodized table salt (NaCl)
  • 10 grams sodium thiosulfate pentahydrate (Na2O3S2 +5H2O)
  • 200g lye (NaOH)
  • Artemia hatching cone and air pump and necessary tubing
  • 100µm mesh bag or filter sieve
  • At least three glass 1l beakers or similar
  • Watch, timer, or stopwatch
  • Squeeze bottles
  • Stir plate and magnetic stir bar

Solutions to be prepared 24h in advance.  All of these solutions should be stored overnight in a refrigerator at ~4°C.

  • 1N NaOH (40% w/v)
    • Add 200g lye (NaOH) to about 400ml purified water and stir till dissolved
      • This is a strongly exothermic reaction and will become very hot
      • Both the dry NaOH and the 1N solution are very caustic and appropriate protective equipment should be worn when handling
    • Once the NaOH is dissolved add additional water to bring the final volume to 500ml
  • 36ppt Salt Solution
    • Add 8g salt to 250ml purified water
    • Stir till dissolved
  • Bleach or sodium hypochlorite solution should also be stored overnight in refrigerator

Solutions which can be made either on the day of decapsulation, or in advance, without refrigeration

  • 1% Sodium Thiosulfate
    • Add 10g sodium thiosulfate to 1l purified water
    • Mix till dissolved
  • Saturated Brine Solution
    • Add 330g salt to 1l purified water and stir till dissolved

Procedure

  1. Hydrate the cysts
    1. Add about 1l water to the hatching cone and begin aeration. purified or dechlorinated tap water are all fine to use
    2. Add the cysts to the hatching cone
    3. Allow the cysts to circulate for 1 hour
    4. During hydration, use a squirt bottle to dislodge the cysts that will accumulate on the walls of the cone.
    5. After 1h check the cysts under a stereoscope. They should be nicely round.  If hydration is not complete, the cysts will have a “dimple”.  If the dimple is present, continue hydration for up to an additional hour, checking periodically to determine when hydration is complete.
  2. Prepare buffered salt solution
    1. While cysts are hydrating, add 15ml of the 1N lye solution to the 250ml 36ppt salt solution, and place back in refrigerator till time for use
  3. Settle and collect hydrated cysts
    1. Turn off air to the hatching cone. Hydrated cysts will settle to the bottom of the cone.
    2. After about 5 minutes, the cysts should be well compacted in the base of the cone.
    3. Gently pour off the hydration water, leaving the compacted cysts in the base.
  4. Decapsulation
    1. Add the buffered salt solution to the hatching cone and restore aeration
    2. Add the chlorine solution and begin timer
      1. This is a time-critical step. Not enough time in the solution will result in incomplete decapsulation, too long will destroy the cysts.
      2. Decapsulation will generally be complete in 5-7 minutes, but can vary based on temperature, strain of cysts used, etc.
    3. Monitor decapsulation progress
      1. Cysts will begin brown in color, then turn grey, then finally, orange. The later stages usually coincide with foam accumulating at the water surface
      2. Once ~80-90% of the cysts have turned orange, decapsulation is complete
    4. Rinsing and chlorine neutralization
      1. Once decapsulation is complete, immediately pour the cysts through the 100µm sieve, and begin vigorously rinsing with purified water
      2. After a few rinses, backwash the cysts into a 1l beaker and add the 1% sodium thiosulfate
      3. Stir or swish the solution gently to ensure all cysts contact the thiosulfate
      4. After ~1 minute, pour the cysts and thiosulfate solution back through the sieve and rinse again with purified water to remove residual thiosulfate
    5. Cyst dehydration
      1. Thoroughly clean the hatching cone to remove any debris and chlorine
      2. Move the cysts back to the cone. Saturated brine can be used to rinse the cysts off of the sieve and into the cone
      3. Add the saturated brine and aerate for 24 hours
      4. Periodically a bit of additional salt (just a few chunks of rock salt, or a few pinches of table salt) to keep the brine saturated as the cysts release their water
      5. After 24h the solution can be stored in the beaker, or suspended and aliquoted into 50ml centrifuge tubes (store the tubes on their sides for maximum viability). Store in a refrigerator