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Decapsulating Brine Shrimp

Please find enclosed an extract from the brine shrimp FAQ, concerning
decapsulation of brine shrimp eggs. You may find this useful. 
- Vahe
Brine Shrimp "FAQ"


  1. Brine Shrimp FAQ 1.1
     by qx01820 at inet_d48.lilly.com (13 Jun 95)


Brine Shrimp FAQ 1.1
by qx01820 at inet_d48.lilly.com
Date: 13 Jun 95
Newsgroup: rec.aquaria


                    Decapsulating Brine Shrimp Cysts

Having had several questions on how and why this is done, I
have decided to include this procedure at the end of this FAQ.
It is an involved process and not many people will choose to
perform it, but it is good information to have in case you get a
wild hair some day, or just want to impress your friends.
      Separating nauplii from their shells may be desirable for
several reasons. Cyst shells are indigestible and can lodge in
the gut of predators causing fatal obstructions, and the shells
have been speculated to be a source of heavy bacterial
contamination.  While in all my years of messing with these
guys, I have never heard of anyone having problems with either
of these two scenarios, quite a few commercial aquaculture
ventures go to the trouble of decapsulation. Decapsulation is
accomplished in four steps: re-hydrating the cysts, treating
with the decapsulation solution, washing and deactivating the
residual chlorine, and the hatching of the embryos.
      Dry cysts have a dimple in their shell which makes it hard
to remove the complete inner membrane. For this reason, the
cysts are first hydrated into a spherical shape. The cysts
should be re-hydrated in soft or distilled fresh water at 25
degrees C for 60-90 minutes. The lower the temperature, the
longer it takes to re-hydrate them. But, no matter what the
temperature, never leave them longer than 2 hours, as this
decreases the percentage of decapsulated cysts and lowers the
hatch rate. Hydration should be done in a container identical to
the one used for hatching regular cysts for the same reasons of
circulation and aeration. Cysts should be filtered on a 100-125
micron collection screen and rinsed, but this step may be
missed if you dont have the screen.  It is best to decapsulate
the hydrated cysts immediately, but they can be refrigerated
for several hours if needed. During the hydrating process, you
need to prepare your chlorine solution.  Either household liquid
bleach or powdered pool chlorine is mixed with salt water.
      In preparation for decapsulation the cysts are placed in a
pre-cooled buffered solution, 4 degrees C and about pH 10,
consisting of 0.33 ml of 40% sodium hydroxide (NaOH) and 4.67
ml of sea water per gram of cysts ( you may have a hard time
finding NaOH, most pharmacies should have it though, I got
some from work so I havent really looked much). The buffer
solution is prepared by dissolving 40 grams of sodium
hydroxide in 60 ml of fresh water. Decapsulation will begin
when you add 10 ml of liquid bleach to the buffer solution. You
will need to have a thermometer in the brew, because the
chemical reaction taking place gives off heat. It is important
to keep the solution between 20 and 30 degrees C. Starting
with pre-cooled buffered seawater makes it easier to keep the
reaction in the right temperature range. If you need to, an ice
cube can be added to help drop the temp.
      A second method is to add 0.70 grams of dry pool chlorine
powder per gram of cysts. In this case the buffer is sodium
carbonate consisting of 0.68 grams sodium carbonate in 13.5
ml water. It is easier to split the water in two equal parts,
add the required amount of chlorine to the first part, and the
sodium carbonate to the second. Allow them to dissolve and
react, which will cause a precipitate. Pre-cool the two
solutions, and mix them together, then add the hydrated cysts.
During decapsulation, stir the brew continuously to minimize
foam formation, and to dissipate heat. Note the color of the
solution, it will change from a dark brown to grey, to white,
and then to a bright orange. This reaction usually takes 2-4
minutes. With the calcium hypochlorite solution, the cysts will
change only to gray, and will take about 4-7 minutes.
      The cysts can be filtered from the solution as soon as the
membranes have dissolved as indicated by the color (bright
orange or grey). The chlorine should be washed off the cysts by
rinsing with fresh water or salt water until you cant smell
the chlorine anymore. The residual chlorine attaches itself to
the decapsulated eggs, and has to be neutralized. Do this by
washing the cysts in a 0.1% sodium thiosulfate (0.1 gram
sodium thiosulfate in 99.9 grams water) for one minute. An
alternative method uses acetic acid (1 part 5% vinegar to 7
parts water). The first method works better, but the second
method is easier as everyone has the materials in their
kitchen. The cysts are then re-washed with fresh or sale water
a placed into the hatching container, and hatched as normal
artemia.  The decapsulated cysts can be hatched immediately,
or stored in the refrigerator for up to 7 days before hatching.
For long term storage, like the expensive stuff you can buy, the
cysts need to be dehydrated.
      Dehydration of the decapsulated cysts is done by
transferring your one gram of decapsulated cysts into a
saturated brine solution of 330 grams salt to 1 liter water.
Aerate this for 18 hours, replacing the solution every 2 hours.
The cysts are releasing their water through osmosis in the
solution, so it is important to keep the salt concentration high.
After 18 hours, the cysts have lost about 80% of their cellular
water, stop the air flow and let everything settle, then filter
the cysts out. These cysts can then be placed in a container and
topped off with fresh brine solution. seal the container and
store it in the refrigerator or freezer. Cysts with 16-20%
cellular water can be stored for a few months without a
decrease in hatching rate. For a longer term storage, you have
to reduce the cellular water content to less than 10%.