[Zbrafish] Bleaching Embryos

Leviathan jason.cockington at adelaide.edu.au
Fri Jun 30 05:25:09 EST 2006


Hi all,

I am about to start a new aquarium facility to work alongside our old
one.  The idea of this facility is to keep it isolated in as many ways
as possible.  That being the case, we want to start bringing emryos
across from the old room (via a bleaching) to establish the new room's
population.

The reason i am posting, is to find out a few pointers.

I am following this protocol:

Embryo Bleaching Procedure

1. Prepare the bleach solution: 100 Fl (0.1 ml) of bleach in 170 ml of
clean system water. Mixing it up ahead of time guarantees that the
bleach is evenly dispersed. The bleach we use is 5% sodium hypochlorite
(NaOCl) from Baker (Order No. 9416-01; 5.3% available chlorine; PO4
<0.0005%; Ca <0.001%). The use of chemically pure sodium hypochlorite
is recommended. Some inexpensive brands of bleach contain contaminants
or additives that are potentially toxic to embryos.

2. Place the embryos in a clean dish, remove the water they are in and
add the bleach solution, or use a pipette to add the embryos to the
bleach solution in a small volume of water.

3. Gently swirl the embryos in the bleach solution and allow them to
stand for 5 minutes.

4. Rinse the embryos two to three times in sterile system water (or
embryo medium) and transfer the eggs to a clean disposable petri dish.

5. The bleaching procedure can be repeated if desired.

6. There are many opinions regarding the timing of the bleaching
procedure for optimal survival. Some people bleach the embryos within
several hours of fertilization (3-8 h), others wait until epiboly is
complete (10 h) and still others wait until they are 24-36 h. In
general, we recommend bleaching the embryos within the first day.
Bleaching toughens the chorion to the point that fish may not be able
to hatch. Because of this, the chorion should be removed either
mechanically or with pronase treatment prior to normal hatching.

7. Embryos that have been properly bleached can be removed safely from
the quarantine room. Be careful not to recontaminate the embryos by
contact with nonsterile water or glassware.


I have been bleaching at about 10hrs, and repeating the bleaching step
3x, rinsing in Embryo Medium.  i am concerned that i might under
bleach, and then the whole procedure wont achieve anything.

The thing i am not sure about, is how can you tell the embryos were
bleached adequately?

Is there any negative side effects in the fish if you overbleach (aside
from increased death)?

If the embryos have been seperated from the original water by 2
petridishes, and 3 steps of bleach, do you think it is safe to assume
that the embryos will not be carrying any of the putative infirmity
across?

I have noticed the bleach hasn't killed fungus in the water.  If i
added methelene blue to the embryos prior to bleaching (to inhibit the
fungus) do you think that would be adequate to prevent any fungus
travelling over, if after each bleach they embryos are transfered to a
new petridish?

any pointers you can offer will be very much appreciated  :O)

many thanks,

leviathan



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