[Zbrafish] Re: Bleaching Embryos

Claudia claudia_hohn at hotmail.com
Wed Jul 5 17:01:52 EST 2006


Hi,
I am working in a zebrafish facility for 2 year now and from my
experience the bleaching method is to harsh on the embryos. I prefer to
just use methylen blue and boiled system water. With this method I
never have problems with fungus and survival rate is great also you do
not have to remove the chorion. Let me know if you wish more detail.

Claudia
Leviathan wrote:
> 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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