Ahoy,
Firstly, sorry for staying somewhat vague but I am not allowed to go into more details about the drug its effects and the wanted outcome.
I am currently conduction an experiment where I place 25 freshly fertilized Zebrafish eggs each per well of a 6 well plate containing a drug solution. Every 12h I remove any dead eggs, embryos, chorions and larvae. Every other day I change 50% of the drug solution. After 6 days of treatment the surviving larvae get released into their tanks.
Unfortunately the drug I am using is quite toxic and has severe effect on early development.
Only about 25% of the eggs/embryos survive those 6 days and of these most display such severe teratologies that they die as soon as their yolk is depleted.
Obviously this means that the media surrounding the eggs is at constant risk of spoiling leading to runaway death.
Eventually I end up with about 5% growing into healthy adults showing the subtle change that we want to see and this cannot be changed. I cannot change drug, concentration, administration timepoint or route.
However because I need quite a few of these 5% fish I have to start off with very, very high numbers of eggs and using the above setup I waste tons of plates and invest hours and hours and hours into sorting and removing dead eggs.
Very often I end up with only a single viable, treated larvae per well at the end of these 6 days.
I do not really care about percentage of lost eggs other than it obviously playing a big role for the amount of eggs I have to start and work with. What I need is to find a way to loose less time on the half daily tidyup, removal sessions.
Categorically I would say the answer is either a way to keep the media from spoiling so easily or to find a super, super quick way for removing dead stuff.
Unfortunately increasing the media volume isn’t an option as the drug is quite expensive.
I tried to place 200 eggs into a well. Every 8h I would move them into a mini strainer rinse them thoroughly with drugfree media and move them into a fresh clean well containing drug solution. This would be super quick and easy and I thought this thorough media replacement would help but unfortunately even the 8h between exchange can lead to total runaway death.
Embryo rearing tidyup is something that everyone working with zebrafish is facing all the time so I thought maybe there are some fantastic small tricks out there.
Can you make dead eggs float or sink faster than live ones, which could be used for sorting?
Can you choose the opening of your pipette so that it will only suck up one or the other?
Would having the eggs on a membrane and thereby allowing for particles of death to fall away from live eggs help?
Does mild agitation reduce runaway death risk? I ask because I feel like it isn’t even the whole media but rather the thin bottom layer the eggs share that spoils rapidly.
Is there a neat chemical enzymatic solution to digest or neutralize whatever it is that makes egg death contagious?
...
I am grateful for any advice.
Best regards
Tim
Ps: While writing this I just had another idea, maybe I should move to 384 well plates and just leave dead ones where they are. Not a great solution as it is heavy on plastic use but it would involve far less time.
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