My lab is doing an experiment where we need to maximize embryo recovery after bleaching. At present we’re washing off ~250+ gravid herms for each bleaching event (pooled from multiple 6cm plates). So, this is a fairly modest number and results in a modest sized embyro pellet when spun down. We want to retain as many of these embryos as we can through the workflow.
We are very good at bleaching generally. Our best protocol is to use 15mL falcon tubes, to watch carefully for when the carcasses are dissolved, then spin, aspirate, rinse, etc. But my sense is that we lose a fraction of the embryos with each wash.
We’re going to try 15mL glass centrifuge tubes next - perhaps this will reduce the possibility of embryos sticking to the side of the tubes and limit embryo loss.
The 15mL size is a bit big for the amount of worms/embryos we are working with. We may try 5mL or 10mL glass tubes if needed, but we don’t have any yet. And might require adapters for the centrifuge?
We find that 1.5mL Eppendorf tubes are hard to bleach in - hard to agitate the mixture due to the small volume size, hard to see within the tube, hard to form a proper embryo pellet, hard to aspirate the liquid without disrupting the embryos. But - perhaps others have high confidence in this method?
Advice? Anyone with experience bleaching in alternative vessels? Other ideas? Where do the lost embryos GO?
Annalise,
I have had problems with worms (not embryos, per se) sticking to the sides of tubes. Plastic tubes more so than glass, but even glass seems a bit sticky. If that is your problem, if look at the sides of the tubes with your dissecting microscope you see the worms (embryos) stuck to the sides. I found that pre-treating the plastic with 1% BSA helps make the sides less sticky. Basically, I make up aliquots of sterile BSA and then put about 1 ml in a 15 plastic tube, vortex vigoroursly, remove the BSA solution, then rinse with sterile water. On the other hand, some of the perceived loss of embryos could actually be due to unfertilized oocytes, small bits, and old egg shells stuck to the sides of the tube.
Hope this is helpful, Janet
For the sake of completeness, I will also mention that we have considered using some kind of detergent (triton/tween) at low concentration during part of the process to reduce sticking, but haven’t followed up on it.
Your point about the perceived loss of embryos actually having more to do with loss of non-embryo stuff is a good one. Certainly this appears to be the case if we stop the bleaching process before all the worm carcasses are dissolved - the pellet after the first spin is huge, then gets smaller with each wash. I think more embryos are lost in each wash in that scenario in addition to the debris getting washed out as well.