For the many patch clampers out there doing experiments under constant buffer perfusion, bath overflow and flooding can be a rare but infuriating occurrence. Whether it ha
ppens because the department's vacuum supply suddenly goes on the fritz, or because an errant piece of fluff has wedged itself up inside the overflow tube, the result is usually the same; a large pool of saline solution with the potential to rust the hell out of various expensive bits of equipment if not policed quickly and efficiently.
Some ideas:
- Inspect and clean the outflow regularly, and keep the work area as dust and lint free as possible.
- Make sure the join between the bath and the stage is water tight, so that any overflow doesn't seep through the cracks and drop straight onto the objective carousel. Electrical tape is handy for this. Also use electrical tape to cover any manufactured holes bored through the stage that are positioned in a manner that might also direct overflow onto the microscope's moving parts.
- If practical, it's also worth smearing a very thin layer of silicone grease around the outer top edge of the bath itself, as this will cause the buffer to form a large bead before spilling out of the bath, buying a little extra time to notice something is amiss and shut off the inflow valve.
- A thin layer of silicone grease or Vaseline on the air table, around the base of the microscope and any free standing apparatus. This way, even if solution does spill out over the stage and onto the air table, it won't get under any bits of equipment that are difficult to access and clean.
- If you get any overflow or splash outside of the bath, make sure it is cleaned up and the area fully dried before progressing with the experiment. A narrow connection between the bath solution and any wet external area below the bath level is often sufficient for gravity flow to take place and cause a repeated flood.
3 comments:
My approach to this has been to avoid constant bath perfusion for the anathema that it is, instead preferring The Pipes-TM. A Bean lab speciality, perfected while using boatloads of expensive peptide calcium channel toxins.
But I hadn't ever seen the vaseline trick, that's useful for those times when bath perfusion is unavoidable.
"My approach to this has been to avoid constant bath perfusion for the anathema that it is, instead preferring The Pipes-TM."
It's a pain, to be sure. We do a lot of stuff with P2X receptors, so it's preferable to minimize the possibility of released endogenous ATP hanging around in the recording chamber.
Can you expand on The Pipes method, or is that a lab secret? ;)
Actually, it's basically a sewer pipes system, but made with quartz glass tubing. Good enough when your exchange timing need not be faster than ~0.5 seconds.
But the solution experienced by the cell is solely whatever's coming out of the pipe next to teh cell. You can be in control soln with TTX at 1000 times Kd and have no reduction in INa (that's just how most people in the Bean lab would test it for themselves).
Plus, you never really need more than 5-10 mL of each soln, which lasts for the entire recording day.
I have to fabricate some new ones soon, and am hoping to blog it when I do.
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