In addition to filtering, an important step is flame blunting the back end of the pipettes to make the glass smooth. If not, the glass edge may be very sharp and cut off small plastic pieces from the pipette holder that can sink out and block the tip. You may also want to check the rubber o-rings in the pipette holders. If they are old and disintegrating, they may be a source of rubber dust falling into the pipette solution when you mount the pipette.

Do you actually see something in the pipette that is physically blocking the pipette?  We use a similar internal solution and occasionally see the resistance increase when positive pressure is applied, though nothing can be seen in the pipette.  This is rare enough that I can just use a different aliquot.  In your case, you may want to try using a filter than can be attached to the syringe used to fill your recording pipettes.  This would block any small particles, such as something that came out of solution, from clogging your pipette.  Millipore is one vendor that supplies syringe filters.

Hi Joe.

We filter solution only with a regular paper filter before freezing. We use a 0.2 um filter between a filament for pipette filing and a 1 ml syringe with intracellular K-gluc solution after it is thawed. We used to have the same problem and it was solved by keeping the syringe in a refrigerator (+2+3C) all of the time. Take the syringe out just for filling pipettes and put it back right after that. It is also helpful to wash the inside and the outside of the filament with distill water and the inside of the filament cover as well. Try do not touch with the filament anything beside the pipette and the inside of the filament cover. We haven't had the clogging problem since that even with the high resistance pipettes (10-15 MOhm) for axonal recordings.

In addition to filtering, an important step is flame blunting the back end of the pipettes to make the glass smooth. If not, the glass edge may be very sharp and cut off small plastic pieces from the pipette holder that can sink out and block the tip. You may also want to check the rubber o-rings in the pipette holders. If they are old and disintegrating, they may be a source of rubber dust falling into the pipette solution when you mount the pipette.

Thank you all very much for your advice. Florian, our pipettes often clog before we patch, ie without positive pressure we can't patch. Also the blockages are quite visible, (although Amber as you mentioned a few years ago I remember the 'phantom blockages' ie the resistance would increase without anything obvious in the pipette tip). We do fire polish the back of the pipettes and have replaced the rubber o-rings. Vladimir, we've also flushed the pipette holders and filaments with dH20 and will now try your suggestion of keeping the pipette fillers cold - will rport back! Thanks again all for your help.

Joe

Dear Joseph:

Do you have the same problem with your patch electrodes filled with filtered (0.2 mkm) 1M KCl? What is the in-bath resistance of your pipette before it gets clogged? If you have no problem with 1M KCl, then you should check your internal solution. Apparently, some of your compounds (K-gluconate of Na GTP) crystallize and block the tip. It this is the case, then without pressure your pipette should get clogged slower.

However, if you have also difficulties with internal KCl, you should look more carefully into the syringe or fillers you use. Keep in mind that the inner of certain brands of 1ml syringes and needles are processed with grease (to make them water-tight, especially those used for inoculation e.g. TB). Hence, if you use these kind of syringes, you will get small drops of oil inside of your patch pipette, which can block its tip. These are quite nasty beasts as they are invisible... Hope this helps.

All the best,

Saak         

Try to change the batch of glass pipette that you are currently using with different  one. We have has similar problem in the past due a dirty glass pipette. If this did not solve the problem.change the filament of the electrode puller. Good Luck

I have had similar problems where a particular batch of internal solution often clogs the pipette regardless of how or when you filter. Sometimes you can even see the clog and it looks like a microscopic gel (not dirt or crystaline), which is likely a percipitate coming out of solution.  I find remaking the internal stock often if not always fixes the problem. You can also consider purchasing new chemicals as contaminates can accumulate over time when many people are sharing stock chemicals.

Hi, I just had this very same issue for days... turns out in my case the glass capillaries were dirty, as suggested by someone already. I am now routinely cleaning them with EtOH, drying them and then fire polishing the ends before pulling. I have not seen any dirt ever since. Good luck!

Hi, Joseph: I see you have many interesting and useful commentaries already. Our problems with pipette clogging have been traced to three main possibilities. 1) dirty pipettes; once we had a batch of dirty capillary glasses that forced us to change supplier. 2) living things growing in the internal solution; this is the most frequent problem and I suspect they may be fungi; they can grow very fast and can contaminate your tubing, so that when you put positive pressure the tip gets clogged; they are essentially invisible, but if the problem is persistent sometimes you can see "filaments" or "globs" coming to the tip; rinsing with alcohol and/or diluted chlorine and abundant distilled water (not necessarily in that order, but much water at the end) may clean your tubing. Change regularly your filters, as soon as they get a little resistant. Now, the weakest link in the system is by force the needle used to fill the pipettes. You must also clean it regularly. More important, don't leave solution for more than 24 hs on it, not even in the fridge: discard it at the end of the day, wash and rinse your needle and let it DRY. Other clogging materials can be dust, grease, particulate glass and rubber, as has been pointed out, but I find this more rarely. Once in a while you will see some living creatures: is not that difficult to get contamination of all kinds. Finally, 3) when the pipette resistance (Re) is higher than 7MOhms, they would tend to clog during recording, particularly if you applied stronger suction to achieve Gigaseals. If the system is perfectly clean, I find the equivalent of 100-200uL of air (suction) is enough to create a strong seal without much membrane going into the tip. If you are using stronger suction, you are likely having an issue with all/some of the above. Remember: "cleanliness is next to craziness..." or something like that: you should find yourself obsessing about it. A last note: my experience is with isolated cell pairs, I do not know your preparation, and there may be other sources of problems which I can not comment about.

By the way, I wouldn't think the gluconate have much to do with your problems.

There are many good comments in this string.  One additional point is that you can back fill pipettes with a PE syringe that has been pulled out to a fine, long tip after heating the shank in a flame.  This avoids having to keep syringe needles clean.  You should consider getting a manometer; use it to apply only as much pressure as you need: this will reduce the volume ejected from the pipette per unit time and reduce the frequency with which junk does find its way to the tip.  

Dear Joseph,

If I were you, I would try to use K-methanesulfonate (K-Meth) instead of K-gluconate (K-Glu). Both K-Meth and K-Glu may have the same viscosity and provide a similar liquid junction potential. However If you still have a clog, I would try to reduce the phosphocreatine concentration, as it could precipitate while setting the holding potential to very negative values (≤Vrest; resting potential). In any case, these internal solution crystals are not a big issue when using a pipette resistance as large as ~5 MOhm, so you can try to use larger pipettes.

Best,

Hey!

I experience similar problems. I'll try out the advices already given here - if something is helping I'll let you know. I've attached an image of one of my clumped pipettes.

Have a nice day,

Richard

I was experiencing same problem with gluconate based internal. I would make it fresh every two weeks and sonicate before experiment.

I am having same problem. This is what I found after having increased Rpip. At the beginning, there was no clogging and IPS streamed out from the tip. Sometimes clogging can be found in the capillary. I need to change recipe of the IPS. 

Hi Joseph, do you see also clogging of pipettes with any other internal solution?

If not, then you have a problem with your solution (not properly filtered, perhaps oil droplets from the tube attached to the back of the pipette or too much pressure applied).  

If yes, then perhaps the problem is not with your internal solution but with your ACSF. E.g. poor bubbling of the media can promote precipitation of Ca2+ in your ACSF with its deposition on the tip of the pipette. Warm weather can also cause troubles... check your ACSF -   

It is very frustrating but there are ways out - good luck! sv