Ultrasonic bath - bacteria lysis using a ultrasonic bath (Feb/15/2007 )
I'm trying to lyse small bacterial samples (1ml) and don't have a convencional sonicator (with a probe). The only thing I have is a ultrasonic bath but I'm not sure how to use it. Did anyone used this for bacteria disruption successfully?
Any advice welcome, thanks!
Do you mean the one with water bath? Apparently, it wouldnt be as effective as the sonicator with a tip. You really need a very high amplitude in order to disrupt the cells. I think you can try by doing about 5 times with short burst on the ice?
So, I just put the eppendorfs on water for short periods of time? I tried that at the maximum amplitude and it didn't seem to work. How long should I leave the tubes on the bath?
Actually I am not too sure as I used detergent lysis instead of sonicator. My supervisor mentioned to me that water bath sonicator will never work because it doesnt have a very high amplitude compared to the tip one. But I tried myself anyway thinking that it might able to lyse the cells. It doesnt really work as effective as detergent lysis. What I did was put my samples in the water bath and sonicate for like 10 seconds and put it back on ice for 20 seconds. Then I repeated about 4 times. So I guess my supervisor is right about it.
ok, thanks. I also think detergent lysis is better but my supervisor wants me to try sonication instead!
If you have the sonicator with the tip. That will be much better. The other method perhaps you can try is the Freeze Thaw method if you are lysing a gram negative bacteria. Hope it helps.
Do you have access to liquid N2, or dry ice? If so, try freeze-thaw (the dry ice needs to be mixed with ethanol, it gives you about -80C liquid). Resuspend your cell pellet in lysis buffer containing some lysozyme and DNase I (and protease inhibitor like PMSF), then freeze. Thaw in a water bath for a few minutes, and repeat the process a couple of times. Spin down hard to clarify the lysate.
I don't think the ultrasonic baths are anywhere near strong enough to disrupt cell walls.
I agree with swanny that ultrasonic baths are as effective as the sonicator.
If ultrasonic bath is strong enough, you will need to wear a ear plug (to prevent damaging your eardrums) when cleaning equipment.
I think swanny is right about using freeze and thaw method to make the cell lyse if you don't have a sonicator.