Tough Bacteria Break Up Method? - Isolation DNA and RNA from Gram Positive Staphy (Aug/08/2005 )

Hello:

I was wondering if anyone can recommend a method or a kit to break up tough Staphylococcus? The bacterial strain is not identified but much tougher that S. aureua. We tried Qiagen, Ambion, Epicentre, Promega, Tel-Test, Invitrogen Kits but none of them worked. We used lysostaphin, lysozyme, proteinase K but all failed to lyse those cells. I never had any problems isolating DNA/RNA from yeast, E. coli, plant and human cells but this particular bacterial strain is so tough to crack.

We don't have liquid nitrogen to grind the cell pellet but we would like to know if there are other mothods or chemicls that can lyze these cells?

Thank you,

James

I haven't tried it, but would imagine that a beadbeater with glass or zirconia beads would trash these cells very effectively.

Hi!

We frequently use a beadbeating kit from Q-Biogene for our samples. For this you need a special instrument.
In my opinion, beadbeating is a very efficient way to lyse the cells, but you can get some DNA shearing.

i guess you also can try to combine freeze-thawn cycles (in -80C fridge) with lysozyme treatment followed by Proteinas K. I've found this to be more efficient than just lysozyme treatment.

Another thing you can try is using cooking (+100C) and freezing cycles (-20C). It's a dirty methode but PCR can sometimes be done immediatly on the sample.