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isolation of intracellular bacteria from human cells - Protocol for release intact intracellular bacteria (Dec/09/2004 )

I am searching a good protocol to release intact intracellular bacteria from human tissue samples (gut samples). Can anybody help me?
Thaks in advance.


don't know if it helps, but we routinely isolate intracellular bacteria from coral tissue and worm tissue by the very simple method of dissecting and crushing it (mortar and pestle), vortexing (top speed three min) and then allowing heavy particles to settle (1 to 10 minutes) and plating/fixing the soup.
trouble is of course you get much contamination from bacteria present on the outside of your sample. one way to minimize this is to incubate sample with gentamycin (200 microgram/ml for 1 to 3 hours) before crushing. Gentamycin cannot penetrate eukaryotic cells and so shouldn't hurt your intracellular bacteria - of course you have to wash it off really well with sterile media/water before crushing. problem is some bacteria are completly resistant to gentamycin!
a different way which has been sugested to me but not yet tried is to use a microinjector to suck contents out of tissue.

good luck

if you come up with a good method could you please post it - i'd be very intrested...