Separating bacteria from nuclei - (Feb/14/2006 )

Dear All,

I work with bacterial pathogens that invade mammalian cell lines. Does anyone know of a protocol or a published paper that describes how to separate out the bacteria from the rest of the cell's subcellular components after homogenisation, particularly the nucleus?

The bacteria are very dense as they are found predominantly at a similar level in a sucrose gradient as the nucleus. Also the bacteria tend to bind strongly to one another so there is a range of bacterial sizes along the gradient. Any hints or advice would be much appreciated.

Paul

hmmm.. I haven't done what you ask before but I have done invasion assays and we would treat the mammalian epithelial cells with triton X to lyse them. This would release the internalised bacterial cells for plate counts. The bacteria (Gram neg) would not lyse, although you should check this with your strain by incubtaing your bacterial cells with Triton X and checking for viability. How about lysing the epithelial cells with Triton X, then treating the sample with proteinase K and DNase. This should remove any contaminating DNA and protein. The DNA from the bacteria will be protected by the cell wall and membrane.