Re: Harvesting bacteria from infected tissue for subsequent flow analysis [Microbial]

Nicholas Lejarcegui wrote (for a third party):

>I was wondering if anyone had any experience with harvesting 
>bacteria from infected tissues for subsequent flow analysis? I'm 
>sure there's a way to do it, but before I jumped into a literature 
>search I thought I'd go to the folks who might know.

A very old paper from BD (Mansour JD, Robson JA, Arndt CW, Schulte 
TH: Detection of Escherichia coli in blood using flow cytometry. 
Cytometry 6:186-190, 1985) describes the use of a proprietary lysing 
reagent containing detergents and enzymes (not otherwise specified) 
to get rid of all the cells in whole blood, enabling seeded E. coli 
to be detected at concentrations of 100/mL or less. Most cells in 
tissue don't have cell walls, as bacteria do, and enzymes such 
collagenase should get rid of a lot of the glue that holds soft 
tissues together, so the overall approach might work for at least 
some tissues, not likely for cartilage, bone, etc. You'd have to be 
careful if you wanted to keep the bacteria alive, and/or to detect 
surface antigens. If you wanted to used rRNA probes for 
identification, you'd probably be OK.

If you're looking for a few bacteria per gram of tissue, you'll 
probably be better off doing the lysis and filtering through a black 
membrane or aluminum oxide filter and then doing image analysis 
instead of flow.

-Howard