[DNA Staining of cells with intracellular bacteria]

Michael Herron herro001 at umn.edu
Wed Jul 28 07:30:58 EST 2004


Some of the live DNA stains partition preferentially into some bacteria 
growing in some host cells.  (How is that for waffling?)  The same goes 
for for  the cell tracking dyes.  I think you need to approach the 
problem empirically, though I am all for throwing in some theory too!

I do this sort of thing every day with the bacteria that I study, but I 
usually do it microscopically rather than by flow.

Mike


On Monday, July 26, 2004, at 08:38  PM, Howard Shapiro wrote:

> Todd Parker wrote-
>
>> Does anyone have experience performing DNA staining using cells 
>> infected with intracellular bacteria (i.e., Preferrable dyes, 
>> combination of dyes).  All information is welcome
>
> What is the objective of the staining? If it is to detect the 
> intracellular bacteria, there is going to be a substantial 
> signal-to-noise-problem using almost all known DNA stains, because 
> bacterial genomes are typically well under a hundredth the size of the 
> human genome. Unless there are a lot of bacteria in each infected 
> cell, the overall DNA content won't increase enough to make the 
> infected cell detectable, even with an instrument CV of 2 per cent or 
> so.
>
> Bacteria exhibit a wide range of ratios of A+T/G+C, whereas, in human 
> cells, the ratio is approximately 1:1. If there were a fairly large 
> number of bacteria in a cell, staining with a combination of A-T 
> selective and G-C selective dyes, e.g., Hoechst 33258 and chromomycin 
> A3, might show a different fluorescence ratio in infected and 
> uninfected cells, possibly improving specificity of detection - 
> although this wouldn't work for the many bacterial species with ratios 
> similar to the 1:1 value for humans.
>
> You would almost certainly have to use highly DNA-selective stains, 
> e.g., the Hoechst dyes and DAPI, as bacteria typically contain about 5 
> times as much double-stranded ribosomal RNA as DNA. The RNA stains 
> with less DNA-selective dyes, e.g., ethidium or propidium and the SYTO 
> dyes, and it is harder to get rid of using RNAse than is intracellular 
> RNA in mammalian cells, because getting RNAse through the bacterial 
> cell wall is difficult.
>
> If you needed to do this without fixation, you'd pretty much have to 
> use Hoechst 33342 as the stain, and there is no guarantee it would get 
> into the bacteria. On the other hand, if you can fix the cells, a 
> better approach to detection might be to use PNA probes to detect 
> ribosomal RNA sequences specific to the bacterial genus, species, or 
> strain in the infected mammalian cells. The rRNA probes offer better 
> specificity than you would usually get using antibodies.
>
> All of the above is predicated on measurements made with flow 
> cytometry or low-resolution imaging. It is relatively easy to detect 
> bacteria - or even mycoplasmas and, in some cases, viruses - in 
> infected cells by looking at (or imaging) a DAPI-stained slide at 
> relatively high resolution.
>
> I'd be happy to expand on this given more information about the 
> desired end results, provided you don't have to shoot me if you tell 
> me.
>
> -Howard
>
>
>
>
>
---

Michael J. Herron,  U of MN, Dept. of Entomology
   herro001 at umn.edu
      612-624-3688 (office) 612-625-5299 (FAX)




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