I am trying to image and quantify bacterial cells on muscles using confocal laser scanning microscope. But I am having trouble quantifying the bacteria as the stains that I used ( live/dead staining) also stains the muscle fibres.
Can anyone recommend a stain that would only stain bacterial cells ? Thank you
Hi Nirmani,
Your question is rather difficult to aswer to as mitochondria, which are abundant in muscle cells, are kind of bacteria.
Many dyes usually used to stain mitochondria stain bacteria and vice versa. So it will be difficult for you to stain bacteria without staining mitochondria.
If you are interested in only one species/genus of bacteria, you can try immunodetection with a specific transport protein for exemple, but if you plan to count the whole population of bacteria, no matter the species, you take the risk to stain organelles as well.
Are you interested in outer bacteria or you seek at inner cell bacteria as well ?
Fresh or fixed muscle tissue ?
You may try permeant DNA dyes (Syto9, Syto83, SYBR-Green) to avoid muscle fibre staining.
Oliv
Use and do not thank :)
The BacLight Green (Cat. no. B-35000) and BacLight Red (Cat. no. B-35001) bacterial stains are fluorescent labeling reagents for detecting and monitoring bacteria. These two dyes are not nucleic acid stains. Bacteria stained with the BacLight Green and BacLight Red bacterial stains exhibit bright green and red fluorescence (absorption/emission ~480/516 and ~581/644 nm, respectively), and can be resolved using the appropriate flow cytometric channels or fluorescence microscopy filters. The BacLight bacterial stains are compatible with formaldehyde or alcohol fixation methods.
From
https://www.thermofisher.com/order/catalog/product/B35000
Thank you very much for your answers. I use fresh muscle tissues not fixed ones.
Yes I have used Syto 9 & Syber green. I can distinguish muscle fibers from bacterial cells but still have not worked out a way to sort out the fluorescence.
There aren't any that I'm aware of. With all due respect to Alexey, I know from experience here at Molecular Probes that the BacLight dyes will stain eukaryotic cells, too, and nucleic acid stains like the SYTO and SYBR dyes will as well. The only choice you would have, I think, would be to use a surface antibody that recognizes proteins only expressed on bacteria, but I don't have examples to suggest.
Jason, what then is the feature (attraction) of the BacLight Green, if acridine orange is cheaper and affordable ?!
HI, Alexey, BacLight Green and BacLight Red are good as a means of determining viability status, with flow cytometry, by looking at the degree of separation of the two populations of live and dead with and without treatment (see this link for the manual, for example data), but not in a mixed population with eukaryotic cells. I'm not as familiar with acridine orange usage with bacteria. I haven't seen comparison data, but if AO does the same thing, then I guess it would be a good choice too. Also, AO is a nucleic acid stain, which can lead to difficulties in some situations, whereas BacLight dyes are not. http://tools.thermofisher.com/content/sfs/manuals/mp35000.pdf
Nirmani, that's a very interesting idea. Do you have some example images (maybe in a Google Docs folder)? I'd love to see them.
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