I'm not sure if that kit would allow for subsequent culture of cells post-treatment though. It may not kill/destroy the cells immediately but they will not continue to live after the treatment. As the kit says you must analyse the cells within 18 hours.

Fixing and permeablising cells, from my experience, is permanent for mammalian cells. I've no idea if this would be different for microbes due to their cell wall and plasma membrane structure.

The action of the fixative "freezes" the cell's physical state and the permeablisation treatment punches holes in the membrane to allow antibodies in. I don't think most cells would be able to survive this!

If you're looking to continue growing the cells after analysing them then don't fix them. Mild detergents are often used to permeabise mammalian cells to probe their internal structure. You could try using very low concentrations of such detergents to see if you could gently permeabilise the membrane. I'm not sure if work has been done on how damaged the membrane can be for microbes to survive but it could be a trial and error thing for you there. Triton X-100 is a common lab detergent used for this. Sigma sell it. By low concentration I'd imagine something like 0.1% v/v or below.

Depending on what you're looking for, do you have to use antibodies? There are plenty of dyes out there that are lipid soluble and so pass across membranes freely. That would mean you wouldn't have to permeabilise the cells though it may not be clear what the dye would do to the metabolics of the cells once in there and staining it's target...

Fix and perm kit from invitrogen, does not kill the cells

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Flow-Cytometry/Sample-Preparation-Assays-for-Flow-Cytometry/FIX-and-PERM-Cell-Fixation-and-Permeabilization-Kit.html

Also to later exclude dead cells, a live/ dead staining kit, which is shown on the DAPI channel.

Not sure if this is good for microbes, hope it helps.

I'm not sure if that kit would allow for subsequent culture of cells post-treatment though. It may not kill/destroy the cells immediately but they will not continue to live after the treatment. As the kit says you must analyse the cells within 18 hours.

Fixing and permeablising cells, from my experience, is permanent for mammalian cells. I've no idea if this would be different for microbes due to their cell wall and plasma membrane structure.

The action of the fixative "freezes" the cell's physical state and the permeablisation treatment punches holes in the membrane to allow antibodies in. I don't think most cells would be able to survive this!

If you're looking to continue growing the cells after analysing them then don't fix them. Mild detergents are often used to permeabise mammalian cells to probe their internal structure. You could try using very low concentrations of such detergents to see if you could gently permeabilise the membrane. I'm not sure if work has been done on how damaged the membrane can be for microbes to survive but it could be a trial and error thing for you there. Triton X-100 is a common lab detergent used for this. Sigma sell it. By low concentration I'd imagine something like 0.1% v/v or below.

Depending on what you're looking for, do you have to use antibodies? There are plenty of dyes out there that are lipid soluble and so pass across membranes freely. That would mean you wouldn't have to permeabilise the cells though it may not be clear what the dye would do to the metabolics of the cells once in there and staining it's target...

Live/dead kit will kill cells

I agree with Piers Gordon-Brown, I don't think you can use the fix and perm kit for sorting bacteria subpopulation that have to be amplified for your next experiment.

I agree with Piers Gordon-Brown again. I also recommend you the use of commercial staining dyes instead of fluorescent antibodies. Anyway, you could try to submit cells to a sonication bath (in the hot spot) in order to permeabilize them previous staining stage. The duration as well as its intensity should be optimized to avoid any deleterious influence on cell viability. In addition to use detergents, you could use or some permeabilization agents such as EDTA, I usually use this chemical to facilitate the uptake of non membrane-permeant dyes by Gram negative bacteria.

It is true that certain Live/dead kits may decrease viability after dye staining, but it is not an established gold standard. From my experience, microbial cells can be easily cultured after staining them and subsequent sorting using FACS with high yield and cultivation recovery. Good luck!!

As with all the above, I would agree with Piers Gordon-Brown. Fixing bacteria will kill them. I would imagine introducing an antibody into them may reduce their viability also. I'm not aware of any previous studies for intracellular labeling followed by viable growth, but I could easily be wrong.

As for permeabilising the bacteria, it would be a good idea to try some MIC's with the bacteria and different concentrations of your detergent. Triton-X 100 is often used to permeabilise mammalian cells to then count the intracellular viable bacteria, but I believe it is better suited for Gram-positive bacteria as they are more robust. I regularly use up to 1% saponin to permeabilise mammalian cells with no loss of viability with the Gram-negative bacteria that I use. I have also seen Digitonin used to permeabilise mammalian cells in the presence of bacteria, and this is again more gentle a detergent than saponin. However, I do not know if the bacteria are tempararily permeabilised or resistent.

Perhaps if you could tell us what it is that you want to look at someone could suggest an adaptation to your method?

Best of luck.

Thanks folks. Saul, using commercial stains isn't an option. It has to be the antibody and nothing else, as I'm looking for a specific metabolic activity which a dye will not detect. The Invitrogen kit isn't an option either unfortunately, I don't want to fix the cells. It seems that many protocols have been developed so that the morphology of the target cells are not changed, but these tend to kill the cells. The search continues!

Dimethyl sulphoxide (DMSO) is very good for taking other chemicals/compounds across membranes without apparent damage.