FISH only reflects presence of rRNA, i.e., potential protein production. The best way to assess viability is to use it as DVC-FISH. This is the combination of the cell elongation assay ("direct viable count", after Kogure et al.). It marks the target organism and indicates cell division which can be taken as a life sign.

Beware of the live/dead kit without calibration. For some organisms, the concentration of propidium iodide as indicated by the supplier is too high. The consequence is "false dead". We had this with Pseudomonas aeruginosa, when we saw that the proportion of culturable bacteria (cfu) during the log phase was 85 % of the total cell count (determined by DAPI) and the "dead" count as determined by the live/dead kit was 80 %. We use PI now in dilution 1:200. Corner points are the counts during the log phase and after treatment with hydrogen peroxide.

That's great, thanks for all that information. I will take a look at that paper. I hope your work is going well.

Also I will approach the Live/ Dead kit with more caution in future...

FISH may also be targeted to the chromosome, in which case it is even more stable than when it targets RNA and less reflective of active metabolism. On the other hand, depending on what you are interested in, the presence of intact cells may be sufficient. There are a few studies out there on how long it takes the DNA to disappear below detection when killed cells were introduced into a sample environment. A similar study might assist you in determining whether the time scales are relevant. Also, be aware that FISH may have trouble penetrating spores, among other things, so that you can face challenges in false negative as well as your presumed concern with false positive.

There is a technique called CLASI-FISH that allows you to visualize diverse microbiota in samples (including soil) at once. Perhaps the DVC-FISH could be combined with this technique. It would be very powerful in understanding microbe-microbe associations.

CLASI-FISH: Principles of combinatorial labeling and spectral imaging.

Alex M Valm, Jessica L Mark Welch, Gary G Borisy

Marine Biological Laboratory, Woods Hole, MA 02543, USA; Brown University, Providence, RI 02912, USA.

Journal Article: Systematic and Applied Microbiology (impact factor: 2.64). 04/2012; DOI: 10.1016/j.syapm.2012.03.004

some excellent literature (open source):

Hammes et al. (2011): Cultivation-independent assessment of bacterial viability. Adv. Biochem. Engin/Biotechnol. 124, 123-150

Maybe a combination of FISH and CTC could tell you something about the activity of specific groups that you can label with FISH (e.g. http://aem.asm.org/content/71/5/2748.short, very easy technique). However, this does not tell you something about viability of the complete community...

1)If you are trying to look at just live/dead in combination with universal probes to light up all bugs then I might suggest using a common gene product like from tca cycle as a fish probe target. But it has the downside of ligting up a lot of bugs, so

2) if you want to look at specific subpopulation viablity, it would be better to target gene products specific from them and overlay with 338probes. With a decent confocal and about 4 cobitorial probes you can answer viablity of specific bugs within a biofilm.

3) As other learned members have pointed out, it all depends on what you are trying to ask scientifically and what answers would be of help.

You can use MAR-FISH, using, for instance, [3H]leucine.

Yes, you can combine FISH with microautoradiography with various kinds of e.g. 3-H labeled organic substrates, uptake is usually quite well visible. However, the method needs some skills and experience.

Dear Dr. In case I have a biofilms of multispecies and I would to perform Class-fIsh on it. Is it that possible to differentiate between the same species such as strp mutans and strp oralis? Is that any other ideas to do so?