Bacterial viability is a crucial issue when we detect bacteria in environmental samples. RNA based RT-qPCR methods are used frequently, but these methods can be difficult to implement due to the instability of RNA, problems associated with extracting RNA directly from environmental matrices, and variation caused by the physiological condition of the cell. However combining Ethidium Monoazide (EMA) or Propidium Monoazide (PMA) with qPCR provide a potential alternative for enumerating viable bacterial cells in environmental samples but have limitations in terms that these dyes approach is restricted to membrane integrity of bacterial cell.
My questions is, what could be the probable methods or improvement in qPCR to detect viable bacteria in the environmental samples like surface or potable water?
although not an exact measurement, we did the following to get approximal CFU counts. 1 liter of potable water was filtered using 0.1µm PVDF filter (Millipore + MIllipore filtersetup). Membranes were carefully scraped and rinsed with 0.5 ml of culture medium and plated onto R2A agar plates. CFU counts were determined after incubation at 25C for 7 days. Spiking experiments with E.coli K12 yielded recoveries of 90-110%. Keep in mind that, using this method, you will only be able to quantify culturable bacteria.
@Jeroen: you don't need to scrap the bacteria, you can simply place the filter on the agar
@Gulshan: by viable you mean alive bacteria, right? How can you distinguish by PCR between alive and dead bacteria?
@Tomáš Hluska
We can distinguish viable bacteria from dead on the basis of mRNA quantification by RT-PCR. DNA based methods like qPCR could not discriminate between DNA arising from dead bacteria or from live bacteria, thats why PMA dye was coupled with qPCR to discriminate between live and dead cells.
Mine interest was, is there any dye present besides PMA/EMA, that can be used to couple with qPCR for estimating viability of bacteria?
@Jeroen van bergenhenegouwen
The method u describe is suitable for culturable bacteria but this will omitt the VBNC (viable but not culturable cells) that still are alive ,only lost their culturability due to some stress...
Hi
At this moment I only know these two dyes, and there exist a lot of papers demonstrating a good correlation between viability in terms of culture / membrane integrity and vPCR.
Somme researchers also suggested that active topoisomerases can correct the effect of PMA/EMA, thus also some enzymatic reaction is evaluated. On the other hand membrane pumps also can extrude, in live cells , EMA.
vPCR with PMA/EMA is an emerging field, still a lot of things needs a lot of science but in my opinion is a good combination with the specificity of PCR protocols.
If you don't need specificity a good approach is ATP measure.
best
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