How sensitive are your probes?

Annelie Pernthaler, Jakob Pernthaler, and Rudolf Amann Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria, Appl. Envir. Microbiol., Jun 2002; 68: 3094 - 3101.

Fluorescence in situ hybridization (FISH) of microbes was a break-through well over a decade ago. However initial researchers found it difficult to apply the technique to anything other than eutrophic systems. As we know most bacteria in the oceans are slow growing, vastly small and starving, which results in the intensities of signal hybridization fluorescence getting lost in background fluorescence. This is attributed to low levels of 16S ribosomal RNA being present, thus causing low complementary base pairs and probe binding. Due to the lack of sensitivity of early FISH methods, they were not ideal for application to marine oligotrophic microbes. Therefore it was a key interest of marine microbial researchers to develop more sensitive techniques (2002).

Researchers from the Max Plank Research Institute, Bremen, Germany further developed an established more sensitive method (which used FISH combined with horseradish (HRP)-labelled oligonucleotides probes and tyramide signal amplification, known as catalysed reporter deposition (CARD)), they advanced the protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. The main problem for them to overcome was high sp.-selective cell loss, as a result of cell wall degradation from HRP penetration following permeabilization. This was observable through decreases in cell counts (using DAPI) following permeabilization, rendering the technique unreliable as a tool for the detection of O.T.U. and for the enumeration of marine microbes.

These Max Plank Institute researchers developed a method where concentrated sample filters were embedded in low-gelling agarose, which resulted in no observable decreases in bacterial cell counts during a 90 min incubation of lysozyme. These modifications in preparation and permeabilization procedures, as well as improvement to staining protocols for CARD-FISH, for the quantification of marine microbes, led to a significant improvement in detection rates compared to previous protocol which involved mono-labelled probes (such as EUB338 mono, compared with probe EUB338-HRP). These researchers, when using their improved method (multi-labelled), reported an improvement of 46% based on the differences between mean values of mono-labelled probes and multi-labelled probes in detection rates of coastal North Sea bacterioplankton. They also reported that the modified technique detects clades e.g. SAR86 which were previously undetectable by mono-labelled probes.

These discoveries led to the authors concluding that oligonucleotide probes are more superior for the staining of bacteria with low rRNA content in the marine environment, provided cell disintegration is minimised during the permeabilization stage of the technique.

In the discussion the authors discuss the potential and limitations to their advancements to the CARD-FISH technique for environmental microbiology. They acknowledge that there are limitations to the technique e.g they are not confident that the technique they developed is suitable to be used on Archea. However they do highlight the potential of the CARD-FISH approach, and suggest it is not just limited to staining of rRNA’s. They suggest a wide range of methods used in histology and cytology could also be used in marine environmental microbiology e.g. the detection of mRNA. The works of these authors demonstrate minor advancements in techniques of molecular methods result in a widening of disciplines for the application of its use. This progress in specialized areas of microbial ecology provides future marine microbial scientists with far more powerful and sensitive tools, resulting in more confidence in the accuracy of researchers work.

On a more negative note the authors failed to produce vital unpublished data which supports their argument for the evidence of the improvement of their method. These omissions cast doubt on the confidence of readers,( such as myself) have in what seems to be such a pertinent development in molecular techniques.

To conclude advancements in molecular methods provided by researchers like Pernthaler et al. result in further superior research being produced provided their protocol is adopted and is as good as stated on the tin.