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environmental microbiology studies, to the authors' knowledge its use in studies relating to
the gut microbiota and probiotic colonization of the fish gut has not been documented. In
metabolically active bacterial cells the CTC is reduced, resulting in the formation of insoluble
formazan crystals after incubation. The enumeration of viable cells as a proportion of the
total can then be facilitated by counterstaining with a general nucleic acid stain such as
Syto 9 or DAPI. However, it is worth noting that whilst it represents a simple method of
counting active bacteria, there are some disadvantages. For example, bacteria exhibiting a
low level of metabolic activity (with a corresponding low level of intracellular formazan)
may be below the limit of detection by some methods, and a lack of standardized methods
makes comparison between studies difficult (Creach
et al.
2003). As previously stated in
this chapter, the association of cells with extracellular material (extracellular polymeric
substances, digesta, sediment etc.) can also lead to difficulties in the reliable enumeration of
cells using fluorescent probing of cells by different methods.
A commonly used non-microscopical method for the determination of active biomass is the
analysis of nucleotide adenosine triphosphate (ATP) levels through bioluminescence assays.
First developed in the 1960s, and based upon the measurement of light emitted due to the
hydrolysis of ATP in the presence of the enzyme luciferase and its co-factor, D-luciferin, such
assays are rapid and extremely sensitive. Another advantage of this method is that while micro-
scopical analyses may sometimes be hampered by the close association of cells with extra-
cellular material, direct extraction of microbial-derived ATP is achievable using less biased
methods. Indeed, many commercially available kits are designed for the specific measure-
ment of microbial ATP from environmental samples. Examples include HS (High Sensitivity)
kits (manufactured by Biothema Ltd., Sweden), the ATP luminescence assay kit (Molecular
Probes Inc.) and the ATP Bioluminescent Assay kit (Sigma-Aldrich, USA). ATP biolumines-
cence assays are sensitive and rapid, with the amount of emitted light directly proportional to
the amount of intracellular microbial ATP. However, there is a requirement for specific detec-
tion equipment and a high risk of contamination from extracellular sources. While analysis of
ATP levels can provide an accurate indication of the level of active biomass within a sample,
it is not species specific and therefore should be used in conjunction with other more suitable
methods where appropriate.
NGS technology can also be used to address questions of microbial activity within the fish
intestine. RNA may be extracted from the intestinal contents and used to prepare a cDNA
library. A cDNA library may be queried for specific transcripts via qPCR, or alternatively
used for a metatranscriptome approach in which the collective transcripts from the intestinal
microbiota are sequenced (e.g. by Roche 454 or Illumina) at sufficient depth of coverage to
provide transcripts even from the rare microbiome. To our knowledge this approach has not
yet been applied to examine the expression of fish intestinal microbiota, but it has been suc-
cessfully applied in other animals (Poroyko
et al.
2010). The cDNA sequences generated by
this approach may be annotated using existing bioinformatics pipelines such as MG-RAST
(
http://metagenomics.anl.gov/) th
at can indicate specific functions associated with transcripts
that have a significant BLAST hit. It should be noted that this approach is inherently limited to
those expressed genes that have been previously described in the literature with an annotated
function, and that a very large percentage of total cDNA sequences will not have a signifi-
cant BLAST hit in GenBank or other databases. This reveals our collective ignorance as to the
expressed functions of the intestinal microbiota and indicates the need for continued research
to understand the contributions of the intestinal microbiota to fish health and disease.
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