Methodological Approaches Used to Assess Fish Gastrointestinal Communities - Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics - page 115

5.3 FLUORESCENCE BASED METHODS
Quantitative means of culture-independent analysis with FISH (Spanggaard et al . 2000; Asfie
et al . 2003; Huber et al . 2004) and direct nucleic acid stains such as DAPI (Spanggaard et al .
2000; Huber et al . 2004; Sugita et al . 2005; Shiina et al . 2006) and acridine orange (Navarrete
etal . 2008; 2009; Abid etal . 2013) have been applied to quantitatively study the gut microbiota
of fish.
Due to its speed, sensitivity and inexpensive nature the FISH technique is an effective way
to derive cellular abundance information and is often utilized in ecological and environmen-
tal studies (Bottari et al . 2006). FISH analysis with rRNA-specific oligonucleotide probes
designed to target bacterial taxa at different phylogenetic levels has proven to be a powerful
tool to assess microbial diversity of the fish GI tract (Spanggaard et al . 2000; Huber et al .
2004). However, due to the viscous and dense nature of partially digested feed (i.e. digesta)
which constitutes the gut contents inhabited by allochthonous microbes there are often diffi-
culties yielding images that can be utilized effectively to quantify microbial populations (refer
to Figure 5.1). Cells may often be too densely packed into digesta material and become clus-
tered, leading to difficulties in reliably enumerating cells. Additionally, Huber et al . (2004)
reported that FISH analysis with the bacteria-specific probe EUB338-mix only accounted for
80% of the total cell count (as determined by comparison with DAPI) in the GI tract of rain-
bow trout. In goldfish this value was reported to be lower, in the region of 50-70% (Asfie
et al . 2003). The non-hybridized cells in such cases may be non-bacterial or it is possible that
the cells were not permeabilized by the applied FISH conditions. In addition, viable but less
metabolically active bacterial cells with lower ribosomal content may not be visualized with
a ribosome-targeted approach. Despite this, with the application of extensive sets of probes,
Huber et al . (2004) revealed high abundances of Gammaproteobacteria, Aeromonas spp. and
Enterobacteriaceae in the intestinal tract of rainbow trout from a number of fish farms. Fur-
ther issues regarding the efficacy and limitations of the FISH technique are discussed in detail
by Amann et al . (1995) and Zoetendal et al . (2004). Direct nucleic acid stains may avoid the
potential problems associated with hybridisation, but the issues relating to reliable enumer-
ation of cells in viscous digestive material remains. Further, direct staining does now allow
for the discrimination of different microbial species (thus providing total cell counts only), or
indeed differentiation between microbial cells and eukaryotic cells.
5.4 ELECTRON MICROSCOPY
Electron microscopy can be an important tool for investigating intestinal mucosal microbial
populations and in recent times the gut microbiotas of several fish species have been inves-
tigated with electron microscopy (for reviews refer to Ringø et al . 2003; 2007). Scanning
electron microscopy (SEM) is useful for screening large sections of mucosal surfaces (see
Figures 4.1 and 5.2) and transmission electron microscopy (TEM) allows for the observa-
tion of potentially endocytosed and paracellular bacterial cells, thus revealing translocation.
These methods also allow the assessment of bacterial-host mucosal cellular interactions, pro-
biotic and indigenous bacterial interactions and the resultant effects on enterocyte morphology.
Despite providing indications of gut colonization by bacteria-, yeast- or Archaea-like cells,