Methodological Approaches Used to Assess Fish Gastrointestinal Communities - Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics - page 106

estimates on the cultivability of gut microbes are restricted to a limited number of fish species,
the cultivable bacterial levels in salmon (using tryptic soy agar (TSA) plates incubated for
10 days at 17 ∘ C) represent ca. 1% of the total bacteria (coho salmon Oncorhynchus kisutch ,
Romero and Navarrete 2006; Atlantic salmon, Navarrete et al. 2009). Culture-dependent
techniques therefore, even when using molecular methods for isolate identification, provide
a rather skewed and restricted representation of the microbial communities in the GI tract
of fish.
Despite the obvious limitations of culture-dependent approaches they are still useful if con-
ducted appropriately, with a high number of isolates selected for molecular identification, and
used in combination with culture-independent approaches. In future studies utilizing culture
based approaches, it is important that microbiologists recognize the unique characteristics of
fish and avoid applying protocols developed for terrestrial homeothermic animals. This is par-
ticularly true with respect to the selection of culture media and incubation conditions. Even
now it is all too clear from both published literature and data presented at international sym-
posia that the culture media used is not always appropriate. The most obvious cases include the
use of selective media for the enumeration of Bifidobacterium spp., which are not common or
abundant members of the gut microbiota of fish, and Escherichia coli , which is not an impor-
tant enteropathogen of fish species. Although these bacteria have been identified in a limited
number of studies in fish there are more relevant bacterial groups, which are more abundant
and have known connotations for fish health status, which should take precedence. In addition,
culture conditions will often be erroneously set at 37 ∘ C for 24-48 h. These methodological
approaches are appropriate for assessing the cultivable microorganisms in mammals but are
not appropriate for poikilotherm animals such as fish.
5.2 MOLECULAR TECHNIQUES
A wide range of molecular ecology techniques are available based on the sequence variability
of the 16S and 23S rRNA genes. These techniques allow for the identification and quantifica-
tion of the intestinal microbiota, and in the past 5 years culture-independent molecular based
techniques have become increasingly utilized for the assessment of the microbiota of the fish
digestive tract (refer to Table 5.2). These techniques, however, are not currently utilized in the
vast majority of probiotic and prebiotic studies.
Which molecular based approach to use depends on the aims of the study: clone libraries
and metagenomics can be used to identify the microbiota composition; microbial community
structure and diversity can be analysed by ingerprinting methods; while dot blot hybridization,
quantitative real-time PCR (qPCR) or fluorescent in situ hybridization (FISH) can be used
to measure the abundance of particular taxa (Zoetendal et al . 2004). This review will only
discuss techniques that have been used in fish gut microbiota, prebiotic and probiotic studies
or techniques with most relevance to future studies. For a broader discussion of molecular
based techniques readers are referred to the reviews of Amann etal . (1995), McCartney (2002),
Zoetendal et al . (2004), Ben Amor et al . (2009) and Roh et al . (2010).
5.2.1 PCR based methods
A key stage in the process of PCR based methods, often overlooked, is DNA extraction.
The DNA extraction process is the foundation of any PCR based method. He et al . (2009)