Methodological Approaches Used to Assess Fish Gastrointestinal Communities - Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics - page 118

mammalian GI microbiota, few studies are available in fish (Clements et al . 1994; Mountfort
etal . 2002; Moran etal. 2005; Fidopiastis etal. 2006). Readers with an interest in the digestive
enzyme activities and SCFA production of the GI microbiota of fish are referred to the reviews
of Clements et al . (2009) and Ray et al . (2012).
The gut microbiotas of fish have also been extensively studied for their ability to antag-
onize bacterial pathogens, either to assess their intrinsic potential benefit to the host in situ
or as a means to identify potential probiotic candidates. Typically, this involves the isolation
of bacterial candidates on appropriate media, often LAB or Bacillus spp., and subsequently
assessing potential antagonism using streak plates, spot overlays, agar wells, mucus adhesion
and displacement assays or co-incubation in media or ex vivo sacs. Depending on the method
used, the extent of antagonism can be determined qualitatively, semi-quantitatively or quanti-
tatively. A plethora of studies are available on this topic in fish and they provide evidence of
gut microbial activity and the potential role in limiting the proliferation and colonization of
potential pathogens in the GI tract (Olsson et al . 1992; Ringø et al . 2000; Balcázar et al . 2008;
Kim and Austin 2008; Askarian et al . 2012). For a review on this topic the reader is referred to
previously published reviews (Ringø and Gatesoupe 1998; Gatesoupe 2008; Ringø etal . 2010).
Other methods of providing useful quantitative data on microbial activity, without the need
for culturing, include the use of commercially available kits and fluorescent stains for the rapid
determination of active microbial biomass and bacterial viability in environmental samples.
The ability to accurately measure such parameters provides a level of understanding beyond
that gained by merely enumerating cells which may or may not be playing an active role within
the environment. To date the application of such methods to the investigation of fish gut micro-
biota has been limited. This is surprising given the widespread application of these methods in
the field of environmental microbiology, and may be due in part to the fact that the cultivability
of probiotic bacteria is generally high, thus enabling detection by more conventional means.
The LIVE/DEAD BacLight Bacterial Viability kit (Molecular Probes Inc., USA) is a
method for microscopical enumeration of bacterial cells and has been developed to distin-
guish between live and dead bacteria. It is more sensitive than the conventional culture based
approach, and is also simpler to perform than some of the molecular approaches described
earlier in this chapter. Based on the differing permeability of the plasma membrane in each
case, the kit consists of two fluorescent nucleic acid stains, Syto 9 and propidium iodide (PI).
Syto 9 is membrane permeable and exhibits green fluorescence, whilst PI is not membrane
permeable and exhibits red fluorescence. The use of these stains in combination therefore
facilitates the quantification of live versus dead cells. This method has been applied in a wide
variety of environmental microbiology studies, most notably in a large number of studies con-
cerning the use of probiotic bacteria in humans that assessed survival rates of human-derived
probiotics during heat treatment (Gardiner et al. 2000), bacteria in probiotic dairy products
(Auty et al . 2001), and GI transit tolerance of probiotic strains of Bifidobacterium (Masco
et al. 2007). Consequently, there would appear to be significant scope for its application
to similar fish based investigations. However, it is important to bear in mind the need to
optimize staining conditions beforehand, as staining efficiency may be influenced by factors
such as pH and the physiological state of the bacteria. This may lead to variation in the level
of fluorescence between individual cells (Boulos et al . 1999). In certain cases, non-specific
binding to extra-cellular DNA may also be problematic if staining conditions are not correct.
Another method of assessing bacterial viability is the use of fluorescent redox stains such
as 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). Whilst widely used in a broad range of