Culture-dependent methods

The evaluation of microbial diversity in fermented food is problematic. It is often difficult to cultivate viable microorganisms using known media, as part of them might not be cultivable in vitro. Some species are outcompeted by numerically more abundant microbial species. Moreover, Fleet (1999) highlighted that adverse conditions in food ecosystem such as nutrient depletion, heat treatments, pH variations, or low temperatures could induce microbial cells in a stressed state characterized by the inability to produce colonies on media even if they are still able to perform metabolic activity. They enter in a viable but noncultivable state, which cannot be detected by culture-dependent methods. 

Nevertheless, detection, identification, and both phenotypic and genotypic characterization of strains isolated from food matrices are still of great importance, especially for the food industry. The selection of new starter cultures with desirable competitive ability and metabolic properties is of paramount importance and starts from the screening of hundreds of isolates obtained from food. This has led to the development of polymerase chain reaction (PCR)-based molecular methods for strain identification and characterization. Here, we will discuss the most widely used advanced techiuques in food fermentation. 

When researchers are approaching the identification of food microbial communities isolated on selective media, species-specific PCR could represent a valid and rapid alternative. Different researchers have optimized protocols for the identification of fermented food recurrent species such as Lactobacillus helveticus (Fortina, Ricci, Mora, Parini, Manachini, 2001), Streptococcus thermophilus (Lick, Keller, Bockehnann, Heller, 1996), Lactococcus lactis (Corroler, Desmasures, Gueguen, 1998), and Lactobacillus delbrueckii (Torriani, Zapparoli, DellagUo, 1999), which have been widely applied in the study of food microbial populations (Dolci et al., 2008 Fonseca, Ouoba, Franco, Carballo, 2013 Fortina et al., 2003 Robert, Gabriel, Fontagne-Faucher, 2009). 

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Culture-dependent methods to characterize antibiotic resistance in the environment are essentially based on the guidelines developed for clinical and veterinary microbiology (e.g. [20, 66-69]). Nevertheless, several adaptations have been introduced. 

The exploitation of marine actinomycetes as a source for new drug leads is in its infancy. Even with the limited screening efforts that have been dedicated to date, the discovery rate of novel bioactive metabolites from marine actinomycetes has recently surpassed that of their terrestrial counterparts.1 5 Culture-dependent methods have demonstrated that actinomycetes with tremendous... 

However, these findings may reflect only a tiny fraction of the true therapeutic potential of marine actinomycetes. Recent culture-independent studies have shown that marine environments contain a high diversity of actinomycetes that are rarely, if ever, recovered by culture-dependent methods.8 Most of these unculturable actinomycetes have very different 16S rRNA sequences when compared with their terrestrial counterparts,6,9 and the ability to cultivate these novel actinomycetes will provide a new source for the discovery of novel drug leads. The future success of bioprospecting marine actinomycetes relies on our ability to isolate and grow novel actinomycetes from the marine environments. 

One culture-dependent method that is now being widely utilized in analysis of community-level physiological profiles (Garland and Mills, 1991 Zak et al., 1994) is available commercially as the BIOLOG system. This system is based on the microbial utilization (detected by the reduction of a tetrazolium dye) of a suite of 95 different carbon sources that can be categorized... 

In TBV, the oxidative fermentation is carried out by spontaneous acetification due to the natural occurrence of AAB in the environment. Recently, the application of selected AAB strains in TBV production has been proposed, and a procedure has been developed for the scale-up of the fermentation process at the vinegar factory scale (Fig. 4.5) (Gullo et ah, 2009). However, the actual basic technology used for cooked must fermentation is without any physical-chemical control therefore, fermentation breakdowns cannot be predicted. Ecological studies on AAB of TBV were first conducted by culture dependent methods. The oldest studies date back to 1970 and 1988 (Sacchetti, 1970 Turtura and Benfenati, 1988) and deal with the ecological aspects of AAB in TBV. 

Diagnostic tests for viruses, bacteria, fungi, and their spores are often based on culture-dependent methods. While these methods are very sensitive and generally simple to perform, they can be time consuming, requiring several days, and possibly... 

Wagner, M., Amann, R., Lemmer, H., and Schleifer, K.H. (1993). Probing activated sludge with oligonucleotide specific for proteobacteria inadequacy of culture-dependent methods for describing microbial community structure. Appl. Environ. Microbiol. 59 1520-1525. 

Due to the shortcomings of traditional, relatively inexpensive techniques like culture-dependent methods, other methodologies have been developed. These methods are based on molecular biology techniques and use the genetic material (DNA and RNA) of the bacteria. Referring to them collectively as genetic techniques , they have the capacity to [32] ... 

In comparison with culture-dependent procedures, culture-independent methods are more sensitive and have an increased potential to survey the diversity of antibiotic resistance genes in the environment. A weakness of these methods is the impossibility to elucidate about the bacteria in the community that host specific resistance determinants. On the other hand, the possibility to explore the genetic environment (mobile element, associated genes, promoter, etc.) in which the resistance determinant is integrated offers relevant clues about the gene transfer potential and gene acquisition history. 

Carraro, L., Maifreni, M., Bartolomeoli, 1., Martino, M. E., Novelli, E., Frigo, R, etal. (2011). Comparison of culture-dependent and -independent methods for bacterial community monitoring during Montasio cheese manufacturing. Research in Microbiology, 162, 231—239. 

Dolci, R, Barmaz, A., Zenato, S., Pramotton, R., Alessandria, V., Cocohn, L., et al. (2009). Maturing dynamics of surface microflora in Fontina PDO cheese studied by culture-dependent and -independent methods. Journal of Applied Microbiology, 106, 278-287. 

Rantsiou, K., Urso, R., lacumin, L., Cantoni, C., Cattaneo, R, Comi, G., et al. (2005). Culture-dependent and -independent methods to investigate the microbial ecology of Italian fermented sausages. Applied and Environmental Microbiology, 71,1977-1986. 

Temmerman, R., Huys, G., Swings, J. (2004). Identification of lactic acid bacteria culture-dependent and culture-independent methods. Trends in Food Science and Technology, 15, 348-359. 

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