## 3 Methods of Isolation and Identification of Genus _Candida_  
In general, the identification and enumeration of microorganisms present in wine involve enrichment techniques [14,15]. These methods are considered indirect, because they do not reflect the number of original cells in the sample, but they are also considered to be progeny, because they are enriched in a selective growth media for cultivating yeast and bacteria from wine. The characterization of _Candida_ at the species level is laborious, since they are widely disseminated, highly variable, change their physiology with varying conditions, and normally are associated with other yeasts,bacteria, and molds. Nonselective media most commonly used for yeast separation, cultivation, and enumeration are composed of glucose such as carbon source, and these media may be employed at the beginning. Examples include dextrose agar (pH 6.9), dextrose broth (pH 7.2), Sabouraud medium, dextrose tryptone agar, rice agar, malt extract medium, or plate count agar. The use of lactic, tartaric, or citric acid (10%, final pH 3.5) for acidification of the media, as well as the incorporation of antibiotics (up to 100 mg/L), such as cycloheximide, streptomycin, chloramphenicol, and gentamycin, enhances their selectivity in order to inhibit the development of acid lactic bacteria and other yeasts. Biphenyl, propionic acid, and dichloran control overgrowth of filamentous fungi. The culture temperatures are also an important factor; those between 25 \({}^{\circ}\)C and 30-32 \({}^{\circ}\)C should be chosen. Incubation times are fixed in the range of 3-5 days and must be increased for osmotolerant and osmophilic yeasts to 5-10 days and 14-28 days, respectively. Many specific commercial media are available for isolating and enumerating the genus _Candida_ in different food products, including the brewing industry and wine industry [12]. Although selecting wine yeast strains have been addressed for decades, the unequivocal characterization has been possible with the knowledge of molecular techniques. [PERSON] et al. [16] applied the PCR amplification and restriction pattern analysis of the ITS1-5.SS-ITS2 regions of the nuclear ribosomal gene complex for species characterization of isolated yeasts based in the techniques developed by different authors [17; 18; 19]. Most PCR-DGGE studies have been employed to discriminate both yeasts and bacteria in wine. [PERSON] et al. [20] were the first to apply PCR-DGGE method in wine fermentation, developing primers for the D1/D2 domain of the large-subunit rDNA amplification of the yeast species. That work demonstrated that the population shifts of different wine-related yeasts could be easily followed using PCR-DGGE [21]. This study also confirmed the persistence of _Candida_ sp. throughout wine fermentation, detecting populations until 104 days later. Supplementary studies on commercial sweet wine fermentation showed that non-_Saccharomyces_ yeasts could be found in late stages of the fermentation process by PCR-DGGE and even a long time after could be cultured on specific media [7]. This fact was particularly evident for the _Candida_ sp. population, _C. zemplinina_[11]. DGGE signatures from both RNA and DNA templates directly extracted from wine revealed _C. zemplinina_ signatures remained throughout the fermentation, even when direct plating manifested clearly a relative low number of cells. Applying RNA dot blot analysis with _C. zemplinina_-specific probes shows that the size of that population could be relatively high (>10\({}^{6}\) cells per mL) at the end of the fermentation, while only 100-1000 CFU per mL could be detected by plating. These results provided some of the first evidence of the presence of metabolically active but nonculturable yeasts in wine fermentation. Endpoint PCR assays have been developed and applied for several wine yeast and bacteria. [PERSON] et al. [22] used a multiplex PCR approach amplifying different segments of the yeast _S. cerevisiae_ COX1 gene to enumerate different starter strains. [PERSON] et al.