Traditional methods for differentiation of production strains and variants
Ale and lager yeast strains can be readily differentiated based on key physiological characteristics. For example, ale strains are able to grow on solid media and form colonies at 37°C while lager strains are not. In addition, the ability oflager strains to utilize meli- biose can be exploited using X-a-Gal based medium. This medium determines the capacity of yeast to cleave the melibiose homologue X-a-gal (5-bromo- 4-chloro-3-indolyl-a-D-galactopyranoside), resulting in the development of a blue/green coloration derived from indol. While this is traditionally conducted using solid media, more rapid results can be obtained using a revised protocol where cells are suspended in liquid media and results obtained within one hour (Box et al., 2012). Differentiation of production strains of the same type is more challenging, particularly if the strains in question are both S. pastorianus. Most ale strains show differences in giant colony morphology when grown for an extended period of time on WLN or gelatin-based media (Morris and Hough, 1958). For differentiation of lager strains, a number of methods have been used with varying degrees of success. Ultimately, without undertaking molecular analyses, often the best means of determining strain identity is to conduct some form of test to examine known fermentation characteristics, such as flocculation capacity or sugar utilization patterns.
Variants create similar problems and typically necessitate precise phenotypic analyses, as is the case for cultures that display enhanced or reduced flocculation patterns. In this instance, flocculation tests such as the modified Helm assay (Bendiak et al., 1996) can be applied. Respiratory-deficient petite mutants (see Chapter 2) can be detected by growth on glycerol-based media since petites are unable to grow only on media containing only nonfermentable carbon sources. Alternatively, these can also be identified using the TTC (triphenyl tetrazolium chloride) overlay technique. In this method, the colourless TTC stain is poured over colonies present on the surface of an agar plate. The TTC dye is reduced by respiratory-sufficient yeasts to form a pinkish red colour. Respiratory-deficient yeast colonies cannot reduce the dye and remain white (Ogur et al., 1957).