I looked at a variety of descriptions of the classic Luria-Delbruck fluctuation assay protocol for determining mutation rates in different organisms (e.g. 1, 2, 3) and noticed that they all start from a cryostock of an organism which is inoculated in medium and grown overnight. Next, the preculture is split into parallel cultures. After another round of incubation the cultures are plated on selective medium and the number of colonies counted. With this count together with the number of generations the mutation rate can be estimated.
However, I'm wondering, why not start with parallel cultures directly from the cryostock? It seems to me that the in between preculture might just introduce additional variability because mutants may arise during this period of time already. This appears especially relevant if the studied organism has a very high mutation rate.
Can someone explain why apparently having an additional preculture does not matter?