Since you mention that you intend to freeze this down for storage and as basis, presumably, for future experiments, I would recommend discarding it and starting over. Differentiation isn't so much the issue as the fact that the population would have been subjected to enough stress (and a founder effect from being diluted) that there will likely be enough heterogeneity from genetic drift alone for it to be a reproducible stock. 

This would generally be true of almost any microbiological stock. 

On Sep 21, 2012, at 1:42 PM, John B. Biggins wrote:

> Hello, I have a question for all.  I am relatively new to using Dicty.
> 
> I had obtained Dicty from the Dictybase stocks & properly grew them up as directed.  
> 
> Due to some unforeseen instances, I had to be away from lab for a few days and as a result, my axenic cultures (in HL5 medium) have become confluent.  I am growing them to both store permanently and to do a bacterial co-culture from log-phase growth
> 
> I know that at some point they start to differentiate at a certain density.  Is it possible to cut these cells into a new fresh diluted media for a new culture for me to use or if once they differentiate, they are no longer useful.
> 
> Thank you
> 
> ~john