Another cryo problem

Richard heath at
Sun Sep 25 15:28:48 EST 1994

In article <4ACCAD2D17 at>, LYTTLE at BOTN.CANTERBURY.AC.NZ writes:
>      Dear all,
>      The comments over the last week or so about cryopreservation 
> of microorganisms have prompted me to put my own problem before the 
> net.
>      I am working with four Comamonadaceae able to degrade 
> in pure culture the nitrogen heterocyclic compound isoquinoline as 
> sole 
> carbon, nitrogen and energy source. Unfortunately they have a nasty 
> tendency to lose the phenotype, especially when grown 
> in a complex medium such as nutrient broth, LB medium and so forth. 
> My problem is that to restore the cultures from stock at -80 C the 
> organisms currently have to be grown in a complex medium or they won't 
> provide a large enough inoculum for study.
>       Does anyone know of a method of cryopreservation and 
> restoration that may get round this? At the moment I am using the 
> "Protect" bacterial preservers from Technical Service Consultants in 
> the UK which use beads and a cryopreservative fluid. Any suggestions 
> would be leapt upon with great glee and gusto!! Thanx in advance.
> Trevor C Lyttle
> Plant & Microbial Sciences Dept
> University of Canterbury
> Christchurch 
> New Zealand

I don't really know the answers to your problems, but here are my preliminary

Can you recover the cells onto a more complex minimal medium?  For example, M9
plus all required supplements plus case amino acids?  Maybe add the
isoquinoline and a small amount of glycerol...

Do you pull your freezes out onto rich medium, then select on defined plates? 
I always do this with _E. coli_ to ensure a defined starting culture.  Even
coli don't like to be plated straight onto minimal medium.  I then go from the
minimal plate to either rich or minimal 'overnight'cultures to be used as
inoculums for experimental cultures.  Do *all* your bugs lose their phenotype
during a single passage on rich medium???  Are the genes carried on episomes? 
I really don't know anything about Comamonadaceae, but this seems pretty

Best wishes,

Richard Heath, Ph.D.
St Jude Children's Research Hospital,
Department of Biochemistry
Memphis, TN 38101
heath at

P.S. What is "Protect"?  We store freezes (of _E. coli_) in 7% DMSO at -80 C.  
This is apparently the prefered method of storage (according to my boss, who 
claims Barbara Bachmann at the Coli Genetics Stock Center uses this method.  
Cells remain viable for _years_.  We have lots of freezes >15 years old, and 
they all recover fine).

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