How to kill groving cells (nystatin summary)

Tomas Drgon tomasd at bdg10.niddk.nih.gov
Wed Aug 30 17:25:16 EST 1995


Hi folx.

This is a summary (incomplete and not well quoted) of responses to the
selective-killing-of-growing-cells question by Lubo Tomaska.

Tomas Drgon
--------------------------------------------------------------------------
An alternative method involves the enrichment for nongrowing cells due to
their resistance to heat killing.  This comes from a paper from the Pringle
lab (MGG 171:111-114).

Charlie Hoffman

At the Lisbon yeast meeting last month, there was a poster that carried an
interesting variation on the standard methods in use for this problem.
Unfortunately, I did not note down which poster at the time, and I now
cannot remember, or trace it in the abstract book! So if the authors read
this, or anyone else who remembers the poster (or uses a similar method)
does, I would be grateful if you could follow this up with details of the
method.

The method was based on the use of ethidium bromide in media which does
not support growth of the required mutant type. Ethidium bromide converts
close to 100% of growing cells into petites. So growing cells are not
killed, but can subsequently be selected against by placing the culture in
non fermentable media (eg glycerol-ethanol based). The petites will not
grow, so only those cells which hadn't grown in the previous media will
now grow.

In practice, the fact that the non-required cells are not actually killed
should be an advantage. The main problem with penicillin/nystatin type
methods is that the killed cells leak nutrients into the media which can
enable previously non-growing mutants to commence growth and hence
themselves be killed. Converting cells to petites should not have this side
effect.

Of course, this is not to say it won't have other problems. How close to
100% conversion do you get? Will the non-growing cells really not be
affected etc. But it sounds good and seems well worth a try. So if the
authors could please get into contact?

About 20 years ago I spent a little time unsuccessfully trying to use the
nystatin method. This followed a period when I had made much use of
ethidium bromide to generate petites! So, I keep asking myself, why didn't
I think of that.....?
Elliot Gingold
South Bank University
---------------------------------------------------------------
An EthBr method is described in McCusker and Haber, Genetics 119: 303-315
1988. They say the method was modified from one provided by D. Perlman.
They claimed they could get 50% of colonies as auxotrophic mutants.

Preston Garrison
garrisonp at uthscsa.edu
Biochem. Dept.
Univ of Texas Health Sci Ctr
San Antonio, Tx 78284-7760 USA
210-567-3702

------------------------------------------------------------------
Alternatively, you can use the method of Susan Henry and Mike Culbertson
(order does not imply anything), starting with inositol auxotrophs....if
deprived of inositol, any growing mutants die (likely from membrane
failure)...mutants whose growth is arrested survive to be recovered on
inositol plus the permissive condition for your mutation.

Refs:
M. R. Culbertson and S. A. Henry,  Genetics 80:23-40  (may 1975)

S. A. Henry, T. F. Donahue and M. R. Culbertson, Mol. Gen. Gen. 143:5-11
(1975)

in short this method is like penicillin enrichment for E. coli mutants.

hope this helps!!

-Micky

-------------------------------------------------------------
I have used the method of Snow as described in:
 
Snow,R. Nature 211:206-207 (1966)
An enrichment method for auxotrophic yeast mutants using the antibiotic
nystatin.

I have had success isolating many mutants based on nystatins ability
to selectively kill non-growing cells.

                -Al(aplummer at sprint.uccs.edu)
---------------------------------------------------------------

Here's yet another method using the cell wall inhibitor, Echino-
candin B.  We haven't used it in our lab for quite a while, but
here's the reference anyway:

Mol Gen. Genet. (1982) 186:295-297.


        Man, this sounds an awful lot like that Parks Lab gang to
        me.....probably drinking homebrew in the back labs, morning noon and
        night.........Oh, by the way, Inositol-less death also seems
        to work with ino-  auxotrophs.
           <Tod---missing the homebrews in the solvent lab>
----------------------------------------------------------

You can use the drug Nystatin.

See:    Nature 211, p. 206 (1966)
        J. Bact. 95, p. 197 (1968)

No, there is no mistake about the years in those refs.

Good luck,

John

--
=========================================================
John Bratty                     bratty at bch.umontreal.ca
Departement de biochimie        
----------------------------------------------------------------
   

Hi yeasteis, and others. Just a note concerning using Nystain as a method
of selective killing of growing as opposed to non-growing yeast cells.
Most, if not all, Nystatin resistant mutants are sterol mutants and they
are easily obtained. Point: t is easy to inadvertently obtain Nystsatin
resistant cells.

---------------------------------------------------------------



end

-- 
I speak for nobody but myself and my pet frog.



More information about the Yeast mailing list