e.coli-killing infection

Rafael Maldonado rafael at howard.genetics.utah.edu
Sat Nov 25 17:39:00 EST 1995


On 25 Nov 1995, Stacy Ferguson wrote:

> In article <49196f$ldp at mserv1.dl.ac.uk>,
> R Hanemaaijer <R.HANEMAAIJER at novell.pg.tno.nl> wrote:
> >Dear netters,
> >
> >Help !!
> >we have a rather big problem with our E.Coli (both XL-1blue MRF' and 
> >JM109). When we grow them, they behave normal untill they reach an 
> >OD550 of 0.4. From that moment on in 10 minutes time the OD550 drops 
> >to less than 0.1; the medium gets clear with undefined precipitates.
> >When we grow them further over-night the culture is full grown (OD550 
> >is more than 2.0), but we can not infect them anymore with helper-
> >phages.
> >Can the reason be a lytic-phage?, and, if so, does anybody know how 
> >can we get rid of them. (We grow M13-phages so we need a F-pilus on 
> >the cells) Any suggestions are welcome!!
> 
> That does sound rather frustrating. If it is, in fact, a phage contamination
> problem, you should be able to figure that out. You might want to ask a 
> neighboring lab to grow up some bugs (at least one of the strains you're
> having problems with) and give them a tiny bit of your unintended lysate
> to titer. If that's it, I suppose you should bleach down your lab and start
> all over with brand new bacterial stocks. Getting new stocks would seem to
> me to be a heck of a lot easier than trying to clean up your old ones.
> They are, after all, common strains.

That's a good idea. It looks like your stocks are contaminated with some 
phage. The easiest way of contamination in a lab is the pipetmans. Don't 
use them with phages, it is very dangerous. Those old fashion glass 
pipettes work fine; they are single use are it cross-contamination is more 
difficult. Instead, you can use those tips with cotton on the top. Or use 
disposable plastic pippetes.

You could save some strains cring them from phages. Growing then in 1mM 
EGTA (used to cure lysogens of P22), or citric acid (curing of T4) could 
save some of your strains. Check specific methods for those phages in
some methods book.

Save your plasmids extracting the DNA from those resistant mutants you 
get (those you cannot infect with helper phage); throw away all the 
media, tubes, etc. from the lab. Autoclave every autoclavable thing, and 
clean the inside and outside of the pipetmans with detergent. Get new 
fresh strain from other lab.

Remenber, phages are easily transported in aerosol particles, but they 
are easy to kill: any detergent will do.


Good luck,


	Rafa

___________________________________________________________________
                                             |
Rafael Maldonado                             |  La cita ha sido
room 6160 Eccles Institute of Human Genetics |  
Department of Human Genetics		     |  retirada por respeto
University of Utah			     |
Salt Lake City, Utah 84112. USA.	     |  a la propiedad 
Rafael.Maldonado at genetics.utah.edu           |
Rafael at howard.genetics.utah.edu	             |  intelectual.
Tel: 801-581-4429			     |
Fax: 801-585-3910			     |
					                  






More information about the Methods mailing list