What's the best temperature for killing E.coli? (View forum version)



Thana

Posted 05 February 2009 - 07:01 AM

Hello,

I want dead E.coli for my experiment and I have used 65oC for 15 min following one of the article. The bacteria were not completely dead and I could see live bacteria under microscope and on LA (next day) when checking the viability (10-20% alive).

Therefore, I have increased the time (20-25 min) and temperature(70oC) but it made the bacteria clumped together which was impossible for me to determine the concentration by counting with Burkur chamber as I need certain concentration for my experiment.

Does anyone know the temperature used to kill E.coli and the bacteria does not clump together?

Thank you in advance for all suggestions,

little mouse

Posted 05 February 2009 - 07:08 AM

Hello,

I want dead E.coli for my experiment and I have used 65oC for 15 min following one of the article. The bacteria were not completely dead and I could see live bacteria under microscope and on LA (next day) when checking the viability (10-20% alive).

Therefore, I have increased the time (20-25 min) and temperature(70oC) but it made the bacteria clumped together which was impossible for me to determine the concentration by counting with Burkur chamber as I need certain concentration for my experiment.

Does anyone know the temperature used to kill E.coli and the bacteria does not clump together?

Thank you in advance for all suggestions,




I have no idea.
could the medium where you heat your bacteria play a role in clumping? Did they heat in LB or in PBS?

GeorgeWolff

Posted 05 February 2009 - 09:47 AM

what is the application?

cellcounter

Posted 05 February 2009 - 09:51 AM

I have no personal experience, but if you resuspend the cells well in water and heat them up, they should not clump.

Perhaps you should not let them settle down during your heat treatment. Just vortex every five minutes so.

basjhj

Posted 09 April 2009 - 06:56 AM

Hello,

I want dead E.coli for my experiment and I have used 65oC for 15 min following one of the article. The bacteria were not completely dead and I could see live bacteria under microscope and on LA (next day) when checking the viability (10-20% alive).

Therefore, I have increased the time (20-25 min) and temperature(70oC) but it made the bacteria clumped together which was impossible for me to determine the concentration by counting with Burkur chamber as I need certain concentration for my experiment.

Does anyone know the temperature used to kill E.coli and the bacteria does not clump together?

Thank you in advance for all suggestions,


80oC for 60 minutes should do the trick. I would culture them o/n, spin them down, wash them 3 times with PBS, heat-kill them and plate some to see if you get colonies. If you would like to add the heat-killed bacteria to cell culture medium containing monocytes/macrophages/DC, first wash & resuspend them in that particular medium.

Kind regards,
Bas