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Autoclaving media twice - (Nov/16/2006 )

I tried to make some 2XYT and some terrific broth the other day, and the autoclave failed halfway through the cycle.

So I ran the cycle a second time, and this time it completed.

The media is notably darker than normal, from the heat or something?

Is it still OK to use, and if so, is it OK for every purpose - including making supercompetent cells?


I think u just burnt your media
Better prepare new one



it will be better to prepare again


Yeah, it would be better to make fresh media, but can we get more quantitative than that?

I mean, I don't want to throw the stuff out, and I've used media of varying shades before. I expect stuff will still grow in it, and I would appreciate some sort of feedback about either what is happening at the molecular level (is it the sugar, or the amino acids, that's "burning" or something else?) or else some experience one way or the other ("I tried making competent cells with dark media once and it worked fine" sort of thing.)


well i think eventhough it might work ,
if ur doing an experiment and going to re do it again or further use it its better to start from fresh since i think after autoclaving for more than 15 min the meida properties might have changed

so whatever grows on it, it will grow in different parameter than in a rightly prepared media

when one is doing an experimentin research we try to keep the parameters related to it constant so that when we recreate it , it should give the same result next time too

like the media ,reagents ,pH, distilled water quality ,temperature etc etc
and we change only the parameters we are using to study

we wouldnt exactly know whats the difference it will cause on the organism, atleast i am not aware of it ,, i dotn konw if anyone has done any studies on that

thisis my view point , of course 2xyt is autoclaved 20min , maybe u should check out media preparations and why the time limits and pressure ,temperature limits were used for autoclaving .


pH changes. Decreases becoming more acidic

Some more volume is lost, so osmotic pressure increases further. The volume lost is variable... though I believe it depends on how loose the cap was place on the bottle when it was autoclaved.

Sugar gets caramalised even more... if you conduct an sugar run, then the main cause by hyroxide ions catalysis. The more basic your solution is, the worse is the caramalisation.

Never played with any other buffer aside from plain old phosphate buffer so unable to comment here.

Some amino acid degredation. probably caused by the hydroxide ions.

But from the extremely limited aspect of growing E coli to amplify plasmids, there is only a slight drop is wet cell weight. So the stuff is okay.

Never tried to find out the effects on double autoclaved media on competent cell production.


Thank you!