LB broth very dark colour - (Dec/02/2010 )

I have just made up a bottle of LB broth, 7.5g yeast extract, 3.125g tryptone, 7.5g NaCl in 750ml water. But it has come out of the autoclave a very dark colour, much darker than I was expecting. It is more the dark brown colour of Terrific broth than the light straw colour of LB broth. Does anyone have any ideas why this would happen? and whether it would still be ok to grow bugs in for a maxiprep?

sounds like the media has been autoclaved for too long or left sitting at high temperature for too long (or autoclaved at too high a temperature - if your machine can go higher than 121C). The brownness is caused by millard reaction product form by sugars reacting to the amino acids in the media. Incidentally millard reaction product is what gives food its nice nutty flavour

What this means is that there is less than usual amount of sugar and amino acids in the media. And higher than usual concentration of millard reaction products in the media. Millard reaction products are growth inhibitory to most bacteria. However E. coli has some tolerates to millard reaction product.

Now the question is can the media still be used?

It depends... how brown is brown here? If it is on the side of dark brown going to black... the media can't be used. If the media is still golden in colour, it is okay. A picture would be helpful.

It's Maillard reaction. Anyway I wonder if there's any acrylamide developing in the LB, according to wikipedia, the formation starts at 120°C...

Did you reverse the quantity of yeast extract and tryptone or just typed wrongly? My recipe is 10g tryptone and 5g YE per litre LB.

donny on Fri Dec 3 08:47:08 2010 said:

Erm I think you might be right. oops. I have just stuck another bottle in the autoclave using the same recipe as well, assuming it was due to the bottle or someone autoclaved it twice or something.

Oh well that clears that up, I should copy down my recipes more carefully next time...

ok now it's getting weird, I just did it again using the CORRECT ingredients and it is still very dark.

can I just check I am getting this right?

10g Tryptone
5g Yeast Extract
10g NaCl
1L MilliQ water

mix, then autoclave with loose lid

because I seriously cannot work out what I am doing wrong anymore