20 replies to this topic

[Sam Wangner]

We need a lot od E.coli for our experiment. But we have problem to grow them more in yeast extract based media. How/what to do to increase the OD and so more E.coli?

[Satiekumar]

Try with Tryptic soy medium.

[TRUONGTHANG]

Quote

Quote: from Sam Wangner on 3:06 pm on Feb. 14, 2001
We need a lot od E.coli for our experiment. But we have problem to grow them more in yeast extract based media. How/what to do to increase the OD and so more E.coli?

[franck]

there is a lot of medium, SOB or LB medium are good.

[nagesh]

sob, lb medium are very good,
importance should be given to maintain pure culture,
proper autoclaving of media, the amount of inoculm
and finally the temperature should be 37 C, with continous shaking

e.coli grows enjoy any media!!!

[Vageesh]

Try Terrific broth. E. coli grows abundantly and happily! Or to your medium, just add 1% sterile glycerol (just add exactly, glycerol is very thick and denies to enter your pippette tip smoothly!). SOB is also very good.

Aeration is very, very important; so also, the speed of shaking. Your container should be a minimum of 5 times bigger (for eg., for every 1 litre of medium, do it in 5 litre flask) and set the speed of shaker to 300 rpm. Then you will thank me for the golden harvest of E. coli!!

[jadefalcon]

Terrific Broth should give you very high ODs of E.coli.
Culture Volume / Vessel Volume should be 1 / 5 as indicated above.

If you still need more bacteria, you may want to get the help of companies or some microbiological institutes have large fermenters, where you can grow 50, 100 or 150L bacterial culture inside.

mike

[amelie]

Hi,
try super broth (same as terrific broth??).
For 1 L:
32 g tryptone
20g yeast extract
5 g NaCl
1,25 ml NaOH 4N

good luck,
a

[jadefalcon]

amelie, on Aug 17 2004, 07:42 AM, said:

Hi,
try super broth (same as terrific broth??).
For 1 L:
32 g tryptone
20g yeast extract
5 g NaCl
1,25 ml NaOH 4N

good luck,
a

terrific broth:

16g/L tryptone
10g/L yeast extract
5g/L NaCl
9,4g/L K2HPO4
2,2g/L KH2PO4

pH=7

so, your super broth seems to be a doubled terrific broth!
(Somekind of "Supersize Me" for bacteria? )

mike

[amelie]

yes it is,
in fact, the time to reach an OD of 0,6 is twice faster than in LB for my strain of E. coli!

[GeorgeWolff]

Doubt your need is a better medium - just increase incubation time and that will bring you to same cfu/ml.

If you want more cell mass - increase culture volume.

[pDNA]

What you need is buffered medium ...that's it! If you want more cfu/ml than you'll have to elongate the time cells are growing exponentially and prevent them from entering stationary phase. Use buffered media like Terrific broth or PDM (see Sciencdirect Link) ...if you want to reach high OD-values.

Another thing is to use Kanamycin instead of Ampicillin of example ...because the beta-lactamase gets excreted and degrades the Ampicillin rapidly ...therefore you won't be able to maintaine a productive culture for a very long time (e.g. longer than over night cultivation)!

Regards,
              pDNA

[biolabwork]

I want to grow Ecoli in M63 Minimal media . I have never seen any growth with only M63 so added T and U. Later I am able to see a very low growth rate .  I want to change the carbon source  to glucose.

Can some one tell me the concentrations I should follow to see a reasonable growth?

[Puteri Q]

I also have the same problem. My research goal is to know when exponential phase of my E.coli is occur. Because, I use non standard media to grow E.coli. It has low amount of protein or Nitrogen source, therefore I added 0,5% yeast extract into it (ust like in Luria Bertani) to optimize the E.coli growth.
But, the growth curve always shows different pattern if I do it in different time. First time I did it, OD600=0.6 could be reached in 4 H. But at other times, E.coli only reached OD600=0,3 in 4 H. It is half of the previous result.
I do really confuse with this.
Is there anyone who could explain why bacterial growth rate in the same media could change?

Advice please....

[pito]

Puteri Q, on 02 September 2010 - 10:41 PM, said:

I also have the same problem. My research goal is to know when exponential phase of my E.coli is occur. Because, I use non standard media to grow E.coli. It has low amount of protein or Nitrogen source, therefore I added 0,5% yeast extract into it (ust like in Luria Bertani) to optimize the E.coli growth.
But, the growth curve always shows different pattern if I do it in different time. First time I did it, OD600=0.6 could be reached in 4 H. But at other times, E.coli only reached OD600=0,3 in 4 H. It is half of the previous result.
I do really confuse with this.
Is there anyone who could explain why bacterial growth rate in the same media could change?

Advice please....

I have had this before too.. its just something that happens. There are many factors that can influence your growthrate: oxigenrate, how you shake the bottles, the temperature (do they open the incubator often or not, your starterculture...