I don't agree with people thinking it is not a problem if the culture needs 5-6 hours to reach the exponential phase (OD = 0.6). In particular, if you need to inoculate successive cultures in the same day (this is the case of NMR spectroscopists when preparing labeled proteins), the working day would be very long...

I've often encountered this problem with some specific E. coli strains. Unfortunately, optimization of the parameters cited above (type of rich medium, oxygenation, pH...) did not speed-up the growth. However, I figured out that the problem was coming from the pre-culture (either inoculated with a colony or a glycerol stock). Starting from an overnight pre-culture in a stationary phase (OD > 2.0) sometimes leads to a very slow growth of the culture (a significant part of the pre-culture may have lost the plasmid during the night due to antibiotic degradation by the bacteria). So when I have this problem, I avoid overnight pre-cultures and use the following protocol:

- Day 1: inoculate a 10-40 mL LB pre-culture with a colony or glycerol stock. Once OD is 0.7-0.8, stop the growth and put your flask in a fridge or a cold room for the night.

- Day 2 : inoculate the LB culture with your 0.7-0.8 OD pre-culture (OD may have increased a little bit).

Using this protocol, it never took more than 2 hours for the culture to go from 0.1 to 0.6 in LB medium. I hope it will also works for you.

Good luck!

Dear Mr. Wani,

Please check pH of your media what ever you are using.........

good luck............

You may increase the amount of bacteria culture you use for inoculation.you must inoculate about 5-10% of the media volume you use for bacteria culture.

Hi Tasaduq,

I have noticed this as well starting from a colony, and I would be interested to hear the answer. It may just be inoculation volume, but from the literature I have seen it doesn't seem to be the whole story. pH could be it as well, but you generally don't see pH changes in fermentation until after log growth has started depleting the oxygen. The traditional media for bacterial growth are not buffered very well, but upon dissolution usually are in the 6.5-7.5 pH range, because the components are generally extracted at physiologic conditions. I am wondering if coming from frozen culture to plating may have influence? Any information you find please share...

Proper temperature and shaking is also must.

There are many reasons for the OD to not reach 0.6 quickly. pH could be one as indicated by Braj. Your transformants may have got sick after you introduced your constructs. Some plasmids make the cells sick and they undergo a lot of stress. If you think about it, you are taking a piece of foreign DNA and forcefully entering it into the cells. It can create a lot of stress. I would be more happy about the good quality of protein that you are getting because that is what matters at the end. Also, rather than inoculating a fixed volume in the secondary inoculum, I would shoot for a fixed OD. Let's say 0.1 as a starting OD. Then take readings every hour. That will give you a better idea about the doubling time of your culture. I am giving an example for calculating how much volume you need to get an OD of 0.1: Lets say your overnight culture is at an OD of 2. You want to get a starting OD of 0.1 in 250 ml fresh medium. 0.1 (O.D.) x 250 (ml) = 25 (O.D.) (ml). Now your overnight culture's OD is 2. To get a starting OD of 0.1, 2 (OD) / 25 (OD) (ml) = 0.08ml. This makes me think that your overnight culture's OD must be really low because if it were around 2 then inoculating 10 ml itself would get the OD higher than 0.6. If that is the case (Overnight OD being low) then that gives you a clue that your cells are really sick and thus are growing slow

Several variables affect the growth rate of your culture. Are you growing in rich media or minimal media? What temperature are you growing at?Have you considered doing a growth curve? The proper way to determine how fast your cells grow is to innoculate with a starter culture, and check the OD600 at different time points for at least 12 hours. You then plot the OD600 versus time. Typically 1h is sufficient time to get to OD 0.6 if you're starting from approximately OD 0.3 and in rich media at 37degrees. But it will take longer if your starting OD is 0.01 or if you're using minimal or SeMet media or at other temperatures! So do a growth curve and play with the variables and optimize your expression of your protein properly.

The OD that you mentioned is corresponds to deep exponential phase or Early standard phase.Its Quite common the exponential phase takes 5 to 6 hrs.

It dose not correlates any mistake, the over night seed culture initial inoculation also matters here.I am not aware of this literature, but the growth curves explains clearly above explanation was correct.So nothing bad or new.

The LB broth, optimum temperature and rpm is enough for getting your OD, beyond that pH also important.

No bacterial culture can give the exponential OD in 2 or 3 hrs, unless you add more inoculumn as a seed (Relatively SAY NO)

there are numerous variable

pH of media

Temperature

media components

Age of the seed

Age of the inncoulum

Age of the colonies

Antibiotic concentration

Agitation and aeration in shake flasks

percent of positive recombinant cells in your innoculum

viability of your innoculum

if the delay affects your protein yield, I will start fresh of everything and try again

Good Luck

All the answers are very interesting, the thing is that I do not see what is wrong about cells taking 4 or 5 hours to gain an OD of .6. Actually for my is kind of normal to let the cells grow 4 hours until induction, maybe it might be because of the strain that I use. Anyways, as I red among the answers, what you should be more concerned about is the quality of your recombinant. Just an opinion.

I don't agree with people thinking it is not a problem if the culture needs 5-6 hours to reach the exponential phase (OD = 0.6). In particular, if you need to inoculate successive cultures in the same day (this is the case of NMR spectroscopists when preparing labeled proteins), the working day would be very long...

I've often encountered this problem with some specific E. coli strains. Unfortunately, optimization of the parameters cited above (type of rich medium, oxygenation, pH...) did not speed-up the growth. However, I figured out that the problem was coming from the pre-culture (either inoculated with a colony or a glycerol stock). Starting from an overnight pre-culture in a stationary phase (OD > 2.0) sometimes leads to a very slow growth of the culture (a significant part of the pre-culture may have lost the plasmid during the night due to antibiotic degradation by the bacteria). So when I have this problem, I avoid overnight pre-cultures and use the following protocol:

- Day 1: inoculate a 10-40 mL LB pre-culture with a colony or glycerol stock. Once OD is 0.7-0.8, stop the growth and put your flask in a fridge or a cold room for the night.

- Day 2 : inoculate the LB culture with your 0.7-0.8 OD pre-culture (OD may have increased a little bit).

Using this protocol, it never took more than 2 hours for the culture to go from 0.1 to 0.6 in LB medium. I hope it will also works for you.

Good luck!

Ludovic idea is perfect to stop growth before they will reach lag phase. Mine trick is simpler, since I’m more lazy;) I just inoculate them to few flasks with different amount, and leave without shaking on the bench ON. In the morning I’m choosing the one I like (my eye likes, since I’m to lazy to use spectrophotometer). I dilute the culture of OD 0.2-0.6 by factor 5, since these little creatures, when are poisoned by the protein feel better in a company:)

Thanks all for your valuable comments. I will surely try to find out what is it that makes my cells go sick.

It would be nice if you let the followers of this question informed about the solution you found to fix the problem.

Thanks in advance!

You had better to check if there is bacteriophage contamination. I had met the similar situtation, too.

Use fresh grown culture for inoculation, don't use stored culture or glycerol stock directally

Sure, Ludovic Carlier I would soon update on the issue.

its a common problem we encounter. If you think this problem not from molecular biologist but as a microbiologist, it makes sense. First for protein expression, you might be using minimal media so basically, you are giving shock to cells plus burden of antibiotics. so, the the cell use maximum energy for sustenance but not for growth. if you change the media to Luria Bertanni broth, which u will find early growth. it has no correlation with how much seed culture u take basic bacterial growth physiology.

Hi Tasaduq, check the salt concentration in your medium. In my experience, if you are using LB broth, adding Nacl up to 10% and 1 mM CaCl2 after autoclaving fixes the problem of poor growth. You can also try swapping to a richer media.

Your insert might be toxic to E.coil which could explain lower growth rates. The Lac operon is know to leak and to give low but significant expression levels.

With some inserts you would expect delayed lag phase and also enrichment of random mutations with less toxicity to E.coli.

Check your construct for spontaneous mutations if you suspect toxicity!

Also, try pLys strains that are more efficient in repressing the Lac operon or simply add ~2% glucose to your LB.

hdh

S.

I'll suggest a simple thing, have you tried with a fresh stock of competent bacteria? I had that problem before and when I switched to a new fresh stock of competent bacteria the problem was solved

just use prewarmed medium

Can anyone tell me the optimum rpm for the culture?

thanks

Hey.

Optimum rpm for bacterial culture if it is to be used for plasmid or DNA isolation is 3000-4500 rpm. But I have seen people going beyond this also without having any effect on post isolation procedure. However high rpm makes pellet too tight and then resuspension takes an effort. So spin down as long as you get pellet tight enough not to be lost while decanting the residual media.

Hope the answer of your question.

hey tasaduq,

i am encountering the similar problem like yours...but in my case i donot get enough cells after centrifugation for resuspending.. do let me know how did you solved the problem.I will try Ludovic method too

@Anam Nayab....

I hope Ludovic C. method worked for you. Reasons for not getting enough cells for pellet include:

1. Less growth of bacteria itself.

2. May be your protein is toxic to cells and won't let them grow.

3. Antibiotic selection is too tight or stringent.

Try using healthy inoculum for secondary culture.

Hiii Tasaduq,

I am also facing the EXACTLY SAME problem... I tried many things but still situation is same.

In my case, I use colony as well as glycerol stock to inoculate the seeding culture (overnight). Then next day morning, I seed the fresh 10 mL LB media at 0.2 seeding OD. But then it takes ~5 hrs to reach 1 OD, which should be ideally ~2 hrs.

Just let me know if you find any solution for that...

OR anyone else if you find any solution kindly let me know.

Thank you.

-Hitarth