This happened with me too. You can Try to grow it in selective liquid media first and then plated on selective agar

Article The pJan25 vector series: An enhancement of the Gateway-comp...

Hello Dr.

It can happen due to scarcity of utilizable Carbon source. In LB broth Carbon sources for E. coli are only catabolizable Amino acids, but the sugars in other media.

Kindly check the attached reference

Regards

jb.asm.org/content/189/23/8746.full

check your media material

it may be have some material that suppress the grow of bacteria

repeat thawing and re-freezing of the stock can reduce viability of the bacteria

@ PERKINS. Thank you very much your information. I used 3 strains of E.coli , O104, O157 and C600 together for several experiments ( 5 or 6)  from same glycerol stock  for each strain and same frequency of thawing and refreezing for each strain of glycerol stock but I found only problem for C600 but 0104  and O157 growing properly. Could you please tell me where is the problem?

In my answer I suppose your C600 strain is not carrying plasmids. The best way to get E. coli growing coming out of -80°C is (i) plate for single colonies on LB (or another general medium) and incubate. (ii) Pick a single colony and transfer that to a broth of choice (LB will do). The plating is often "forgotten" as it adds work and time, yet it is a very important step as it gives your bacteria the occasion for resuscitation and allows you to evaluate colony phenotype and speed of growth. You are probably aware of the following: E. coli is a facultative anaerobe, nevertheless it will grow much faster aerobically. So don't overfill your culture flasks (10 % of max flask volume is a rule of thumb) and don't close your culture flasks tightly or use cotton plugs. Many people grow E. coli directly in centrifuge tubes, but this is kind of horror for traditional microbiologists. 

In order to answer your question, let me ask you as to why do you suspect that this E.coli will not grow in LB broth?

Try a powerful medium under shaking conditions then inoculate on your LB medium.

@Ghosh, I would like to inform you that actually it is growing in LB but very slowly compared to previous time subculture  after overnight incubation although I inoculated one single colony  growing on fresh MacConkey agar in fresh 3 ml LB broth( same procedure that I did previously).

I would not start from MacConkey agar plates. They are selective for gram negatives, yet the bile salts and christal violet impose pressure on the cells. MacConkey (and other selective media) in traditional microbiology were developed for wildtype bacteria mostly in a clinical or food safety setting. The E. coli strains used in molecular biology are far from wild type and might get severely stressed by these media. Even when this is not the case, you are introducing a medium change in the protocol, which introduces need for adaptation and hence slows down growth. If not absolutely necessary stay in the same media or at the very least in very similar media.

Also starting in extremely rich media, like suggested by collegue EL-Sayed, I would not recommend. Even if it solves your problem (it might), it is only curing symptoms. E. coli C600 is supposed to grow well in LB. Like it was beforehand. Meaning something is wrong, you should solve the problem in this kind of work and not only cure the symptom.

@ Dardenne , Thank you very much for your information.

debes antes de sembrar tu conservado incubarlo de 20 a 30 min. a 37 grados y sembrar en medio sólido, incubar, aislar una colonia y realizar una segunda siembra para que reanimes el cultivo. Debes tener en cuenta el crioprotector , ya que el glicerol puede resultar en algunas concentraciones citotóxicas. No olvides pener presente el volumen de medio de cultivo, el pH, la aereación, y no menos importante la concentración del inóculo para disminuir la latencia. otra alternativa es adicionar el contenido del conservado en un caldo enrriquecido por unas horas y después inocular el medio, preferiblemente en erlenmeyer y en agitado.

Strain C600 carries  some markers, does require threonine, leucine and thiamine to grow on minimal medium + glucose.(Minimal medium: Davis &Mingioli or Vogel-Bonner).   . .Main markers of the C600 or any other basic E.coli strain genotype  should be rather regularly tested .

I couldn't agree with Freddy Dardenne more. It will work just fine. But if your C600 has a plasmid with recombinant protein and after following the steps, you still have the same slow growth result; its a case of plasmid instability or basal expression of toxic product (protein). So that should guide you on what next to do!

If the same frozen stock grew well a few months ago but not currently, then the first thing I would check is your LB medium. It may be that you have changed ingredients or someone has made the recent batch incorrectly. It might also be you just picked a bad colony, I would restreak freshly on a LB plate from the frozen stock and pick 2 or 3 different colonies to see if there is a difference. 

You might concurrently pick some other strain from your freezer and see if that grows similarly or different to the C600 stock to determine if it is the strain or some exogenous issue. 

A former colleague with a background in biophysics once told me that when he started out he tried to grow E. coli with medium made with ultrapure water. Of course it failed since trace elements are needed. I assume you are not using ultrapure water, but thought I would add this as a precautionary note.

i suggest you put streak plate and you preserve that in normal fridge temp or -4c . from that plate u can pick colony u can try that . otherwise be issuse with your incubator there may rise in temperature or low temp. for that u keep and external temp and check whether you incubator working showing correct reading or not .

@ Pravin, Thank you very much

As I understand it , your C600 was OK but has now stopped growing properly?  It  also seems that you have been following good practice by first streaking for single colonies and then using a single colony to inoculate your broth culture.  In that case ensure that you pick colonies from fresh plates and not old plates that have been stored for a while.    If those conditions are met then it seems your problem is either in the media, the culture vessels or the incubator temperature consistency.  Regarding media: It is important to use deionised water (source water may contain traces of elements that are toxic - especially Pb or Cu from piping). Secondly there are two types of LB recipe - low salt (5g NaCl pet litre) and high salt (10 g NaCl).  Try using the low salt recipe - some strains prefer it.  Regarding culture vessels - check that the glassware has been thoroughly washed and rinsed with deionised water - any trace of detergent will cause your culture to grow slowly. Ensure that your autoclave is working correctly - that  it is clean, calibrated and serviced.  Regarding incubator temperature: the optimum temperature for E coli is 37C. Good incubators will operate at 37 +/- 0.5C overnight when the door opening is much less. There should be a temperature monitoring protocol in place for such equipment - check with your department. Good luck!

Normally C600 will grow well from 30 to 42oC with good aeration in LB, low and high salt. I completely agree with former answers but may add this: we used to sometimes have problems with so called 'flying' phages. C600 being TonA is T1 resistant (T1 is the most aggressive among these phages) and F- and hence M13 resistant. But we had problems with T7... and there may be other phages as well. Getting rid of those is sometimes difficult: need purification through at least two rounds of single colony isolation on LBagar and possibly sterilization of the room you're working in with UV light. It seems unlikely someone in your lab or institute works with T7... but I thought it may be useful to remember this kind of trouble occurs! In any case going through isolated colonies and checking that the colony you recover has C600 genetic markers is the way to go. Good luck!

Thanks all who gave kindly answers of my questions.