Antibiotic is a secondary metabolite product and cannot be produced if you do not reach the trigger conditions. One of the conditions is filamentous or mycelium formation of cells in dispersed form that is always happening on agar medium but in liquid medium the cells typically creates pellet forms or if agitation too high the cells just broken down and in both cases antibiotic production is too low or even close to zero.

Dear Mr Sangma, Here In case of solid media or surface the metabolites produced by organisms are diffused in to the media and hence not readily available to the same or other organism as a food. while in case of liquid media it can be readily utilized and easily available to the organisms. Moreover in liquid media the pH of the medium can be drastically changed due to fast metabolism in compare with solid media, that may be the second reason. So, you can try liquid media with good buffering capacity to solve this problem.

Media components and pH are the main affecting factors on production of secondary metabolites..how did u check antibiotic production in liquid media?

There are two things to be tested. First in solid media, the relative concentration of active metabolite around the producing colony would be higher and as a result it will show zone, while in liquid media the antibiotic would be distributed in bulk and if you are directly testing the antimicrobial activity without concentrating it, you would not get the activity....I would suggest you to go for some sort of concentration, which may involve precipitation followed by solubilization, solvent extraction, lyophilization, etc...and then test for activity in case of liquid fermentation.

Another possible reason may be you may need to incubate for longer periods, to allow production of bioactive metabolites.....Because secondary metabolites would appear faster on solidified media rather than in liquid media.....

If you have any querry or need further assistance please feel free to contact me. my mail ID is [email protected]

I do agree with Keharia ... u need to concentrate the liquid medium before testing the antibiotic activity

Thanks for replies..

I have tested the broth by direct well diffusion method using 60ul of fermented broth (of 6 days, 10 days and 15 days). And i also tried solvent extraction by extracting with chloroform followed by concentration and assay by sterile paper disc diffusion method. But results were same. Regarding pH, The pH kept same as it was for the respective agar. And nutrients also same as in solid medium but without agar.

The antimicrobial agent you are looking for may not be soluble in chloroform...you need to use other solvents and if it is a polar compound then solvent extraction will not work......

Further, you need to identify the organism you have isolated....it will give us further leads........

thanks Hareshji.. I will concentrate the broth and do it again.. and try to identify the isolates..

Hi thnx Andre.. wl see to it

This Antimicrobial metabolites are not constitutive genes express always but only when needed. in agar plate they have to express due to the competences and cell signalling. In broth if you are fermenting it is not necessary to express antimicrobial compounds.

What exactly is the perpendicular streak method? Are there multiple strains in this assay, and if so, do you grow these two isolates in co-culture in liquid as well?

in the middle of sterile agar plate test organism will be streaked as a line, incubated for 3-5 days till it produce any ry antagonistic metabolites and the bacteria on which the antagonistic effect to be noted will be streaked perpendicular to the central streak. The susceptible bacteria will not grow in the diffusion zone around central streak.

No.. in broth the isolates grown singly and not as coculture. But co culture signal induction seems to uninstrumental as the agar piece from singly streaked isolate also had the activity.

Hi Pradeepa....this subject is so interesting for me...because I am working on taxol producing endophytic fungi...

my question is " are you sure about the production the antibiotics?" if not isn't possible to pursue presence of their gen using PCR amplification or separation and structure elucidation?

I mean at first you should be sure about them?

Good luck!

liquild fermentation sometimes got different result from the solid fermentation. some actinomycetes showed anti-microbial activity with solid fermentation but opposite with liquid fermentation. Meiji company in Japan studied on this, they isolated 6500 actinomycete strains from soil, 1300 showed antimicrobial activity with solid fermentation, but when these strains were fermented with liquid medium, only 1275 strains exhibited activity, 25 strains no. the reason maybe: 1. when fermented in liquid medium, the mycelium broken and affected the strain producing antibiotics ;2. when antibiotics produced in the liquid medium, another agents which can decompose the antibiotics produced at the same time, that is related to permeance ability in the liquid. 3. some certain element in the agar is necessary for the production of the antibiotics . in this case you can fermented with agar plate, after fermentation collect all the agar ,pound to pieces and extract them with solvents.

Antibiotic is a secondary metabolite product and cannot be produced if you do not reach the trigger conditions. One of the conditions is filamentous or mycelium formation of cells in dispersed form that is always happening on agar medium but in liquid medium the cells typically creates pellet forms or if agitation too high the cells just broken down and in both cases antibiotic production is too low or even close to zero.

I agree with Caixia Chen, I had the same problem with actinomycetes as antagonists to plant pathogenic bacteria, they showed great activity on the agar media against the tested bacteria when conducted by the perpendicular method but their broth culture filtrate did not exhibit any activity. I had to extract the agar culture with solvent. The solvent fractions when evaporated, their residues showed antibacterial activity against the tested bacteria. Then I separated the active ingredient with chromatography and identified it by spectral analysis.

Thank you Huda badr, actually i had faced the same problem regarding the Actinomycetes. I will follw your method. But it also shoud take in concern that always liquid fermentation could not be useful for Actinomycetes. We should use solid state fermentation optimize the production.

Dear pradeepa,

your working plan was good try to culture in a differ broth because some of the broth couldn't contain the nutrients which if favor for growing the bacteria, in the case you just try some other broth like luria broth and check again the activity test. i am sure their might be a activity don't let it down continue everything is possible., good luck.

some potential gene are not expressed in some kind of culture, thus different sencondary metabolites are produced. Different compounds determine different bioactivities. There are many scientific papers introduce this, and you can find some in the googlescolar