I have a bacteriocin producing lactic acid bacteria... How can I perform confirmatory test for it? Meaning how can I check for clear zone of growth inhibition in a petriplate. Thanks
You need to isolate a cell-free supernatant and perform agar-well diffusion assay with the indicator strain that you want to be inhibited. Bacteriocins are generally proteins. You have to be careful that it's not the organic acid that's killing the pathogen indicator strain.
You need to adjust pH to almost neutral and treat it with proteolytic enzymes.
You could try using an absorbent - like a filter paper with a known diameter. If you know the strength of the liquid, and how much the paper as absorbed you will know the dose. Place this on a agar surface already seeded with the sensitive organism.
It will be very similar to those generated by an antibiotic test used in hospitals to test for microbial susceptibility to antibiotics.
You need to isolate a cell-free supernatant and perform agar-well diffusion assay with the indicator strain that you want to be inhibited. Bacteriocins are generally proteins. You have to be careful that it's not the organic acid that's killing the pathogen indicator strain.
You need to adjust pH to almost neutral and treat it with proteolytic enzymes.
I agree with Dr. Edward.
There are a few papers with this kind of tests. For example, this is the classical one: http://aem.asm.org/content/21/5/943.full.pdf
Here you have a article in which you can read a comparison of some methods: A comparison of methods for the measurement of bacteriocin activity. J Microbiol Methods.
In order to test the nature of the substance you must do some assays with proteolytic and lipidic enzymes.You can read an example in this paper: Physical chemical and biological characterization of a new bacteriocin produced by Bacillus cereus NS02
Good luck!!
Hi Sreeram: as Edward pointed out, it is very important that you include a control of the non-inoculated broth that you used for culturing the LAB, WITH THE pH ADJUSTED TO THE FINAL pH THAT YOU FIND IN YOUR INOCULATED CULTURE. This will let you know if the inhibition is caused by the production of organic acids or by any other substance, like a bacteriocin.
It is also important that you check the proteinaceous nature of the inhibition, so another alliquot should be treated with protease and then checked again whether the inhibitory activity remains or not. A good result would be that you lose the inhibitory action.
And do not forget to check that the inhibitory activity is not caused by hydrogen peroxide productions. So do not forget to treat another alliquot with catalase and check that the inhibitory activity is not affected.
Best regards
How can I check the bacteriocin activity in native PAGE ? I have spred plated with pathogen and kept the gel on it, to get a clear zone of growth inhibition in a petriplate. But I am not getting any zone. the CFS shows good activity as well, before subjected to native PAGE.
please help
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