Hi everyone,
I'm working on the isolation of possible bacteriocin-producing lactic acid bacteria from fermented food. After incubating my samples for 48 hours, I noticed a very large colony/colonies with clear zones surrounding it. My question is, is it considered clear zone? And do i need to incubate the sample for another 24 hours because (I think) the size of the clear zone is not big enough. I'm a bit worried, because this is my first time isolating lactic acid bacteria from fermented food. Thank you so much
In order to help you, more detail would be needed. What is the target bacterium you are using? Is the target bacterium incorporated in the agar or surface inoculated? What agar medium are you using?
When you see an inhibition zone, depending on how you performed your test, it may not be due to bacteriocin production. Lactic acid bacteria have other inhibition mechanisms towards other bacteria. You will need to perform further testing (thermal stability, degradation by certain proteolytic enzymes) to be sure that the inhibition you are seeing is indeed due to bacteriocin production.
When you see an inhibition zone, depending on how you performed your test, it may not be due to bacteriocin production. Lactic acid bacteria (LAB) have other inhibition mechanisms towards other bacteria. You will need to perform further testing (thermal stability, degradation by certain proteolytic enzymes) to be sure that the inhibition you are seeing is indeed due to bacteriocin production.
Another comment: is you are using a medium containing a target bacterium to isolate inhibitory LAB, you need to make sure that your colonies are indeed LAB. Usually, culture media for LAB are more elective that really selective, which means that you can get other bacteria growing, such as Bacillus (if using aerobic incubation). A simple confirmatory step I would do before embarking on deeper identification schemes would be to check whether your colonies harbour gram+, catalase-, oxidase- bacteria.
The inhibition zone you are observing is indeed small and it usually doesn't get larger upon prolonged incubation .
Good luck with your experiment! LAB are an exciting group of bacteria.
Hi Maria de Lurdes Enes Dapkevicius, thank you for answering my question! The medium I use is MRS agar supplemented with 1.0% calcium carbonate and 0.05% sodium azide.
I homogenized the food sample and 0.9% NaCl as diluent, diluted until 10^-7, and I spread 300 microliter of the sample on the medium. After it was dried, I laid MRS soft agar (0.6%) on top of the medium and incubated it for 48 hours.
Because I’m not sure what to do first, I decided to streak the colony on another MRS agar, hoping I’d obtain small single colony.
I will definitely do the gram+, and catalase-, oxidase- test after this. Thank you so much for your suggestions😀
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