We are screening antimicrobial activity of endophytic bacteria.Some of it have shown antimicrobial activity against Staphylococcus aureus by agar overlay assay. We also tried from supernatant but no activity found. what can be the reason.?....if it is due to intracellular metabolite activity.. how to determine it?
In general, microbes produce extracellular products to compete against others. These will have a secretary signal. As in my case when i clone a antimicrobial gene into pET28vector, it dsnt hv dat signal so i have to lyse the cell to get my protein though the wild type protein is secretary.
So,U cn simply pellet down your cells n isolate the compounds in your media and simultaneously lyse the cells(be sure that u use different type of solvents to get proteins as well as chemical metabolies). It would hv been easy for you if you had a multivapour where u cn evaporte 6/8 solvents at d same time. For both extracellular componds and intracellular lysate u cn conduct the same xperiments as u did n u will know jst by positive results if it is intra/extra. I hv d composion of lysis buffer if u need.
How are you sure that is an antimicrobial (bacteriocine, antibiotic, or else) and not that that bacteria grows better and their metabolism product change the culture condition in their favor (like changing pH)? Lots of acid lactic bacteria do that and they displace other bacteria in the culture.
Many of the antimicrobial compounds are extracellular. But if you have doubt, then you can sonicate the bacterial culture in cell lysis buffer and after centrifugation check the activity.
Dear Badrul Haidar,
I think You can do one thing to determine the extracellular and intracellular antimicrobial compounds. For extracellular antimicrobial compounds you can use the supernatent for antimicrobial assay and for intracellular compounds you can sonicate your bacterial culture for cell lysis and then centrufuge it. After centrifugation collect the supernatent and check the antibacterial activity.
If you are seeing activity from an agar overlay assay it is most likely an extracellular compound, which are most common as several of the other answers have said. But also be careful in your terminology, if the compound is excreted it is extracellular, if is 'intra'cellular it could not affect other organisms unless they phagocytize the cells, but it could be some compound that is put into the membrane that could affect other bacteria when in contact. Also, if you simply sonicate and lyse a bacterial culture you then have both intra- and extra- cellular chemicals in the assay and cannot tell which is the cause (separate the bacteria first, then sonicate/lyse and test that material).
use filtered sterile supernatant of the bacterial culture will tell if is extracellular.
Check and adjust the pH to eliminate the pH effect.
Simply, you can grow the bacterium on liquid medium then separate the culture supernatant (CS) from the biomass by centrifugation. CS can be used as a source for the extracelluar activity. On the other hand, the biomass can be sonicated to obtain the cell-free extract (CFE) that can be used as a source for the intracellular activity. Do not forget the control experiments using sterilized distilled water with pH values adjusted at the pH values of either CS or the CFE.
Dr. Ghareib is correct on finding out whether a chemical is intracellular or extracellular, but remember that if it is found intracellularly it will not be able to affect other organisms (unless they eat the bacteria) unless it is released and becomes extracellular.
Take the cell free culture supernatant (membrane filtered) and check its antimicrobial activity against same bacteria.
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