Hello Hyun,

There can be a few factors that introduce variation in your results. According to the steps you described here are only some factors that I can think of.

In step 2.: You need to be able to tightly control the concentration of the inoculum for the 24 hour incubation. 1% is a relatively rough assesment of how many cells you are introducing into your fresh medium. A better way would be to determine and adjust the Optical Density (OD) of your overnight culture before adding a 1% inoculum to your fresh medium. Furthermore your growth medium needs to be tightly controled. Overcooking your medium may produce Maillard reaction products which may affect the growth rate of your bacteriocin producer and/or the residual antimicrobial activity in the medium at the end of the fermentation.

In step 3: Each time you start the "crude" purification of your Bacteriocin Like Inhibitory Substance (BLIS) by centrifugation and removal of cells you need to make sure that your culture has reached the same stage of growth. Undergrown culture may mean not enough production of bacteriocin, overgrown culture may mean cell lysis, release of proteases and degradation of your peptide BLIS.

Step 4.: The well diffusion itself is prone to variations. Important factors here are the concentration of agar in well diffusion plates, the time you let your plates to dry before addition of the BLIS solution in the wells, the pH of your supernatant and others. Not to mention of course the inoculum level in the agar (that of your target microorganism). For the target microorganism you need to follow the same precautions as in step 2 and 3 for your producer microorganism. In general the number (amount) of cells (target or BLIS producing) and the physiological state of the cells (young or old, stressed or not) may affect the final activity result. Last but not least you need to ask yourself whether other antimicrobial activity factors may be present in your supernatant. If you are talking about lactic acid bacteria, organic acids, diacetyle, hydrogen peroxide and many more factors may be present and vary your antimicrobial activity effect.

By the way it is just not "right" to refer to your antimicrobial activity as bacteriocin activity since I suppose you do not know yet whether this activity does come from a bacteriocin molecule or any other factor with antimicrobial activity.

I hope this helps.

All the best,

Panos

Dear Hyun

In first this is not bacteriocin , it is cell-free supernatant of bacteria.

Confirm the concentration of cell-free supernatant added to wells of agar.

Before add, please mix the cell-free supernatant.

Thank you. So I can't say bacteriocin activity through cell-free supernatant. Is this right?

Hello Hyun,

There can be a few factors that introduce variation in your results. According to the steps you described here are only some factors that I can think of.

In step 2.: You need to be able to tightly control the concentration of the inoculum for the 24 hour incubation. 1% is a relatively rough assesment of how many cells you are introducing into your fresh medium. A better way would be to determine and adjust the Optical Density (OD) of your overnight culture before adding a 1% inoculum to your fresh medium. Furthermore your growth medium needs to be tightly controled. Overcooking your medium may produce Maillard reaction products which may affect the growth rate of your bacteriocin producer and/or the residual antimicrobial activity in the medium at the end of the fermentation.

In step 3: Each time you start the "crude" purification of your Bacteriocin Like Inhibitory Substance (BLIS) by centrifugation and removal of cells you need to make sure that your culture has reached the same stage of growth. Undergrown culture may mean not enough production of bacteriocin, overgrown culture may mean cell lysis, release of proteases and degradation of your peptide BLIS.

Step 4.: The well diffusion itself is prone to variations. Important factors here are the concentration of agar in well diffusion plates, the time you let your plates to dry before addition of the BLIS solution in the wells, the pH of your supernatant and others. Not to mention of course the inoculum level in the agar (that of your target microorganism). For the target microorganism you need to follow the same precautions as in step 2 and 3 for your producer microorganism. In general the number (amount) of cells (target or BLIS producing) and the physiological state of the cells (young or old, stressed or not) may affect the final activity result. Last but not least you need to ask yourself whether other antimicrobial activity factors may be present in your supernatant. If you are talking about lactic acid bacteria, organic acids, diacetyle, hydrogen peroxide and many more factors may be present and vary your antimicrobial activity effect.

By the way it is just not "right" to refer to your antimicrobial activity as bacteriocin activity since I suppose you do not know yet whether this activity does come from a bacteriocin molecule or any other factor with antimicrobial activity.

I hope this helps.

All the best,

Panos

Hello,

Panagiotis Chanos did a nice enumeration of factors. One important thing, make sure that you are adding exactly the same volume of agar medium to your plates (also mix it well before pouring into the plates), otherwise the diffusion of whatever antimicrobial you have there is different, an important source of variation. One more thing, you should make several replications and average them to control the variations a little bit. I hope this helps.

Best,

Alex

Dear Hyun Woobin

You are using crude bacteriocin (cell free supernatant) to determine the activity against pathogens. 1 % inoculum should not be a problem, you need to add fixed amount of agar including soft agar and pathogen culture to petri plates for all three replicates. Make sure you add precise amount of cell free supernatant to agar wells.

High Regards

Muhammad Saleem

Really Thank you everyone.

Dear,

The crude cell free supernatant (CFS) may contains antimicrobial substances such as (1) bacteriocins (2) bacteriocin like inhibitory substances (BLIS) & (3) low molecular weight non-proteinaceous antimicrobial molecules. The activity obtained may be because of the presence of any one or more than one of these above mentioned antimicrobial molecules.

The difference in the inhibition zones in the plate may have the following reasons:

(i) The CFS amount added to the wells are not equal in all wells

(ii) The thickness of the medium in the plates is not uniform

(iii) The growth of the indicator microorganism in the plate is unequal

You must estimate the protein concentration in your crude CFS . An equal amount of CFS added to the well would give almost equal zone of inhibition.

The crude CFS may be treated with proteases; if CFS treated with protease gives any antimicrobial activity then it may be due to any nonproteinaceous substance or protease resistant bacteriocin / BLIS.

Regards,

Dr. A. K. Dubey