Help: Antibiotic sceening problem

[MarySanabawy]

I am a graduate student who is interested in identifying new antibitoics isolated from strain in marine sediments.
I collected samples from marine sediments from the red sea coast and grown them on marine agar plates. I found some colonies formed zone of inhibition around themselves as they killed the other bacteria diffusing around it, I isolated these colonies and grew them again on marine agar. However, when I add a top layer of soft agar with the test strain, they don't form zone of inhibition or any trace of it although the original plate shows that and its very clear. I repeated this several time.
I thing I noticed in the soft agar is the test strain grown on the top of it, with a loop i was able to remove the bacteria on top and found the soft agar layer below it is totally clear (I dont know if this is normal, i never did soft agar before)

I made another technique, where i cultured these colonies in marine broth and then added a drop of them on top of marine agar seeded with the test strain and yet no sign of zone of inhibition.

I dont have so much experience in this screening, so can anyone help me and tell my why the original plate showed ZOI and not anymore.

[jazzlynblu]

It is possible that during the initial screening, your bacteria still possess the antimicrobial activity (as in, the antimicrobial gene is activated). However, as you culture them, they have no need for their antimicrobial activity because of the environment they are in and the antimicrobial gene becomes deactivated.

I would count the number of transfers you have made from your original sample and maintain the original culture in storage. Because the more transfers you make, you are selecting for more strains that are comfortable in the media you use, and thus will have slightly different biochemical profiles from your original sample.