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.