Do you mean adding ethyl acetate to the growth medium before inoculation? That's not very useful - it's pretty volatile and will be lost during fermentation, it's not miscible with water - will form a layer over the medium that could inhibit oxygen exchange and growth. Use it after growth to extract. btw - please do not attempt to "prove antimicrobial activity" - "test" the capability.

Dear Jorge,

Phil is absolutely correct.

If you want to know whether your Aspergillus produces antimicrobial activity I recommend you grow them in a number of liquid as well as agar media –with diff ingredient at diff concentrations. Sample daily and test for antimicrobial activity from D2. 1 ml samples can be spun in an eppy so that you can test the supernatant as well as the mycelium (after extracting with 500ul methanol). You can cut small plugs from the agar plates and extract them with 3x volume of methanol.

When you observed the activity, if it is the supernatant you need to check that it is extractable in EtOAc. You can do this by extracting the supernatant in a separating funnel and testing the original, the EtOAc extract (upper layer) and the ‘spent’ aqueous layer. If the activity is in the mycelium as well as the broth or only/higher in the mycelium then extract the mycelium with methanol. Sometimes the compound in the mycelium is different from the cpd in the supernatent.

Good luck!

As part of the growth curve of the fungus, you should expect it to grow during the logarithmic or exponential growth phase and that is when the stationary phase begins when the fungus produces the secondary metabolites. You can check these phases by monitoring the optical density of the crops. Any additive that interferes with the normal growth of Aspergillus fumigatus will negatively affect the production of the products you are testing.