Possible factors:

i. Poor Solubility and Bioavailability: Ellagic acid is poorly soluble in water, which can limit its ability to penetrate microbial cells or reach effective concentrations at the target site.

Ii. Concentration Used:

Iii. . Ellagic acid can degrade or oxidize under certain cond

Several factors could contribute to ellagic acid's lack of antimicrobial activity in your agar-well diffusion assay. Here are some considerations to explore. Solubility Issues: Ensure that ellagic acid is fully dissolved in your solvent. Suppose it is not soluble at your current concentrations. In that case, it may not diffuse properly into the agar.

2. Medium Compatibility: Check if the agar medium you are using is suitable for the growth of the bacteria in question. Some media may inhibit or promote bacterial growth differently.

3. Concentration Range: The concentrations you tested (1 mg/ml, 2 mg/ml, and 3 mg/ml) might be below the minimum inhibitory concentration (MIC) for the specific bacterial strains you are testing. Consider testing higher concentrations if feasible.

4. Bacterial Strain Sensitivity: The bacterial strains you selected may not be sensitive to ellagic acid. Reviewing existing literature to identify which bacteria previously showed sensitivity to ellagic acid can be helpful.

5. Test Conditions: Ensure that the incubation conditions (temperature, time, and atmosphere) are optimal for bacterial growth. Improper conditions can affect the results.

6. Control Samples: Include positive and negative controls in your assay. This helps to confirm that the method is working correctly and that the bacteria are viable.

7. Time of Exposure: Consider the time allowed for the ellagic acid to interact with the bacteria. Sometimes, longer incubation times may be necessary to observe effects.

8. Interactions with Agar: Ellagic acid may interact with components of the agar medium, potentially affecting its bioavailability.

By evaluating these factors, you can identify the reason for the lack of antimicrobial activity and adjust your experimental design accordingly.

I am going to take a different approach to the answer:

First, are you sure that ellagic acid is antibacterial? The reports I noted were not studies, and the study I looked at did not even discuss how ellagic acid was dissolved. If dissolved in a solvent (be careful with this suggestion), the solvent could be the actual cause of the "antibacterial" effect. If you use a solvent other than water, make sure you use a control of only the solvent to determine if it is the cause of the antibacterial activity.

Second, a simple mistake that I have even seen in posters is that the bacteria is spread on the agar surface, allowed to grow, and then the test material is added. Of course since growth already occurred the test material can not then prevent it from growing.