Hi Vince – good question. I believe the best way for us to get a more conclusive result would be to replicate all of our tests, controlling for compound preparation. Specifically, we would want to replicate the max dose, dose response, and solid media culture tests on both longifolene and thymoquinone with various compound preparation methods. Namely, we would want to perform these tests with the compound diluted in water vs. DMSO and with and without a 24-hour refrigeration period. This is because most of the volatility we saw in our data appeared to be a result of different dilution bases (water vs. DMSO) and different refrigeration periods before testing (immediate vs. 24-hour).
Great presentation!
I might have missed it, but what does it mean when you say that the results show a ring of inhibition. What does the ring mean in terms of effectiveness?
Hi Arly, thanks and good question! I did quickly go over the ring of inhibition in my presentation so I apologize for the lack of clarity there. Essentially, in a solid media culture test like the one we performed, you would expect to see a visible ring – a ring of inhibition – around any compound which is effective at inhibiting the growth of the bacteria you are testing it against. In our case, the bacteria we are testing compounds against is Salmonella Typhimurium (S. Typhimurium). A good example of a ring of inhibition in a test against S. Typhimurium would be ampicillin since it is a known effective antibiotic for it. So, we would expect to see a ring around it in a solid media culture. Indeed, the ampicillin from the solid media culture we performed does show this, having a clearly visible ring around it (as can be seen in the top right of Image 3 in our “Results” section).
In our case, thymoquinone did not produce a ring of inhibition which would be expected if it was an effective antibiotic against S. Typhimurium. However, longifolene (the other compound we tested) did yield a ring of inhibition, indicating it does have an antibiotic effect. That said, the ring of inhibition seen around the longifolene is far less visible than the ring around ampicillin, indicating (at least in that trial) that longifolene is not as effective as ampicillin.
Spoorthy, great question! As you are pointing out, we see there is a large difference in how thymoquinone performed in the max dose test vs. the dose response test at the same concentration of 10um. The first of which shows the compound has a similar effect to the negative control of water and the second of which shows the compound has a similar effect to the positive control of ampicillin. We believe this most likely has to do with how we are preparing our thymoquinone, as that is the major difference between the max dose tests and the dose response tests we performed. Specifically, the thymnoquinone tested in our dose response tests had a 24-hour refrigeration period with more rigorous vortexing while the max dose tests did not. Due to time limitations, we were unable to test this hypothesis which is why we urge replication of our tests that control for compound preparation as a viable path for future study.
curesymposium.org 
What could you have done better to get a more conclusive result, that would reject of agree with the hypothesis?
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Hi Vince – good question. I believe the best way for us to get a more conclusive result would be to replicate all of our tests, controlling for compound preparation. Specifically, we would want to replicate the max dose, dose response, and solid media culture tests on both longifolene and thymoquinone with various compound preparation methods. Namely, we would want to perform these tests with the compound diluted in water vs. DMSO and with and without a 24-hour refrigeration period. This is because most of the volatility we saw in our data appeared to be a result of different dilution bases (water vs. DMSO) and different refrigeration periods before testing (immediate vs. 24-hour).
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Great presentation!
I might have missed it, but what does it mean when you say that the results show a ring of inhibition. What does the ring mean in terms of effectiveness?
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Hi Arly, thanks and good question! I did quickly go over the ring of inhibition in my presentation so I apologize for the lack of clarity there. Essentially, in a solid media culture test like the one we performed, you would expect to see a visible ring – a ring of inhibition – around any compound which is effective at inhibiting the growth of the bacteria you are testing it against. In our case, the bacteria we are testing compounds against is Salmonella Typhimurium (S. Typhimurium). A good example of a ring of inhibition in a test against S. Typhimurium would be ampicillin since it is a known effective antibiotic for it. So, we would expect to see a ring around it in a solid media culture. Indeed, the ampicillin from the solid media culture we performed does show this, having a clearly visible ring around it (as can be seen in the top right of Image 3 in our “Results” section).
In our case, thymoquinone did not produce a ring of inhibition which would be expected if it was an effective antibiotic against S. Typhimurium. However, longifolene (the other compound we tested) did yield a ring of inhibition, indicating it does have an antibiotic effect. That said, the ring of inhibition seen around the longifolene is far less visible than the ring around ampicillin, indicating (at least in that trial) that longifolene is not as effective as ampicillin.
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Besides human error, why do you think there could be fluctuating absorbances of the well as concentrations of the compound increased?
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Spoorthy, great question! As you are pointing out, we see there is a large difference in how thymoquinone performed in the max dose test vs. the dose response test at the same concentration of 10um. The first of which shows the compound has a similar effect to the negative control of water and the second of which shows the compound has a similar effect to the positive control of ampicillin. We believe this most likely has to do with how we are preparing our thymoquinone, as that is the major difference between the max dose tests and the dose response tests we performed. Specifically, the thymnoquinone tested in our dose response tests had a 24-hour refrigeration period with more rigorous vortexing while the max dose tests did not. Due to time limitations, we were unable to test this hypothesis which is why we urge replication of our tests that control for compound preparation as a viable path for future study.
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