You would have to infect the organism with the bacteria and then treat by injection. The other option is infecting STEM cells with the bacteria and then treating with the compound. This allows us to test how the drug work in living tissue.
A time kill experiment essentially is where you grow the bacteria in a negative control for 24 hours and then incubate with your compound for 24 hours. The point is to see whether the bacterial population is frozen (it stays the same over the course of the incubation with the compound), indicating the antibiotic is bacteriostatic, or if it reduces (it goes down after being incubated with the compound), indicating the antibiotic bactericidal. Bacteriostatic antibiotics basically just prevent bacteria from reproducing (thus allowing the immune system to kill them), and bactericidal antibiotics kill bacteria actively.
I’m sure there is, but I’m not sure how to articulate it! We can see a very, very slight curve consistently with increasing concentrations, and in both experiments, the 1.25 uM concentration always notably increases.
Is the 1.25uM concentration the fourth bar on the yellow graph? It seems strange the absorbance would increase consistently with a higher concentration!
To answer your second question you posted on here as well, yes! This is a pattern we noticed consistently with every experiment was a slight increase at the 1.25 uM level. No matter what the results were, 1.25 uM always went up a little bit, which would indicate more bacterial growth. This just shows that it is less effective as an antibiotic at this concentration.
If a future step would be to do in vivo testing with animals, is there a way to make sure that it is done humanly or is there a negative effect on the animals tested on?
Yes, there are several regulations imposed on animal testing, particularly for vertebrates (mice). Most of them revolve around the necessity of using animals and the absolute reduction of any suffering on the animal’s part. There are actually special committees that must be formed to ensure there is no animal cruelty for each in-vivo experiment, they’re called IACUC’s if you want to look more into that. The possibility of a negative effect is unfortunately there, which is awful, but it is nearly impossible to pass a drug without animal testing. In mice, Salmonella typhimurium causes Typhoid fever.
Yes! There are a LOT of regulations involving treating lab animals humanely. Particularly, a committee called an IACUC is formed for each experiment involving vertebrates (mice), in case you’re wondering what to look for. Unfortunately, there is the possibility of negative effects on animals. It’s very sad, but Salmonella Typhimirium causes Typhoid Fever in mice, which can cause them to become fairly sick. It’s very sad that they have to experience that for the sake of our research. It raises a lot of very interesting ethical questions.
It was really just a guess. Any drug that requires over 10 uM to be effective is simply just not possible because that high of a concentration in the blood is pretty much fatal. So knowing it has to be under 10 uM for that reason (to be effective), we took a guess at a middle ground. Thank you!
How would you test this drug in living organisms? Is there any specific technique you can think of?
You would have to infect the organism with the bacteria and then treat by injection. The other option is infecting STEM cells with the bacteria and then treating with the compound. This allows us to test how the drug work in living tissue.
Great job overall! What is a time kill experiment just out of curiosity?
A time kill experiment essentially is where you grow the bacteria in a negative control for 24 hours and then incubate with your compound for 24 hours. The point is to see whether the bacterial population is frozen (it stays the same over the course of the incubation with the compound), indicating the antibiotic is bacteriostatic, or if it reduces (it goes down after being incubated with the compound), indicating the antibiotic bactericidal. Bacteriostatic antibiotics basically just prevent bacteria from reproducing (thus allowing the immune system to kill them), and bactericidal antibiotics kill bacteria actively.
Do you think there is any significance to the lack of differentiation in absorbance with different concentrations of your drug?
I’m sure there is, but I’m not sure how to articulate it! We can see a very, very slight curve consistently with increasing concentrations, and in both experiments, the 1.25 uM concentration always notably increases.
Is the 1.25uM concentration the fourth bar on the yellow graph? It seems strange the absorbance would increase consistently with a higher concentration!
To answer your second question you posted on here as well, yes! This is a pattern we noticed consistently with every experiment was a slight increase at the 1.25 uM level. No matter what the results were, 1.25 uM always went up a little bit, which would indicate more bacterial growth. This just shows that it is less effective as an antibiotic at this concentration.
If a future step would be to do in vivo testing with animals, is there a way to make sure that it is done humanly or is there a negative effect on the animals tested on?
Yes, there are several regulations imposed on animal testing, particularly for vertebrates (mice). Most of them revolve around the necessity of using animals and the absolute reduction of any suffering on the animal’s part. There are actually special committees that must be formed to ensure there is no animal cruelty for each in-vivo experiment, they’re called IACUC’s if you want to look more into that. The possibility of a negative effect is unfortunately there, which is awful, but it is nearly impossible to pass a drug without animal testing. In mice, Salmonella typhimurium causes Typhoid fever.
Yes! There are a LOT of regulations involving treating lab animals humanely. Particularly, a committee called an IACUC is formed for each experiment involving vertebrates (mice), in case you’re wondering what to look for. Unfortunately, there is the possibility of negative effects on animals. It’s very sad, but Salmonella Typhimirium causes Typhoid Fever in mice, which can cause them to become fairly sick. It’s very sad that they have to experience that for the sake of our research. It raises a lot of very interesting ethical questions.
Great job! Why in particular did you assume that the lowest effective dose would be ~5 micromolars?
It was really just a guess. Any drug that requires over 10 uM to be effective is simply just not possible because that high of a concentration in the blood is pretty much fatal. So knowing it has to be under 10 uM for that reason (to be effective), we took a guess at a middle ground. Thank you!