Science Buddies: "Ask an Expert"

Hi,

I grew a bacterial lawn on sterile nutrient agar petri dishes, and although the lawn didn't seem all that dense, I am pretty sure there was bacterial growth. On the agar, i also put diffusion disks that had essential oils on them (I was testing antibiotic resistance). However, there were zero zones of inhibition. You can hold the plates at an angle and see some sort of circle around the plates, but I doubt that's actually any zone of inhibition. Did the oil not diffuse correctly into the bacteria? Thanks.

Hi Kershaw,

Did you put the paper disks with the oils on the agar right after you spread the E coli? The problem might be that oil and water don't play together very well, so the oil probably stayed in the disk instead of diffusing into the agar.

You could try adding a tiny amount of detergent to the oil to emulsify it before you put it on the disk. Be sure to include a control with just detergent to see if that kills E coli on its own. If the detergent inhibits the bacteria too much then use a smaller ratio of soap to oil.

Do you have a positive control disk with some antibacterial solution like mouthwash [not the alcoholic variety as the alcohol will kill the bacteria by itself]? It is always good to have a control that you know will show the effect that you are trying to measure.

Good luck,

Sybee

I actually put them in a day after. They didn't look all that dry even then, but it wasn't dripping.
My bacteria look like the second one Essential Oil Girl grew (https://www.sciencebuddies.org/science- ... =28&t=4508). If you scroll down, there's a powerpoint attachment with pictures, my plates all pretty much look like the second one.
Was the oil the problem? I put in the plates a day after, so what do you think went wrong?

You should apply the test strips right after you spread the bacteria. What are you using for a spreader?

Try it again using some detergent to allow the oil to diffuse into the agar. Make sure you spread the bacteria well. How much of the liquid culture did you put on each plate?

Try to incubate the plates at a temperature as close to 37C as possible. E coli needs warmth to grow well.

Take pictures, put them in a powerpoint and attach it to your next post.

Hi there,

I agree with SciB that for the disk diffusion assay, you should apply the antimicrobial disks right after you spread the bacteria. SciB's suggestion about emulsifying the oils in detergent is a great idea, but make sure the detergent and the concentration at which you're using it isn't actually killing the E. coli because that will make it difficult for you to interpret whether the antimicrobial effect was due to the oil or the detergent. How do your lawns look without treatment with the disks at all? If you show us some pictures, we can provide more advice on what you can troubleshoot for your procedures if necessary.

Best,
Connie

unfortunately, it won't let me attach any files. It says, "Sorry, the board attachment quota has been reached."
Would you say that the timing of the placing of disks was the main reason?

You have two possible causes here--one is the lack of solubility of oil in water and the second, and more important problem, is that the E coli were allowed to grow before you placed the disks on the lawn. Do you have time to repeat the experiment? If so try solublizing the oil with a bit of detergent and place the disks on the agar right after you spread the bacteria on the plate. Use the LEAST amount of detergent possible to emulsify the oil because detergent alone can kill bacteria. Be sure to include a control disk with the same amount of detergent WITHOUT oil.

If you want to show more photos you can put them into a shared google doc and post the link.

Hi there,

I agree with SciB about the potential reasons why your experiment didn't work out the first time. If you have enough reagents, and the main problem turns out to be the timing of when you put the disks onto the bacteria, I would just try the disks dipped in oil that has or hasn't been emulsified with detergent, just in case that the detergent concentration you tried killed bacteria on its own. That way, you can increase your chances of having at least one condition working out. How much time do you have left, and when do you ideally want to get your data so that you still have enough time to analyze it and put your project together? If you have some time left, I would suggest doing a quick pilot experiment where you use disks dipped into different detergent solution concentrations (this is without the oil) and see which could kill bacteria and which would leave them intact. I would then use the detergent concentration that doesn't kill bacteria to try emulsifying your essential oil. SciB, do you think that is a good idea?

Good luck!
Connie

Yes, I would do a test experiment with a range of concentrations of detergent alone to determine how toxic it is to the bacteria. You don't have to have zero growth inhibition, but you don't want a wide zone around the disk with the detergent because then you probably won't be able to see the inhibition that may be caused by the oil.

And as Connie suggested, when you do the actual experiment, include two controls--detergent only and oil only.

I hope you have enough E coli broth culture left to do the experiment. You can get by with spreading just 50 microliters per plate so you should have enough. Try to incubate your plates in a warm place--around 32C.

Good luck!

Sybee

No, unfortunately i dont have enough time for a second run. How should i proceed from here? (In the report)

Hi there,

That is completely fine if you don't have time for a second run. I'm sorry that the experiment didn't work out, but I hope you take this as a good learning experience because scientific research is actually a lot of trial and error! Oftentimes, an experiment has to be done multiple times before all the conditions have been optimized to give clear results, and I think in your report, you should definitely provide explanations as to why you think your experiment didn't work out this time (i.e. not adding the disks immediately after spreading the e. coli, the oil didn't diffuse well into the plate, etc.). One important thing your readers/audience will be looking for is how you reason through these problems, and how you plan to address these problems if you redo these experiments in the future. This experience may have also shown you that planning and doing an experiment takes a lot of work, and the earlier you get started on it, the better because it will give you time to troubleshoot things before your deadline if things don't work as planned! However, I think it's great that you have made the effort to try this experiment, and even though it didn't quite work out as expected, you've learned a lot about the process of coming up with a question and hypothesis, selecting the proper method to test your hypothesis, and analyzing your results--that is the important part!

Let us know if you have anymore questions in analyzing your results and preparing your report.

Best,
Connie

How should I word the results section? I understand that I have lots to talk about in the discussion/conclusions section (error analysis, alternative methods, explanations, etc.), but I'm not sure what do to in results. What kinds of charts/graphs am I supposed to put in? I was thinking generic charts of E. coli growth curve over 24 hours, and pictures of inhibition zones of other experiments (both of these found online), and also put in my own pictures for comparison. How should I properly credit these pictures found online? Also, I was thinking of maybe putting in data tables of the outcomes that I expected, and then explain why they didn't come out they way I thought they would in the conclusions section.

Thank you everyone that tried to help me, you guys have all been such a great help, and I can't thank you enough! Although this didn't really work out, it was such a learning experience for me.

Generic charts of the E. coli growth curve will be great if they were from your actual experiment. Did you actually measure the concentration of E. coli in your liquid culture overtime? Because if the growth curve you're going to show is not your own data, you should not put it in your results section because your results section should show your own data, and not data from others. If you wanted to give others an idea of how E. coli growth looks overnight for a liquid culture, you can definitely put it in your background section if it's important for explaining your experiment. Pictures of inhibition zones from other experiments should also go in your background or methods section if you want to show others what your assay set up will look like and what the read out of your experiments will be (the zones of inhibition). Since this is not your own data, I would not put that in the results section. To cite these pictures, I would just put the website link if you found it online. If you were able to find the name of the person who did those experiments, you can also cite that person's name. Other experts, if you have better ways of citing pictures found online, please chime in! For your results, I would simply show the pictures of your own plates and explain what conditions have been tested for each plate and just say what you observed--small to no zones of inhibition. You can also compare it to pictures of zones of inhibition from other experiments that you have shown in your background or methods section (if you're going to do that). Then in the discussion section, you can elaborate more on why you think the experiment didn't work out. This is also a great place to talk about what you expected your results to be based on the background research you've done.

I'm glad that even though the experiment didn't quite work out as expected, you still had a great learning experience going through the process of planning and doing it! Let us know if you have anymore questions.

Best wishes,
Connie

Am I allowed to put the online pictures in the conclusions section? It would be easy to refer to them in there.

It depends on how you're going to tell your story. If it makes more logical sense to put it in your conclusion, then put it there. However, be sure to make it very clear that this is data from someone else and not from your actual experiment, and properly cite those pictures.

Alright, that makes sense.
Would I say that my hypothesis was proven wrong? It wasn't "right", but i don't actually have data to prove that it was "contradicted."

Nope, you wouldn't say that because you do not have any data that disproves your hypothesis. Therefore, you wouldn't conclude anything about whether your hypothesis was validated or not, but rather say that troubleshooting the problems that you encountered with this experiment will hopefully get the experiment working in a way where you could address your hypothesis.