Science Buddies: "Ask an Expert"

Hi,

I'm interested in microbiology and thought that this project might be interesting: https://www.sciencebuddies.org/science- ... p022.shtml
Unfortunately, this project idea was an abbreviated project so I am a bit lost. Does anyone have a link to some studies so I could kind of understand the procedure (for example, how to dilute extracts to certain concentrations, how to prepare bacteria strains). Are there certain bacteria that I could use that would be safe enough to test at home and where could I buy them? Also, does anyone know which antimicrobials I could actually use? I know the project listed some like coffee, tea, herbs, spices, and I've read about some studies using honey, but I was curious if there was anything out there that is fairly easy to access. Lastly, do you think I should compare natural antibiotics to prescription antibiotics or the effectiveness of natural antibiotics using just natural ones, or is that completely up to me? What is your suggestion? Thank you so much!

Hi kershaw,

Welcome to Science Buddies!

First of all if it were me, I would probably approach this experiment as testing the efficacy of the "natural" antibiotics, and using either a clinical antibiotic test disc, or even an alcohol or iodine solution as my control (anything you know will kill the organism you're working with). As for what organism to use, have you considered yogurt as a source for bacteria? Easy and cheap to access. Alternatively you could purchase live organisms from an online vendor. But I always figure why pay for what lives pretty much everywhere you could think of, haha.

I thought this might give you some ideas though.
http://www.hometrainingtools.com/bacter ... AodE14AXg/
It's a link to a bacteria project kit. It includes everything you might need, except for the organism itself. It even includes iodine for the antibiotic, along with project instructions. *Please note that I am not in any way affiliated with this store, just trying to offer some suggestions.

And here's a link to a PDF with detailed instructions on how to isolate and culture bacteria from yogurt (if that's what you choose to do, but it should be helpful regardless).
https://docs.google.com/viewer?url=http ... yogurt.pdf
There's probably a lot of info in there you don't even need, but it should at least help you get an idea of how the procedure works. You shouldn't need a bunsen burner if you use plastic disposable loops.

And if you don't have access to an incubator, you can pretty easily rig one up out of an aquarium and a desk lamp. Other materials work well too, don't be afraid to Google and get creative with what you've got. Here's a link just one example.
http://www.ehow.com/how_7487725_make-in ... ology.html

So anyway I hope I've been somewhat helpful. Good luck with your project!

Wow thanks a lot! The incubator maker and bacteria kit should be helpful. The yorgut idea is interesting, but do you think E. coli and pre-prepared bacteria would be easier to handle? And there are still a couple questions I still have, liste on the first post. Thanks cbrambley!

Hello there,

I think cbrambley gave you some really great tips! Although I think that taking bacteria from yogurt is an interesting idea, it might be difficult trying to extract the bacteria from the yogurt and plate it onto some agar plates without getting some chunks of yogurt on there. In addition, it may also be difficult to figure out the type of media agar plates the bacteria in the yogurt would optimally grow on, as well as whether a specific temperature or oxygen condition is necessary. For those reasons, I would probably just go with normal E. coli that you can purchase from vendors, which is really easy to grow and work with, and we also know that E. coli can be grown in Lb media at 37 degrees (if grown at liquid culture though, you may need a shaker). As for more ideas for natural antimicrobials, I've attached one paper to this reply (and also another in the next reply, since we're only allowed to attach one file per comment). Another interesting idea for natural antimicrobials is that humans and other animals actually naturally produce molecules called antimicrobial peptides, which are secreted in many areas of our bodies and have broad spectrum effects as they can kill many different species of microbes (http://en.wikipedia.org/wiki/Antimicrobial_peptides), and I do know that many people are interested in developing these into broad spectrum therapeutics for microbial infections, so perhaps you can do a bit of research to see if there are any products that contain antimicrobial peptides.

Let us know if you have anymore questions!

Connie

Here is the other paper attached.

Thanks connief!
Looks like the antimicrobials from plants would be good for projects (fairly easy to access, inhibitory to a wide range of bacteria).
The "Novel Natural Food Antimicrobials" study was especially helpful
Alright, I guess my project will be pretty much testing the effectiveness of antimicrobials of plant origin on E. Coli and...
Do you have any suggestions for some bacteria?
Also, it looks like most projects involving microorganisms need to be done in a regulated research institution, according to Intel Rules Wizard, unless the project passes the safety assessment.
Do you guys think this project will require a lab? I was really hoping to be able to do it at home...
Thanks!

Also, does anyone know how these natural antibiotics are applied to the bacteria? Obviously I can't just put a garlic in the middle of a petri dish. I think it might be extracts, but would I need to do that myself or is it possible to order those herbs, spices, etc in a form that is already acceptable to apply to the bacteria? Any help is greatly appreciated

Hi Anthony,

Your project does sound like something that would be best done in a lab, since you'll need to use sterile technique, grow bacterial cultures in an incubator, etc., but you might be able to get by with your high school lab. I think some people are doing some similar projects with bacteria at home, but I'd be somewhat concerned about the safety issues. Maybe another expert can help me out on this one.

As for what kind of bacteria to use, a common, non-pathogenic strain of E. coli called K12 is commonly used for these sorts of experiments and is readily available from Carolina Biologicals, so that would probably a good choice.

As for how to test the effectiveness of inhibition, probably the most common way is a zone of inhibition assay. There are good procedural instructions in one of our projects you can use. https://www.sciencebuddies.org/science- ... ml#summary This link for a project idea on Natural Antimicrobials might also be useful (https://www.sciencebuddies.org/science- ... ml#summary), and that link includes a link to Microbiology Techniques & Troubleshooting which could also come in handy. (https://www.sciencebuddies.org/science- ... ques.shtml).

As for whether or not to make your own extracts, I think that is really up to you and the availability of your materials. A quick google search suggested that garlic extract is readily available, but if you want to try to make your own, that would be okay too. Just remember to document exactly what you used and/or how you made your extracts and include that information in your report.

Hope this helps!
JMP

Thanks JMP for the reply!

I asked my teacher if I would need to get special forms signed for the safety issues and he said my project was fine and didn't need them, which is great. Maybe the lab won't be necessary since this project is fairly simple.
Thanks for the links! They were really helpful!
E. Coli would probably easiest to work with, but do you guys think I could also use staphyloccocus (staph infection bacteria)?
Does anyone have a link to a study where they use these extracts and possibly how they prepared them ?
Thanks for all the help so far everyone!
I'm really liking Science Buddies!

With regards to E. coli v. Staph (I assume you mean S. aureus, the kind that causes most infections and not S. epidermidis which is largely commensal), the only potential problem I see with using staph instead of E. coli in your experiments are biosafety concerns. E. coli K12 is a BSL1 organism, which means that you don't need much in the way of safety precautions. S. aureus is BSL2 so will require some more safety precautions. I recommend you check with your teacher and see if you have the ability to work with a BSL2 organism either in your high school lab or at home and to learn in more detail what the safety concerns are and how you will need to work with the bacteria. If your teacher thinks it is okay then there is no reason you couldn't use staph for these experiments. It is certainly no harder to work with than the E. coli.

JMP

Thanks JMP!
I'll be sure to post questions if I have any.

Hello there,

Like JMP mentioned, I would also suggest working with E. coli K12 if you were to do this in your own home because Staph aureus will require more safety precautions since it is potentially infectious and could cause serious disease. If you do want another strain of bacteria to compare to E. coli that is safe to work with in your home, I would probably suggest Bacillus subtilis, which is another model bacterium that's often used in research and is BSL1 (just like E. coli). Furthermore, Bacillus subtilis is a Gram positive bacterium, while E. coli is Gram negative, so it will be interesting to see whether these extracts you use will be equally effective against Gram positive and Gram negative bacteria.

In the paper that I had sent you (Novel Natural Food Antimicrobials), did they not mention how they made the extracts in the materials and methods, or perhaps where they purchased the extracts? I think it will be helpful to look at papers that have looked at antimicrobial effects of these plant/food extracts because they will likely outline the procedures of how they obtained the extracts and also how they measured bacterial inhibition. If you were to make your own extracts, if it requires resuspending the contents in certain chemicals like methanol or chloroform, then you definitely need to do that in the lab because the fumes from those chemicals can be harmful. Otherwise, if there were a way that you can purchase extracts resuspended in harmless chemicals, then by all means work on it at home!

Let us know if you have anymore questions!

Connie

Thanks connief!
Would this be an okay science project?: Testing to see if Natural Antimicrobials are effective on gram-positive bacteria as they are on gram-negative bacteria.

Hi Anthony,

I think testing whether certain natural antimicrobials can inhibit Gram positive and/or Gram negative bacteria is a great idea. E. coli and B. subtilis will be good representatives of Gram negative and Gram positive bacteria, respectively, but you should definitely be careful when you interpret your results about whether the natural antimicrobials have specific effects on Gram positive versus Gram negative bacteria. This is because you only have one strain of each type of bacteria, and it may be difficult to make generalizations for all Gram positive or Gram negative bacteria based on data obtained from experiments done with one strain of each type. For example, let's say you take some Extract "X" and it kills B. subtilis but not E. coli. That result would suggest that Extract "X" is effective in inhibiting B. subtilis but not E. coli, but you cannot conclude that Extract "X" kills Gram positive and not Gram negative bacteria. Perhaps Extract "X" has a species-specific effect on B. subtilis, and that if you try putting Extract "X" onto other Gram positive bacteria, it may not kill them. Likewise, it could be that E. coli is resistant against Extract "X", but other Gram negative bacteria may be killed by Extract "X". Hence, if you want to look broadly at the effects of these natural antimicrobials against Gram positive and Gram negative bacteria, you may want to choose a couple more different bacterial species that are Gram positive or Gram negative, but that would likely require you to work in a lab. If you just want to test the antimicrobial properties of your extracts of interest on bacteria, it's fine if you just do it with E. coli or B. subtilis alone, but you should also note that when you interpret your results, the antimicrobial effects will be specific to either E. coli or B. subtilis, but may not apply to other bacteria.

By the way, here is a project idea from the Science Buddies site where they used garlic tablets to make garlic solutions. Perhaps this is one way you can make your own garlic extract if that's one of the substances of interest that you plan on testing!
https://www.sciencebuddies.org/science- ... #procedure

Let us know if you have anymore questions!

Connie

You have a good point there, Connie.
Hmm...this really changes things...how would you restruct this project?
And thanks for the link!

Quick question, are plant extracts different from volatile oils from plants?

Hi Anthony,

"Plant extracts" is a very general term that is basically used to describe any substances that are obtained by separating and processing the juices of various plants. Depending on how you process the plants, different substances can be extracted from them. Essential oils (or volatile oils) are one thing that you can extract from plants, and they are basically a hydrophobic liquid that contains volatile, aromatic compounds from plants. The following wikipedia pages give a pretty good overview of plant essential oils:

http://en.wikipedia.org/wiki/Essential_oil
http://en.wikipedia.org/wiki/List_of_essential_oils

As for how to re-structure this project, it's completely up to what question you're interested in answering most. If you want to look at whether certain natural antimicrobial compounds can have a wide range of inhibitory effects on both Gram positive and Gram negative bacteria, I would suggest that you should probably choose a couple more bacterial strains that are Gram positive or Gram negative in addition to B. subtilis and E. coli. If you just want to look at the antimicrobial effects of different compounds, I would suggest just working with one strain of bacteria (probably E. coli since it's a widely-used model microbe and is very easy to grow and work with). Do realize though that the antimicrobial effects that you see from these compounds have only been tested on E. coli though, so you can't make conclusions about whether these compounds are widely antimicrobial, but you can definitely propose future experiments in your conclusions where you can look at other different types of bacteria, or Gram positive versus Gram negative bacteria, etc. to probe whether the compounds you've tested have effects on a wide range of bacteria as opposed to just E. coli.

Keep us posted on how your project goes and don't hesitate to ask more questions if you need help with developing your project ideas, conducting your experiments, or analyzing your data!

Connie

Hopefully the essential oils are easy to prepare or access...what do you think?
I was really liking the idea of using both B. subtilis and E. coli...
I wish I could construct a project involving those two without having to choose more different bacteria (because it would require a lab)

The essential oils should be easy to access--you just need to find out where you can purchase them. I think you can still do the E. coli vs. B. subtilis experiment without having to choose more bacterial strains. You can say that you're using E. coli as a model for Gram negatives and B. subtilis as a model for Gram positives, and whatever results you obtain will provide interesting implications about the effects of these compounds on certain types of bacteria. Then in your concluding remarks, you can say that more bacterial strains could be tested in the future to see if there really is a Gram negative versus Gram positive effect. For example, if some extracts kill both E. coli and B. subtilis, it'll be interesting to see whether they can also kill a bunch of other bacteria--in other words, does this compound kill bacteria regardless of whether they're Gram positive or Gram negative? If some extracts kill only E. coli and not B. subtilis, it'll be interesting to test other Gram negatives to see if they are sensitive and other Gram positives to see if they are resistant, and vice versa.

Hope that helped!

Connie

Thank you so much connie!

I thought about it, and what would you think of restructuring the project like this?:
Using natural antibiotics that are already known to be effective against E. Coli, see if changing the concentrations of the essential oils affects the effcacy of inhibiton?

According to one study, the essential oils of black pepper, thyme, oregano, clove, and nutmeg are all effective against E. coli.
I found them all online to buy.
The one question I have right now is, would it be okay to use these oils directly? (I'm just wondering if companies added certain chemicals to the oils before selling).
It says that these oils are diluted in a "carrier" oil, which helps to dilute them before actual application on skin, etc.
Does anyone know if using essential oils diluted in carrier oils would create different/inconsistent results with pure essential oils?
Also, when manipulating concentrations, would one normally use water to dilute it, or just use less of the oil?
And finally, one study made a hole in the agar and put in 15 microliters of the oil while a different study put concentration disks...
Which one would be right for this experiment?
Thank you!

Hello Anthony,

I think the way you thought about restructuring the project is a good idea, but you may obtain results that are to be expected. For studies where the researchers just apply the natural antimicrobial onto E. coli without any indication of what concentration they used, it will be cool to demonstrate what is the lowest concentration necessary for inhibition. However, for studies where the concentrations of the oils used for inhibition have been carefully analyzed, I'm not sure if you can show something that is different from the results already obtained, and decreasing the concentrations from whatever they used will just mean there will be less inhibition and increasing the concentrations from whatever they used will just mean there will be more inhibition. But if this is what you're interested in doing, by all means go for it! This is your project so you should try to answer questions that intrigue you the most.

I'm not sure whether essential oils diluted in "carrier oils" would have any effect on your experiments--it really depends on what the carrier oil is. One possible problem is that the carrier oil itself may also be toxic to E. coli and hence it will be difficult to tell whether it's actually the essential oil or the carrier oil that is killing your bacteria. One way to test that is to actually get a pure stock of the carrier oil they used and test that on E. coli to see if that has any affect on the bacteria. Another problem is that if the carrier oils for all of the essential oils you're purchasing are different, it is quite possible that the carrier oils may have their own effects on E. coli, and it will be difficult to compare between different essential oils and say which is more effective at inhibiting E. coli.

As for manipulating concentrations/diluting, if the essential oils are hydrophobic, they won't dissolve very well in water. In the studies that have done work with essential oils, do they mention in the materials and methods what they use to dilute the essential oils to different concentrations? Very common solvents for more hydrophobic things can be alcohols, chloroform, etc. If you do have to end up using these chemicals, you would need access to at least a lab space that has a fume hood because the fumes from some of these chemicals can be harmful.

As for the hole in the agar versus the concentration disks, I would probably going with the concentration disks. I'm not sure how putting a hole in the agar is a good idea in testing a compound's ability to inhibit bacterial growth--I actually never heard of this technique before. The concentration disks are much more commonly used.

Other experts please chime in if you have any other thoughts! Let us know if you have anymore questions!

Best,
Connie

Thanks connie for the information. Well, I honestly don't know what to do now. I had no idea that the manipulation of concentrations would be so difficult and that the carrier oils would cause lots of complications. Any ideas or suggestions? I know I've asked a lot of questions so far, sorry about that.

Just read a study that used cinnamon, garlic, wasabi, and pomegranate (prepared by adding 75 mL)
Maybe I can shift away from essential oils (although I really wanted to test them) and use natural antibiotics that are not hydrophobic?
That way I can easily change the concentrations.
What do you think?

Sounds good to me, as long as you know how to prep the solutions. You mentioned adding 75 mL. 75 mL of what solvent, water? As long as you keep the solvent for all the natural antimicrobials consistent, then it should be okay.

And honestly, I'm not 100% sure that the carrier oils will cause a huge problem, but it's probably better to be safe than not. If any other experts know a bit about carrier oils and how they may affect bacterial inhibition, please chime in.

And no worries about asking lots of questions! It's a good thing that you thinking about all aspects of your experiments to make sure that your results and clear cut and that you can interpret them with as little trouble as possible. Let me know if there's anything else you need, and if you need help once you do the experiments and/or interpret the results.

Connie

Yeah sorry I forgot to add "water" there next to 75 ml.
Even if the carrier oils DIDN'T cause a problem, I still wouldn't be able to do the experiment unfortunately. I would need to work in a lab to change the concentrations
The proposal is coming up soon and I don't want to mess up on that!
Thanks so much connie!

Hi,
I am writing up my procedure now and have a couple questions...
Carolina Biological Supply sells E. coli strains in tubes and E. coli strains in dishes. If I were to choose the dishes, would that mean that I wouldn't have to actually inoculate the bacteria, only incubate? Would I just directly work with the essential oils on sterile disks? Also, I am working to see if different dilutions of essential oils affect their ability to inhibit bacterial growth. How many total samples should I work with? I was thinking of making my control 2%, the standard dilution for aromatherapy applications, and then testing .5%, 1%, 3%, and 3.5%. Should I do four sterile disks per dish, and three test runs through the whole thing? So 4 * 3 + 1 (control)? Is that ok? Do you think that the 2% as the control is fine since it is usually the standard dilution, or is the control supposed to be 0%?
Thanks!

Or should I try an experiment testing the MIC and MBC?
Thanks

I would leave your control at 0%. If you do dilute your oils down I would use the liquid you use to dilute and treat the disc with just that. That will control for the solvent itself having a MBC/MIC value you may not have been aware of. I would do another plate with no disk, or a dry disk to compare your experimental plates to. I would set the plates up in the following manner:

Plate 1: Control 1 - Dry disk, or no disk, just a inoculated plate.
Plate 2: Control 2 - Plate with disk treated with only solvent, no essential oils.
Plate 3: Dilution 1
Plate 4: Dilution 2
Plate 5: Dilution 3
And more plates as needed for more dilutions if you plan on testing more than three.

Thanks medigi!
For plate 2, what concentration would be ideal for the solvent?
If the solvent DID have a MIC/MBC, would that be okay? Could I continue with the experiment?
Hypothetically, if I put 3 disks of a concentration A in dish 1, 3 of B in dish 2, 3 of C in dish 3, and 3 of D in dish 4, would that be considered testing multiple times? Or would I actually need several plates for each concentration?
How many disks can fit in a petri dish? I have five different oils, and hoped to be able to test them on the same plate. If not, should I cut it down to three?
And also, does anyone know about the plates vs. the tubes?
Thanks!