tvithlani
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S. epiderimidis v. s. aureus

Postby tvithlani » Thu Nov 15, 2018 2:22 pm

Hi there,
My grade 7 son is conducting a science experiment for his science fair on the antibacterial effect of turmeric vs. polysporin vs. fusidic acid. He cannot get his hands on s. aureus to conduct the experiment given that it is biosafety level 2, but can obtain s. epidermidis. Based on his research, both bacteria are present on the skin, and both are gram positive (which I take means that their cell walls are similarly structured). Would s. epidermidis be an appropriate substitute for s. aureus for purposes of his experiment?

On a related note, he needs to first complete a research paper prior to designing and conducting the experiment, and is having difficulty finding any prior experiments/research on the effectiveness of tumeric (or its bioactive compound, curcumin) on s. epidermidis, but has found some limited research on the effectiveness of turmeric/curcumin on s. aureus.

Are s. epidermidis and s. aureus simiilar enough that he could reasonably extrapolate that the results/findings of the effectiveness of turmeric on s. aureus could potentially apply to s. epidermidis as well? Is the fact that they are both gram positive bacteria sufficent to draw this conclusion in and of itself, at least for Gr. 7 purposes?

Any help/advice/pointing us in the right direction would be greatly appreciated!

Thanks,
Tanvi

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Re: S. epiderimidis v. s. aureus

Postby SciB » Thu Nov 15, 2018 7:29 pm

Hi and welcome to Scibuddies!
Thanks for the very detailed post with the questions. As far as I know Staph epidermidis is an equivalent substitute for Staph aureus in antibiotic experiments as they are both Gram positive and in the same bacterial genus. Staph aureus has some especially virulent properties that Staph epidermidis lacks, but otherwise they are fairly similar.

For the literature review I would suggest trying to determine exactly what the effect of curcumin (the active component of turmeric) is on bacteria. Pay attention especially to the difference between bacteriostatic (stops bacteria from growing) and bacteriocidal (kills bacteria) properties. The general term that includes both these properties is 'antibacterial'.

Curcumin is a chemical compound extracted from the roots of Curcuma longa, the turmeric plant. It is chemically known as a polyphenol which just means that structurally the molecule is made up of several (poly) phenol ring structures. The chemical structure is important because it can provide clues as to how curcumin (usually abbreviated Ccm) acts on cells--either bacterial or human.

Do a search for 'curcumin antibacterial' and see if you can find out exactly what Ccm does to bacteria and how it does it. The action is probably similar on most bacteria, so if you find a paper about the effects of Ccm on S. aureus, it would most likely be applicable to S. epidermidis.

If you get stuck on technical concepts or terms, let us know and we will be happy to explain--or at least try to.

Good luck!

Sybee

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Re: S. epiderimidis v. s. aureus

Postby tvithlani » Sun Nov 18, 2018 11:59 am

Thank you so much! That’s very helpful.

Tanvi

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Re: S. epiderimidis v. s. aureus

Postby SciB » Sun Nov 18, 2018 6:21 pm

You are welcome, Tanvi. I think you have a very interesting project. Be sure to check with us about the best way to design it and do ht experiments. It is a good idea to run your plans by an expert because we can tell you if something needs to be changed, done a different way or added to.

Be extra careful in choosing which brand or type of Ccm you use. The chemical is not very water soluble, but some brands (BCM95, for example) are better than others. Also, if you can get a brand that has ONLY curcumin, that would be better for your experiments. Sometimes, the Ccm is compounded with a black pepper extract called 'bioperine' which supposedly improves its activity. If you have two compounds in your test solution, then you can't prove which one was the active ingredient. You need to have a source of Ccm alone.

Good luck!

Sybee

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Re: S. epiderimidis v. s. aureus

Postby tvithlani » Tue Nov 20, 2018 7:53 pm

Thanks again Sybee. My son has hit a slight snag. The school is uncomfortable ordering and using S.epidermidis. They are suggesting e.coli, but isn’t that quite a different bacteria? Can you suggest how my son could go about finding another non-staph gram positive bacteria that could be a reasonable substitute?

Thanks
Tanvi

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Re: S. epiderimidis v. s. aureus

Postby SciB » Tue Nov 20, 2018 8:18 pm

You are correct, Tanvi, E coli which is Gram negative would not be a suitable stand-in for Staph aureus. Experiments with bacteria are easy and safe if students follow the clear instructions for handling microbes. S. epidermidis requires only BSL 1 working conditions, the same as E coli. You might point that out to the school.

The only other thing I could suggest is to try and find a university microbiology lab that would allow your son to use their facilities temporarily in order to do the experiments. The project to compare the antibacterial properties of the compounds is a good one and some researcher may be interested enough to allow your son to do the experiments there with supervision. Working with scientists who know the techniques for testing antibacterial properties of specific compounds would be a valuable educational experience as well as allowing him to do an excellent project.

You will have to make a proposal to the professor outlining exactly what you want to test and how you plan to do it. Take the time to learn the jargon and understand the methods in detail.

Let me know what happens. I wish you luck so that you can do the experiments as planned.

Sybee

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Re: S. epiderimidis v. s. aureus

Postby tvithlani » Mon Dec 10, 2018 1:03 pm

Hi Sybee,

Thanks for your help. Good news - the school agreed to order the s. epidermidis, so the experiment is a go!
Quick (I hope) question. My son has made his own paper filter discs for his experiment by using a hole puncher to punch holes in coffee filter paper. He read that the filter discs could be sterilized by either dipping them in ethanol, or by baking them in an oven at 300 degrees F. He decided to go with the oven method, and "baked" them in tin foil for 10 minutes. Do you think that is sufficient to sterilize the discs?

He was intending to grow the bacterial lawn on agar (tryptic soy) but the lab technician at the school made him grow it in some liquid broth. He will have to change his procedure to match what he actually did, but is unclear what the next step would be. His original plan was to spread the bacteria on agar dishes, insert the medicated paper discs on the dishes, incubate for 24 hours and then measure zones of inhibition. But now he is not sure what the next steps would be - i.e., how would he get the bacteria from the broth onto the agar dishes? Would he just measure out using a pipette? And then would he still need to incubate for 24 hours?


Many thanks,
Tanvi

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Re: S. epiderimidis v. s. aureus

Postby SciB » Mon Dec 10, 2018 8:35 pm

Hello, Tanvi. That is great news that your son will be able to do the experiment with S. epidermidis.

The answer to your first question about sterilizing the filter disks is yes, baking for 10 mins at 300 will kill everything except the most resistant microbes, and there should not be any of those on the disks anyway. Just tell him to be sure to handle the disks with sterile tweezers. These can be sterilized by dipping them in rubbing alcohol and igniting the alcohol in a candle flame. The alcohol will burn off and when the metal cools, you can pick up the disks and apply them to the agar surface.

Please inform the teacher that the Kirby-Bauer disk diffusion test requires growing the bacteria on agar: https://en.wikipedia.org/wiki/Disk_diffusion_test

I think perhaps the teacher meant that you should first grow a broth culture of S. epidermidis and then use that to spread a lawn of the bacteria onto the agar surface. The disks soaked in the appropriate solutions are then placed onto the agar and the Petri dishes are incubated at 25-37C.

Check Youtube for tutorials and demonstrations of how to spread the bacteria evenly, apply the disks and measure the zone of inhibition. There are spreaders that you can make and sterilize for evenly spreading a drop of bacterial culture on a plate and the videos will show you how to do that.

Good luck and do let me know whenever you have any questions.

Sybee

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Re: S. epiderimidis v. s. aureus

Postby tvithlani » Tue Dec 11, 2018 9:13 am

Thanks Sybee!

I think the plan is indeed to spread the bacterial culture on agar plates later this afternoon and proceed with the disc diffusion method. The only thing my son is unsure of is how to get the bacteria from the broth onto the agar plates. He was unable to find any relevant videos. Does he use a dropper of some kind and then a spreader? He knows what to do once the bacteria is on the agar plates.

Thanks!
Tanvi

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Re: S. epiderimidis v. s. aureus

Postby SciB » Tue Dec 11, 2018 11:28 am

Hello again,

Sounds like you're on track for the inhibition test--that's good!

To make an even 'lawn' of S. epidermidis on the agar surface you will need a spreader. In the lab we make these out of glass rods but that's not practical at home. You can also buy the from Carolina Bio, but I believe that it is better to learn how to make things yourself. Before I describe how to make a spreader that you can do, here's a video that shows the technique: https://www.youtube.com/watch?v=Rye6DfMv66Q&t=2s

In the video you can see the spreader is made from a bent glass rod. Also, the lab worker is using a device called a micropipettor to dispense a certain volume of bacterial culture, but you can just use a glass dropper with a rubber bulb. I specify glass because you will want to sterilize the end by placing it in a small cup of rubbing alcohol. Just before you are ready to use the dropper (one drop per plate) take it out of the alcohol then carefully put the tip into a candle flame to ignite the alcohol. Before you do this you will want to put on your safety glasses and work in an area that is free from all flammable materials.Place a lid over the cup of rubbing alcohol when not using it so that you don't accidentally set it on fire.

I would recommend making your spreader out of a straightened-out paperclip. I tried finding a video of exactly how to do this, but could not locate one, so you will have to just bend the metal to approximately the shape you see on glass spreaders: https://www.youtube.com/watch?v=Rye6DfMv66Q&t=2s
https://en.wikipedia.org/wiki/Cell_spreader

The metal spreader is sterilized the same way as glass ones--by letting it sit in alcohol and then flaming it to burn off the alcohol just before use. The paper clip metal is rather thin and you need to hold it and use it gently so as not to dig into the agar when spreading the drop of bacteria.

I think that's all I need to tell you for now. Here's a similar Scibuddies procedure for doing a zone-of-inhibition test in case you haven't seen it: https://www.sciencebuddies.org/science- ... on#summary

I'm sure you'll have more questions and do keep us posted on your progress.

Sybee

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Re: S. epiderimidis v. s. aureus

Postby tvithlani » Tue Dec 11, 2018 12:10 pm

Perfect, thank you! This is exactly the type of guidance my son was looking for.
Tanvi

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Re: S. epiderimidis v. s. aureus

Postby SciB » Tue Dec 11, 2018 12:57 pm

You are very welcome--glad to help. Post again if you have any other questions. There is a lot of information on Youtube, but it is often quite technical and subject to misunderstanding by non-scientists. It is better to ask more questions than make an error in procedure that might invalidate your data.

Good luck!

Sybee

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Re: S. epiderimidis v. s. aureus

Postby tvithlani » Mon Jan 28, 2019 11:22 pm

Hi Sybee - just wanted to let you know that my son was awarded 1st prize in the biology category at his school fair and has now been invited to participate in the regional science fair! Thanks so much for all your help & advice - it was really useful and he learned so much.

Tanvi

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Re: S. epiderimidis v. s. aureus

Postby SciB » Thu Jan 31, 2019 8:54 am

Wow! That is great news, Tanvi. Thank you so much for taking the time to tell me about your son's success. Please congratulate him for me and be sure to tell him that I will be here to help whenever he has questions about the next project. It is always a good idea to talk to me when you are planning a project because I can make suggestions for improvements in the science and to avoid any potential problems.

Think of us as your science consultants! Helping the next generation of young scientists on the path to success is our pleasure and our duty. I decided to become a scientist at the age of five and it was the best choice I could have made, in my opinion. In this age of misinformation and manipulation of the 'facts', the scientific method is the one shining star that maintains the goal of searching for the truth.

Best wishes,

Sybee


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