Hegeman
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How to grow safe bacteria to test the effectiveness of essential oils

Postby Hegeman » Mon Nov 09, 2020 7:28 pm

I have read many of the posts on here about different agar plates and also different ways to grow bacteria. My son is doing his project on the effectiveness of essential oils in killing bacteria. I love science and he is very interested, but I need a project example he can follow or easily tweek to make his own. I looked at "Germs" and "How Well do Disinfectants Work?" He would like to grow the bacteria. We have Pre-poured-LB agar plates to do this with and the same brand, yet different types of organic essential oils / no fillers. Will the pre-poured agar plates work? What is the best source of bacteria to grow without being harmful? Can we swab something from the same place in the house, shoe, fridge, garage? (as long is it is the exact same place) to grow the bacteria? Also, after it is grown, he wants to use a pipet and put a drop on the agar plate to watch and see if it kills the bacteria and how rapidly. Will the oil affect the agar?

One really important question, we may be moving in the middle of this. Will it suffice to put the agar plates in a cardboard box and keep them there to move them, so the temp doesn't change too drastically, if we do move before he is done with the experiment, so that will not be a factor? Any thoughts welcome. Thanks!!

probiotics
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Re: How to grow safe bacteria to test the effectiveness of essential oils

Postby probiotics » Mon Nov 09, 2020 7:59 pm

Hi!

I think that this sounds like a great project for your son! I'm sure that he will enjoy the experimental process. I am glad to hear that you've explored some Science Buddies resources! Now, to answer your questions:

Pre-poured agar plates will work great! Pouring agar plates can get complicated without a sterile environment, so pre-poured plates are a great option. If you want non-harmful bacteria, you could try ordering a k-12 strain from a reputable website like Carolina Biological. For example, they offer a k-12 strain of E. coli. K-12 strains are specially designed to not be harmful to humans - however, you must still follow proper precautions when working with bacteria. When buying, make sure that the bacteria is in a liquid medium! Nutrient broth is a common liquid medium that you may encounter. Here's a link to the E.coli:

https://www.carolina.com/bacteria/escherichia-coli-living-k-12-strain-nutrient-broth-tube/155068.pr

You could also swab areas like suggested. However, a drawback to this method is that there may be some variability in the amount and type of bacteria plated each time. Since his goal is to completely cover the agar plate and create a lawn of bacteria, this may not be an issue.

Here's an excellent project outline that he can easily follow and adapt to his hypothesis!

https://www.sciencebuddies.org/science-fair-projects/project-ideas/MicroBio_p014/microbiology/measuring-antimicrobial-effectiveness-with-zones-of-inhibition

This project goes over the Kirby-Bauer disk-diffusion method, which is commonly used to determine antibacterial activity. Instead of pippeting a drop onto the agar, you would dip a small circle of paper into the essential oil and place it onto the plate once bacteria has grown. Bacteria around the circle will die, and depending on how far the death spreads, you can determine how efficient the essential oil is. The link provided goes into much more detail!

You shouldn't have a problem with the oil affecting the agar! There have been many past scientific studies in the past with similar questions, and they have utilized the same procedure without impacting their results. A great learning activity would be going through one of these articles! The content will be very high level, but it would be exciting to see how researchers are using essential oils to help solve problems right now! Here's a link to a potential read:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206475/

In relation to moving and storing your agar plates, bacteria is normally incubated at a specific temperature. Every bacteria has its own preferred growing temperature. You could try creating a DIY incubator using a carboard/styrofoam cooler and a lightbulb inside the box to reach the preferred temperature. However, bacteria will still grow at regular temp, just at a slower rate. As long as the temperature inside the box is kept somewhat constant, I don't anticipate any problems!

Hope this helps, and let me know if you have any further questions!

Thanks, Probiotics

lmp1341
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Re: How to grow safe bacteria to test the effectiveness of essential oils

Postby lmp1341 » Tue Nov 10, 2020 12:37 pm

Will the pre-poured agar plates work? What is the best source of bacteria to grow without being harmful? Can we swab something from the same place in the house, shoe, fridge, garage? (as long is it is the exact same place) to grow the bacteria? Also, after it is grown, he wants to use a pipet and put a drop on the agar plate to watch and see if it kills the bacteria and how rapidly. Will the oil affect the agar?

Hello and welcome!!
It sounds like you have a really cool experiment underway!
My fellow expert Probiotics has done a great job at providing you lots of resources to explore, but I thought the more information, the better, so here's my input for your project:

Yes! The pre-poured agar plates will work, if they are in sterile packaging. You will not need to make your own agar plates unless the pre-poured plates are not sterile.

The best source of bacteria would be educational grade K12 E.Coli, which you can find here:
https://www.bio-rad.com/en-ch/sku/16604 ... 1660408EDU

They are lyophilized pellets, so you would have to suspend them in an LB Broth medium which we can help you with too. Here's a step by step on how to do that:
https://www.microbiologics.com/core/med ... 4&_xt=.pdf

Swabbing something from your house might introduce extraneous variables, so if you can use the K12 strain of ecoli just to keep everything consistent, that might be best, as you might pick up multiple strains of bacteria if you swab in your house.

Please refer to Probiotic's section on the Kirby Bauer method! :)

Best of luck and please don't hesitate to reach out again!

Stay Nerdy,
lmp1341

Hegeman
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Re: How to grow safe bacteria to test the effectiveness of essential oils

Postby Hegeman » Fri Nov 13, 2020 9:44 am

Thank you for the great information!
I already have LG-Agar plates, they are ones you use to swipe different areas of the house. Will these work for the lawn too?
I love your idea of having the constant of one type of bacteria. I read through the material on how to do it and am unsure of the process.
Do you put a cup in boiling water to sterilize, remove the cup with the water and let the water cool.
While it is cooling, put a couple of drops of the bacteria on the agar plate.
Once the water is cool (not sure-room temp?), take tongs with a cotton ball and dip it in the water and rub it around on
the agar plate that has the drops of bacteria?

It seems to me the cotton ball would soak up too much water and then the agar plates would be soaked. Am I misunderstanding something?

With this bacteria, do you have to use it right away when I order it? (I want to time it right)
How long does it take to grow a lawn? Do I have to have some sort of "heating lamp" to grow it?
If so, what type of lighting should he use? I found this on Amazon...is the lighting something like this? https://www.amazon.com/gp/product/B08CZ3SNZ1/ref=ox_sc_act_title_1?smid=A2D918VL93QU47&psc=1

Also, what is the drawback of doing a drop with a pipet as opposed to using the small round paper and dipping them in the oil and placing it on the agar? (I'm just trying to keep it easy for his 1st time, but also really want him to see correct results.)

I read on the link you'd sent they are supposed to be stored upside down, so not sure how that would work with putting a drop of the oil on the agar, except to leave them right side up. Maybe use a heating pad with a towel between so it doesn't get too hot.

Would love your thoughts on all this too. Thank you!!

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Re: How to grow safe bacteria to test the effectiveness of essential oils

Postby probiotics » Fri Nov 13, 2020 10:42 am

Hi!

These are all excellent questions!

First off, do you mean LB-Agar? If, so, LB agar is a great growing medium for E. Coli! You should be able to easily grow a lawn of bacteria on the plates you currently have!

The science fair project link that I initially sent used dried E. coli, which is why there is a long process to get the bacteria ready.
However, Carolina Biological sells E.Coli suspended in a liquid medium. This means that you don't have to use water or go through a special sterilization process. Instead, you can plate it directly from the tube! However, considering that this sample is highly concentrated, you may want to dilute it by adding a few mL of distilled water (around 9 mL). Dried E.coli and liquid E. coli are both the same bacteria and will work in the same way.

However, if you do want to use dried E. coli, you can follow the steps in the project procedure! The only difference between your interpretation and the project procedure is that they use a sterile cotton swab, not a cotton ball. A cotton swab is like a q-tip, except it will come in sterile packaging. Since the cotton swab is much smaller than a cotton ball, you don't have to worry about soaking the agar plate! The cotton swab will only pick up a small amount of water.

With the liquid suspension of bacteria, it's best to use it as soon as possible. However, you should be able to keep 2-3 days safely after arrival. When you get the tube, loosen the cap and keep the tube upright. Keep the tube at room temperature, 68 to 73° F.

The length of time it takes to grow a lawn depends on the temperature that the agar plates are at. I anticipate that at room temp, the longest it would take would be two weeks, but the time may be shorter. For your incubator, all that you need is a thermometer, a styrofoam cooler, and a lightbulb! You don't need a grow lamp, as all we need from the lighting is heat. Therefore, it may be better to get a cheaper lightbulb, as those will probably heat up more. As long as the temperature isn't above 37 Celcius or below room temperature, your bacteria will grow!

One of the drawbacks of doing a drop with the pipet is that it is hard to see measureable results! There wouldn't be a specific zone of inhibition, and it is likely that the oil would spread across the plate. Therefore, you may see an overall reduction, but getting numerical data (such as radius of zone of inhibition) would be difficult. In addition, it would be difficult to compare the different essential oils, since there isn't a constant measure of efficiency. Agar plates are also usually stored upside down as you mentioned!

I hope this helps!

Thanks, Probiotics

Hegeman
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Re: How to grow safe bacteria to test the effectiveness of essential oils

Postby Hegeman » Mon Nov 23, 2020 12:36 pm

Again, thanks for the guidance!
We like your idea of using sterile disks for the Kirby-Bauer disk-diffusion method. Thank you for the very helpful information.

We have run into a few more questions.
What type of paper disks should we get. I am unfamiliar with this product. What I am finding is incredibly expensive for the small ones in a vial. The ones I think we can use are a little better in price, but way too big for our use, I mean bigger than the agar plate itself. Are there other options, less expensive that would still be sterile? The only thing I could think is to use the bigger ones, sterilize my hole punch, and make them smaller. lol. I'm not sure this would introduce bacteria. I am really wondering if there is something I am missing as far as price, or if I'm looking for the wrong thing. All suggestions are welcome.

Just now saw these, are these it?:
https://www.carolina.com/microbiology-s ... erile+disk

Also, he and his dad built an incubator, yay. We have read multiple experiments, is the bacteria supposed to be at 90-98.6 degrees Fahrenheit? And are the agar plates ok in the incubator by themselves, Or should we put them in a plastic bag too for extra protection? Or use saran wrap for extra protection?
We were thinking ahead about a workspace to be sterile. Any suggestions for doing it at home? Maybe a plastic serving tray we can wipes down before we start with disinfecting wipes? or alcohol?

One more thing,
For the agar plates, we brainstormed how to mark them so we can do this experiment twice. We have 20 disks. We came up with three cenarios:
1. one agar plate be the control, one plate have just oil (to be sure the oil doesn't produce bacteria), then the other plates divide into 1/4 pie sections with sharpie on the back of the bottom. each section marked with an individual oil he chose to use.
- this would let him choose 4 oils
- it would allow him to have 4 results from the same oils
- and it would allow him to do his testing 3 times. (one being someone following his procedure, which is required.) (This would give a total of 12 results from each oil)

2. one agar plate be divided in 1/2 with the left side as the control, the right side with just oil (to be sure the oil doesn't produce bacteria), then the other plates divide into 1/2 sections with a sharpie on the back of the bottom of each section, the same oil in both sections per plate.
- this would let him choose 5 oils to test
- this would give him 2 results from each oil
- and allow him to test 3 times too (so 6 results altogether)

3. The last option is to have one agar plate as the control, another place be with just oil (to be sure the oil doesn't produce bacteria), then use 1 plate per oil, the plate marked on the bottom with oil used.
- this would let him choose 5 oils to test
- this would give him 1 results from each oil
- and allow him to test 2 times (so 2 results altogether)

What are your thoughts?
Is it better to have more results? so you know your experiment is verifiably consistent? or are we overthinking. We like a portion of the last example we came up with because we could potentially add another disk and see if the potency of the oil would change the outcome, but that may change his goal. So, trying to keep it simple.

Thanks again for helping! We really do appreciate it. He is looking forward to getting started and doing it after all this prep we have done and advice your have given!
Truly...thank you every time!

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Re: How to grow safe bacteria to test the effectiveness of essential oils

Postby probiotics » Mon Nov 23, 2020 1:37 pm

Hi!

I'm glad to hear that he's had some fun building an incubator! It's often interesting to see how you repurpose everyday materials for scientific uses!

Regarding temperature, the optimal temperature for E.Coli to be incubated at is 37 degrees Celcius, which is 98.6 degrees Fahrenheit. The range of 90-98.6 degrees Fahrenheit would be a perfect range for E. Coli to be incubated at! A common problem that people encounter when working with agar plates is condensation on the upper lid. This is actually why scientists advise storing agar plates upside down so that the condensation will not pool on the surface of the bacteria. However, since you are using pre-poured plates, I don't anticipate this being a large problem.

The opposite problem can also occur, where the agar dries out in the incubator. In the lab, scientists use this special type of clingfilm called parafilm to wrap their agar plates. If you want to see how it works, here's a short video of it being used to wrap agar plates! Parafilm is used because it creates a seal where it keeps the moisture in but allows for the exchange of oxygen.

https://www.youtube.com/watch?v=f47Hq2uiJIs

However, saranwrap/clingfilm can also be used for the same purpose! Just cut a strip, and wrap it around the side of the agar plate like done in the video.

The link you provided for the paper disks is exactly what you need! I think a few of the options you may have encountered were either sold in bulk or were already coated with specific antibiotics, which is why they were so expensive. Here's another link for the same item, however, it may be easier to just buy everything from the same supplier (Carolina Biological). I had a friend who used the Kirby-Baur method for her project, and actually used a hole punch and coffee filter, so your idea isn't that far off! However, I doubt it'd be sterile enough, so I recommend going with either of the links.

https://www.homesciencetools.com/product/sterile-sensitivity-disks-50?refSrc=12479&nosto=nosto-page-product1#description-anchor

For a sterile workspace at home, I like your idea of using a plastic serving tray and wiping it down prior to experimentation. Either alcohol or a disinfecting wipe could work. You could also get a plastic trash bag or table cloth, and lay it under your workspace to make sure that you don't contaminate anything. It also makes for easier cleanup! You would also need a pair of gloves for everyone working with the bacteria, as well as a sterile tweezer to pick up the paper disks. You don't need to buy a new tweezer - you can just soak a tweezer you already have in some rubbing alchohol for around 15 minutes to sterilize them. The last thing I can think of right now is sterile cotton swabs, to plate the initial lawn of bacteria.

I like the options you have for plating and trials! The control for this experiment would be an empty paper disk, without any extra oil. The control must be tested on a plate that has bacteria so that you can see how much bacteria is killed without any oils added. In the link I provided in the initial reply, a control quadrant is included in every plate. I don't think that you need a control for just oil, as we are testing bacterial death rather than bacterial growth. Given these statements, here is a plan that I think could work well:

Split each plate into quadrants, leaving you with 80 different quadrants/trials. Save 20 of these quadrants as control quadrants on each plate, leaving you with 60 trials. Given that you are testing 5 oils, you can have 12 trials for each oil. If you want to test 4 oils, you can have 15 trials each. You do not need to plate the same oil in the remaining three quadrants of a plate. You can combine different oils within the same plate. What is the diameter of the plate you have? Normally, plates are around 90 mm in diameter. If you have significantly smaller plates, it may be difficult to split into quadrants, and splitting them in half would be the better option.

Having more trials is always a better option! The more trials you have, the more reliable results you can achieve. I think that the number of trials above for both 5 or 4 oils is more than adequate! I'm really enjoying seeing this project take form, and I hope that he gets to have some fun and learn more about science! Let me know if you have any more questions!

Thanks, probiotics

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Re: How to grow safe bacteria to test the effectiveness of essential oils

Postby cnoonan180 » Sat Nov 28, 2020 8:17 pm

Hello!

Here is some more information about the types of agar and how the different types are used in science fair projects:

https://www.sciencebuddies.org/science- ... %20rows%20

Additionally, here is another forum about how a safe bacteria solution can be made for growing bacteria on agar plates:
viewtopic.php?t=21654

For storing bacteria on an LB agar plate, a home refrigerator should have a temperature cold enough for a few weeks' time (4 degrees Celcius or about 39 degrees Fahrenheit). Make sure to keep the bacteria away from food, and be safe when handling organisms like bacteria, taking special care to make sure bacteria does not come in contact with food items!

Hope this helps!

Hegeman
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Re: How to grow safe bacteria to test the effectiveness of essential oils

Postby Hegeman » Tue Dec 01, 2020 10:32 pm

Hello again,
So we came across a few more things as we were typing out his procedure to follow to do his experiment. And had a couple of questions for clarification about your previous post.

INCUBATOR ??
We had a question about the incubator. We finally found a low enough wattage for the temp to stay at a constant 95 degrees and can turn it up with our dimming switch if needed.

The model of incubator we mimicked shows cutting holes in the top sides of the box. I imagine this is to let some of the heat escape so the correct temperature could be reached without going over. We went with a lower wattage bulb and an enclosed box the box at the right temp.

Will having an enclosed box dry out the agar if we do this?
Is there anything else we should be concerned about if we use it as is, enclosed?

We were thinking with our enclosed box, the E-coli bacteria would be contained and not "spread". And it would keep out any other bacteria, keeping the environment around it clean. (No cross-contamination of bacteria)


HUMIDITY?
The plan we used also gives instructions to put a silicone tray in the bottom to keep the humidity constant, so it won't dry out. We put little plastic Tupperware cups about 2"x1" in the bottom and covered it with a drawer liner. This will allow him to put a little water in the bottom of the box as he needs to keep the humidity constant. Something I have not read or seen is what percent humidity bacteria grows best in. We don't want too much - that it would create condensation, but don't want the plates to dry out either.

Do you have a link to something with this info?

PLATING?
I think we understand what you mean by plating, but is this what you mean?
We put "C" in the upper left corner of each agar plate. (10 plates) (The Control would have one sterile disc without oil in it)
We put "CL" for clove on the remaining quadrants of two plates.
We put "TT" for teatree on the remaining quadrants of two more plates.
We put "OR" for oregano on the remaining quadrants of two more plates.
We put "EU" for Eucalyptus on the remaining quadrants of two more plates, and then
We put "LG" for Lemongrass on the remaining quadrants of two more plates.
This allows us to do 5 oils and get 6 tests per oil, and then have enough to run one more experiment. with the same amount of result. Ending in double results when finished.

OVERLAPPING OILS?
We understood about being able to put different oils in the same plate but were concerned the oil may spread into other quadrants and mess up the experiment. Or we wouldn't be able to get a good read if the zone of inhibition was too large.

MULTIPLE DISCS?
What are your thoughts about adding another sterile disc with oil, and then another, after the inhibition of the bacteria stops, to see if potency would make a difference in the "death" of the bacteria? (This seems like another hypothesis testing strength of essential oil...is it? Or could it just be an added question to answer in our experiment? We don't want it too complex, but might be interesting to see)

SARAN WRAP?
So we can use saran wrap/clingfilm. Can we also use a zip lock bag? Or I have also seen tape used on the side, I think they said not the put it all the way around, just enough to hold the lid in place. Is tape ok?

EXPERIMENT?
With the experiment "The End Zone: Measuring Antimicrobial Effectiveness with Zones of Inhibition"
when applying the bacteria to the agar plates, it says to wait 5 min to let the bacteria dry? Then add the paper disc with the oil to the center of the quadrant. And then put it in the incubator and observe. We were thinking of growing the bacteria to a lawn first, and then after the lawn is grown, adding the paper disc and watching to see the "death". Is either way ok? Or is there a reason one way is better over the other?

Thanks for the link for the paper disks and the idea for a clean workspace. It reminded me of some black plastic sheets I have that I use for art with my daughter. We are going to use that instead. =) We also have art gloves we use too, but maybe we need to get sterile ones? I just don't see how you keep them sterile after you open the box. Do you?

Would using alcohol wipes to wipe off the forceps in between each oil work for disinfecting them and not getting mixed oils?

For the Sterile cotton swabs, we saw an experiment that used an "L" spreader to spread the bacteria. It looked much easier and like it would come out more consistent, so we got those. Is there something else we would need cotton swabs for? I believe we have a few that came with our kit (maybe 6)


The diameter of the agar plates we have is the standard size, I think 90mm.

Thank you again for all your help....we really appreciate it!!

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Re: How to grow safe bacteria to test the effectiveness of essential oils

Postby probiotics » Wed Dec 02, 2020 9:48 pm

Hi!

There are all great questions! Let me try to answer as best I can.

Incubator: The dimming switch sounds like a great idea! I've looked over a few DIY incubator ideas, and I've seen mostly ones without holes! I think having an enclosed incubator will work great considering professional incubators are usually sealed! It will help create a more contained environment!

Humidity: I haven't been able to find something specific on the best humidity levels for E. Coli. I think what could be the best method to prevent condensation is to allow the bacteria to grow without the water cups, and if you see any slight drying of the agar plates, then you can add in the water cups as needed.This way, you wouldn't need to worry about extra condensation. I think the issue of dried out plates won't be as prevalent with agar plates that are bought pre-poured.

Plating: Yes, that is what I mean! I think the labels are a clever way to keep track of everything! The control also sounds good!

Overlapping Oils: Based on what I've seen with past projects with a similar theme, the zones of inhibition will not expand past the quadrant where the discs are. Therefore, it wouldn't be a problem to overlap oils within the same plate. However, you can also plate the same oils on the same plate for peace of mind! Either option works. Considering that your plate size is 90 mm, it would be large enough for separate quadrants.

Multiple Discs: This is a new idea, I've never heard of this before! It would add another variable to your experiment (potency). Will you be adding the disc on top of a previously plated disc with an established zone of inhibition? If so, the starting conditions for your 1x potency and your 2x potency will be different, which could make it difficult for a direct comparison. Nonetheless, it wouldn't take much extra work, and these potential sources of error can be noted in the science fair report. I think it'd be a cool idea to investigate!

Tape/Saran Warp: Tape can also work! Make sure to leave a gap so that oxygen can enter and exit the plate, which affects the growth of the bacteria. Saran Warp, which can be wrapped around the entirety of the plate, may be better since it would keep the humidity in and would still allow for oxygen to enter and exit the plate. If you have access to Saran Wrap, I would go with this choice.

Experiment: Both methods work, but I'd go with growing the lawn first. This gives you the peace of mind that the bacteria is already growing, and you don't have to worry about the initial bacteria not growing. The zone of inhibition will be comparable for both methods! Also, art gloves will work fine since you won't directly be touching the bacteria. If you are using an alcohol wipe, make sure that it is completely dry for the next use since we don't want the alcohol to end up on a disc and affect the results. L spreaders work great! You wouldn't need sterile swabs in that case.

Hope this helps! Let me know if you need any clarification!

Thanks, Probiotics


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