Natural Antimicrobials against Bacteria

[deleted-140482]

Hi Anthony,

For plate 2 you would add the solvent directly, without dilution in order to control for that in your dilution experiments. In other words, if you intend to dilute your essential oils in water, then you would use a disk treated only with water. If you are diluting your essential oils in a carrier oil then you would use a disk treated with that carrier oil. This serves as a control so you can attribute any inhibition in growth to your essential oils and not what they are diluted in. If it turns out that your solvent does have MIC/MBC you can absolutely continue with the experiment. By performing the control we just discussed (i.e. disk treated only with solvent) you have all of the information you need to determine how much of the inhibition you see is due to the essential oils vs. your solvent. You can just subtract the amount of inhibition seen with your solvent from the amount seen with your essential oils and thus determine the amount of inhibition that is due to your essential oil.

I think using 3 disks at each concentration on the same dish would be considered testing multiple times.

I don't know how many disks you'll be able to fit on the petri dish, because it will depend on the size of your petri dish and the size of your disks. I've seen people do upwards of 10 disks on a plate, but that may not work for your sizes. You'll have to see how may disks will fit on a petri dish with a good amount of space around them for the zone of inhibition. You don't want to put them so close together that their zones of inhibition overlap or you can't determine whether the inhibition is coming from one test or a different one.

As for the tubes v. the plates, I couldn't find what you were talking about on Carolina Biologicals website. From what I saw, you would buy the culture as a slant culture in a tube (this is basically agar in a tube that has some bacteria on it). Traditionally you would use this to inoculate a liquid culture and then plate from that liquid culture. I didn't see any pre-inoculated plates, but it's possible that I missed them, so if you want to post them on here with a link, one of the experts can take a look and let you know.

Good luck!
JMP

[deleted-160001]

Thanks JMP!
For control plates (one with dry disk, one with solvent disk), do we also need multiple tests? Or is one concentration disk on those two plates ok?
Here are the links for the plate vs. tube: (first link is to tube, second to plate)

http://www.carolina.com/bacteria/escher ... ve+e.+coli
http://www.carolina.com/bacteria/escher ... ve+e.+coli

Thanks!

[deleted-132180]

Hi Anthony,

Megidi and JMP already gave you some really excellent advice! I also have a couple of suggestions.

I think you have some great negative controls set up (the dry disk and the disk dipped in solvent only). Have you thought about adding a positive control? For example, something you know for sure has very strong antimicrobial activity to make sure that inhibition is occurring if you dip the disk in that particular compound and then stick it onto a plate with bacteria? If any of the papers in regards to the essential oils used a particular high concentration that totally inhibited bacterial growth, then I may use that same high concentration as my positive control, or you can choose any other compound known to have strong antimicrobial activity (but make sure it's soluble in the solvent that you're using to dilute your essential oils just to keep it consistent). This isn't necessary, but just a suggestion! Also, if you are going to do multiple tests for your different dilutions, I would probably do the same number of tests for your controls just to keep things consistent. Also, it's always a good idea to do multiple tests anyway because for all you know, the one disk that you put on your plate may be messed up and would give you results that you may not have anticipated, so just to be sure that your results are consistent, you should do multiple disks for your controls as well.

It's interesting that Carolina Biologicals sells E. coli on plates? I have never heard of vendors selling bacteria directly on nutrient agar plates before--I have only heard of the agar slants. I would probably go with the agar slants just to be safe since I don't exactly know what they mean by having the bacteria on plates, but you can always contact Carolina Biologicals and ask them about both products and see which they would recommend for your experiments.

You also mentioned that you have 5 essential oils. I would probably test disks of different oils on separate plates, because for all you know, if you put different oils on the same plate, they could potentially interfere or interact with each other and would make your results difficult to interpret. So to be safe, I would probably use separate plates. I know that means that you have to use A LOT of plates, but it's better to be sure that your setup is as clean as possible to make analyzing your results a lot less painful.

Hope that helped! Let us know if you have anymore questions!

Best,
Connie

[deleted-160001]

Thanks connie!

I'm a bit confused on the positive control concept. All of the oils I'm testing have already shown that they are effective in inhibiting E. coli in previous tests; I'm testing the concentration. So, would the positive control be an essential oil that is effective, but not going to be used in my project?
Yeah, the slants are probably safer to go with. I should add the steps for inoculation in my procedure as well, right? If I based my procedure off of the experiments on the Science Buddy website, should I cite it later in the works cited section?
I cut down the essential oils to 3, but if I followed your suggestion, that would mean that I would have 3 plates per oil, 9 in total because I'm testing 3 concentrations and 3 oils?
Thanks!

[deleted-132180]

Hi Anthony,

If you are testing oils that have already been shown to be antimicrobial, a positive control that is a different antimicrobial wouldn't be necessary. Just use a concentration of the essential oils that you're testing that you know has been shown to be inhibitory, and if you do see inhibition at that concentration, you would know that your experimental setup is working. And yes, you should add the steps for inoculation in your procedure and also cite Science Buddies as your resource. 3 plates per oil sounds good, but if a known inhibitory concentration of those oils isn't one of the ones you're going to test, you should probably add an additional plate for that for each oil just as a "positive control" to make sure that your experimental system is showing inhibition at a concentration known to be inhibitory. Also, don't forget your two other negative controls with the dry disks and the disks dipped in solvent only, so that's two other plates!

Let me know if there's any part of my explanation that you didn't understand, and if you have anymore questions!

Connie

[deleted-160001]

Thanks a ton everyone!

I have one quick question. I ordered the nutrient agar plates from Carolina Biological Supply (associated with Science Buddies) and apparently it was backordered.
Does anyone know how long this will take?
Thanks!

[deleted-140482]

Hi Anthony,

I think your best bet would be to call Carolina Biological and ask them when they expect to have the nutrient agar plates back in stock. I don't think anyone here will be able to tell you that.

JMP

[deleted-160001]

Ok thanks! I just received the E. Coli, and the box is labeled "perishable." I won't be able to start all that soon, so is there a proper storage method till then?

[deleted-140482]

I assume you ordered a slant of E. coli (i.e. a culture in a tube with agar). If that's the case, refrigerated, the E. coli should last for quite some time without a problem.

[deleted-160001]

Thanks JMP.
Do I need to "transfer broth culture to fresh broth" first or do I go directly to "streaking broth culture to fresh petri plate"?
Here's a link to the paper describing the sterile technique (from Science Buddies), but I'm not sure where I'm supposed to start.
I have the E. coli slants.
Thank you!

https://www.sciencebuddies.org/science- ... hnique.pdf

[deleted-140482]

Hi Anthony,

What you have in the E. coli slants is not a broth culture, but rather an agar culture (essentially a petri dish in a tube, instead of a dish), so you can't really start with either of the techniques you described, although the sterile technique is essentially the same. You could proceed in one of two ways. You can follow the protocol for streaking from one petri plate to a fresh petri plate. Instead of touching one colony on a plate (with your inoculating loop), you would contact the bacteria in your slant and use that to streak the fresh plate. Alternatively, and perhaps faster, you can use the bacteria in your slant to inoculate a broth culture. Just use the same sterile technique described in the paper from Science Buddies and touch your sterile loop first to the culture in your slant and then into your broth. Don't worry if you don't see anything visible on your inoculating loop. It doesn't take much bacteria to start a fresh culture, so as long as you have actually touched the colony, you'll be fine.

Hope this helps!
JMP

[deleted-160001]

So if I did the alternative method, I would make the inoculating loop contact the slant, then put it into the broth, then go from the broth to a petri dish?
The sterile broth would have to bought separately right?
Thanks!

[deleted-140482]

Exactly right on both counts. You would have to let the bacteria grow in the liquid culture (usually overnight) before transferring to a petri dish. And make sure when you contact your loop to the slant, you are touching where you can see bacteria and not just in the agar.

[deleted-160001]

oh ok thanks JMP!
The broth method would require a centrifuge right?
Since I don't have access to one, I would probably need to go with the petri dish to petri dish method.
When streaking using the four-quadrant streak method, is it one bacterial colony per quadrant?
And a new colony every time you flame the inoculating loop?

[deleted-132180]

Hi Anthony,

From what I know, the broth method wouldn't require a centrifuge. You can just inoculate the broth with a loop that you had touched your slant with and let your culture grow overnight. Since it's E. coli, it will grow pretty quickly and your broth culture should be very turbid the next day. You can then take a loop, dip it into your broth, and then swab it onto your plate to get bugs growing on there (anyone correct me if I'm missing something or if I'm wrong).

Also, for the four-quadrant streak method, it's not necessarily one bacterial colony per quadrant. What you do is you first take a loop and swab up some bugs from broth culture or a plate, and then streak the plate in one quadrant. Afterwards, you flame the loop, wait for it to cool, and run the loop through a bit of your first quadrant and expand onto a new quadrant. You repeat these steps until you've reached the fourth quadrant, and typically, your bugs are now "dilute" enough that they can grow up as single colonies, making it a lot easier for you to pick single colonies for your experiments. Here is an image depicting this method:

http://www.personal.psu.edu/faculty/k/h ... streak.gif

Your streaked-out plate should look something like this when your bugs grow up: http://0.tqn.com/w/experts/Biology-664/ ... E-Coli.jpg. Notice the single, isolated colonies on the left side!

Let us know if you have anymore questions.

Connie

[deleted-140482]

I'm with you Connie, no need for a centrifuge for the broth method. I'd do basically exactly what you described.
JMP

[deleted-160001]

Is there a certain broth and method for E. coli?
I'm not so sure if the broth can come on time, so the petri dish to petri dish method is still ok right?
Thank you so much for the quadrant streak method description!
Do I swab up more "bugs" every time after I finish streaking the first quadrant and flaming?
So swab, flame, cool, then swab with more bacteria again? Or swab, flame, cool, and swab without more bacteria?
Thanks!!!

[deleted-132180]

Hi Anthony,

People grow E. coli in regular Lb broth--super simple. If the broth can't come on time, then the petri dish to petri dish method is still fine. And yes, for the quadrant streak method, you swab up more bugs from your previous quadrant after flaming, and then streak that onto a new quadrant. The point is to dilute the bugs out enough so that you can isolate single colonies.

Here's a very helpful video if you still have trouble visualizing how to do it, although in your case, you would take your initial swab of bugs from a plate of E. coli you grew up instead of from a broth culture like in the video. http://www.youtube.com/watch?v=AaG3Pt3nwLQ

Hope that helped!

Connie

[deleted-160001]

Hello,

Thank you so much. That was extremely helpful.
I'm going to be testing to see if there is any resistance from the bacteria.
In one of the procedures on Science Buddies, it says to do the selection four times.
I'll be working at the school lab, so I don't want to take up the teacher's space the whole week.
How many times do you think would be acceptable enough to test for resistance?

Thanks

[deleted-132180]

Hi Anthony,

Which procedures are you looking at? Why don't you paste the link here so I can take a look and then we can discuss how many times you should test for resistance?

Best,
Connie

[deleted-160001]

Here's the procedure:
https://www.sciencebuddies.org/science- ... #procedure

I was reading the previous thread, and found that the person's experiment is similar to mine (https://www.sciencebuddies.org/science- ... 28&t=12920). I was getting kind of nervous, because I already ordered my bacteria (http://www.carolina.com/bacteria/escher ... ion=e+coli). Did I do something wrong? Would I not be able to perform the experiment by streaking?

Also, my friend mentioned that I might need media to help keep the bacteria alive and grow. Could someone help with this? I know if I do need it, I should use Lb media. I looked around Carolina Biological and found this (http://www.carolina.com/biotechnology-c ... tion=media). Are these any different than the ones I ordered? (http://www.carolina.com/prepared-biolog ... ?question=).

Thank you so much!

[deleted-132180]

Hi Anthony,

As far as I know, all of the things you have been doing are fine. As for the nutrient agar plates vs. Lb plates, you should probably contact Carolina Biologicals to ask them what the nutrient agar is actually composed of because I know on their website they don't really mention much. Traditionally, E. coli is grown on Lb plates, but for a lot of experiments on Science Buddies, it seems like many people use these nutrient agar plates though, so it should be okay. But like I mentioned, you should contact Carolina Biologicals and ask them if there's a difference between these two types of plates.

For your assay, it seems like you need to make plates with lawns on E. coli on them in order to do the disk diffusion test. In that case, I would follow the instructions in this video, which really nicely shows how you prepare plates for disk diffusion assays: http://www.youtube.com/watch?v=sx1uDYSfINA. Also, in that thread you sent me, SciB also posted a nice link to how to make bacterial lawns (https://www.sciencebuddies.org/science- ... #procedure).

The streaking method that I had mentioned to you is useful in isolating single colonies of E. coli, but not for actually generating lawns to do your experiments. You would need to take one of your single colonies and grow that up in liquid broth in order to make the lawns. What you would basically do is pick one of your isolated colonies, inoculate it into Lb broth, and let that culture grow overnight. The next morning, your culture would be super dense--you would then have to dilute that back down and wait for the culture to grow to what we call mid-log phase. To backup a little, bacterial growth can be characterized into several phases: 1. lag phase, 2. log phase, 3. stationary phase, 4. death phase. Lag phase is when the bugs are still adapting to their environment, maturing, and are not ready to divide yet. Log phase is when the bugs are starting to divide. Stationary phase is when the nutrient media is starting to become depleted of a certain nutrient that the bacteria needs, and so you would stop seeing bacterial cell doubling like you would in lag phase. Finally, death phase is when the bacteria run out of nutrients and they start dying. Hence, your overnight culture, which would be super dense, is probably in stationary phase. What you want to do is to dilute that culture in fresh Lb broth to replenish the nutrients, and let the bugs get back to mid-log phase, which is when they are healthy. Usually, an OD600 of 0.5 is indicative of E. coli being at mid-log phase--do you know what OD's are and have you had any experience in measuring them? If you have access to any machine that can measure the OD for you, that would be awesome, but if you don't, you can always estimate. Let me know what materials you have access to and we can figure out how you can do this. So once your bacterial liquid culture is in mid-log phase, you can then follow the video by aliquoting a constant volume of bugs onto each of your plates (this is to make sure that all of your plates have the same number of bugs, at least as similar as possible), spread the bugs out to create a lawn, and then do your disk diffusion assays.

As for the selection of resistant bacteria portion, I'm not exactly quite sure why four times was specifically instructed. I haven't actually done disk diffusion assays myself (I've always done antibiotic resistance assays in another manner), so I don't know whether the selection step is necessary or not or what the purpose of that is. Other experts, if you do know, please help!

If I mentioned anything incorrectly or if other experts have any other advice, please chime in!

I hope this is helpful!

Best,
Connie

[deleted-160001]

Wow, that is much more complicated than i thought.
Tell me if I got this correct:

Swab up some bugs from the culture tube, make contact with some media (how much?), have it grow overnight, then dilute it (how?), and wait for it to grow to mid-log (I don't have a machine to measure), and then inoculate the broth to the plate? Is this done through the quadrant streaking method? Does the bacteria grow to become isolated colonies? Because if not, I'm not sure how I'm supposed to test for resistance

I would appreciate fast replies (thank you connie and others who have replied), because i need to finish before the second week of February.

[deleted-132180]

Hi Anthony,

If you want to follow the procedures as in the link you sent me, you are going to be testing for resistance to essential oils using the disk diffusion method. This method is described very nicely in this wikipedia article (http://en.wikipedia.org/wiki/Agar_diffusion_test), but to quickly give you an idea, the principle of this technique is to use small disks that contain your antimicrobial compound (in your case, the essential oils) and see if they kill bacteria. The way this works is that you need to have a lawn of bacteria on an agar plate (basically, your entire plate is uniformly covered with bacteria like a blanket, with no isolated single colonies). You will put your disks containing the essential oils onto your lawn of bacteria. If your bacteria are susceptible to that essential oil, then you'll see an area of clearing around the disk where the bacteria aren't capable of growing (this is called a zone of inhibition). It should look something like this (http://upload.wikimedia.org/wikipedia/c ... B_test.jpg). So far so good?

So... once you see the zones of inhibition show up, you would then measure the diameter of these zones. According to the Science Buddies procedures, the bugs would be resistant to your compound if the zone of inhibition's diameter is 10 mm or less, whereas if the zone of inhibition's diameter is 16 mm or greater, that means the bugs are susceptible to your compound.

So the most important step is... how do you grow up these bacterial lawns? First of all, you would need to get your hands on some E. coli. Like you said, the bugs you ordered come in the slanted agar. What JMP and I had mentioned to you before is that you can touch the bacteria in the slanted agar with a loop, and then use the quadrant streaking method to streak the bugs out onto a plate. Your plate should look like so: http://www.personal.psu.edu/faculty/k/h ... streak.gif. The only purpose of doing this is because you want to isolate a single colony that you will use for your experiments. The reason why we like using single bacterial colonies for experiments is because when you further grow this colony up, you will know that it has come from a single E. coli cell and you will get to test the exact same cells with different conditions, which allows you to compare the behavior of the bugs in these different conditions. So far so good?

However, like I mentioned, you need to grow your bugs in broth conditions in order to make the bacterial lawns. I realize that this may be a little complicated given the limited resources that you may have in your high school lab, but if you want to make things simpler, you can definitely order the E. coli liquid broth culture from Carolina Biologicals. This will basically send you some bacteria in liquid culture. I do realize that you did order your bugs in an agar slant already... so you can call Carolina Biologicals to see if you can switch this product with the liquid form. If not, you can always just grow your own liquid cultures... or if you'd like, purchase the liquid culture of E. coli. I would call Carolina Biologicals if this were the case to confirm that the liquid culture you're ordering will be good for disk diffusion assays.

So... if you're sticking with the agar slant bugs, this is what I would do next. After you streak your bugs out and get single colonies, I would take a loop to pick one of your E. coli colonies and swab that into some volume of Lb broth. Whatever volume you use will depend on how many plates you need to make into lawns. If I recall correctly, you said you have 3 essential oils that you want to test at 3 different concentrations, and you will have two negative control plates (one for dry disks and the other for disks dipped into solvent only), so that's 11 plates total? I would probably take the loop that you swabbed your E. coli colony with and inoculate about 10 mL of Lb broth with that. I would then let that culture grow overnight at 37 degrees Celsius, shaking overnight (an overnight culture is typically 13-15 hours). Does your high school lab have a shaker and warm room available for you to grow E. coli broth cultures? If not, you can always make your own incubator (http://www.umsl.edu/~microbes/pdf/Incubator.pdf). After you let your bugs grow overnight, the culture should be pretty dense and turbid, and the bugs will be in stationary phase, like I had mentioned. The bugs have basically stalled and aren't really doubling anymore overtime. What I would then do is probably dilute that culture 1:10 (that is, take 1 mL of your overnight culture and put that into 9 mL of fresh Lb broth). Now, your culture is not as dense as before, AND you have fresh nutrients so your bugs will start growing and be healthy again. I would then let this culture grow at 37 degrees Celsius until mid-log phase, which should take about 4-5 hours. It is EXTREMELY important that you are using healthy, growing bugs for these antimicrobial assays, because if you use bugs in their death phase, how do you know whether a lack of growth is due to your compound or if your bugs are all just dead? If you decide to get the liquid broth cultures from Carolina Biologicals instead, we can talk about how you can go about prepping those. Nonetheless, this link has some information on the same exact method, and if you take a look at it, I think it will give you lots of help (https://www.sciencebuddies.org/science- ... =28&t=4508).

After you get your mid-log phase liquid culture, I would take a sterile pipette and put equal volumes of the culture onto each of your plates (0.5 mL each should be good). Then, I would use a spreader (here's a link to how to make one out of a paper clip and instructions on how to spread out a lawn: https://www.sciencebuddies.org/science- ... #procedure) and spread an even lawn of bacteria on each of your plates. I would then let the plates dry, and then I will put your disks onto these lawns. If I recall correctly, you mentioned that you wanted to use 3 disks for each condition to basically equal doing the experiment multiple times. Make sure that these disks are firmly attached to the agar. Then, I will incubate these plates overnight at 37 degrees C, and the next day, you should see zones of inhibition if your compounds do have some sort of antibacterial effect. Then, you can measure the diameters of the zones of inhibition to see if your bugs are resistant to the essential oils at specific concentrations or not!

I hope this explanation clears things up and is helpful. Other experts, if you have additional advice or any corrections, please share your thoughts.

Let us know if you have any more questions. I hope that video I sent you in the previous reply will give you more of a visual aid of how to prep the plates for this assay.

Good luck! I know that the procedures may have been a little more complicated than you had expected, but it will be totally worth it when you get some really cool data in the end--you'll be able to find out whether the essential oils you picked are actually antimicrobial! If everything goes well, this experiment shouldn't take you more than 2-3 days (most of the time will be spent waiting for the bugs to grow up overnight!).

Connie

[deleted-160001]

Thank you.

Would this be the correct broth? http://www.carolina.com/biotechnology-c ... n=lb+broth

Did I understand this correctly?:
Day 1: Go to lab, swab up bacteria, put into 10 mL broth, put into incubator
Day 2: Go to lab, get 1 mL of overnight, mix with 9 mL of new, put into incubator
Day 3: Go to lab, get dilute broth, pipette (?) mL onto agar plate, use spreader, put in disks, wait for drying, incubate
Day 4: Go to lab, take out, measure, done?
Day 5: Any way to test for resistance?

OR

Day 1: Go to lab, get out E. coli in broth form, pipette into agar plate, use spreader, put in disks, wait for drying, incubate
(or do I need to refresh the broth that I receive?)
Day 2: Any way to test for resistance?

Also, you mentioned that to get from the stationary phase to mid log, it takes around 4-5 hours. I wouldn't be able to stay that long at the school, so would I be able to bring it home? And you said that the broth needs to be shaken in order for agitation. Does the incubator do that for you?

It certainly looks easier to get the E. coli in broth form in the first place...I will see if I can change my order.
I would love to know where Essential Oil Girl got hers...(https://www.sciencebuddies.org/science- ... =28&t=4508)

Thanks!

[yvetteds]

Hi -
I found this explanation to answer the question - what is the difference between plant extracts and volatile oils - hope it helps.

http://bubbleandbee.blogspot.com/2013/0 ... racts.html

good luck!

[deleted-132180]

Hi Anthony,

That broth does look correct, but again, I would call Carolina Biologicals just to make sure. About purchasing the bacteria in liquid culture, I'm slightly concerned that when the bacteria arrive, they will already be in late log to death phase (it depends really, you would have to ask Carolina Biologicals how they will ship this product and in what state the bacteria should be when they arrive)... in that case, you still would have to supplement that with fresh broth and let it grow overnight so the culture can recover.

The first set of procedures that you mentioned to me sounds correct, except for on Day 2, you would wait 4-5 hours, and then pipette the bacteria on the agar plates. However, I do know that you have restrictions on to how long you can stay at school, so what I would do is probably just use your overnight cultures (which should be in late log phase) to pipette onto your agar plates... Like I mentioned earlier, take a look at this page: viewtopic.php?f=28&t=4508. I think it will give you lots of help in what they did with their bacteria.

So... a brief schematic on what you would do...

Day 1: Go to lab, swab up bacteria, put into 10 mL broth, put into incubator to grow overnight (12-15 hours). Yes, it would be nice if you can grow the bacteria shaking... you would need a shaker to do that, so do you know if your high school teacher has that available? If you have to build your own incubator, the shaking is not available for that specific protocol I sent you.

Day 2: Go to lab, pipette 0.5 mL onto each agar plate, use spreader to spread liquid uniformly over plate, let the plates dry, put the disks onto the plates (make sure they firmly attach to the agar), put plates in incubator. In the case of plates, you don't need to grow them shaking--only for the liquid culture you do.

Day 3: Go to lab, take out plates, measure diameters zones of inhibition, use measurement parameters provided in procedures to determine if the bacteria are resistant to susceptible, done.

Let us know if you have more questions.

Connie

[deleted-160001]

If my teacher doesnt have an incubator that shakes, is that a huge problem?
And is there a way to test for resistance?

I think once these two questions are solved, I'll be good to go!

[deleted-132180]

Hi Anthony,

I have personally never grown E. coli liquid cultures without shaking them before, so I don't know how they would grow without shaking. Perhaps you can try a quick trial of swabbing some bacteria in broth and growing them in the incubator overnight and see if you have a more turbid culture the next morning (indicating growth). Since E. coli grows pretty well, I would assume that they can probably grow without shaking, but it'll probably be slower? Other experts, if I am wrong, please correct me.

As for testing for resistance, I mentioned before that that is the purpose of the disk diffusion assay. Once you see zones of inhibition show up on your plates, you can measure their diameters. Like I said before, the zones of inhibition around your antimicrobial disk are there because they are areas that contain enough of your antimicrobial substance that it's not allowing any bacterial growth around it. If the diameter is above a certain length (check Science Buddies procedure link you sent me), that means that the bacteria are SUSCEPTIBLE to your antimicrobial agent. However, if the diameter is below a certain length, that means that your bacteria are RESISTANT to that antimicrobial at whatever concentration you tested it. Did you have something else in mind to test for resistance? Is there something that I didn't explain very clearly? Let me know!

Best,
Connie

[deleted-160001]

Oh i meant to test to see if the bacteria DEVELOP resistance, like after repeated exposure to the essential oils.
Without the shaking, would it be okay to take the broth out after 24 hrs? Or should i wait more?