How to test for anti-bacterial and drug resistance properties of Curcumin

[CaptainHumanLogic]

Hello! I am a freshman in high school conducting an experiment to try to create an anti-bacterial ointment using curcumin with an inactive ingredient to make it in a cream or liquid form. I want to first test how well curcumin kills bacteria as well as if bacteria don't become resistant to curcumin over time. Finally, I will try some inactive ingredients with curcumin and see if the cream or liquid produces the same results. However, I have not worked with bacteria before and I'm unsure how my design of the experiment is. So, I wanted to see if my experiment is sound and then I had a few questions afterward:
My experiment will start with preparing the petri dish with a small 0.5-centimeter diameter hole in the middle of the top. Then I will grow 3 types of bacteria (E. Coli, Staphylococcus, and Streptococcus) in agar in a Petri dish. Then, after 24-48 hours, I will put Curcumin through the hole and wait for some time, another 24-48 hours, I will find the zone of inhibition with the ring created (Kirby-Bauer Method). I will reuse the dishes to test for the next generations of bacteria to see if they develop any resistance to the curcumin. I will test around 10-12 Petri dishes to see with testing to the 4th or 5th generation.
1) Is my experiment OK?
2) Is it OK to reuse a bacteria colony in the same petri dish?
3) How visble are the rings created in the Kirby-Bauer Method and is that a good measure of inhibition?

[SciB]

Hi and welcome to the Scibuddies project help forum.

Curcumin (Ccm) has been in the news a lot over the past several years because of its powerful antioxidant, antibiotic, anti-inflammatory and anticancer properties. One big problem with using Ccm therapeutically is that it is a molecule that is almost insoluble in water. This means that Ccm may not be available in sufficient concentration to have an effect on bacteria.

Many scientists have struggled to chemically modify Ccm to make it more water-soluble or encapsulate it inside a nanoparticle that is water-soluble, without losing its activity. There is a form of Ccm called BCM-95 that has been patented I believe and is supposed to be more absorbable than unmodified Ccm: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792534/

I found what I think is a really good description of the Kirby-Bauer antibiotic disk diffusion method: http://www.asmscience.org/content/educa ... tocol.3189

Note that the K-B method uses a kind of agar called Mueller-Hinton. Other kinds of agar may be used, but I would use the recommended type if you can. Carolina Biologicals sells prepared M-H agar plates: https://www.carolina.com/microbiology-s ... /822922.pr

I see by your description of the method that you planned to create a well in the agar into which you put the Ccm preparation. The standard K-B diffusion procedure uses 6 mm filter paper disks placed on the agar surface. The disks contain the test chemical which diffuses into the agar and, if it inhibits the growth of the bacteria on the plate, creates a clear zone around the disk that is called the zone of inhibition.

While you can use a well for containing the antibiotic, I don't see any advantage to doing it that way. How will you make 5 mm diameter, exact-sized holes in the agar?

To get back to the problem of insolubility of Ccm, you can test some of the brands that are supposed to have enhanced water solubility and see if they actually do inhibit bacterial growth in an area next to where they are placed on or in the agar. The problem with Ccm is its lack of solubility in water which will prevent diffusion. The bacteria cannot be affected by the Ccm if it does not reach them. You will have to do some experimenting with different brands and amounts of Ccm to see if any of them inhibit bacteria. Remember that you need at least two controls--one that is a disk with only sterile saline (blank) and one that contains a known antiseptic like Bactine (positive).

You said you will test Ccm against Staph and Strep bacteria. These are human pathogens and must be handled under BSL-2 safety conditions. Check with your teacher whether your high school lab is equipped for such experiments: https://www.sciencebuddies.org/science- ... egulations

If you use only E. coli K12, a harmless strain, then you can do experiments under BSL-1 conditions. You will also need an incubator for the agar plates that can be set to a temperature of 35 degrees centigrade.

If you haven't looked at this project on bacterial resistance, you should: https://www.sciencebuddies.org/science- ... resistance

Your project is doable provided you can work out the details. Since you haven't worked with bacteria before I suggest you get someone to show you how to use basic aseptic techniques. If you do a search for how to work with E coli on Youtube you will find many helpful videos. Watch a few of those and you will have a much better idea of how to set up and perform the experiments.

Please post again and we will continue to help you plan the experiments and see that they give you statistically useful data.

Sybee

[CaptainHumanLogic]

Hello! Thank you for the advice with my experiment! But however, I still have a few questions:
My experiment is more focused towards creating a liquid antibiotic ointment made with curcumin with some other inactive ingredient, however, I am mostly considering using an oil for my inactive ingredient. Some oils in mind are coconut oil, olive oil, and tea tree oil. I have read that curcumin is actually fat-soluble (http://lpi.oregonstate.edu/mic/dietary- ... s/curcumin). Since my original intent was to test for the ointment, I thought it would be important to test for the antibiotic property of curcumin in isolation, and then later with the inactive ingredient mixed with curcumin.
1) So, then could I test Curcumin with the inactive ingredient to the begin with? Would that make my results valid? How do I confirm that the oils are inactive without testing curcumin separate?
2) I see how film paper is better then what I thought, but I am still confused on how to use it. How will the film paper effectively diffuse the solution? So could you tell me in detail how it will work in the experiment, just to make sure that when I use it, I use it correctly?
3)I realized that E. Coli is in a different phylum and class than both Staphylococcus and Streptococcus. So, will E. Coli be a good test on Curcumin's ability to kill bacteria on the skin?
Thanks Again,
Captain Human Logic

[SciB]

Hi,

You are right that curcumin (Ccm) is much more soluble in oil than water and that is the problem. The Kirby-Bauer (K-B) antibacterial test depends on diffusion of the test substance into the agar. Ccm or oil will not diffuse into the agar. That is why I suggested using the modified form of Ccm that is supposed to be more soluble in water.

Did you go to Youtube and watch a video of how the K-B test is done? (https://www.youtube.com/results?search_ ... usion+test)

I think that would help answer a lot of your questions. The bacteria in a liquid suspension are spread onto the surface of the agar and then filter (not 'film') paper disks wet with saline (negative control) or test substances are placed on top of the bacteria on the agar. The plates are then put into an incubator at 35 degrees centigrade for 24 to 48 hours. The bacteria will grow under these conditions unless they are killed by the test substance in the filter paper. This killing (or inhibition of growth) is seen as a clear zone around the paper disk where the substance has diffused into the agar. Now do you understand why there is a problem with testing a substance like Ccm that is not water-soluble?

I suggest that you concentrate on testing Ccm by itself first. I know the goal is to make an antibacterial skin cream, but first you have to demonstrate that your preparation of Ccm kills or inhibits bacteria.

The harmless E. coli K12 strain that you can buy from Carolina Biologicals is perfectly acceptable as a test bacterium for Ccm.

[CaptainHumanLogic]

Hello!

First I would like to thank you for all the help you have given me with my project. I would have failed without your help. Thank you!

I bought Curcumin-95 from Jarrow Formulas and tested it along with normal Turmeric. As you can see from the image, it seems it hasn't formed a solution for 5 days. Do you think, by the picture, I should use Turmeric or should I go with Curcumin 95? If not either, is there another form or brand of Curcumin I can use?
https://drive.google.com/file/d/0B1R2Ra ... sp=sharing

Also, for testing for the resistance of Bacteria to Curcumin, should I reuse the same petri dish, or should I take a swab of bacteria that was resistant?

Finally, I read some of this paper that used Kirby Bauer with different oils successfully. So, if Curcumin is oil-soluble, then can I use it for the test? https://drive.google.com/file/d/0B_yLOM ... sp=sharing

Thanks,
Captain Human Logic

[SciB]

Hi,

I'm glad my answers were helpful to you. Hope I can keep steering your experiments in the right path so you can get some good data.

As you found out from doing the experiment, neither curcumin nor turmeric will dissolve in water. In Indian cooking where turmeric is used there is always oil added and the turmeric dissolves in that. I did a search for how to dissolve Ccm and found one reference where it was dissolved in a variety of food oils: https://margaret.healthblogs.org/2007/1 ... -curcumin/

You should be able to find a suitable oil to dissolve enough of the Ccm-95 in and also dissolve turmeric for comparison. The active ingredient in turmeric is Ccm but it only has about 2%. Be sure to do some negative control filter disks with adding the same amount of oil alone to make sure that is not inhibiting bacterial growth. Positive control disks have to contain a known antibiotic. You can check Carolina Biologicals to see if they sell antibiotic disks or you can use some over-the-counter antibiotic solution.

You'll have to experiment to find out how much of the Ccm and turmeric oils to put on the disk. Always do at least three replicates of each disk so that you can average the results. The data will be much more accurate that way.

I am still wondering how an oil can be tested using the disk diffusion method because neither the oil nor the Ccm will dissolve in water. When I work with Ccm I dissolve it in dimethyl sulfoxide (DMSO) which IS water soluble, but you can still have a problem with keeping the DMSO concentration high enough, 5-10%, without the DMSO itself killing the bacteria.

The one paper you sent apparently just put essential oil onto filter paper disks so try it. I guess enough will migrate into the agar somehow to inhibit the bacteria.

Once you get some resistant bacterial colonies, I would transfer them to separate MHA plates. In order to keep non-resistant bacteria from growing you will need to spread some of the Ccm oil on the surface of the agar before you inoculate it with the resistant colony. I don't know how much you need to use, however. You will have to try a measured amount and see what happens--too much and even the resistant bugs won't grow, too little and the non-resistant ones will grow.

Good luck working out the details. We will try to help.

Sybee

[CaptainHumanLogic]

Hello!
I have an updated procedure for my experiment, could you review and let me know if the procedure is satisfactory?

Procedure:

Prepare an agar dish by drawing a vertical and horizontal line forming four quadrants.

Prepare the E. Coli culture into a test tube and flame sterilize the rim. 

The bacteria is dipped into the bacterial broth and the E. Coli culture is added and spread around with a swap gently side to side. Rotate and repeat twice

Dip the swab in isopropyl alcohol and then burn on the Bunsen Burner to disinfect. Then dis

Put 5 ml of canola oil in an empty petri dish. Soak 4 disks in the canola oil. Take one of the prepared Agar Plates and label it 'Canola Oil'. Lift the disks with forceps and gently place each in separate quadrants marked on the Agar Plates. With each disc, dip the forceps in the alcohol and burn it. Close the lid.

Repeat the above step for coconut oil, tea tree oil, and olive oil and Bactine.

Put the agar plates in an incubator for 24 hours.

Take the agar plates out of the incubator and record the diameter of the zone of inhibition in each quadrant of each agar plate.

Repeat Steps (prepare an agar-Take the agar plate) with CCM95 mixed with the 4 types of oil, canola, coconut, tea tree, and olive oil with the ratio 1 milligram to 1 milliliter. Use 5 milligrams: 5 milliliters solution. 

After the experiment, use a swab to take 2 of the quadrants' bacteria in all 4 and transfer them to a new agar plate, then disinfect the forceps. Repeat the test with the same 4 mixtures until the 5th generation. Record data constantly for each generation to look for resistance.

A couple of more questions -
1. I am using Bactine as Negative Control and Canola Oil as Positive Control. Is that ok?
2. should I test with different brands of CCM95?
3. Is 1:1 concentration of CCM-95 to Oil ok?
Thanks,
Captain Human Logic

[SciB]

Your procedure looks great! That's the way to set up an experiment--step-by-step--so you think through exactly what you need to have ready and how you will perform the operation.

It is very important to have as even a culture of bacteria as possible over the surface of the agar. When you swab the surface go carefully so as not to dig into the agar but try to cover every bit of the surface evenly.

I am a little unclear as to how much of the Ccm that you are dissolving in the oil. You said 1 mg per ml, but what does the 1 mg consist of--material from a capsule? I have Ccm-95 capsules from Jarrow that contain 500 mg of curcuminoids/turmeric extract per capsule. How did you decide on how much Ccm to use per disk? In your other questions at the end you say you will use 1:1 Ccm--what do you mean?

In answer to your first question. Bactine is your POSITIVE control because you expect it to kill the bacteria, a positive result. The oil alone is your NEGATIVE control because you expect that it will have no effect on the bacteria.

I would stick with just the one brand of CCM95 for now. Demonstrating that bacteria can develop resistance to CCM95 would be a major result and that's what you should focus on. If you had lots more time it would be worthwhile to test more brands of Ccm to see how they compare. You can list that in your report as something to do for a future study.

Post again with your clarification of the Ccm dosing.

Sybee

[CaptainHumanLogic]

Hello SciB! How are you?
I am about to start my experimentation in my school lab. But however, I have one question about the concentration of CCM-95 in the oils. I want to know, is the ratio 100mg of CCM-95: 3 ml of an oil a good ratio? I wanted to check to prevent the concentration from being too high or too low.
Thanks,
Captain Human Logic

[SciB]

Sorry, I didn't see your query until now.

I don't know how much Ccm is enough or too much. You can search Google Scholar for scientific journal articles in which Ccm is used in experiments similar to yours and see what concentrations they use. Otherwise just try some and see what happens.

If you get a good response then reduce the amount by half, test it again and keep doing this until you find the lowest active concentration. This is known as doing a 'dose response' in scientific lingo. In fact if you did a search for "curcumin antibacterial dose response" you might find the does range information you need.

Good luck and do let us know how it turns out.

Sybee

[CaptainHumanLogic]

Hello again!
I have completed testing for my experiment and the only thing that should inhibition was Tea Tree Oil with Curcumin95, but none of the others did besides bactine. Not even Tea Tree Oil on its own did so. I did 8 trials with it and 2nd generation tests and it shows that together they are effective, so I have created a conclusion based on my experiment, and I wanted to know if the conclusion is OK:
The only group to show inhibition besides bactine was Tea Tree Oil and Curcumin 95 mixture, which the rings of inhibition on average were around 12 mm, where on average it was more effective than Bactine on average. The reason for this is that both Tea Tree Oil and Curcumin fulfill each others' weaknesses. Curcumin can only kill cells from the inside of the cell, but it takes curcumin longer to enter and kill bacteria as it can only enter by vesicle, so at times it is not effective on its own killing the bacteria in a quick period of time, which is why none of the other Curcumin Oil besides Tea Tree should any inhibition. Tea Tree Oil is very effective at destroying the cell membrane, but it cannot kill it from the inside and cells have the ability to repair their cell membranes, which is why Tea Tree oil alone didn't inhibit growth. Both are effective together because the Tea Tree Oil can destroy the cell membrane which allows for the Curcumin to kill it from the inside easily. With this, both work together and can kill bacteria quickly and effectively.
Thanks,
Captain Human Logic

[CaptainHumanLogic]

Hi SciB! How are you?
I ended up getting 3rd place at my school fair, and I want to thank you for the tips you gave me for my science fair! I could not have done it without you, which I included your name in my acknowledgments. I wanted to share my final conclusion with you:

There is a problem that most people would hardly even think would happen that are affecting many today: contact dermatitis from antibacterial ointments. What is contact dermatitis? Simply put, when a material interacts with your skin and the immune system sees it as a threat and attacks back, which leads to a rash. This is not the worst of what antibacterial ointments can do. Antibacterial ointments, over a period of time, can also cause bacteria to become resistant to antibacterial ointments. This is similar to the process of natural selection. There is an environmental pressure from the antibiotics that kills almost all bacteria, but there is a genetic variation that allows for some bacteria to become resistant to the antibiotic. Those bacteria grow and spread and eventually give their genetic material to other bacteria, creating a superbug. This problem is becoming worse in fields such as dentistry, as they are in a need of a safer and non-resistant antibacterial ointment. However, there is something that can potentially stop this, Curcumin. Curcumin is the main active ingredient of Turmeric powder, a staple spice of Indian cooking, that has been shown to have much fewer chances of causing contact dermatitis, it is edible, and has antibacterial properties. So this was the idea to make an antibacterial cream out of Curcumin and an oil, as Curcumin is oil-soluble. Canola, Olive, Tea Tree, and Coconut oils were the main candidates for the ointment base. The oils would be tested separately and then mixed with a more water-soluble form of Curcumin called Curcumin 95 (CCM95 - 95% Curcumin). The test was done as E. Coli K12 being the bacteria tested and inhibition of bacterial growth was tested using the Kirby-Bauer method, a method of using agar to grow the bacteria and soaking discs with the liquid desired to be tested. Then the discs are placed on the agar with the bacteria and are incubated for 24 hours at 35 degrees Celsius. After the 24 hours, rings were formed showing how effective the inhibition was. The larger the zones or circles, the more effective the liquid is at inhibiting bacterial growth. Initially, it was thought that Curcumin 95 with either Olive oil or Tea Tree oil would be most effective, as both the oils have antibacterial properties themselves.
The data observed was very peculiar and unexpected. At the end of the first round of testing, neither CCM95 or the oils showed any zone of inhibition independently. Furthermore, out of the four CCM95+oil combinations, the inhibition of about 12mm was evident only in the MH Agar plate with CCM95+Tea Tree Oil. Neither Tea Tree Oil or CCM95 independently showed any zone of inhibition, but the mixture showed a significant zone of inhibition. The second round of testing for CCM95+Tea Tree Oil confirmed the validity of the results in the first round that the combination of CCM95+Tea Tree is much more effective than each of them individually. Finally, the second generation of E-Coli bacteria was tested and surprisingly, the solution was even more effective at inhibiting growth in the second generation than in the first generation. The average zone of inhibition in the second generation was 16mm which was about 4mm larger than the first generation.
Overall, even though the hypothesis was supported by the data, it still was unexpected that Tea Tree oil alone and Curcumin 95 with other oils besides Tea Tree oil showed no inhibition. However, there is a very interesting explanation of this phenomenon and it has to do with a curcuminoid within Curcumin called Curcumin I, the properties of essential oils such as Tea Tree oil and their interaction with gram-positive vs negative bacteria. Bacteria are different based on how the macromolecule called Peptidoglycan is positioned in their outer structure. It is a molecule unique to the bacteria it surrounds and protects them. It helps define their shape and admits essential nutrients and large compounds. If Peptidoglycan forms a thick, uniform layer around a bacteria, it is called gram-positive. If Peptidoglycan is a thinner layer surrounded by thin layers of membrane, it is called gram-negative. Gram-positive bacteria are weaker as the Peptidoglycan absorbs too many molecules from the environment around the bacteria, including toxins, but develops resistance very slowly. Gram-negative bacteria, such as E. Coli K12, are very good at repelling attacks against invaders and antibiotics and develops resistance quickly. Another factor is Curcumin I, which is an active ingredient of Curcumin I that has an interesting property, where it inserts itself into the membranes of bacteria and makes the membrane much more permeable, which enables it to let things into the bacteria. The final factor is the properties of Essential oils, such as Tea Tree oil. Since gram-positive bacteria often accept more toxins and molecules nearby, Tea Tree oil can easily kill gram-positive bacteria. But against gram-negative bacteria, Tea Tree oil is less effective, as the membrane has trouble accepting large and dangerous molecules such as the molecules of Tea Tree oil, so it has a lot of trouble killing gram-negative bacteria. This explains why Curcumin and Tea Tree Oil were very effective against E-Coli K12. Curcumin made the membrane permeable and leaky, but that may or may not kill the bacteria if there is no material accompanying it that actually kills it besides curcumin. Tea Tree oil independently couldn’t kill E. Coli K12 as it is a gram-negative bacteria, and hence it can’t penetrate the membrane and Peptidoglycan. But together they can. Curcumin and Curcumin I are effective on gram-positive and gram-negative, so it can open up the membrane and Tea Tree oil and Curcumin jointly kills it. Phenolic compounds in Tea Tree oil enter the bacteria and denature the proteins and enzymes within the bacteria, killing it from the inside. Curcumin inhibits mainly by membrane leakage or by inhibiting bacterial cytokinesis by inhibiting the FtsZ protein. Together they can destroy bacteria not only efficiently, but quickly, which is the probable reason why other Curcumin 95 combinations had no effect.
This finding could have major impacts on many different fields. This ointment can potentially be used as a safe replacement to a triple antibiotic, as it has been shown to be more effective than Bactine, a standard antibiotic. It can also be used in dentistry as it is quick and effective, however, it needs to be only in a very small amount in the mouth as it is considered poisonous in too high of an amount. For further study, the ointment will be tested more, it might be tested against a gram-positive bacteria, and there will be 3-5 more generations tested to ensure that the bacteria do not develop resistance, but however, as of the second generation, it doesn’t cause resistance.

I can share my final report with you in a private chat.
Thanks,
Captain Human Logic

[SciB]

Hi CHL and congratulations on winning third place with your science project! You did all the work--I just made some suggestions.

Your results are really interesting. I am also a little surprised that tea tree oil alone or Ccm alone did not inhibit E. coli. Your hypothesis as to why the combination of TTO plus Ccm is effective is plausible but needs evidence to back it up. It is a perfect example of good science. Each set of experiments should give you information that you use to make another prediction that you then test with more experiments. That's exactly what we do in the research lab and hopefully each experiment builds on the previous ones until we have a reasonably complete picture of whatever it is we are studying.

I hope you find science and invention so interesting and enjoyable that you decide to make your career in that direction. There are still so many great things to be discovered in the world and the scientific method is still the best way to find them out.

Private communications are not allowed on Scibuddies but I am always here to answer your questions.

Good luck!

Sybee