Science Buddies: "Ask an Expert"

How would you suggest comparing tea tree oil again chemical disinfectants for a student who has never done any kind of chemistry or microbiology before? Thanks for any "step by step."

I take it that you are wanting to compare the effectiveness of commercial disenfectants and tea tree oil in killing bacteria. Is this correct?

What grade are you in?

Please answer these questions and I'll get back to you with some answers!

Hello,

This is for a sixth grade project. I have a few things on my mind, as you will see:

It has been noted that several essential oils are effective in reducing bacteria. We are trying to stay away from harsh chemicals in our house.

First, would it be best to pick only two oils for the project, more than two, or compare them with a chemical such as bleach or rubbing alcohol?

What would be the best way to go about this project? If I chose to use a hard surface for the contaminant, would it be best to sterilze the area, (kitchen tile for example), then apply a controlled contaminnat, like a certain smearing of food, take a swab of the contaminated surface and spread on petri dish, then come along later and apply soaked blotter paper disks on the agar plate to see if there has been any reduction in any way?

If I was to use a kitchen sponge for obtaining the bacteria, would I need to soak equal pieces of the contaminated sponge (with a controlled substance or not?) in a test tube with saline or could I just take a swab of the sponge and do the same thing as the hard surface?

By the way, when and why is each method used (soaking item in test tube with saline and then taking sab from there vs just a swab on the surface itself?

Thanks in advance for any information you can provide. As you can see, my head is spinning. In additon, I am new to all of this, so thank you for your understanding. I do have more questions, but I will save them for later so your heads will not spin. Thanks again.

First of all, make sure that cultivating bacteria is allowed in your school (and higher-level) fairs. Many fairs have, at the very least, extra forms that need to be filled out, and some require that such work be done in a lab.

For your antibacterial substances, I would certainly use bleach or rubbing alcohol (or another chemical known to kill bacteria) as a control. You will also want a control petri dish that is not treated with any oils or chemicals. This plate will tell you if things have gone wrong in your procedure.

You can compare the effectiveness of tea tree oil with other natural antibacterials, or you can compare tea tree oil with industrial chemicals, or you can use a combination of the two. It depends on what you want to do with the project.

I would suggest Q-tips for collecting swabs, since you don't need many bacteria to start a colony. Q-tips are also easier to work with on agar.

The problem with working with swabs of bacteria is that so many different kinds will be cultivated, even if you are using the same substance to obtain your bacteria. You might consider working in two steps-- make one petri dish with your substance or surface, then use one colony on that plate to make your experimental plates. This will make it more likely that you cultivate only one kind of bacteria.

You can add the blotter paper disks to the petri dishes right after smearing the bacteria if you want to test how well the oils/chemicals keep bacteria from growing; or you can wait until the bacteria have already grown before applying the oils if you want to test how well the bacteria are killed.

Have you thought yet about how you are going to measure the effectiveness of each oil?

I hope this helps clarify things for you. Post if you have more questions.
Sonia

Thank you very much Sonia. For measuring, would you use the zone of inhibition. I have never done the other way where you count to measure. (how do you do that if there's a lot?) Also, how would you suggest going about getting some surface bacteria? Just swab different areas in the house, then repeat in other places for the repeated trials? Thanks very much. My message keeps cancelling. This is my third time posting this.

A zone of inhibition is probably the most effective way to measure results. It also allows you to get multiple repetitions from the same plate (if your blotter paper disks are small enough). If you want to count bacterial colonies, you would need to first find out the size of the swab that would give you a reasonable counting number. Dividing the plate into quarters or eighths can also help with counting.

I would suggest swabbing surface bacteria from a surface that is commonly used (such as a doorknob or a faucet handle). You can also take swabs from foodstuffs or from your mouth.

Repeated trials give more results for the same experimental conditions and are used to tell how valid your results are. (Ideally, the results for all trials will be similar, but that does not always happen in experimental science.) So for the repetitions, if you are repeating swabs, they will be taken from the same location. (Bear in mind, though, that there will not necessarily be the same bacteria there on different days.) You can also make repeated trials from a single swab by adding, say, 3-5 disks of the same oil/chemical on a petri dish, or by having 3-5 petri dishes that receive the same treatment.

Sonia

Thank you very much for helping me. I have copied the info and am reading it over and over to better understand all of this and to figure things out. I wrote another post earlier about bacteria, but now I see that the answer lies here:If I am understanding correctly, it is fine to get the bacteria from the same area/same surface on the same day for all the experiments to keep everything constant, except for the products being tested. Am I understanding this correctly? If so, then it makes things much more simple than making a homemade, controlled bacteria like they did on Myth Busters. Again, I am going to use the zone of inhibition like you mentioned. You said something about putting the disks on the plate at the same time as putting on the bacteria or waiting for the bacteria to grow. I want to see if my products will actually kill the bacteria, so I think I am going to put the disks on later. However, my mom and dad are not too happy about that becasue they don't want me to be around growing bacteria. So, I may have them put the disks on the plates. Anyway, does it sound like I have the right understanding of what you have been trying to help me with? Let me know if I am off. Thank you again for helping me. I am thankful for your information and time.

Yes, it is best to get your bacteria from the same surface on the same day so that the only variables are the herbal/chemical substances you are testing. As I mentioned earlier, you can ensure that you work with only one kind of bacterium if you take a swab from your surface, let the bacteria grow, and transplant a single colony to your experimental plates. If you are not able to do this (or if your parents aren't happy about you doing it), bear in mind for your writeup that you will be growing many kinds of bacteria, and they may not all respond the same way to your treatments.

Your understanding of the project and the methods is right on. Best of luck with your experiment!
Sonia

Hi! I spread my bacteria from the same source on my agar plates, then put one soaked disk in the center of each plate. Therefore, I have a total of 18 dishes incubating in a dark, 35 degree Celsius room. I used three dishes as a positive control, putting 8% household bleach on the disk. Then, for the negative control, I used nothing on the blotter paper. (Am I correct here?) Four essential oils have three separate dishes with one disk on them. Bacteria has been growing for five days. However, there are only a few visible bacteria colonies on each disk. (2-9 for each dish, on average.) I'm bummed beause I thought I would get more activity than this by now. I wanted to measure the zone of inhibition, but I don't know if that is going to be able to happen. What could I do next? Count the number of colonies to measure or should I take one of the colonies and start over on some new plates? (My agar dishes are approx. 1/8" deep. Is that too thin?) Thanks. Please tell me if I should be more patient about this bacteria growing. Have a nice holiday season!

Sunn,

Bacteria grow very fast; you should not need to wait five days to get results. Your procedure, however, is sound.

If you are only getting a few bacterial colonies on the plates with the essential oils, you should be fine; it means that your oils are effective. If there are only a few bacteria on the negative control plates, however, something went wrong in the experiment.

It may be that the area you chose to get your bacteria did not have many bacteria on it. This might also be the case if you used bacteria from a single colony on your experimental plates. I also notice that you put blotter paper on the negative controls. You might try plates without any blotter paper--maybe the blotter paper has a chemical that is killing the bacteria?

If there is a significant difference in the number of colonies between the positive and negative controls (say, 1 colony on the positive control and 9 on the negative control), you can still count colonies and use those as your results.

Whether or not you want to restart is up to you, and will depend on exactly what's on your plates. If you restart and want to get a lot of bacteria, you can try culturing the bacteria in a test tube of LB broth for one night, and then plating them to the experimental petri dishes.

Experimental science is not always clear-cut, and it is not uncommon to have to do "trial runs" before you get usable results.

I hope this helps you. Feel free to post if you have more questions.
Sonia

Thank you very much for your help, Sonia. I went back again and looked at what's happening so far. for each oil, I did three trials with all disks in separate dishes. Thankfully, the oils vs the bacteria count seem pretty consistent for each experimental group. Now if I wanted to retest to make sure things are accurate and get more bacteria to possibly measure zones of inhibition....> you mentioned culturing the bacteria in LB broth for one night. How would one go about doing that? Also, what happens if the agar dish surface gets wet after plating? Should you wait until it is dries out a bit? Would you suggest putting all my disks from each oil, plus controls (including one blotter paper without anything) on one dish this time, repeating this two more times? Should I have one dish with nothing at all for the control or is the blotter paper with nothing on it enough for the control? Thanks again, Sonia. I appreciate all this amazing help! One more thing> if I do this additional test, do I just add it my procedure list, but in a separate part, saying"procedure #2? THANKS!

Sunn,

For your LB broth culture, you would first swab the area where you want to get the bacteria from. You can then put the Q-tip directly into the test tube with broth and swirl it to get the bacteria off. The test tube is incubated overnight, and the bacteria can be plated the next day. Ideally the same amount of culture should be plated on each petri dish; are you able to find a pipet? A dropper (such as you find in medicine bottles) would also work too, just be sure to clean it out thoroughly first. You shouldn't need more than a few drops.

The LB broth culture ensures that there will be lots of bacteria to plate. Of course, you will also be getting all different kinds of bacteria.

How is your agar getting wet after plating? Are you making the plates yourself or buying them? I remember when I was making agar plates, there would sometimes be condensation from the hot liquid agar (or after incubation). In this case, the water should not make a difference to your results. You can stack the plates when you're making/ incubating them to minimize condensation.

I would keep the same oils on the same plate and not mix oils on one plate. If you want to use fewer plates, though, you can put multiple blotter papers with the same oil on a single plate. Each blotter paper counts as a "repetition." You should be able to fit three or four small ones on a single dish.

For your negative control, you can either use blotter paper with nothing on it, or a petri dish without any blotter paper. You can also do a plate of each, if you want to be extra-exact. If it seems like the control plate from your previous trial gave you uninhibited growth, you can stick with that method.

For your writeup, I would use the finalized procedure as your procedure. In the results and discussion, I would present two trials (a "preliminary" one and a "final" one) and describe briefly any changes in procedure between the two trials and reasons for making those changes.

Good luck!
Sonia

Hi Sonia,thanks for the information. Since you are talking about the plates having different kinds of bacteria, I think I am going to get some E.coli k-12 if I have the time. I was also thinking of keeping the results that I have and measuring the distance from the disk to the closest visible colony. (Should I not count the fungus looking thing as it is not a bacteria, or can it be.? (I know that is hard for you to answer without seeing it. )When I make the graph, do I need to put the positive and negative control on the graph, or just the experimental group? (or just the positive control with the essential oils?) What kind of graph would you suggest? One bar graph with the average or one showing all the trials?(Is each experiment within each group called a trial? If not, what is that called?) What is the difference (or advantages) between using a sterile swab and inoculating loop when using bacteria in a tube? Thanks in advance for any more information. I'm hoping to make some good lawns of application so I can see some zones! Happy New Year1

It's great that you are looking at using E. coli. Just make sure that you understand your school's or fair's requirements-- many strains of E. coli are pathogenic (disease-causing) and may have restrictions attached to them in fairs.

You do not have to include the fungus in your graph, but I would at the very least mention that it appeared on the plate. You might bring abnormalities like that up in the discussion section of your writeup. You could, for instance, use it as a reason to explain why you switched to E. coli.

Innoculating loops are useful for picking up bacteria from colonies for culturing. You would use them when you have bacteria growing on a plate that you want to make a culture of. You would scrape some of the bacteria off the plate with the loop, put in in LB broth, and swirl it around to get all the bacteria into the broth. You can also use innoculating loops to spread drops of culture on a plate. You would spread the culture all around the plate going up and down, then all around going left and right.

Innoculating loops are good if you know exactly where your bacteria are. If you are not sure (for instance, when you are first getting bacteria from a surface), I would stick with the sterile swab.

For the graph, you should certainly include the positive and negative controls. Both of them are useful in telling you how effective your essential oils are in killing the bacteria. I would, however, make separate graphs for this set of results and your next one.

All your results (the zone of inhibition for every plate) should appear in your writeup, at least in table form. Averages should be fine for the graph, as long as it is clear that you are graphing averages. It might also be interesting to show the variation between plates for the same essential oil. Bar graphs should work fine for your purposes--they are the most straightforward way to compare categories.

I don't think "trial" has a specific association with it in science. I tend to use "trial" when my results don't turn out the first time and I need to make changes to the procedure; each repetition of the experiment is a "trial". (So this first set of results that you have is one trial; the next trial will be with E. coli.) But I've also used it in place of "repetition." As long as it's clear what you mean by a "trial," it shouldn't matter how you use it.

I generally refer to the experimental groups simply as experimental groups, or by their variables (for instance, the "rosemary plate" or the "tea tree oil plate").

You seem to be well on your way with this experiment, and it's great that you are working so hard to conquer all its details! Feel free to post if you have more questions,
Sonia