Essential oils and their effects on bacteria - what solvent?

[SciB]

You got it GG! Your calculations are correct, at least for the hypothetical 6% T20. To make your EO dilutions, you could prepare your highest concentration of EO as you said, but for the lower concentrations, instead of mixing more EO and T20 each time, just make up 100 ml of 6% T20 and use that to make serial dilutions of your 2% [or whatever] EO. For example, if you want 1% EO then take 5 ml of the 2% EO plus 5 ml of 6% T20. For 0.5% EO, take 5 ml of the 1% EO plus 5 ml of 6% T20, and so on...

I knew you would ask the question about how large a zone of inhibition [ZOI] is too big, and I have to tell you i don't know. You have to have a certain amount of T20 to dissolve the EO, but not too much. It is going to depend on how sensitive E coli is to T20. If that paper i sent you is correct and 4% causes 50% inhibition then 6% T20 would probably be the maximum amount to test in your toxicity experiment.

What i don't know is how large of a ZOI you will get with 6% T20. You'll just have to try it and see. I am hoping that 6% T20 only gives maybe a 0.5 cm ZOI, while 6% T20 plus 2% EO gives a ZOI of 2 cm. If you knew the answers ahead of time then there would be no point in doing experiments!

Good luck and if you have any questions at all about doing the experiment be sure to ask. This test is critical to the success of your EO experiment. Be extra careful to pipet the same amount of E coli culture onto each plate. Swirl the tube a little before you pipet to make sure you have a uniform suspension of the bacteria. Be sure to spread the drop of culture immediately and keep spreading evenly over the entire plate until the liquid is absorbed into the agar. If your lab has a laminar flow hood for biosafety level 2 work it would be best to use that to keep everything as sterile as possible. You should wear disposable rubber gloves, safety glasses and a lab coat. Make sure you have everything ready and set out before you begin.

Do you have a 37C incubator that you can use to incubate the plates? This is the preferred temperature for E coli and it will grow fastest if incubated at 37C. It will grow at room temperature, 20C, but it will take 2-3 times as long.

Sybee

[GalaxyGirl4]

Yes, everything really does depend on the Tween 20 concentration I can use.... hopefully it works out!
Yes, I have a professor at the university who's willing to provide me with the incubator and a lab technician who will be there with me.
I'm planning on putting 100 microlitres of bacteria (so 0.1mL into each dish, and spreading as evenly as possible).
I have one more question on the nutrient agar. How exactly do I heat it? I'm not sure if i can use a microwave there as I would at home, and I've seen people putting the agar in these hot water baths to boil the agar. Have you ever heard of a simple way to do it? I will probably have to ask the lab if I can use some more of their equipment to melt the agar.... approximately how long would you say it takes agar to cool into gel before I can spread my culture? Around 15-30 minutes, perhaps?

[SciB]

Oh. I thought you had the plates made already. Most university labs have access to a shared autoclave, so ask the tech who is helping you to show you how to use it. You have to do a SLOW EXHAUST cycle because you have liquid.

You can boil the agar solution for 15 minutes if you have to, but you will have to do it on a hotplate-magnetic stirrer with a spinbar and constant stirring so that the agar doesn't burn and you have to watch it carefully so it doesn't boil over. Use a 500 ml flask if you are making 100 ml of agar--1.5 g of powdered agar medium in 100 ml of distilled or deionized water. This is enough for 4-5 plates.

if you do use the boiling method, wait about ten minutes for the agar to cool before you pour it into the plates. Wrap the neck of the flask with a paper towel if it is too hot to handle. Make the plates about one hour before you need them. There will be moisture condensed on the lid so shake it off into a sink so that the surface of the agar is not wet. If you aren't sure about the whole plate-pouring procedure, check youtube for a video.

Good luck!

Sybee

[GalaxyGirl4]

Yes, I know there is an autoclave available in the lab.
I could also boil my agar (follow the instructions given on the bottle when I received the agar) at home and use sterile techniques to pour my agar, right? And then let them cool for a bit, before storing them upside-down in a fridge until I want to take them to the university and use them tomorrow. It might be easier to be more careful at the university, but I've still looked at a lot of videos and researched a lot about how exactly you need to pour the agar and flame the mouth, in order to be completely sterile, so would it be a bad idea to do it at home and then take the plates to the lab?

[SciB]

Sure, you can pour the plates at home. Boiling at 100C will not kill some spores that ARE killed by autoclaving at 121C, but this only matters if you are keeping the plates around for weeks or months which you aren't doing. Since you are going to use them tomorrow, i would store them at room temp in a zip-lock bag, not in the fridge. If you refrigerate the plates there will be a lot of water condensing on the inside of the dish and that can make your agar surface too wet. Also, if you spread the E coli on cold agar it will take a lot longer for growth to start. You only need to store agar plates at 4C when you are keeping them for more than a week. Are you making enough plates to do both the T20 toxicity experiment and the EO experiment? You should do that so you only have to pour plates once.

The volume of agar needed per 10 cm diameter petri dish is 20-25 ml. If you have a spare plate pipet 25 ml of water into it and use that as your guide for pouring. It does not matter if you have a little more or less agar because the E coli only grow on the surface.

Will you be able to incubate your plates at 37C in the lab? Try to do that because the bacteria will grow much faster at that temperature. You need an answer right away to your question of how much Tween20 inhibits the growth of E coli. If you can, use sterile water to make the T20 dilutions.

[GalaxyGirl4]

Yes, I'm using distilled water to make my T20 dilutions. I'm going to be testing from 30-5% in 5% increments, then 3% and 1% and see what happens from there.
OK, so I can boil some water and carefully melt my agar, take it out and let it cool for a while. Then when it's slightly warm I can pour it into my plates and let the agar in those solidify for a couple minutes before putting each one into a baggie, turning them upside down and leaving them overnight at room temperature. What if condensation forms on the lid overnight? When I bring the plates to the university tomorrow, I would just have to carefully shake off the water on the lids before spreading my bacteria?

[SciB]

Do you have a clean glass bottle? I would use that to melt the agar because remember, you are trying to keep the agar sterile so you don't want to expose it to the air, which contains lots of spores and bacteria, any more than necessary.

It takes a few minutes of swirling the agar in the hot water to make sure it is all dissolved, and this is important. If you see any particles of agar remaining in the liquid, keep swirling it--gently though--you don't want to make bubbles in it. You should cover the top with a piece of plastic wrap and heat the liquid in the microwave to just boiling, but don't let it foam up. The plastic wrap will keep the liquid from evaporating and prevent air from contaminating the agar. Oh, and DO remember to use oven mitts to handle the hot glass! Keep boiling the agar suspension and swirling it for at least 15 minutes to dissolve and sterilize it.

You could heat the liquid in the jar to boiling then put it in a pan of gently boiling water on the stove and let it heat for 15 minutes or longer, swirling it from time to time.

When you pour the plates, the bottle should be still quite warm to the touch and I would let the agar harden for half an hour just to make sure it is completely set. Once you pour the plates DON'T move them! If you do you may cause a ripple on the surface of the agar that will make it very difficult to spread the bacteria evenly.

And, yes, you should just shake the water off the lids into a sink. Just don't leave the lids off any more than necessary so you don't expose the agar to air.

[GalaxyGirl4]

I got my plates back from the lab yesterday and I examined them to find zones of inhibitions.... however, I couldn't see any real "zones of inhibitions". I'm afraid they're not noticeable because of the colour. I may have also did a few things wrong (scraped surface of agar when spreading, lumps in agar?). I'll try to attach some photos, but it might not be too visible in the photos.... My question is, there are a few white white dots and speckled things near a lot of the disks. Are those bacteria growing close to the disks? If that is the case, either something went wrong or the Tween 20 really isn't bactericidal? I couldn't see any obvious circles like I've seen in pictures of sample disk diffusion tests and pics on google. I guess you can see a few "faint" circles but they don't look like pictures I've seen of this test. Either way, I went to the lab yesterday and re-did my experiment, this time trying to be a bit more sterile, remembering to turn my plates upside down in the incubator, and not gouging the agar.

[GalaxyGirl4]

https://docs.google.com/document/d/1pCB ... sp=sharing

I went to the lab to get my new results and the same thing happened - I can't tell if these are zones of inhibitions because there are little white dots in them (are those colonies of resistant bacteria?), but I feel like I can see a circle forming - but they don't seem "clear" like a zone of inhibition, so are they zones of inhibition? I have the new photos of the recent re-done test, and "circles" can be seen again but not like a clear one.

[SciB]

OK. Before i can try to figure out what happened can you please answer these questions:

How old is your culture of E coli and how have you been storing it? How does it look? Is it quite cloudy or not? Remember to swirl the tube gently before pipetting to make sure all the bacteria are suspended evenly throughout the liquid.

What kind of agar medium are you using and what weight of it did you boil with what volume of water and for how long?

What volume of E coli culture did you spread on the plates? What did you spread with?

Were the plates at room temperature when you spread them?

Did you incubate the plates at 37C, and if so, for how long?

Looking at your pictures i can see that there is a ZOI around some of the disks but you did not get an even spread on the E coli. That is the main problem. And it looks like they haven't grown long enough.

It also looks like you may have waited too long to pour the agar so that it formed lumps--or else you moved the plates before they were set. You have to pour the agar while it is still hot, which means wrapping the neck of the flask or the bottle with a paper towel so you can hold it without your fingers becoming uncomfortably warm.

To get a perfectly even lawn of bacteria, the agar has to be completely smooth. Did someone show you how to spread the plates? It is quite easy to dig into the agar with the spreader if you don't hold it loosely and at the right angle. I like to use a glass spreader. It is easy to sterilize by dipping it in alcohol; and flaming it, and the smooth surface of the glass rod does not dig into the agar.

E coli colonies on nutrient agar are shiny and translucent; but you should not be getting separate colonies. The agar surface should have a uniform growth of E coli completely covering it with no breaks anywhere. Your white spots could be yeast colonies. Yeast spores are in the air everywhere and they like to grow on nutrient agar. It won't be a problem if there aren't too many, but some yeasts can produce chemicals that inhibit bacterial growth, so you really don't want them on your plates.

If you still have agar left, pour some more plates and make sure the surface is smooth. I would go ahead and try your EO this time, but also have some disks with T20 alone. Make sure your disks are nice and wet with the solutions when you apply them to the agar [don't blot them!]. T20 definitely inhibits bacterial growth but the dose the bacteria are getting from the disks may not be enough to inhibit them very much. Hopefully the T20 + EO will have a greater effect.

Try to keep everything as sterile as possible. Work in a hood at the lab if they have one. Otherwise, expose the agar to the air as little as possible.

This ZOI assay does work so don't give up. You just didn't get a good lawn of bacteria. It takes practice to spread plates well and to work aseptically.

Good luck,

Sybee

[GalaxyGirl4]

I got my culture of E.Coli last Thursday (so 3 days ago). I have been storing it in a lab refrigerator at the university. It's in a nutrient broth, in a tube. It's yellow, and mostly not cloudy - it's pretty clear. I believe it said there were 5ml of bacteria for 15ml on the website.
I'm using nutrient agar, from Science Stuff. I basically followed the instructions given with the bottle - loosened the cap slightly, put it in the microwave and heated it up on low, swirling to melt any clumps every minute or so, until the bottle was very hot and it looked completely liquid. When I re-poured the plates and repeated the experiment on Friday afternoon, I went to go collect these new plates yesterday evening. The new plates were smooth, with no clumps of agar. The same thing basically happened - I can't tell if these are zones of inhibitions as they aren't "clear" and there are small white colonies around the disks.
I dropped two drops of the E.Coli from a dropper onto the middle of each plate, then closed the plate. I sterilized a paper clip that I bent into 90 degrees and opened the dish, and carefully spread the E.Coli as best as I could. I ripped the agar in the pictures I posted, but in the new test I observed yesterday I didn't rip the agar and it seems like I have a better lawn.
Yes, the plates were at room temperature. The E.Coli, however, was taken straight out of the fridge, swirled around for a few seconds, and then spread. The plates were then closed and I then picked up the disks that were soaking in the different dilutions and placed them, with tweezers, onto the fresh plate.
I did incubate the plates at 37 degrees, upside down in an incubator for 24 hours.

Should I perhaps use a different agar? I know that E.Coli grows especially well on MacConkey agar since it's meant for gram - bacteria. Will a ZOI be more visible? Or can I just use nutrient agar, I don't see how that would be a problem.
If those are ZOI, and based on the new ones I observed yesterday from my new set of plates, I don't see a big difference in the ZOI for all the dilutions. I would now like to use 3% Tween 20 (so 1% EO), 1.5% Tween 20 (so 0.5% EO) and 0.5% Tween 20 (so about 0.16% EO). I'm going to the lab today again because I want to see the definite difference between no ZOI and a ZOI. I want to try to split my plate into 4: have one section for 3% Tween 20 again, one section for 1% EO (with 3% Tween 20), one for a sodium benzoate and water dilution, and one with nothing. I just want to see what a real ZOI might look like, and compare it to the Tween 20 ZOI.

I can post the pictures from my most recent experiment that I observed yesterday, which was a little better.

[SciB]

Hi GG,

Yes, do post your photos. Well, you ARE doing everything according to the book, and E coli grows fine on nutrient agar so I am beginning to suspect the culture that you received was not grown dense enough. You should be able to see a distinct turbidity when you swirl the liquid culture in the tube. Is there any debris in the bottom of the tube?

Your proposed experiment including the sodium benzoate should give you the information you want--what does a true ZOI look like with your bacteria on your plates. I hope you do see a quantitative difference between the ZOIs with EO and those without. Try incubating the plates longer at 37C and see if you get a denser lawn and clearer ZOIs.

Once you do get some believable data, you will have to repeat the experiment several times and average the ZOI values so you can do some statistical tests to prove that the difference is significant.

Good luck!

Sybee

[GalaxyGirl4]

https://docs.google.com/document/d/1e-n ... sp=sharing

Is 48 hours too long to incubate? Will my bacteria dry out?

[SciB]

No, 48 hr isn't too long at 37C. The agar contains a lot of water. I would check the plates at 30 hr or so and see how they look.

[GalaxyGirl4]

I just went to the lab and completed the dish and placed it in the incubator. I'll check it on Tuesday this time. And to answer the question about my bacteria, it was pretty cloudy and I had to shake it up before I used it. I also added a little bit more bacteria - about half a drop more and waiting five minutes before putting everything in the incubator.
I'm still trying to find someone at the university who knows enough about this assay to help me. There's one professor I've contacted that might be able to help. But, I just want to thank everyone at science buddies for guiding me through my project.... I have definitely gotten further than I thought I would have, without knowing much starting this.

[SciB]

I am very happy to help you with your project. I know how hard it is when you have so many questions and there's no one there to ask. When I do a new kind of experiment in the lab I expect it to fail the first couple of times until I get the hang of it. That's why it's so important to have all the controls so you can try to figure out what went wrong. Hopefully this time it will work!
Sybee

[GalaxyGirl4]

It's been a few days since I posted, but I did got to the lab again and I measured the effects of 1% EO with 3% Tween 20, 3% Tween 20 alone (again), and some diluted sodium benzoate. I am now even more confused than ever because it seems that my Tween alone has a LARGER zone of inhibition than my tween + oil. My sodium benzoate did have a slight ZOI, but my oil and tween barely had any visible circle. Does anyone know if it is possible for Tween 20 to inhibit the effects of the antimicrobial (in my case, clove EO)? I talked to a graduate student at the university and she was confused as well because there were colonies growing inside the ZOIs. So, she told me I might want to try counting the number of colonies in a plate instead. I should streak a plate with bacteria, let that grow overnight, then drop my antimicrobials onto the surface of the plate and spread uniformly, incubate and look at the appearance of the plate afterwards. I'm not exactly sure if this method will work, however, because if I spread the antimicrobial, won't the colonies already growing be spread? How can I compare the two? And, what if there are no visible colonies that I can actually count?
I read some stuff online and I don't know if what I understood from them is correct, but I might have another idea if the method above doesn't work as well. perhaps I can mix a particular amount of antimicrobial into my agar, pour the agar into the plates and let that solidify, then streak bacteria across the surface? Could I could incubate that overnight and count the number of colonies I get afterwards? How do I get the plates/bacteria to look like this:
http://www.moldbacterialabs.com/index.p ... ab4d9e791e
The picture with all the colonies in the plate.

[SciB]

Hi GG,

Your results are telling you something, but what it is i'm not sure. You can't or shouldn't base conclusions on one experiment, however.

Your 3% clove oil plus T20 had a smaller ZOI than T20 alone. Your conclusion would have to be that the EO is protecting the E coli from the damaging effects of the detergent and that is possible. Detergents kill bacteria by dissolving the bacterial membranes which are made of fatty acids. If the T20 detergent is saturated with EO then maybe it is not able to break up the bacteria as effectively. So, what the experiment may be telling you is that clove oil is not a good antibacterial under these conditions. Plain old soap and water works better.

You mentioned doing an experiment where you count colonies. That is a more difficult method but it is the way I always used to measure effects of antibacterial treatments because it is much more quantitative than the ZOI assay. The cytotoxicity experiments in the paper that i sent you about the T20 effect on E coli were done that way. in order to do the colony count method you need to grow the bacteria in L broth (Luria-Bertani medium) which is just like nutrient agar except it doesn't contain agar. To do the experiment, you would grow about a 100 ml culture of E coli to mid-log phase then split it up into sterile tubes--as many as you need for a range of treatments. Then you would add T20 alone to give a range of concentrations and T20 plus EO at a range of concentrations. lastly you would have one culture with no treatment.

To do this expt properly you would need a shaking incubator at 37C. Since you have access to a university lab, i would expect them to have such an incubator for growing E coli. The bacteria need oxygen--that's why the culture tubes or flasks need to be shaken--to aerate the cultures. You would incubate the E coli for 1 or 2 hours with the various compounds, or without anything, then you would do serial dilutions of the cultures and spread 0.1 ml on a nutrient agar plate. The plates are then incubated for 24-30 hrs and the number of colonies per plate are counted.

At the mid-log phase of growth, the cell density of E coli is about 2-3 x 10^8 cells/ml so you need to make quite a large dilution. I would take 0.1 ml of culture and add it to 9.9 ml of L broth. This gives a 1:100 dilution--still way too high for individual colony counts. If you take 0.1 ml of the 1:100 dilution into 9.9 ml of LB you will then have a 1:10,000 dilution. If you multiply the dilution factor times 2 x 10^8 cells/ml you get 2 x 10^4 cells/ml. Now if you spread 0.1 ml of that dilution on the plate you will be putting the equivalent of 1000 bacteria on the plate which is TOO many to count.

The most accurate count is 100-200 colonies per plate, so you would need to do one more dilution, 1:10 this time. Take 1 ml of your 1:10,000 dilution and add it to 9.0 ml of LB. This will give you a 1:100,000 dilution or 10^-5. If you multiply that times 2 x 10^8 you get 2 x 10^3 and if you spread 0.1 ml of that on a plate it should be about 200 colonies which will give you an accurate count.

Now, remember, the dilutions i just gave you are for the CONTROL untreated E coli. If your treatments kill some of the bacteria or prevents them from growing and dividing, then your culture will have fewer cells per ml. For these cultures you might want to do a 1:5 final dilution instead of 1:10.

I hope this dilution thing makes sense to you. If you look on youtube you can find videos that show you how to do serial dilutions. you need to watch someone doing it to know the way to hold the tubes and the pipet, how to vortex the tubes to mix the suspension well before pipetting, etc. This is a lot of work and takes a lot of plates and liquid medium, but it will give you accurate, publishable results that you can do statistics on and that is the goal of science.

Let us know what you decide to do. Send some pictures of your ZOIs. I'd like to see what they looked like.

Good job so far!

Sybee

[GalaxyGirl4]

Hello,

Do I have to count the colonies specifically? I've mostly learned about the method you described myself through research and have had some of it explained to me.... however, I don't think I can find someone who will be able to help me with the procedure in a short notice. I read a few papers/websites that did something similar.... but they put their antimicrobial into the agar itself before plating their bacteria. I'm assuming they diluted their bacteria because you can see nice, visible colonies in the dishes in the pictures at the beginning. I also read a few papers that suggested using agar as a stabilizer for oils instead of Tween because it does not have effects on the bacteria (however, I don't know if they meant nutrient agar, or the agar I use to grow the bacteria on??), but I don't know if you can do that? And also, do I have to use Luria Bertani medium? I don't know if this was a misunderstanding on my part, but is it ok to use sterile distilled water as a solvent (9ml each time) with my E.Coli grown in the nutrient broth?
At this point, I'm not sure if I have the time, knowledge or people to help with a detailed colony count experiment, so do you think it would work if I could just observe the effect different concentrations of the antimicrobials have on the bacteria, and not have to dilute them detailed enough to count colonies (i.e. higher concentration shows a plate with less colonies, by observation)? Maybe it would be easier to observe this by streaking bacteria and not spreading?
This is assuming that I cannot get the disk diffusion to work.... and now, I can't see the point of making different dilutions of EO because disk diffusion only tells you if the bacteria is susceptible to the antimicrobial, and you can't actually observe anything else. and I will definitely post the pictures tomorrow morning.
Thank you so much!

[SciB]

Hi GG,
Yes, if you treat the E coli with EO in liquid culture then counting the number of colonies that appear on the plate is exactly what you have to do. I forgot to say this, but what you are actually counting is the number of bacteria per ml in the culture. Each bacterium, when you put it on the plate and incubate it, grows and divides by binary fission to make two daughter cells. These two grow and divide to make four and so on until they form a visible colony. so what you are counting is actually the number of bacteria that you spread on the agar.

Unless you can find someone who has everything ready to do bacterial colony counts and can help you to do it, then you better just stick with what you have done with the ZOIs. A quick assay like that is often done in the lab when we don't need the quantitation of colony counts so it is perfectly valid for your project.

I don't know about using agar to 'stabilize' [what does that mean??] an oil, but let me clarify one thing. The agar that you use in your nutrient agar plates is exactly the same as any other agar. The powder that you use to make nutrient agar plates contains agar plus yeast extract, glucose, salt and some other things bacteria need to grow. The purpose of the agar is just to act as a support for the bacteria to grow on. It is the 'nutrients' that they eat.

I suggested LB broth because that is the most common liquid medium for growing E coli. You don't have to use that. If you do a search you will find other growth media for special purposes. When i was doing mutation studies on E coli, I used what is called a 'minimal' medium (M9) that contains just glucose and salts and a few amino acids. The bacteria grow slower on this but they do grow.

You should not use water alone for making serial dilutions because it stresses the cells. You could use water with 2.5 g/L of sodium chloride but the M9 medium is best.

You could try streaking or swabbing bacteria on agar that contains your EO but it is not a scientific method because you cannot control the amount of bacteria you put on the plate. If you are going to make dilutions and spread these on the agar to get individual colonies then you might as well just do the expt and treat the E coli in liquid culture.

You should use different concentrations of EO because this is how scientists get what is called a 'dose-response curve'. In your case, you would incubate the bacteria with a range of EO concentrations from 0% up to maybe 15% then make serial dilutions, plate them, incubate the plates and count the colonies. What you will see is a normal number of colonies at 0% and a decreasing number as you increase the concentration ['dose'] of EO. I am thinking that in this case you might try the treatment without T20 as long as you can put the liquid cultures on a shaker. Vigorous shaking will bring the oil into contact with the bacteria even though it is not dissolved in the broth and you will eliminate one variable by removing T20. You couldn't do this with the disk method because the oil would not diffuse out of the disk into the agar, but shaking the bacteria with the oil for an hour might work. In your report, you could explain about the T20 effect and say that is why you tried using the oil alone.

Ok. I think I have gone on long enough! Good luck in whatever you decide to do.

Sybee

[GalaxyGirl4]

http://www.ncbi.nlm.nih.gov/pubmed/8120787

^So they used 0.2% agar instead of the usual emulsifiers, I don't know if it would make a difference if I used it....
Also, do you possibly think the reason I do not have noticeable zones of inhibition is because the bacteria is too concentrated? If I could dilute my culture, spread it, and then place disks soaked with the diluted oil on the surface would it possibly be easier to measure the ZOI?

[SciB]

Yes, the number of bacteria that you spread on the plate could have an effect on the ZOI. If you have time, you should try diluting your E coli culture 1:10 with sterile water (saline would be better, but for such a short time it won't matter) and spreading 50-100 microliters of that. If you have enough plates you should also do a 1:100 and 1:1000 dilution. If your culture has 10^8 viable E coli per mL and you make a 10^-2 dilution and plate 0.1 mL of that, then you are spreading about 100,000 bacteria. For the 1:1000 dilution you are spreading 10,000 cells.

I am impressed by your reasoning, ideas and persistence in making this work. I'm giving you an A+++!!

[GalaxyGirl4]

I've gotten some results with a 10% dilution of oil while diluting my bacteria, with the disk diffusion assay! I added a little less Tween, and with 10% oil I had a ring approximately 11.5mm wide. The only problem is, I just can't get a complete emulsions because I don't want to add that much Tween 20. I will just have to explain that I couldn't get a perfect dilution because I would rather have proper controls and a fair experiment, but my oil IS still diluted because I added the right amount of solvent and solute and mixed as well as I could. Now, I just have to dilute with some other concentrations and measure ZOIs. I am also going to try a colony count method now, just to see if it works and I can get a little more quantitative results. My only question is, would I match these concentrations with sodium benzoate concentrations? The accepted amount of sodium benzoate in meats is apparently only 0.1%. I want to test at only 0.1% so that i can show that this is the maximum amount permitted, but you can add more of a dilution when it comes to clove EO. is it ok if I only test SB at 0.1%?
Also, quick questions - this experiment would be an experiment, and not an innovation, correct?
Thank you so much!!

[SciB]

That's good news, GG! I'm glad you are finally seeing some inhibition. Don't worry about the lack of complete solubility. When you explain to the judges how you tried to solublize the oil and what happened with the T20, they will understand completely. Working scientists run up against problems like this frequently.

I'm also glad you will try the colony counting method. This is a a useful technique to know if you plan to work with bacteria again in the future.

The sodium benzoate control is a good idea, but just to satisfy my curiosity, why don't you try 0.5% or 1% SB. I know you said 0.1% is the max safe amount for adding to food, but naturally one wonders if higher concentrations would inhibit the bacteria even more.

Here's a question for you. You've probably heard of MRSA, right? Methicillin-resistant Staphylococcus aureus? http://en.wikipedia.org/wiki/Methicilli ... cus_aureus

These bad bugs have mutated so that their enzymes that catalyze the synthesis of building blocks for their cell wall are not inhibited by methicillin or penicillin as are normal Staph aureus. What i am wondering is whether or not E coli or other bacteria could become resistant to sodium benzoate. My guess would be no because otherwise, the SB would have become ineffective long ago. But the question is could E coli become resistant to clove oil? Do you know how clove oil acts to inhibit or kill bacteria? If not, then you should do some reading and find out what is known about how it works. Then you would be able to answer a judge if they asked you whether E coli could become resistant to clove oil.

In answer to your question, I would say your comparison of clove oil and SB is an experiment because you are simply comparing a known bacterial inhibitor, SB, with clove oil which is not routinely used as a bacteriocide. One other point that you should keep in mind and be prepared to answer questions about, should the judges ask you, is that clove oil is not a single chemical as is sodium benzoate but a mixture of chemicals. Try to find out what part of the essential oil is thought to have antibiotic properties. Some of the chemicals in clove oil can be anesthetic--that's why it is an old remedy for toothache. Which of the chemicals in clove oil are thought to be antibacterial?

Your liquid culture experiment to test clove oil should be very interesting. Be sure to have your bacterial cultures in sterile tubes on a shaking incubator during the treatment otherwise the clove oil will separate out. Also when you do the serial dilutions, shake or vortex the tubes well before pipetting to distribute any oil evenly in the liquid as an emulsion. If you don't do this you may get some invalid data if too much or too little oil is transferred when you pipet. Be sure to plate a couple of dilutions so you are sure to get plates with the right number of colonies [100-200 per plate] to count easily.

Good luck and keep us posted on your progress. Take lots of photos and post them on google or dropbox in a shared folder so we can see what the ZOIs and colony counts look like.

Sybee

[GalaxyGirl4]

Hello!
I have almost finished my project, I now need to complete my backboard and finalize my discussion and conclusion. I did not test using a plate count method, unfortunately. My oil was extremely insoluble and when I tried to incorporate it into the agar after I couldn't get anything to work, it did not dissolve into the agar and I was afraid of inconsistency, so I tried the ZOI method instead. I used a vortexer and made sure I tried to coat the disks as evenly as possible with it. Oil solubility is definitely an issue. I did test 0.1%, 0.5%, 1%, and even 5% sodium benzoate to see if any effect occurred. I got absolutely NO effect at 0.1% or 0.5%. 1% SB may have inhibited the E.Coli, but only by a very minute amount, so I could not measure it. 5% sodium benzoate was also not too measurable - there was an extra 1.5mm around the disk. This was all for my first test for sodium benzoate.
I then re-tried the sodium benzoate testing once more to obtain average values. Still nothing at 0.1% or 0.5%. 1% still only had a very, very small ZOI. 5%, however, had a huge zone of inhibition! When I measured, I was surprised to see it had a diameter of about 16mm.... which was higher than my actual oil values, which I don't believe was supposed to happen. Is it possible contamination may have occurred, for example? Seeing that this is not consistent with the other values, however, whereas the oil was consistent with its values at every dilution, can I discard that 5% SB ZOI? Or do I have to calculate it in my average 5% SB ZOI, along with the first value? Can I put maybe, an asterisk, for example in the table and explain outside in my observations that this was inconsistent and not included in the average value?
I think this overall shows that sodium benzoate is not as effective as the clove oil was, however. Seeing as clove oil A.) had much more consistent, measurable values for all dilutions and B.) had actual, valid zones of inhibition but sodium benzoate had to be used BEYOND its maximum concentration in food to actually show any kind of effect, and even when it did the results were quite inconsistent.

[SciB]

Hi GG,

Congratulations on completing your project! I think you learned a lot about how real science works in the lab.

Your second result with 5% SB is interesting but, as you say, inconsistent with your first trial. But, considering that 1% SB gave only a barely measurable inhibition, the huge ZOI with 5% can be discarded from your calculations. If you had time, you would have repeated the assay once more to prove that the large ZOI was an error [or that it was correct--it's risky to assume that you know what is true without the experimental results to back it up]. I don't know what could have 'contaminated' your disk to have caused that degree of inhibition. Are you absolutely sure the SB concentration was 5%? Now you see why experiments have to be repeated many times by different people before we can be confident of the results. That's what makes science so great. It's not based on someone's opinion. It's proven by repeatable tests that can be done in many different labs.

I hope you continue to ask questions and do experiments--even if they are only thought experiments. We look forward to working with you again in your next science project.

Best wishes,

Sybee