Science Buddies: "Ask an Expert"

Hello!

I am working on the Science Buddies experiment "Which Acne Medications Really Zap that Zit?"

As I was researching acne, its causes, types, etc., I found that the main cause if acne was Propionibacterium. Why does the experiment say to use e. coli instead? I understand that e. coli is easier to grow, but since it is a Gram-negative organism, and P. acnes is a Gram-positive organism, how are they the same?

I am just worried that my results may not be accurate. So, to sum everything up, my question is what makes e. coli able to substitute P. acnes, and how does e. coli affect acne?

Thank you in advance! My project is due in 5-6 weeks so a quick answer is much appreciated!

Hi - great questions on this project. I suspect the main reason e. coli is used instead is because of ease-of-use and safety. You are right - if you wanted to study the BEST model system, you may pick a different bacteria. However, for our learning purpose here, the model organism is similar enough.

Tonya

Thank you for the reply Tonya!

I understand why e. coli is substituted now. Thank you

Do you know of any other easy to grow/use bacteria that may be even more similar to P. acnes? I am just trying to get the most accurate answer possible

-Madelyn

Hi Madelyn,

Bacteria can be harmful to humans and even deadly, so growing certain species has to be done by experienced persons under what are known as Biosafety Level 2 or 3 conditions. These regulations require the use of special isolation hoods and personal protection equipment. E coli K12 has been genetically engineered to be safe for use and it has been so well studied over many decades that it is the 'lab rat' of bacteria.

I understand why you want to do the experiments on bacteria that cause acne because that is what a scientist would do. You could do this but you would have to find a university microbiology lab that does research on acne-causing bacteria and ask them if you could work there under supervision. This usually takes a commitment of at least 6 months and not all labs are able to spare one of their people to be your mentor.

I would stick with E coli as a model organism. You can do some online research comparing E coli and P acnes and list the similarities and differences as they relate to the action of the antibacterial compounds. This would make a good project.

Let us know if you have more questions.

Good luck!

Sybee

Thank you for replying Sybee!

Obviously, with my science fair in a month I don't have time to contact a university for the use of a lab, and I am I homeschool student so I do not have access to a high school laboratory either.. But I will definitely do some more in depth research on P. acnes and e. coli and list their similarities.

I'm very excited to start on my experiment! Thank you very much Tonya and Sybee for the quick and extremely helpful replies! I will be sure to let you know if I have any more questions

You are most welcome, Madelyn. If you need more help with the bacterial methods or with reading and understanding microbiology papers online, do let us know. We are part of your virtual education and happy to teach you whatever you need to know!

Good luck,

Sybee

Hi there,

As the previous experts mentioned, E. coli is substituted for P. acnes for this project most likely due to its ease of use. You are correct in that E. coli is Gram negative and P. acnes is Gram positive, and since the membrane and cell wall composition of those two bacteria are different, these factors could potentially influence how well these bacteria deal with different medications. However, E. coli is harmless and super easy to grow and work with, whereas P. acnes much more difficult to culture because it grows really slowly and requires an anerobic environment. If you are concerned about the fact that E. coli is Gram negative while P. acnes is Gram positive, you can certainly try to use Bacillus subtilis, which is a Gram positive bacterium that is harmless, easy to grow and work with, and also has been studied extensively as a model organism, much like E. coli. You can also buy Bacillus subtilis from Carolina Biologicals.

Let us know if you have anymore questions!

Connie

Hello!

I am working on the experiment "Which acne medications really zap that zit?".

I have grown the e. coli as instructed, and on my 3 controls I have tons of growth evenly spread across the dishes. On the plates that contain antimicrobial discs with medications, there is little growth of bacteria and no zones of inhibition! I do not have access to a 37C incubator, so I have been letting them grow in the laundry room next to the dryer since it tends to be a warmer room. Its been about a week now, and I'm still just not seeing any signs of the zones of inhibition... Maybe I did something wrong? The only thing I did differently from the instructions was purchase a nutrient agar kit where you mix up the agar, pour it in petri dishes and then let it set instead of purchasing pre-made agar plates.

Please let me know if you need any more information!

Thanks in advance!

Hi love2fly,

I'm sorry that you don't think your experiment worked properly. That happens to all of us, but it doesn't make it any less frustrating. I have a couple of thoughts and questions.
1) You say that you have tons of growth evenly spaced across the dishes in your control plates, but would you describe your growth as a bacterial lawn (i.e., completely covered with bacteria to the point that you can't distinguish individual colonies) or do you have lots of colonies, but still separated by a fair amount of agar in between each one? You really need pretty good bacterial coverage of the plate to see a zone of inhibition, so if you didn't have enough bacteria even on the controls, I would say that you may not be able to see the zone of inhibition.

2) Another possibility is that you have very little bacteria on your antimicrobial plates because of the antimicrobial medications. If they were too strong, they could be inhibiting bacterial growth in a very large zone, essentially preventing bacterial growth.

3) My third thought has to do with you pouring your own plates. You said you purchased a nutrient agar kit where you mixed up the agar and poured it yourself, which is fine, but if you added the antimicrobial discs while the plates were not entirely solidified, the medication could have diffused throughout the plate somewhat, leading to overall lower growth, but no zone of inhibition.

4) Certainly a 37C incubator would be better for looking at this, since that is the ideal growth temperature for E. coli, but as you know, the E. coli will eventually growth even at low temperature, but it can take awhile. Possibly the low temperature and the antimicrobial discs combined to make bacterial growth much poorer on the experimental plates compared with the effect of low temperature alone on the controls, but since you did get growth on your control plate, I wouldn't worry about it too much.

I hope this helps you come up with some thoughts. If you'd like, you can post pictures of the plates here for me and other experts to look at.

JMP

Hello JMP! Thank you so much for your help!

After reading your first suggestion, I've discovered that while I may have lots of growth, it is defiantly not a bacterial lawn!

I don't think I placed the discs on the agar too soon... The plates were in the refrigerator as the instructions said to do for about a week.

Will I have to try the whole project again? What can I do to prevent this from happening a second time?

I tried to post some pictures of my plates but the message board kept saying that the file extensions were not allowed. I tried .jpg, .bmp, .tiff, and .pdf Maybe the system is having some problems.. I will try again tomorrow.

Thank you so much!!

Hi,

JMP has given you lots of good suggestions. I would just like to ask about your procedure for spreading the E coli on the agar. You said that you did not really get a ‘lawn’ of bacteria so that made me wonder whether you plated enough culture on the agar or if your E coli culture is not viable. Did you get the E coli from Carolina Bio? Was it a liquid culture or an agar slant? How did you store the culture? How much of the culture did you spread on each plate and how did you spread it?

These details of how you plated the bacteria are important because they can affect the growth. Do you have an accurate digital thermometer? I would check the temperature where you had the plates. As JMP said, E coli grow fastest at 37C. You can improvise an incubator by putting a small lamp with a 40W incandescent light bulb inside a cardboard box. Check the temperature and open or close some of the flaps until you get it to between 30 and 37C. Don’t let the temp get above 37 as E coli can be inhibited at 42C. Put your plates into the box and cover them with aluminum foil. You should get a nice lawn in a couple of days.

If your E coli culture is not healthy or you don’t have enough to put a good drop (about 100 microliters) on each plate for spreading, then you should purchase a fresh culture.

You may not know what a bacterial lawn should look like so here’s the wiki that shows a picture of a plate with antibacterial disks and zones of inhibition: http://en.wikipedia.org/wiki/Bacterial_lawn

I hope this helps and you are successful in your next experiment. Keep us posted.

Good luck!

Sybee

Hello Sybee!

Yes, I did get the e. coli from Carolina Biological Supply. It is a liquid culture. I stored the culture in its original packaging for about 3-4 days until I received my other supplies from another company.

I put about 10 drops (500 μl) of the culture on each plate, and spread it as instructed by the experiment plans with a sterile cotton swab.

After looking at the picture you provided as well as some others, I realized that I certainly do not have a bacterial lawn! Do you know what file extensions are allowed on the forum? I have tried several and it still wont let be upload pictures of my plates.

Thanks again for all the help

Hi,

Well, you did put enough of the culture on each plate but a cotton swab is not the correct tool for making a bacterial lawn. What you need is a spreader which you can easily make from a large paperclip: https://www.sciencebuddies.org/science- ... #procedure

To sterilize the spreader hold the end in a candle flame for a few seconds then let it cool without touching anything. Take the tube that has your bacterial culture in it and shake it a bit to resuspend all the cells then put a couple of drops on the plate (I think 5 drops is too much) and immediately spread the liquid around on the surface until it covers it uniformly. Be VERY careful not to dig into the surface of the agar with the spreader. If you want to see how to spread bacteria on agar go to youtube and check out videos on making bacterial spread plates: https://www.youtube.com/watch?v=esunMqaHmbU

Spread your plates one at a time because if you put the drop of culture on a plate and let it sit for too long, the liquid can get absorbed and you won't get a good spread. Try to incubate your plates at 30 to 37C so the E coli will grow better and form a nice lawn.

Do you still have E coli culture left? You should store the tube in the refrigerator (but don't freeze it!) because the bacteria will eventually die if kept longer than ten days or so at room temperature.

Rig up an incubator then try the experiment again using the paper clip spreader. You should get a lawn with clear areas around the antibiotic disks.

Good luck!

Sybee

Thank you so much Sybee!!

I will definitely try to rig up my own incubator and spreader with a paper clip. I used the cotton swab because the experiment plans said that if I did not have a bacteria spreader, I could use a sterile cotton swab from an unopened package, and also that I would have to put more drops of the culture so the swab wouldn't soak anything up.

Again, thank you both so much for the help. I will order some more e. coli culture, and try again.

I'm happy to help! If you have any more questions don't hesitate to ask. We're here to guide you in avoiding problems and doing the best project you can.

Sybee

Hello!

I am working on the experiment "Which Acne Medications Really Zap than Zit?"

While waiting for more supplies for my project, I stored nutrient agar plates in a refrigerator for about a week. Now, a couple of them are frozen. Am I still going to be able to use them once they are at room temperature? I ordered 10, and 5 out of the 10 are frozen. I suppose worse case I will only use 2 controls rather than three.

This is the second time I am attempting this experiment due to a couple of minor errors that resulted in an unsuccessful outcome It is due in two weeks, so any quick responses are greatly appreciated!!

Thank you so much!

Hi there,

Are you storing the plates in a regular fridge or in the freezer? If you store them in a typical fridge, it should be at about 4 degrees celsisus, and agar plates typically shouldn't freeze at this temperature. However, I don't think there should be any problem with using them after thawing them at room temperature--just make sure they're dry before you plate any bacteria on it. If other experts have any experience with frozen agar plates, please chime in. If you are concerned that the plates may not work, you can always order another set if they are going to arrive on time.

Let us know if you have any other questions. Good luck with your experiment!

Connie

Hello. We noticed you had multiple threads going for the same topic. We merged them into a single thread. Please keep all questions to one thread so that our experts can best help you based on what has already been discussed.

Good luck, and thanks for using Science Buddies!

Hi,

I wondered what happened when you thawed the frozen agar plates. I think freezing agar is like freezing Jello--it's not in very good shape when it thaws! It tends to form chunks with liquid around them and this would not work for making a lawn of bacteria.

You said you had 5 plates that were not frozen. Isn't that enough if you put several disks on each plate? I would include a control disk on each plate for comparison. How are you setting up the replicates--on the same plate or different plates? Let us know how you are doing the experiment and maybe we can make suggestions so that you can get the most data out of your 5 plates.

Hopefully everything will come out perfect this time!

Sybee

Thank you everyone for the help!

Connief, I did store them in a regular refrigerator.. the temperature must have been to low. I will adjust it.

SciB, When the plates were almost thawed, it looked to me that there was ice inside of the actual agar. I am testing 3 medications on three different plates. Each plate has four sections: water, 1A, 2A, and 3A, the other medications labeled as B and C. The original experiment plans included 3 other plates as control plates to be sure that the bacteria was growing successfully. Since I only had 2 other plates available, I have 2 control plates instead of 3. I also constructed an incubator as you had previously suggested.

I hope this kind of answers the questions.. Let me know If you need any more info and thank you so much for your help yet again!

OK, that sounds good. You have enough plates to do your experiment. Just be really careful when you are doing the spreading of the bacteria with the paper clip so that you don't dig into the surface of the agar. This is easy to do with a wire so you might want to sterilize the spreader and practice on one of the frozen plates if you can. You need to hold the spreader somewhat loosely and turn the plate with your other hand as you are spreading. Watch the video I sent you a while back to make sure you know exactly what to do.

The other thing you should pay attention to is the bacterial culture that you are using to transfer to the agar. I am assuming that you have a liquid culture of E coli--is that right? Then before you remove any of the culture shake the tube gently for a few seconds to make sure the bacterial cells are uniformly suspended in the liquid. Shake the tube each time before you remove the culture. Also, it is better if you spread each plate immediately. If you put culture on all the plates first, by the time you get to the last one the bacteria in the drop may have started to adhere to the agar and you won't get a good lawn.

It is also useful to remove your plates from the fridge 30 minutes or so before you are going to do the plating so that they can warm up. Check the plates and if water has condensed in the lid remove it and shake the water out into the sink. Put the lid back on immediately to maintain sterility. Plates that are too wet can cause uneven bacterial growth and ruin your experiment.

Good luck and do let us know how this comes out. Hopefully your diy incubator will make them grow well and you will get a nice lawn of happy E coli--except around the antibacterial disks. Remember to take good, clear photos of the plates with the lids off to show the zones.

Congratulations, you're almost done!

Sybee

Thank you Sybee!

Yes, my e.coli is a liquid culture.

I think it worked this time! The plates have been in the "incubator" since Saturday afternoon, and there is a big difference in the results! There is a nice bacterial lawn, and the zones of inhibition are faint, but I can see them!

I will keep you updated! Thank you so much for the help! Couldn't have done this without y'all!

Ok, so the results are:
Tea Tree oil (A): 1A-2 1/2cm, 2A- 2 1/2cm, 3A-2cm
10% Benzoyl Peroxide (B): No zones of inhibition!
Acidic Water (C): No zones of inhibition!

Since I still have some plates and culture left, I would like to repeat the experiment with the benzoyl peroxide, acidic water, Noxzema, as well as salicylic acid. Are the plates that were frozen still useable?

Is there any way that I could pigment the e. coli without harming it? It would be a lot easier to see the zones of inhibition if it were colored a little.. especially when taking pictures. Any tips on taking pictures of the plates and still getting the zones of inhibition to show up?

This has been so much fun!! I think the judges will love it! As a bonus, I may be able to get rid of my acne!

Thank you Science Buddies!

Hi,

Yaay! You finally got some actual data. Can you put your photos into a Word document and either upload it here or put it on Google Docs? I really would like to see what your plates look like. A zone of inhibition is usually very clear. Sometimes there will be some resistant colonies growing within the zone but that doesn’t count. How long did you let the plates incubate before you measured the zones?

I’m glad you are going to try the experiment again. As long as the agar surface of the frozen plates is smooth and not wet they are usable. I am surprised that benzoyl peroxide did not prevent bacterial growth. Maybe it just doesn’t work against E coli. Remember, E coli are not the zit bacteria. Those are a different species and they may not respond the same way to an antibacterial. The most common acne species is called Propionibacterium acnes (http://en.wikipedia.org/wiki/Propionibacterium_acnes) and is probably the one most acne medications are made against. When you write up your report be sure to mention that you did your experiments with E coli and not P acnes and discuss whether you think there might be a difference in inhibition.

As for staining the bacteria to make them show up better, you would have to experiment with this. You want to stain the bacteria but not the agar and that can be a problem. You can try food colors using the plates you just measured. Certain kinds of ink may work also.

Since you don’t have much time or plates, I would just stick with adjusting the lighting of the plate to make the zones appear best. Don’t use the camera’s flash and take the lid off the dish while you are taking the photo. Get as close as you can but make sure your camera can focus at that distance. I think putting a piece of black paper under the plate will help show the clear zone. You can always google ‘how to take pictures of bacterial plates’ and see if there are any other suggestions.

Keep us posted on your results. Good luck!

Sybee

Hello!

I let the plates incubate for about 60-72 hours.

Here is the link to the Google Doc: https://docs.google.com/document/d/1HVg ... sp=sharing I hope you wil be able to see everything clearly.. I had trouble getting a clear picture.

Tomorrow I will try the experiment again and keep you posted!

Thank you so much for all of the help!

Hi,

I looked at your photos and they look fine. The zones are clear and measurable. It looks like some of the plates may have been a little too wet when you spread them because you can see excess bacterial growth in some places instead of an even ‘lawn’. If you see a lot of water droplets then shake them off as I said.

I would not bother with trying to stain the bacteria. I took a look on Google images for bacterial zones of inhibition and the photos all seem to be taken against a black or gray background with no stains or special lighting that I could see: https://www.google.com/search?q=bacteri ... w#imgdii=_

Good luck with your next experiment—and HAPPY HOLIDAYS!

Sybee