Microbiology Science Fair Advice Needed! Thanks.

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David Howards
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Microbiology Science Fair Advice Needed! Thanks.

Post by David Howards » Wed Jan 10, 2007 6:07 am

I'm a sophomore doing a microbiology experiment. The focus of my project is on investigating the causes and remedies of antibiotic resistance through Microbiological experiments working with benign yogurt bacterial cultures (Lactobacillus acidophilus) and a generic antibiotic (Cefalexin). So what I'm trying to experiment on is creating living cultures of bacteria and finding approximately the maximum antibiotic tolerance dosage that still allows surviving bacteria that can grow and multiply (MIC). I also want to grow new generations of bacteria that will tolerate antibiotics better than the original bacteria (Grow antibiotic-tolerant bacteria). Finally, I want to find different methods to achieve this goal. If introducing antibiotics to more or less bacteria will result in greater percentage of survival was also one of my goals and finding the amount of time required for bacteria to regenerate and become more tolerant to antibiotics was another.

I've already worked 2 days on this experiment starting on Monday. Basically, I've created agar for 50 petri dishes, but I only ended up filling 40. I mindlessly added bacterial culture to ALL 40 plates - thereby eliminating a control for this step in the project. I did not have the antibiotics at the time and have put all of the plates into the incubator. Assuming I meticulously followed sterilization techniques, I should have parallel lines of bacteria on each of my petri dish by Wednesday. The teacher suggested I add the antibiotics at the same time of introducing the bacteria, which I didn't do. My only solution for now is to freeze half my cultures and refrigerate the other half, thereby generating some time for me to get the antibiotics while creating a side experiment of seeing if lower temperatures kills the bacteria, which I'll find out when I reuse them. I will also leave 4 petri dishes in the incubator to assess maximum potential bacterial growth, which also establishes a control for my experiment. What should I do next?

Is this a good idea? I need responses ASAP by 5am PST Jan. 10, 2007, otherwise I'll be at school and will be following my initial guidelines. Feedback is appreciated and needed.

Oh yes, I have minimal experience in the field of Microbiology, hence the mistake I made. I've taken chemistry for my lab class, but that's only vaguely related to biology. :(

The real big problem I have right now is NO WRITTEN PROCEDURE!!! I'm relying on wherever my initial instincts guide me mainly because I thought my general guidelines were enough for doing the experiment. Now, I found out I've tossed myself out of the frying pan and into the fire. :(

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Post by canalon » Wed Jan 10, 2007 3:24 pm

Sincerely you have a big problem:
- No control is bad, very bad.
- Once your bacteria started growing it is too late to add the antibiotic because you will not be able to see the differences between growth and no growth (and also because antibiotics are less effective when bacterial populations are already grown)
- Maximum bacterial growth has no significance whatsoever, this is useless.

In short you'd better restart everything from scratch.
Patrick

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any proof. (Ashley Montague)

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Post by mith » Wed Jan 10, 2007 7:12 pm

Well it's good that you're in contact with the protocol-online folks, best of luck to you.
Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
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Post by David Howards » Wed Jan 10, 2007 11:52 pm

I have good news!!! The petridishes did not grow bacteria. I think I didn't let the boiled-distilled water cool enough to allow the bacteria culture to survive. I've checked 17 hours after exposure, 19 hours, and 21 hours and all results were the same: ABSOLUTELY NO IDENTIFIABLE GROWTH. I diluted 10 loopfuls of yogurt in 100 ml of boiled-distilled water and used this solution to stain the agar. I know this procedure works because that was my experiment for last year, this year is a continuation based on antibiotic-resistance.

Anyway, what I did for today is put all petridishes in the refrigerator save 6, which I still put in the incubator, just in case the bacteria takes a long time to grow. I thought of freezing a couple but -20C might add wrinkles in my agar, so I decided against doing that. I have not put protective film around the dishes so there is a slight chance they can be contaminated. They were all set lid side down.

So, what should I do next? I will get antibiotics this weekend. Should I postpone experimenting till next Tuesday?

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Post by canalon » Thu Jan 11, 2007 3:00 am

Yes wait until you have your antibiotic, it's better.
Freezing agar would not have been a good idea. But if you have plastic bags or saran wrap, it is better because otherwise agar tend to dry out.

As for your experiment I would try different dilutions of your bacterial cultures to get a better idea of the bacterial population on your plates. Too much bacteria is NOT good. In fact, the ideal is to have just enough bacetria so they cover the agar by making independant colonies. But it might be hard to find the best dilution for that...
Patrick

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Post by David Howards » Thu Jan 11, 2007 2:22 pm

Thanks for the suggestion. My dad (scientist) suggested that the bacteria may be exponentially growing, but doing so slowly. What he means is that bacteria is present but in small quantities. He may be right because the yogurt culture I used was many days before expiration. There might be a slight chance that not enough culture was able to grow in 24 hrs to be visible to the naked eye. Even my experiment last year might confirm this because I used expired yogurt. This is only an assumption though. I still have to check every day to see if this has actually occurred.

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Post by David Howards » Fri Jan 12, 2007 10:07 pm

I checked the petridishes in the incubator and refrigerator again and bacteria did not grow. I'm planning to take pictures tomorrow and do another check before retiring to the weekend. So far, the bacteria had over two days to grow but growth is still not visible to the naked eye. Since the yogurt isn't expired, growth might take a long time to establish verses expired yogurt. I can tell the solution I swabbed onto the petridishes is still visible under the light when reflected off at an angle. The right temperature for growth is already established and that is 37C or 98.6F.

My plans are as follows: After pics and recheck, I'll retire for tomorrow. I'll write my procedures up, type my notes, and then I'll get my antibiotics during the weekend. If bacterial growth is not established in the incubator control variables on return, I will simultaneously mix the antibiotic solutions and reintroduce new yogurt cultures. If growth did establish in the control variables, then I will add antibiotics to the refrigerated petridishes and half of the incubated dishes, leaving three to yield maximum growth.

My only questions: how should I distribute the antibiotics in order to introduce antibiotic resistance? How am I going to measure concentrations to anything? Bacteria in water for my cultures for example. Should I do gram per liter or parts-per-million what ever that is? How about for antibiotics? mg per L? I'm confused. Somebody help me.

My thought is to evenly coat the entire surface of petridish with a concentration of antibiotic and do the same to other dishes but with different concentrations. I will then find which one yields growing bacteria and use that concentration as my basis for further antibiotic experimentation.

How's that sound? Btw, what am I forgetting? I have a feeling I'm forgetting to say something....

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Post by canalon » Fri Jan 12, 2007 11:10 pm

The recommended breakpoint for antibiotic resistance for cefalexine is 32 mg/L, you should be around this kind of concentration in your petri dishes. This means that you should have a good idea of the volume of agar in each plate, and add enough antibiotic to each plate to reach the concentrations you want to have. You dilute your antibiotic in enough liquid (200µl/plate should be good) plate that and let dry. You can even keep the plates 1 night at 4ºC in the fridge to let diffusion happen and be sure to have an homogeneous distribution of antibiotic in yourrplates.

I would test (with repeats for each) a wide range of concentration , mostly below that starting from 2 to 64 mg/L could be a good idea, at least 3 plates for each concentration). Resistant bacteria will be growing in plates containing 32 or 64 mg/ml cefalexin.
Patrick

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Post by David Howards » Sat Jan 13, 2007 12:07 am

Thanks for the advice canalon. However, the ratio between the human body and the petridish weight is enormous. 250mg of cefalexin is the average amount for a 50kg body. Assuming a petridish with agar takes 100g, I'd only need .5mg concentration per dish according to the ration 500:1. Since I'm modeling my experiment to the human body, it would be more logical this way. However, you said between 32-64mg is needed. This is 64 to 128 times the amount that I calculated. How can antibiotic resistance still develop under these conditions?

Wait a sec. Could it be the volume of blood versus the volume of 1 L of water? I think that might be the case.

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Post by canalon » Sat Jan 13, 2007 2:52 am

A petri dish is 20 to 25 ml. You have to reach a concentration of 64 mg per Liter of agar. You will not need much antibiotic per plate.
Believe me those numbers are not coming out of the blue but from the directions for laboratory diagnostic given by the french society of microbiology (which unlike its US equivalent, formerly known as NCCLS, do not ask you to pay to have the critical values for Antibiotic resistance).
Patrick

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Post by David Howards » Sun Jan 14, 2007 7:20 am

But why does that high of a concentration produce resistance when the bacteria should have been killed? Do you have the site where you found the info?

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Post by David Howards » Wed Jan 17, 2007 12:23 am

It's semester finals week and I want to leave experimenting alone for this week. Should I let the bacteria grow on its own anyway? Btw, BIG QUESTION: When I let the bacteria grow, how many plates out of 40 should coat bacteria on? What should I do with the remaining plates? Should I administer antibiotics straightaway? If so, since I need to see if antibiotic resistance is created, should I leave a few empty plates to retry growing the bacteria? Btw, how should I administer the antibiotics? Pour it on the whole culture before I allow it to grow while I leave a few controls to justify growth actually occurs, or use antibiotic paper disks? If I use the disks, how would I know antibiotic resistance occurs? And if so, how should I know the entire plate contains antibiotic-resistant bacteria as opposed to normal bacteria? Should I just take a sample out of the antibiotic ring kill zone and attempt to regrow it? How would I know if a certain dilution of antibiotic is effective or not if I use antibiotic disks? The bacteria will still be killed in the same radii due to equal diffusion of the antibiotic. How would I know what concentration is good? Would I just conclude that all bacteria dies after a certain threshold?

Please carefully answer my questions, thanks.

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