Microbiology Science Fair Project, Advice Appreciated - Any and all help is appreciated. (Jan/09/2007 )

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. :(

hi!
first be sure that the bacteria isn't resistent to the antibiotic, because that will be a really BIG PROBLEM. You could freeze the bacterias if you use a mix of liquid media and glycerol. For the protocol the antibiotic should be added in one of two ways: wait until the media is cold enough (to hot will damage the antibiotic, to cold will solidify the agar) and add the antibiotic to it and then plate(keep in refrigerator until use) or after you spread the bacteria in the agar(should cover all plate) put paper disk soak with the antibiotic. The important thing is that the bacteria is put to grow WITH the antibiotic. do not grow the bacteria and then add the antibiotic. hope this help.

That's my problem. I have grown the bacteria without the antibiotic and today I will refrigerate the bacteria before it spreads through the entire petri dish. You see, I currently do not have my antibiotic needed for the experiment. I still have to pick up the prescription.

If you refrigerate the petri dishes it will slow down the bacterial growth, but they will continue to grow...remember the growth plot!! and the cultures still in risk of a contamination(to avoid this put parafilm around the petri). If you will not get the antibiotic in the next days I recommend that freeze(-20 is ok, but if you find a -80 freezer that will be better) some of the cultures with media and 25-50%glycerol so you could always have bacterias. I recommend that you donot grow more bacteria until the protocol is write and revised. If you want email it to me so I could give more advice.

I have good news Merlav!!! 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?

Btw, thank you very much for helping me on this, Merlov.

Note on earlier post: I can assure you the bacteria is not antibiotic resistant!

Check my profile for my email. :)

When dilute the cells use the dH2O at room temp to make sure that you are no making lacto soup. Wait until you have all the reagents and the protocol to begin the experiment. Make sure that the cells are alive. Check what is their generation time and the best temp for culture them. Is super mega important that before begin an experiment you don't have any question or doubts about the protocol, thats a very common mistake in research.
ahhh..and before I forgot, never put a petri dish at -20C, to freeze the bacteria must prepare liquid media (the same for the petri dish but without agar) and culture them, then add 300-500microL and 300-500microL of 50%glycerol to a microcentrifuge tubes (if have screwcap is better). when you have to store a petri dish in the refrigerator use alway parafilm.

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....

there is a way to count bacteria and expression is cells/mL I don't remember the math equation but exist check a microbiology book. antibiotic could be mg/mL
just add the antibiotic to the agar can be done before plate, but the agar must be a little bit cool or after plating add some microL(100-200) to the agar shake and let it absorb for 10-15min. the important thing is that if added to the agar then it must be added before the cells. the other way is using some paper disk9they come for this purpose) soak in antibiotic, put first the bacteria covering all plate and then with a forcep the disk with antibiotic. the pros that this have is that could divide the petri in 4th and place a disk on each quadrant it will save time and materials and just need to measure the distance between the disk and were the bacterial growth begin and there is tables that explain the results.

I took pictures today! Yeehah! Still no bacterial growth. Guess the bacteria is dead.

My condolence...RIP to the bacterias ....definitly they are dead...try to dilute them at room temperature.