Antibiotics Against Bacteria

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dahae7134
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Antibiotics Against Bacteria

Post by dahae7134 »

Hi,
I am considering starting a science project on either how long it takes for antibiotics to kill out all the bacteria on the agar plate or how much antibiotics are needed to kill out the whole lawn. I would specifically like to use penicillin and Lactobacillus in yogurt. However, how would I add the penicillin (disk or liquid) and when would I add it during the bacterial growth? Second, how would I be able to know when all the bacteria are dead?
jskanderson
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Re: Antibiotics Against Bacteria

Post by jskanderson »

Hi Dahae,

That's so awesome that you're thinking about doing a science project on this; microbiology is amazing!

If you were planning on testing how much antibiotic is needed to completely wipe out a bacterial lawn (specifically using penicillin and Lactobacillus), then I would suggest doing a procedure similar to this one: http://www.benchfly.com/text-protocols/ ... e6ab86.pdf . You would get several plates of agar, and plate each one with the Lactobacillus by swabbing the bacteria on the agar plate. Then, set one agar plate with the Lactobacillus as the control. Then, on the others, vary the amount of disks soaked in penicillin on the agar plates (ex. on one plate, only put two disks soaked in penicillin, and increase by twos after that plate). That will show you how much penicillin is needed to wipe out a plate of bacteria based on the # of disks used.

However, if you were planning on testing how long antibiotic takes to completely wipe out a bacterial lawn, then I would suggest using different types of antibiotics, not just penicillin, as your independent variable. Maybe you could test the effectiveness of different antibiotics on Lactobacillus by measuring time? Remember to have a control plate, and keep the number of disks soaked in antibiotic consistent!

Hope this helped, and please let me know if you have any other concerns/questions!

P.S. I just realized that I forgot to answer two of your questions! You add disks carefully with tweezers by placing them flat on the agar plate with the newly created bacterial lawn (that you just spread on the plate). To answer your other question about being able to tell whether all the bacteria will be dead, the antibiotic disks will create a zone of inhibition devoid of bacteria, and that represents the amount of bacteria killed/effectiveness of the antibiotic! Something like this: http://www.pmbio.icbm.de/mikrobiologisc ... s/x059.jpg Hope this answered your other questions!

-JSK
~ Call me JSK :D
"Be the person who you wish to see in the world."
asyed1229
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Re: Antibiotics Against Bacteria

Post by asyed1229 »

Hi Dahae,

To add to what jskanderson has said, make sure you put the disks in right after you apply the bacteria and I recommend you seal the agar plates shut with transparent scotch tape. It can be very harmful and dangerous to your health to open the agar plate after some bacterial growth and then placing the antibiotic discs.
dahae7134
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Re: Antibiotics Against Bacteria

Post by dahae7134 »

Thank you both so much for your replies!
However, why must you place the antibiotic disks on right after the bacteria is spread? Could you not wait until the petri dish is covered with the lactobacillus colonies and then place the disks? I guess a related question would be how would you be able to tell when ALL of the bacteria are dead? I understand that the antibiotic disks will create a zone of inhibition but will the zones cover all of the plate, signaling the death of all the bacteria?
asyed1229
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Re: Antibiotics Against Bacteria

Post by asyed1229 »

Hi dahae, it takes time for the petri dish to be covered with the lactobacillus colonies (roughly 24 hours with an incubator and 48-72 hours using a lamp) You can't leave the agar plate open and wait for the bacteria to grow you have to close it. When the colones cover the petri dish, you should never reopen them for several reasons. One is for your health, another is that they may get into the air, and another is that the air in the room has bacteria and particles interfere with your results and pose as a extra variable. Therefore you should apply the bacteria and then within five minutes place the disks into the agar plate. After doing this you should tape them shut, so you or somebody else doesn't accidentally open the plate.

It is also highly unlikely that the zone of inhibition will cover the whole agar plate if you use one to three disks and that your antibiotic will kill all the bacteria. I'd test with 3 disks in an agar plate, four disks in another agar plate, and five disks in another agar plate. The more disks you put the more surface area of the agar plate will be inhibited.

The bacteria will naturally die over time after running out of food, which is the nutrient agar.

I am confused on certain things about your project. How are you inoculating the bacteria from yogurt? Also are you using premade penicillin disks or are you using coffee filters and putting them into penicillin liquid?

This site explains the phases of bacterial growth:
http://www.actforlibraries.org/what-are ... al-growth/
allisontu
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Re: Antibiotics Against Bacteria

Post by allisontu »

Hi Dahae,

To add on to what asyed said, you need to add the antibiotic disks right after you plate the bacteria since the purpose of these disks is for the bacteria to grow in the presence of the antibiotic, rather than be killed after growing. It's unlikely that the antibiotic will kill all the bacteria on the plate, but the zone of inhibition will be pretty clear. I've pasted a link below with a helpful photo and more information. The official name of this procedure is called the Kirby-Bauer method, which might be helpful for future searches for more information.

http://www.uphs.upenn.edu/bugdrug/antib ... al/bk.html

Hope this helped,
Allison
dahae7134
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Re: Antibiotics Against Bacteria

Post by dahae7134 »

Thank you once more, but my question is how long it will take for the antibiotic disks to kill all of the bacteria on the plate. However, since it is unlikely that the antibiotic will kill of the bacteria on the plate, how would I go about conducting my experiment? Would you recommend changing my question? Also, I planned to use the pour plate method and to use premade penicillin disks. However, I am not so sure if my question is even one that can be clearly tested.
probiotics
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Re: Antibiotics Against Bacteria

Post by probiotics »

Hi. I am using lactobacterius in yougurt too, but if you are applying it on a agar plate, do you have to isolate the bacteria? Or can you just use yougurt?
allisontu
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Re: Antibiotics Against Bacteria

Post by allisontu »

Hi Dahae,

Since it's very unlikely that one antibiotic disk will kill all the bacteria on the plate, I suggest you use a method like what Asyed suggested earlier: increasing the number of antibiotic disks on each plate until all the bacteria are killed.

Using the Kirby-Bauer method, the antibiotic immediately begins to act on bacteria once you put in on the plate. It's hard to tell how long it takes to kill all the bacteria because you likely won't be able to see a clear zone of inhibition until the other bacteria on the plate being to grow out, which can take as long as overnight. If you'd like to stick with your current question, you could try a modified method like you proposed earlier, in which you place the antibiotic disk on the plate after the bacteria have grown out.

The only issue with performing your experiment like this is that you may not be able to tell if the bacteria are dead or not from just looking at the plate. I would suggest testing this method with just one plate to confirm that it works before you perform your actual experiment. Additionally, I would test a plate using the standard Kirby-Bauer method, then measure the zone of inhibition. Then, use this zone of inhibition as the stopping point for your experiment. Once all the bacteria within the zone of inhibition are dead, then you know to stop timing.

Finally, I would also make sure you have an independent variable that you change from experiment to experiment. I would suggest changing the number of disks on each plate, then measuring the time to kill bacteria and the amount of bacteria killed (which you can find by calculating the area of the field of dead bacteria). You could also try varying the concentration of the antibiotic.

Hope this helped, and let us know if you have more questions!
Allison
jskanderson
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Re: Antibiotics Against Bacteria

Post by jskanderson »

probiotics wrote:Hi. I am using lactobacterius in yougurt too, but if you are applying it on a agar plate, do you have to isolate the bacteria? Or can you just use yougurt?
Hey probiotics,

If you are trying to grow lactobacillus from yogurt, you would take a tiny amount of yogurt and use a sterile inoculation tool (or other tool) and streak the bacteria on the agar plate using the streak-plate method. (here's a link explaining what the method is: https://en.wikipedia.org/wiki/Streaking_(microbiology) ).

Hope this helped!

-JSK
~ Call me JSK :D
"Be the person who you wish to see in the world."
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