student2021
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Help with my project/bacteria

Postby student2021 » Thu Dec 03, 2020 6:24 pm

For my project I plan on conducting a research where I find the inhibition potencies of garlic verses the synthetically manufactured antibiotics I chose, gentamicin and penicillin, and seeing how they affect the gut microbiome by testing them on E. coli and Bifidobacterium animalis subspecies lactis.

I was going to purchase my Bifidobacterium animalis subspecies lactis bacterium from atcc, although it seems to be very expensive. I found the bacterium at NRRL at a cheaper price, but I'm not quite sure if it would work with my project or if I could still use it. The only difference I saw from the one on atcc and NRRL was that they were isolated from two different things. I was wondering if you could tell me your thoughts on it and if I could use the NRRL bacterium in my project.

Here is the information for that bacterium from NRRL.
Bifidobacterium animalis subsp lactis
NRRL B-59135
Source: Vivolac Cultures Corp.
Isolated from (substrate): fermented dairy product
Substrate location: Germany
Growth media: MRS

Optimum growth temperature: 37C

If I can't use this bacteria, do you guys have any suggestions on what other BSL 1 bacteria I can use in my project. Besides that, I plan on making my own garlic extract and was wondering how to proceed in doing so. I am having a hard time in searching how I should make my own garlic extract. Also should I dilute with distilled water or should I just keep it plain. Also what size tip should I use with the pipette when I am inoculating the garlic into the antimicrobial susceptibility disk.

brandimiller610
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Re: Help with my project/bacteria

Postby brandimiller610 » Sun Dec 06, 2020 6:01 pm

Hi Student2021,

Your ideas for your project sound interesting so far and it seems as though you have really thought about the direction you would like to take with it.

I believe E. coli and B. animalis are good choices for your project, as they are both very abundant in the human gut. Because many Bifidobacterium strains are considered "probiotics" (meaning they confer benefits on the host) and are lactic acid-producing bacteria, they are commonly cultured in fermented milk or dairy products. Therefore, I think it is perfectly okay to use the B. animalis strain that was isolated from a fermented milk product; all of the information you provided about it from the NRRL site looks okay. I have experience working with probiotics (including Lactobacillus and Bifidobacterium strains) and I have read a lot of studies that isolate their strains from breast milk and dairy products. These bacteria can also be isolated from the human/animal gut or fecal samples, but I don't think the source will affect your project outcome dramatically.

I do not have much knowledge or experience working with garlic extract, but here is some information I found on using it as an inhibitory agent/antibiotic:

https://www.ncbi.nlm.nih.gov/pmc/articl ... 20bacteria.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458355/

These papers describe methods to prepare garlic extract; one study dilutes the homogenized garlic, while the other does not. I think you should use pure garlic extract as your highest concentration and then do serial dilutions of the extract to determine the minimum inhibitory concentration of garlic extract on your test organisms. I would use a 200 ul micropipette and pipette 50 ul of extract onto the plates at the time of your antibiotic susceptibility disc assay (just be sure to match these concentrations with that of the antibiotics you have selected).

I hope I have helped answer your questions. However, if something is unclear or you have additional questions, please feel free to add them on this forum. Good luck with your project!

--Brandi

student2021
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Re: Help with my project/bacteria

Postby student2021 » Mon Dec 07, 2020 8:57 am

I decided to switch my bacteria to Lactobacillus acidophilus. It was much more cheaper than the other bacteria, as I have budget. Thank you so much for the help and suggestions on the garlic extract!!!

Another questuon pertaining to the tip size of the micropippette, as I am getting disk already innoculated with the antibiotics (grntamicin/penicillin) at Carolina and each disk is 10 mcg, would I have to innoculate 10 mcg of garlic extract to disk? Would the tip size be the same as you suggested? Was that what you meant by the same concentration?

brandimiller610
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Re: Help with my project/bacteria

Postby brandimiller610 » Mon Dec 07, 2020 8:46 pm

L. acidophilus is also a good choice!

If you are using pre-inoculated discs, then ignore my comment about the different concentrations of garlic extract. You should try to match the concentration of garlic extract on the discs to that of the gentamicin and penicillin as much as possible (this may be challenging since the antibiotic discs are already inoculated -- that way you quantitate the zones of inhibition relative to the concentration of antibiotic/extract used. The most important thing is to make sure that you take into account all dilutions and know the concentration of the extract you are using for the discs -- both of the papers I included in my previous comment use a final volume of 50 ul. Be sure to consider how much garlic you start with and how much dH20 you dilute it in for the final volume. So yes, I would still use a 200 ul pipette and aliquot 50 ul for each disc.

I hope I have answered your questions! Again, please feel free to reach out if any more questions arise. Good luck! :)

--Brandi

student2021
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Re: Help with my project/bacteria

Postby student2021 » Tue Dec 15, 2020 2:28 am

When I am performing the disk diffusion assay method and swabbing the bacteria onto the agar plate would I have to use a new swab ever agar plate? I am using 20 agars for each bacteria. I purchases 1 tube of both of my bacterias. I was wondering if thats enough bacteria?

student2021
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Re: Help with my project/bacteria

Postby student2021 » Tue Dec 15, 2020 4:27 am

How would I match the concentration of the garlic extract to the antibiotics?

student2021
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Re: Help with my project/bacteria

Postby student2021 » Tue Dec 15, 2020 5:42 pm

What is the BSL rating for inoculating the antibiotics to a antimicrobial susceptibility disc?

student2021
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Re: Help with my project/bacteria

Postby student2021 » Tue Dec 15, 2020 6:07 pm

I was planning on just inoculating the antimicrobial disks with pure garlic extract. And finding the difference in inhibition potencies in garlic extract, penicillin and gentamicin.

student2021
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Re: Help with my project/bacteria

Postby student2021 » Tue Dec 15, 2020 6:09 pm

My independent variable is the different types of treatment (penicillin,gentamicin,garlic extract) and my dependent variable is finding the zones of inhibition of the three treatments.

student2021
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Help please

Postby student2021 » Thu Dec 17, 2020 2:43 pm

For my project I plan on conducting a research where I find the inhibition potencies of garlic verses the synthetically manufactured antibiotics I chose, gentamicin and penicillin, and seeing how they affect the gut microbiome by testing them on E. coli and Bifidobacterium animalis subspecies lactis. I plan to use the disk diffusion assay method.

My independent variable is the different types of treatment (penicillin,gentamicin,garlic extract) and my dependent variable is finding the zones of inhibition of the three treatments.


Questions I have for my project:
When I am performing the disk diffusion assay method and swabbing the bacteria onto the agar plate would I have to use a new swab for every agar plate? I am using 20 agars for each bacteria. I purchased 1 tube of both of my bacterias. I was wondering if that's enough bacteria? What is the BSL rating for inoculating the antibiotics to an antimicrobial susceptibility disc?

I was planning on just inoculating the antimicrobial disks with pure garlic extract. And finding the difference in inhibition potencies in garlic extract, penicillin and gentamicin. How would I match the concentration of my garlic extract to pre inoculated antibiotics disc?

kgrivera
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Re: Help please

Postby kgrivera » Thu Dec 17, 2020 4:14 pm

Hello!

To answer your first question, I would recommend using a new swab for each plate. This prevents contamination of your plates with unwanted bacteria. After swabbing a plate, you will have touched the sides of the plates, the tube with bacteria and potentially gloves or other surfaces. When doing any type of cell culture or microbiology, sterile technique is very important to prevent contamination of your samples.

Could you provide a link for the tubes of bacteria purchased? You should have enough bacteria for your experiments, but I want to be certain that I give you accurate information.

Both of your strains of bacteria should be BSL-1 even when using antibiotics.

Additionally, could you clarify your question about matching the garlic extract concentration? I am unsure if you mean you want the concentration of your garlic extract to match the concentration on the penicillin or gentamicin disks or something else. Either way, the two types of antibiotic disc likely have differing concentrations of antibiotic. Each antibiotic has a different concentration at which they are most effective, so some antibiotics may have a higher concentration on the antibiotic discs than others. What I recommend is finding the a concentration of garlic extract that has been shown in other research papers to have some antimicrobial effect. You may have to try different concentrations of garlic extract for your project as well.

Let me know if you need any clarification or have any further questions!

student2021
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Re: Help please

Postby student2021 » Thu Dec 17, 2020 4:36 pm

Thank you so much for your help!

Here are the links of the bacteria! :) both were purchased from carolina!

https://m.carolina.com/bacteria/escheri ... dir_155068

https://m.carolina.com/bacteria/lactoba ... ion=155110

I was recommended to match the concentration of my garlic extract to the concentration of my antibiotic disk, alrhough I am not sure if I should or not and how to do so. As my independent variable is differnet treatments would they have to be the same concentration? As i am getting the results of their effectiveness from measuring their zone of inhibitions.

brandimiller610
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Re: Help with my project/bacteria

Postby brandimiller610 » Sun Dec 20, 2020 10:29 am

Hi Student2021,

Thank you for your questions!

(1) Yes, you should use a new swab for every agar plate, to ensure that you don't contaminate your bacterial stocks. I am not sure of the volume of bacterial suspension you purchased, or the colony-forming units per mL; bacteria grows at an exponential rate, so it really shouldn't take too much to inoculate your plates. I know you're using swabs to inoculate, but, just for reference, I have inoculated plates with as little as 100 uL of bacteria and gotten incredible growth. You likely have plenty of stock for your experiment!

(2) To match the concentration, you would have to do dilutions of your extract. Like I mentioned in the previous comment, this may be difficult since you are using pre-inoculated discs. However, in your new posts you made it more clear to me that you simply just want to assess the differences in inhibition of garlic extract and your antibiotics. I am sorry for the confusion -- I have previously done this assay and used similar concentrations for my treatments. Using the pure extract and your pre-inoculated discs is just fine and no dilutions are necessary. Just make sure you know your concentrations of extract and antibiotics so they can be considered when interpreting your data.

(3) Is your strain of E. coli nonpathogenic? If so, the bacteria you are using are safe for a BSL-1 laboratory. Otherwise, if your E. coli strain is pathogenic, then you should follow BSL-2 protocol.

I hope I have answered your questions sufficiently. Again, feel free to reach out if something is unclear or you have additional questions! :)

--Brandi

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Re: Help with my project/bacteria

Postby MadelineB » Sat Dec 26, 2020 4:10 pm

Hello Student2021,

I've merged your posts on this topic so they will be in the same thread. Keeping your posts in the same thread helps the experts who have been helping you see that you have new questions.

Thank you,
Madeline

student2021
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Re: Help with my project/bacteria

Postby student2021 » Sat Jan 09, 2021 10:43 am

Thank you so much for the help!! Okay so I have already started my project and I am now measuring the zones. Although my garlic treatment looks off. it has two zones of inhibition or two rings around the disk. What does this mean?


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