brandimiller610
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Re: Help with my project/bacteria

Postby brandimiller610 » Sun Jan 10, 2021 9:26 pm

Hi Student2021,

I am not too familiar with two rings in a zone of inhibition; are there two distinct rings on the plates that were treated with garlic? Also, the two manuscripts I included for you earlier have images of the plates with each treatment -- do your plates treated with garlic look like the plates in those papers?

Here are a few more manuscripts you could use to compare results:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784133/
https://files.eric.ed.gov/fulltext/EJ876521.pdf

I think you should include the length of both "rings" as your zone of inhibition for the plates treated with garlic extract. However, if you could provide some additional details about the rings you are seeing, it may help me better understand your results and guide you better.

--Brandi

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Re: Help with my project/bacteria

Postby student2021 » Wed Jan 20, 2021 1:11 pm

My zones do not look like the garlic disk on the Science Journals that you included. It is more of a foggy ring around the antibiotic disk with another ring around that foggy ring.

Also for my Garlic treatment on the L. acidophilus bacterium (different treatment) the zones were very unclear and inconsistent. After 24 hours of incubating the disk there was no growth of bacteria. I also didn't see any zone because of zero bacterial growth. On the 48 hour mark of incubating there was bacterial growth and sign of zones. The zones were there, but I had to guess to measure them. On the third day of incubating the plates the bacteria started to grow inside the zone where there was no bacterial growth. I was wondering how do I measure the zones if the bacteria has grown into these zones. Would it be N/A or 0mm. I could not get a clear zone on many of my plates.

Here is a link with pictures of what my plates look like.

https://docs.google.com/document/d/10RN ... sp=sharing

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Re: Help with my project/bacteria

Postby brandimiller610 » Sun Jan 24, 2021 11:47 pm

Thank you for including photos of your results thus far so I can better assist!

The zones for the garlic extract on E. coli look very distinct, but I do see the "foggy ring" that you are talking about. I think the zone of inhibition would simply be from edge to edge where bacteria did not grow. I do not think that the "fog" you are seeing is bacterial growth. Instead, I think it might be from the garlic extract itself. How long are you incubating the plates (E. coli plates in particular) after inoculating them and adding the disks? Optimal incubation time is 16-20 hours, but no more than 24 hours, especially when using bacteria that grow quickly. It might be worth it to repeat if time and resources allow.

For the Lactobacillus plates, it is strange to me that no bacterial growth is seen until Day 3 after inoculation. Did you prepare working bacterial stocks from the vials that were purchased? When looking at publications, it seems that garlic extract may not have antibiotic properties against L. acidophilus. Previous disk diffusion assays for L. acidophilus have not detected any zones of inhibition when treated with garlic extract/powder. These papers (below) also incubated the plates for 48-72 hours before measuring zones, which aligns with the growth period you reported. When reporting the diameter of the zones, I would say "ND" (not detected) instead of 0. If there are distinct zones of inhibition, do report these (an example might be disc 10 from day 3 -- last image in the document you shared). It is okay if the numbers vary for your replicates, but if there is too much variation from plate to plate, I would definitely consider repeating the experiment for L. acidophilus, if time and resources allow. Make sure to use the below manuscripts to double-check your methods, as they both also used L. acidophilus, if you decide to repeat your experiments.

https://academicjournals.org/journal/AJ ... 46DD320292
http://www.scielo.br/scielo.php?script= ... 9000400897

I am sorry this is kind of a lengthy reply, but I hope I have been able to help. I am happy to look at more literature to try to help you interpret your data, if necessary, but I think these papers (and previous ones I've attached) are very helpful. Please feel free to follow up with more questions!

--Brandi :)

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Re: Help with my project/bacteria

Postby student2021 » Thu Feb 04, 2021 10:03 am

I had stored the agar plates in a fridge. Before using them I had waited for them to get to room temperature, so about an hour, before inoculating them and adding the disks. I did not incubate them.

For my [i]L. acidophilus[i] bacteria I purchased them live from carolina. As for the growth, there was bacteria growth on the 3rd day. The only day that I didn't see much bacteria growth on the agars was the first day. It was like that for all of my agar plates even the ones with my antibiotic discs. The first garlic disc was weird tho because it didn't contain much bacteria till the 3rd day. Furthermore, garlic does contains some probiotics components that help feed the bacteria in the gut. I think that is what is happening in after the 3rd day when the bacteria had started to grow into the zones.

Thank you so much for the help!!! My next question is about how to graph my data. I plan to use an Anova test when comparing the averages of zones for each treatment, although I am not sure how to do it. I have all my data and the averages calculated. I am just confused on how to do it with the discs that didn't show zones. Also how would I get an error bar onto google sheets.

Here are the links to my data table and graphs:
https://docs.google.com/spreadsheets/d/ ... sp=sharing
https://docs.google.com/spreadsheets/d/ ... sp=sharing

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Re: Help with my project/bacteria

Postby student2021 » Fri Feb 05, 2021 1:07 pm

I have figured out how to do the error bars. Now I was just wondering do you have anything about how to run an ANOVA test(preferably videos). I haven't really taken stats and it is very confusing.

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Re: Help with my project/bacteria

Postby brandimiller610 » Sun Feb 07, 2021 8:21 pm

Your interpretation of the results for the garlic does make sense -- it may have promoted the growth of the lactobacillus 2-3 days after inoculation of the plates. As far as ANOVA is concerned, I have attached some links to help you understand the test and run it in Excel. It is fairly easy to run the analysis in Excel. Simply put, ANOVA is a statistical method that is used to compare means when there are more than two groups used for comparison. For your data, a one-way ANOVA seems the most appropriate.

https://www.youtube.com/watch?v=0V5scynrVjY
https://www.youtube.com/watch?v=ITf4vHhyGpc

I hope these links are helpful. A quick search of "one-way ANOVA" on YouTube will likely give you a lot of videos on the subject if you would like more. If you have any further questions, please reach out! Good luck running your statistics :)

--Brandi

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Re: Help with my project/bacteria

Postby student2021 » Wed Mar 03, 2021 3:18 pm

Thank you so much for your help! I was wondering if you could look into my paper! And give me some suggestions on what to fix! Or anything at all!

Paper Link!
https://docs.google.com/document/d/1lbd ... sp=sharing

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Re: Help with my project/bacteria

Postby brandimiller610 » Fri Mar 05, 2021 8:47 am

I would be happy to look at your paper! I'll let you know of any suggestions I have.

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Re: Help with my project/bacteria

Postby brandimiller610 » Sun Mar 07, 2021 7:44 pm

Hi Student2021,

Let me start by saying I really enjoyed reading your paper and I think it was really well written! Your paper was very nicely organized, clear and easy to follow, and your Figures/Tables were very nicely constructed. Nonetheless, I do have some suggestions for you. Some are stylistic, while others are related to the content of your paper.

1. Define "prebiotic" in Introduction.
2. I think you should be more specific about the Gram-negative and Gram-positive species garlic extract has been tested on in previous studies (in Introduction).
3. In the second to last paragraph in the Introduction add 1-2 sentences stating how your study is novel or how it contributes to science (i.e. why is it important?).
4. Use "hypothesized" instead of "predicted".
5. Include a "Statistical analyses" subsection at the end of Materials and Methods instead of in the Results section. Here, talk about ANOVA, p-value significance threshold, and indicate how data are expressed in graphs (i.e. mean +/- SD).
6. Get rid of the red background in your tables. I suggest a light gray. If you want to use color, use a very light color as opposed to a bright one (the red is very bright!)
7. On your graphs, indicate what the error bars represent (i.e. standard deviation or standard error of the mean). I would assume SD, but this should be indicated.
8. For figures with multiple pics, label them as A,B, C, and D. An example would be Figure 5. Label as Figure 5A, 5B, 5C, and 5D, respectively.
9. I suggest including your ANOVA p-values on the table instead of stating them in the "Statistical analyses" subsection underneath Results section.
10. I think the Discussion should start by rephrasing the purpose of the study.
11. I think you should add some strengths of your study in the Discussion section.
12. For future studies, I think testing on other probiotic species (i.e. Bifidobacterium spp.) would be interesting to compare the effects of the garlic extract and antibiotics on these microbes. You might want to consider adding this as a future project as well.

I hope these suggestions are helpful and not overwhelming. If anything I suggested is unclear, please reach out and I'll be happy to clarify! Again, I think you've done a great job on your paper and thank you for letting me read it!

--Brandi :)

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Re: Help with my project/bacteria

Postby student2021 » Wed Mar 10, 2021 5:35 am

okay! thank you for your help once again!

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Re: Help with my project/bacteria

Postby student2021 » Thu Mar 11, 2021 5:49 pm

Hi, again.

I wanted to let you know I became on of the finalist for JHSH!!!

I was hoping if you could give me more suggestions on my presentation slides over my project. With my project not being all original I was also wondering how would I share to my audience why this project is important. Should I include a slide to where I have compared my project with other studies? Also what else should I include into my presentation to make more sense and to deliver my information clearly. How do I make it better. I could also use suggestions over how to make it look better or the structure of the presentation.

Here is a link to my presentation
https://docs.google.com/presentation/d/ ... sp=sharing

Thank you again so much!!!

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Re: Help with my project/bacteria

Postby brandimiller610 » Sat Mar 13, 2021 9:54 am

Congratulations on becoming a finalist for your project!!

I will certainly look at your presentation and offer some suggestions for it as well!

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Re: Help with my project/bacteria

Postby brandimiller610 » Sun Mar 14, 2021 12:37 pm

I took a look at your presentation and have included some suggestions below:

1. I think the structure of the first few slides should be changed. I think the flow of information will be better like this: overview of gut microbiome (mention strains/genera used in this project) --> overview of antibiotics and their impact on the gut microbiome --> antibiotic resistance --> garlic extract as an alternative and previous studies that have used garlic extract on microorganisms. These topics should flow straight into your slide that presents your research question. With that being said, I think you should omit the "Rationale: Bacteria" slide.

2. You should only have one research question (i.e. no need to have separate research questions for each bacterium).

3. Again, I think the red color on the tables should be changed to a more neutral color. I also don't really like the blue color of the slides. I usually like to keep it simple with a white background and black text. But a light gray or blue would be okay.

4. Try to incorporate more images, especially on the first few slides. When I make presentations, I like to have a good balance between the amount of words and the number of visuals with the PowerPoint.

5. Before your limitations slide, I think it would be good to include a slide that highlights the strengths of your study and how it contributes to science. You can relate it to previous studies by stating how your project expands on these previous studies.

6. Indicate what tests you used to generate your p values (ANOVA/t-tests).

Please let me know if you have any questions! Congratulations again on becoming a finalist and good luck on your presentation!! :)

--Brandi

student2021
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Re: Help with my project/bacteria

Postby student2021 » Tue Apr 13, 2021 12:59 pm

Thank you for all your help!!!!!


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