Hi, i am doing research on the potential antimicrobial activity of Bacillus against s.aureus.i have tried various methods for six months.. like soft agar overlay method but my bacillus colony keep overgrowing the whole plate and even colony risen up to the surface when pouring the s.aureus overlay! i also tried disk diffusion method but i had the same problem of over growing and getting this unusual shapes and i am not seeing the inhibition zone i expected. i know that my bacteria is inhibiting s.aureus but i am not sure if i can publish the data if i do the calculation with image j! Did any one faced this problem before ? can any one offer advice on how to optimize my experiments? are my data presented in picture well enough to report and be published?
The initial step is to centrifuge the bacillus broth and use the supernatant. Then, take a filtered culture of Bacillus and add the filtered broth to the cultured S. aureus well. (Remove the bacillus)
Thanks for your advice, we were actually trying to see the inhibition potential of B.subtilis while alive, isn't there any method to use it alive and not its supernatat? we tried soft agar overlay method as described in an this article(Jung H-S, Lee H-W, Kim K-T, Lee N-K, Paik H-D. Anti-inflammatory, antioxidant effects, and antimicrobial effect of Bacillus subtilis P223. Food Science and Biotechnology. 2023:1-9) but as you saw in the picture we didn't have much luck with this method!@Sinai Waleed Mohammed
Dear Atena Sayahi,
you also could tried the agar plug or the agar spot method. The first one is discussed in:
Article Methods for in vitro evaluating antimicrobial activity: A review
The agar spot is used in this work:
https://academic.oup.com/jambio/article-abstract/115/1/133/6716311
In addition, you could test different culture media and incubation temperature.
Good luck.
you can culture S.aureus in a sheet or in tight streaks over the entire surface of the agar.
then you culture in a single streak of Bacillus and this in the center of the surface previously sown with S.aureus then incubate.
If there is an effect, there will be an appearance of a hallo all around the bacillus streak.
you can use chapman medium or blood agar medium.
Certainly! Demonstrating the inhibition zone of Bacillus against Staphylococcus aureus (S. aureus) can be done through a simple laboratory experiment called the agar diffusion assay, commonly known as the Kirby-Bauer method. Here's how you can set it up:
Materials Needed:
Procedure:
Tips:
- Ensure that all equipment and materials used in the experiment are sterile to prevent contamination.
- Use appropriate controls such as a plate with only S. aureus inoculation and a plate with Bacillus culture alone to compare and validate the results.
- Repeat the experiment multiple times to ensure reproducibility and reliability of the results.
- Consider testing different concentrations or volumes of Bacillus culture to determine the optimal conditions for maximum inhibition.
By following these steps, you can effectively demonstrate the inhibition zone of Bacillus against Staphylococcus aureus in a laboratory setting.
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