Microbiology Science Project

[deleted-139172]

Hello All,
I would like to do a microbiology project where I test the effectiveness of several herbal extracts using the Kirby-Bauer method on one of the following bacteria strains:
Acinetobacter baumannii,
Escherichia coli
Klebsiella pneumoniae bearing extended-spectrum β-lactamases (ESBL)
vancomycin-resistant enterococci (VRE)
methicillin-resistant Staphylococcus aureus (MRSA)
vancomycin-resistant MRSA
extensively drug-resistant (XDR) Mycobacterium tuberculosis

I have no previous experience in microbiology, and do not have access to any lab other than the one in my school. I have several questions. How would I obtain any of the above bacteria? I have read that there is a much weaker K-12 strain of e.coli, but I would like to do a project that had real-world significance. Is it alright to do the Kirby-Bauer method in a school lab? I have read several descriptions of the method and they are all different. How exactly would you describe the method? Do I need a teacher who is well-versed in microbiology to supervise me?

Thanks, and sorry for the long post,
hello1910

[SciB]

Hi,

Sorry to take so long in answering your post. The Kirby-Bauer method of testing substances for antibacterial activity is an excellent way to do a screening of herbal extracts.

I would stick with E coli K12. It has been the workhorse for research for over fifty years and results with K12 are accepted as indicative of real antibiotic activity. Yes, the other bacteria are more relevant to public health in the real world, but you would have to work in a biosafety level 2 lab with special training to use them. i got my first experience with bacteria using E coli K12 and Bacillus subtilis, neither of which are pathogens.

As for the K-B method, if you can find someone who has done it to show you how that would be the best way to learn. You will need nutrient agar plates, sterile bacterial growth medium, sterile pipets, filter circles, a spreader and incubator (37 degrees centigrade). Does your school lab have all these things? Check out YouTube for methods of spreading the bacterial lawn and placing the discs.

Let me know what extracts you will use and how you mean to prepare them. Remember to add controls where you just use water or saline for the negative control and an antibacterial mouthwash as the positive control.

Best wishes,

Sybee

[deleted-136398]

Good project idea! As stated in the comment above, one starting point is to make sure that your school lab has most of the item that you need (listed in the previous post).

The following you tube link (http://www.youtube.com/watch?v=sx1uDYSfINA) provides a good video of how the methods works, and also gives you an idea of the basic materials you may need.

[deleted-139172]

Thanks so much! Sorry for the very late response, I'll definitely use Ecoli and link was very helpful
Would it be possible for me to apply the kirby bauer method to test the antibacterial properties of a bioplastic instead of a herbal extract?

[deleted-140078]

To be clear, by bio-plastic you mean a plastic or polymer surface treated to have anti-microbial properties, and not a plastic that is produced from biological sources rather than petroleum products?

I am not sure if that would in fact work the same way. Most of the commercially available anti-microbial hard plastic products aren't designed to kill off bacteria in the same manner soap, or antibiotics do. They are instead designed to reduce the number of microbes on their surface, usually by not killing them, but preventing their reproduction. I am not sure you would see an inhibition zone if you placed one of these objects onto a cultured plate. The better approach may be to grow a plate of your selected bacteria, and then place in on the surface your testing, and a control surface. Give it a determined amount of time, and then swab and culture the surfaces and compare the difference.

[deleted-139172]

By bioplastic I mean plastic I would make directly in my kitchen from biological source-would this change the result?
Thanks,
hello1910

[deleted-140482]

In this case, I think megidi's idea would work best in this situation. The Kirby-Bauer method relies on diffusion, which you are unlikely to have from your bioplastic, I think. I'm attaching a paper that looked at the effectiveness of a seed extract incorporated into a plastic. They used the Kirby-Bauer method for their control of testing the effectiveness of the seed extract alone relative to a number of antibiotics, but they used different techniques for the bioplastic itself. You may want to try more than one method if you have the time, since different methods may be testing slightly different things (i.e. whether the plastic inhibits bacterial growth on a plate or whether it will prevent bacteria from growing on it).

Hope this helps!
JMP

[deleted-139172]

Thanks for the great advice! I went through the document, and will perform the "Halo Test" like you suggested. Am I correctly understanding the procedure for this test?
1)Put agar on a bioplastic cut in a circle and do the same for a commercial plastic. (is this in a petri dish?)
2)Incubate for x hours at y degrees
3)Measure size of halo.
Any suggestions for time and temperature of incubation?
thanks,
hello1910

First, we adopted ‘Halo Test’. Generally, this method is adopted to evaluate the effect of antimicrobial activity of fibers. In this method, the test piece puts on agar containing microorganisms. After incubating in proper time and temperature, no microorganism area (halo) expand around test piece. The size of halo indicates strength of antimicrobial activity and the diffusion of antimicrobials from film. We placed the film which was cut into circle having 6mm diameter on a solid agar containing bacterial culture. The agar plates are incubated at 35°C for 24 h. After then, we observed if Halo expand around the PHB sheets or not.

[deleted-140482]

Hi hello1910,

1) Yes, this will be on a petri dish. You would have a petri dish (with a bacterial growth medium, like LB agar), inoculated with bacteria. Then you would put your bioplastic onto the petri dish and measure the size of the halo. This is essentially also called a zone of inhibition. As for your control, that will depend somewhat on exactly what you are testing about your bioplastic. If you are incorporating something antimicrobial into your bioplastic (as they do in this paper) then you would want to run controls that included the bioplastic alone (without the antimicrobial), and the antimicrobial not incorporated into the bioplastic. If you are just interested in how well this specific bioplastic will inhibit bacterial growth, then yes, I think an appropriate control would be a commercial plastic that is as close as possible to the bioplastic you are making. Can you think of any other controls that might be worth including?

2) You will want to incubate your bacteria at 37 degrees Celsius (this is body temperature), because that is the ideal temperature for these bacteria to grow at.

3) In general, incubating for 24 hours (at 37 degrees) should be sufficient, but you can always let it go a little longer if you are not seeing bacterial growth.

Good luck!
JMP