Detecting Antibacterial Resistance

[deleted-340684]

Hi guys,

I am doing the silver nanoparticles neutralizing E. coli science fair experiment and I wanted to detect antibacterial resistance. I was thinking that if I leave the paper filter with silver nanoparticle concentration in the petri dish over time the bacteria will become resistant. I could detect this by seeing if the inhibition zone begins to get smaller. Can anybody confirm if this would be the right way to observe antibacterial resistance or is there a proper method to do it? If my question is too vague I can provide more details. Thank you for all the help!

Ryan

[deleted-284605]

Hi Ryan,

Interesting idea. First off, I'd use the term "antimicrobial resistance" or even "silver nanoparticle resistance" instead of "antibacterial resistance." This is just convention, since I've never heard anyone use "antibacterial resistance" this way.

Second, once the bacteria are plated and those in the zone of inhibition are dead, they won't spread back into this area even if some cells are resistant to the nanoparticles. Thus, this wouldn't be the best way to test for resistance.

A better way to test this would be to take bacteria that have already been treated with a low level of silver and expose them to a higher level of silver. You will need additional materials for this because it will require you to take plated bacteria that you've grown for the main experiment and culture them (let them grow more) in a liquid culture. Thus, you'd need LB or some other media for the E. coli to grow in, additional tubes, etc. Ideally, you'd grow the culture at room temperature (or even better, at 37°C) and have it shaking. Perhaps you have some resources available at school?

If you're able to do this, here's a more detailed version of what I think you could try. In the Science Buddies version of the experiment, you test serial dilutions of your colloidal silver, and should see that at some low level, the bacteria are able to grow around the disk, but at some higher level(s), they cannot grow and are therefore susceptible to the silver. I would take a colony that grew close to the disk at one of the low concentrations (the highest concentration where there was still bacterial growth is best!) and inoculate a liquid culture (basically, you touch a single colony with a toothpick, then touch growth media with the end of that toothpick to transfer a tiny amount of bacteria). Do the same thing with a colony that was not exposed to any silver (as a negative control for resistance), and grow both cultures overnight (preferably shaking at elevated temperature). The next day, plate each culture and add a diffusion disk to the plates. I would try the same dilution series and have multiple plates for both your silver-treated and control cultures. The control culture should be sensitive to approximately the same silver concentration (though this could vary depending on how many bacteria you end up plating since your culture may grow longer and denser than the initial tube you ordered, that's why this is an important control. If your school has a way to measure this like taking an OD reading with a spectrophotometer, then you can control exactly how much bacteria you plate). The culture that was exposed to silver may look just like the control culture (meaning it did not develop resistance), or it may be able to grow at a higher silver concentration than the control (it developed silver resistance).

Hope that's not too complicated and I'm happy to follow up to clarify things. It's a really cool idea, I'm just not sure you'd have all the equipment needed to test it properly! Good luck!
Megan

[deleted-340684]

Hi, Megan!

Thank you very much for your helpful response. I have a few follow-up questions. When I test the control culture and the culture suspected of resistance, do I subject both cultures to the silver nanoparticle concentration that the resistant culture was initially exposed to in the previous test? Also, what is the purpose of the spectrophotometer in this experiment? Would there be a source of error without the use of this?

Again, thanks for all your help.
Ryan

[deleted-284605]

When I test the control culture and the culture suspected of resistance, do I subject both cultures to the silver nanoparticle concentration that the resistant culture was initially exposed to in the previous test?

I would subject both cultures to the same range of silver concentrations that you use in the basic initial experiment as described on the Science Buddies page.

Also, what is the purpose of the spectrophotometer in this experiment? Would there be a source of error without the use of this?

Sorry I didn't explain that part very thoroughly since I guessed that you wouldn't have access to this instrument and it's not THAT crucial! Basically, this machine lets you estimate the optical density (O.D.) of your culture, which is a fancy way of measuring how cloudy it is. Generally, the more bacteria in the culture, the cloudier the liquid looks. If you dilute both cultures so that they have the same O.D., and then plate the same volume of each, then you're plating approximately the same amount of cells on each plate.

If you are unable to do this, it should still be okay. Because one culture could grow a bit faster than the other, you might scrape up a few more cells of one colony compared to the other, etc. you may end up plating different amounts of bacterial cells, and end up with lawns of bacteria that are different density (i.e. one might be a solid patch of bacteria while one is more spotty). This could make it hard to interpret whether or not you have a zone of inhibition around the disc or if your cells are just plated to sparsely.

I'm guessing that as long as both cultures are grown overnight and look pretty cloudy (not clear yellow like un-inoculated media), then you'll get a nice enough lawn to see the zones of inhibition clearly.