Science Buddies: "Ask an Expert"

Hi everyone,

I'm doing an experiment project with my group at school where we are to search for a mutant found in the zone of inhibition of a Kirby Bauer test. The strain we used is e. Coli and this type has been known to be resistant to some of the bacteriostatic antibiotics we used. Our plan is to inoculate the area within the zone of inhibition, because according to our knowledge, even though there's no growth, that doesn't mean the bacteria is dead. We want to reintroduce it to the same antibiotic to see if there's a change in the size of the zone of inhibition or if the mutant shows signs of resistance the second or third around its exposed to the same bacteria. I can't seem to find any studies related like this nor can we give an example to how this can be related to real life situations. We haven't finished the experiment yet, but any expert on the topic is helpful.

-Minnie

Hi Minnie,

Great project!
You can carefully scrape the surface in the inhibited zone of no growth with a sterile swab and use it to streak fresh plates with and without antibiotic. Ideally if there are resistant bacteria in that zone, they will grow definitely on the antibiotic-free plate but may not grow (or grow weakly) on the antibiotic plate.

You can then pick single colonies and inoculate in liquid medium to check for growth (turbidity). You should aim to divide a single colony in three or fours parts (you need very very little) for inoculating in different types of medium: one without antibiotic (control) and the others with your antibiotic at various concentrations (original plus lower concentrations). You will be able to tell if you have enriched antibiotic resistant E. coli in your isolate and if they are completely resistant to lower concentrations of the antibiotic.

Lab strains of E.coli are non pathogenic but you should make sure you follow good lab practices at all stages of this experiment (use gloves and disinfectant, don't come into direct skin contact with any of your experimental material, wash hands etc.)

Regarding real life relevance, antibiotic resistance in bacteria is a huge problem in medical science. If antibiotics are not used correctly they lead to resistant strains (like what you are trying to find) which are then very difficult to treat and can lead to life threatening conditions. You will find a lot of information on the internet about antibiotic resistance, multi-drug resistant bacteria, hospital acquired drug resistant infections etc. which will give you a good idea of why this is important (and why I recommended the good lab practices).

Good luck!
MS