Simple Disc Diffusion Assay

 
     
 
 
 
 
 
  
 
 
 
 

This is a simple disc diffusion assay that we conjured up. The aim here is to see the effects an antibiotic has on a bacterial colony. In this case, we plated E.coli on PDA plates with no antibiotics in the media. We made a stock solution of 1mg/mL chloramphenicol in absolute ethanol and soaked small pieces of filter paper in various amounts (0ug, 5ug, 10ug, 20ug and 50ug) and placed them on plates that were freshly coated with E.coli. We incubated at 37C overnight.

The plate on the left is the before shot, prior to incubation. On the right is the plate after incubating at 37C overnight. You can clearly see the growth of E.coli around the 0ug disc (containing no chloramphenicol) and the lack of growth around the discs that do contain chloramphenicol. You can also see that the inhibition of growth is larger next to the 50ug disc versus the 5ug disc, as expected.

It’s also worthy to note that chloramphenicol is a bacteriostatic antibiotic, meaning that it does not kill bacteria, but prevents them from reproducing. If we took a plate that already had a full lawn of E.coli, we wouldn’t see complete absence of colonies after incubation, just less growth from those colonies exposed to the antibiotic. On the other hand, a bactericidal antibiotic (such as penicillin) will in fact kill bacterial colonies.

Overall, this nicely demonstrates how a disc diffusion assay works. In the future, we plan to isolate our own compounds and use this same setup to test for bioactivity and potency of our isolates. Stay tuned…

Below you can see another plate which had no antibiotics in the media and was not exposed to a disc diffusion assay.

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