Background: Microbiological assay is the gold-standard method for analyzing antibiotics,
since it ensures the success of antimicrobial therapy. However, this method may be time-consuming and it
remains almost unaltered since it was first developed. Here, we present the design space of a rapid microplate
bioassay for bacitracin.
Methods: Triphenyltetrazolium chloride was used in the bioassay to provide faster response. Factorial
design and response surface methodologies were used to optimize microorganism, inoculum proportion,
culture medium composition, bacitracin concentration, and triphenyltetrazolium chloride concentration.
Results: Yeast extract amount in culture medium was a key factor in reducing incubation time and it allowed
the development of a faster bioassay. In addition, inoculum proportion was a critical parameter. On
the other hand, triphenyltetrazolium chloride concentration did not affect the bioassay response. The bioassay
was linear, accurate, precise and robust.
Conclusion: Besides being equivalent to an agar diffusion assay according to statistical analysis, the colorimetric
bioassay requires less culture medium and solutions, and provides faster results.