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. 2016 Apr;6(2):71-79.
doi: 10.1016/j.jpha.2015.11.005. Epub 2015 Dec 2.

Methods for in vitro Evaluating Antimicrobial Activity: A Review

Free PMC article

Methods for in vitro Evaluating Antimicrobial Activity: A Review

Mounyr Balouiri et al. J Pharm Anal. .
Free PMC article


In recent years, there has been a growing interest in researching and developing new antimicrobial agents from various sources to combat microbial resistance. Therefore, a greater attention has been paid to antimicrobial activity screening and evaluating methods. Several bioassays such as disk-diffusion, well diffusion and broth or agar dilution are well known and commonly used, but others such as flow cytofluorometric and bioluminescent methods are not widely used because they require specified equipment and further evaluation for reproducibility and standardization, even if they can provide rapid results of the antimicrobial agent's effects and a better understanding of their impact on the viability and cell damage inflicted to the tested microorganism. In this review article, an exhaustive list of in vitro antimicrobial susceptibility testing methods and detailed information on their advantages and limitations are reported.

Keywords: Agar diffusion method; Antimicrobial gradient method; Thin-layer chromatography (TLC)–bioautography; Time-kill test.


Fig. 1.
Fig. 1
Agar diffusion methods: (A) disk-diffusion method of microbial extract using C. albicans as test microorganism, (B) agar well diffusion method of essential oil using Aspergillus niger as test microorganism, and (C) agar plug diffusion method of Bacillus sp. against C. albicans.
Fig. 2.
Fig. 2
Broth microdilution method of plant extract against B. subtilis using resazurin as growth indicator.
Fig. 3.
Fig. 3
0.5 McFarland microbial inoculum preparation by the direct colony suspension as recommended by CLSI guidelines.
Fig. 4.
Fig. 4
Broth microdilution for antibacterial testing as recommended by CLSI protocol.

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