Background: There is an urgent need to discover new antimicrobial compounds or extracts to address the crucial problem of increasing microbial resistance against current antibiotics. Plant chemical biodiversity is a valuable potential resource. Although compounds from plants are used as basis for several human drugs, no commercially successful antibiotic has yet been discovered from plants, despite more than a thousand publications in this field per year. This may be due to wrong methods that have been used or wrong plants that were investigated. A lot of energy is wasted by using techniques such as agar diffusion that do not work well with plant extracts. Many manuscripts are rejected before sending to reviewers because wrong methods are used. Antimicrobial activity of plant extracts based on agar diffusion studies have limited value.
Methods: Results obtained from several hundred of our publications in this area as researcher and experience as editor was used to identify difficulties in generating reproducible data. Other publications were also consulted and procedures used were evaluated.
Results: Because many of the antimicrobial compounds in plant extracts are relatively non-polar, these compounds do not diffuse well in the aqueous agar matrix used in agar diffusion studies. So many other factors also influence the zone of inhibition, that results between different laboratories are not comparable. The different methods used to determine the minimal inhibitory concentration (MIC) in serial dilution studies have been discussed. Using p-iodonitrotetrazolium violet to indicate growth provided the best results. Factors such as inoculum size, solvent, selection of positive controls and selection of plants to investigate also play a role. A method developed to determine antibacterial and antifungal activity of plant extracts work very well and is widely used based on > 1830 citations.
Conclusions: By using proposed methods manuscripts will provide reproducible information that may be published in good journals. The publications could contribute to a rational basis for finding compounds or extracts from plants that may address the problem of antimicrobial resistance. Random screening of a large number of plant species using this technique have already led to some commercial applications and identification of a potentially new antifungal framework compound.
Keywords: Agar diffusion assay; Extractant; Plant extracts; Serial microplate dilution. p-iodonitrotetrazolium violet.