Agar diffusion techniques are used widely to assay plant extracts for antimicrobial activity, but there are problems associated with this technique. A micro-dilution technique was developed using 96-well microplates and tetrazolium salts to indicate bacterial growth, p-lodonitrotetrazolium violet [0.2 mg/ml] gave better results than tetrazolium red or thiazolyl blue. The method is quick, worked well with Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli and with non-aqueous extracts from many different plants. The method gave reproducible results; required only 10-25 µl of extract to determine minimal inhibitory concentrations, distinguished between microcidal and microstatic effects, and provided a permanent record of the results. Using S. aureus, and a Combretum molle extract, the technique was 32 times more sensitive than agar diffusion techniques and was not sensitive to culture age of the test organism up to 24 hours. The S. aureus culture could be stored up to 10 days in a cold room with little effect on the assay results. This method was useful in screening plants for antimicrobial activity and for the bioassay-guided isolation of antimicrobial compounds from plants. MIC values determined for sulfisoxazole, norfloxacin, gentamicin, and nitrofuratoin were similar to values indicated in the literature but values obtained with trimethroprim and ampicillin were higher with some bacteria.

The development of antibiotic resistant bacteria stems from a number of factors, including inappropriate use of antibiotics in human and animal health and their prolonged use as growth promoters at sub-clinical doses in poultry and livestock production. We were interested in investigating plants that could be useful in protecting humans or animals against diarrhoea. We decided to work on extracts of nine plant species with good activity against Escherichia coli based on earlier work in the Phytomedicine Programme. Leaves of nine medicinal plant species with high antibacterial activity against Escherichia coli were extracted with acetone and their minimal inhibitory concentration (MIC) values determined using a microplate serial dilution technique against Gram-positive (Staphylococcus aureus, Enterococcus faecalis and Bacillus cereus) and Gram-negative (Escherichia coli, Salmonella Typhimurium and Pseudomonas aeruginosa) bacteria. Bioautography was used to determine the number of bioactive compounds in each extract. In vitro safety of the extracts was determined using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide reduction assay on Vero cells. The extracts were active against all the pathogens with average MICs ranging from 0.02 to 0.52 mg/ml. As expected E. coli was relatively sensitive, while E. faecalis and S. Typhimurium were more resistant to the extracts (average MICs of 0.28 mg/ml and 0.22 mg/ml respectively). Cremaspora triflora and Maesa lanceolata leaf extracts had higher activity than the other extracts against Gram-positive and Gram-negative pathogens with mean MICs of 0.07 mg/ml and 0.09 mg/ml respectively. Extracts of Maesa lanceolata and Hypericum roeperianum had the highest total antibacterial activity (TAA) at 1417 and 963 ml/g respectively. All extracts with the exception of that of Maesa lanceolata, Elaeodendron croceum and Calpurnia aurea had relatively low cytotoxicity with LC50 > 20 μg/ml. Cremaspora triflora had the best selectivity index (SI) against S. aureus and E. coli of 2.87 and 1.15 respectively. Hypericum roeperianum had a SI of 1.10 against B. cereus. Bioautography revealed 1–6 visible antimicrobial compounds that were generally non-polar. There was a weak positive, but statistically non-significant correlation between the potency of the extracts and their cytotoxicity (R = 0.45, ρ > 0.05). The activity of the extracts on the test bacteria was in some cases not correlated with cytotoxicity, as shown by selectivity indices >1. This means that cellular toxicity was probably not due to compounds with antibacterial activity. Some of the extracts had a good potential for therapeutic use against the bacterial pathogens or for application in treating diarhoea. It does not appear that activity against E. coli is a good predictor of activity against Gram-negative rather than Gram-positive bacteria. Further investigation is in progress on C. triflora and H. roeperianum, both of which had promising activities and potential safety based on cytotoxicity.

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. 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. 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. 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.