Rapid screening of the antimicrobial activity of extracts and natural products.

Abstract

A spectrophotometric method has been developed for the rapid measurement of the antimicrobial activity of natural products, including crude extracts or pure materials. The assay depends on the measurement of non-specific esterase activity using fluorescein diacetate (FDA) hydrolysis in broth cultures of microbes after they have been treated with test compounds. The assay is accurate, reproducible and economical in both time and materials. The speed and economyof the method make it suitable for the rapid screening of manysamples and the bioassay directed purification of antimicrobial substances. The assay can also be used with a wide variety of micro-organisms since most micro-organisms are FDApositive. Applications are described in the fields of marine natural products chemistry and essential oils research. 1295 The objective of this study was to develop a simple, rapid, inexpensive, accurate and reproducible method for the determination of the minimuminhibitory concentration (MIC) of large numbers of test samples ranging from crude extracts or fractions of natural products to essential oils or pure substances.Methods used for the determination of the MICare varied although they can be grouped into either a single disc diffusion assay (Bauer et ai, 1966) or the dilution plate/broth methods (Haltalin et ai, 1973). The former relates the zone of inhibition produced to the varying concentration of a test compound applied on to a paper disc for the determination of the MIC. There are several problems associated with the disc diffusion method for comparing the relative antimicrobial activity of different compounds. The assay does not always give a sharp demarkation between bacterial growth and inhibition. The time taken for incubation (18~24 hours) may render it unsuitable for volatile or unstable antimicrobial agents. The zones of inhibition may only be compared among antimicrobial agents with similar physical properties such as diffusion rates in agar, volatility or solubilities in aqueous solutions.The dilution plate/broth group of methods measure the bacteriocidal/bacteriostatic effect of a potential antibiotic at a series of concentrations on a given microbial population. While being accurate, these methods are laborious due to the inclusion of viability counts in the assay and are liable to contamination of either the broth or the viable count agar plates or from test samples that often cannot easily be sterilised. Again, the time period involved (1~2 days) can be a major drawback.The method described here is ideal for implementation in a chemistry laboratory by non-microbiologists, can be run under non-aseptic conditions, requires only small amounts of materials and gives results in two hours.Non-specific esterases occur widely amongst microbes (Lundgren, 1981) and have been shown to hydrolyse colourless fluorescein diacetate (FDA) to fluorescein, a yellowish green compound, which absorbs strongly in the visible (/lmax 498 nm; £ l x 105, methanol -potassium hydroxide; Grasselli and Ritchey, 1975). FDAhydr...

Estudo comparativo de técnicas de screening para avaliação da atividade anti-bacteriana de extratos brutos de espécies vegetais e de substâncias puras

“…O método de difusão em ágar pode ser realizado através das técnicas do disco, do poço ou template. [2][3][4] Diversas modificações já foram realizadas nas metodologias de screening a fim de se obter resultados mais confiáveis, uma vez que alguns fatores, tais como composição do meio de cultura, microrganismos testados, método de extração, pH e solubilidade das amostras no meio de cultura, podem alterar os resultados, sendo difícil, portanto, padronizar um procedimento. 1 Quando se utiliza o método de difusão vários fatores podem se tornar fontes de erros, tais como composição do meio de cultura, preparação incorreta do meio de cultura, espessura do meio de cultura, densidade do inóculo incorreta, uso de swab com excesso de caldo para inoculação das placas, temperatura e tempo de incubação inadequados, interações entre o antimicrobiano e o meio de cultura, utilização errada da atmosfera de CO 2 quando necessária, leitura prematura, erro na medida das zonas de inibição ou uso de culturas mistas ou contaminadas.…”

The antibacterial activity of traditionally used <i>Salvadora Persica</i> L. (miswak) and <i>Commiphora Gileadensis</i> (palsam) in Saudi Arabia

“…Minimal Inhibitory Concentration (MIC) was determined for each extract using Fluorescein diacetate (FDA) method (Chand et al, 1994), in ELISA plate.…”

Synthesis and evaluation of antifungal and antibacterial activity of ethyl 3,5-diarylisoxazole-4-carboxylates

“…The antimicrobial activity of the synthesised compounds was determined by the paper disc diffusion assay, 12,13 Briefly, a lawn of the organism was prepared by spreading 50 ml of overnight cultures (conc. 10 6 ∼10 7 CFU/ml) onto agar set in petri dishes.…”