Testing of antifungal natural products: methodologies, comparability of results and assay choice

Abstract

Antifungal testing of filamentous fungi generally suffers from incomparability of results. In order to illustrate this problem, the polyacetylene falcarindiol and the naphthoquinone juglone, two known antifungal natural products, were assayed in various dilution and diffusion bioassays against three selected microfungi, Botrytis cinerea, Cladosporium herbarum and Fusarium avenaceum. Broth microdilution, based on the 96‐well microtitre plate format, can be scored by various direct and biochemical methods. As examples, direct observation with image analysis and the fluorescein diacetate scoring method are compared. Of the other methodologies used, results obtained by thin‐layer bioautography and the radial growth rate method clearly deviated. Disk diffusion results, however, matched microdilution. In conclusion, microdilution offers the greatest potential of all bioassays to become the future general standard methodology. Copyright © 2000 John Wiley & Sons, Ltd.

Antibacterial triterpenes from Syzygium guineense (Myrtaceae)

“…Extract (100 g) was dissolved in water and successively extracted with hexane, ethyl acetate (EtOAc) and n-butanol to yield, respectively, 20, 45, and 18 g. The resultant extracts were tested for antibacterial activity via the disc diffusion method (Hadacek and Greger, 2000) against Escherichia coli and Bacillus subtilis.…”

“…All fractions were tested for antimicrobial activity by direct bioautography on TLC plates (Hadacek and Greger, 2000) against Escherichia coli and Bacillus subtilis. Ethyl acetate extract (40 g), the main active fraction, was subjected to a column chromatography (75 × 5.2) filled with silica gel (63-200, 60Å) and eluted with a gradient of MeOH in CH 2 Cl 2 .…”

“…After evaporation of the solvent, two paper disks were placed in Petri dishes of 9 cm diameter containing nutrient agar previously inoculated with 0.2 ml of suspension of bacteria (10 8 -10 9 CFU/ml). After 2 days of incubation at 30 • C for Bacillus subtilis and 37 • C for Escherichia coli, the inhibition zone for the active extract was measured (Hadacek and Greger, 2000).…”

“…Plates were incubated for 2 days at the corresponding temperature of each bacterium in the dark. The appearance of blank after spraying the plate with a solution of thiazolyl blue tetrazolium bromide indicated antibacterial activity (Hadacek and Greger, 2000). Minimum inhibitory concentrations (MIC) against Escherichia coli, Bacillus subtilis, and Shigella sonnei were determined from the lowest test compound concentration causing recognisable bacterial growth inhibition on nutrient agar.…”

Interference of heating on the antimicrobial activity and chemical composition of Origanum vulgare L. (Lamiaceae) essential oil

“…Initially, 1 mL of the microbial suspension was uniformly spread on sterile nutrient (for bacteria) or Sabouraud (for yeasts) agar Petri dishes. After the absorption of the inoculum by the agar, wells were made using sterile glass tubes (6 mm diameter) and were filled with 50 µL of the essential oil solutions (HADACEK; GREGER, 2002;SOUZA et al, 2005). The system was incubated at 35-37 °C/24 hours for bacteria and 25-28 °C/48 hours for yeasts.…”