Methods of molecular identifications and lab. protocols
Carbon assimilation and mitochondrial activity were investigated using Biolog FF MicroPlatesT. The FF MicroPlate test panel comprises 95 wells with different carbon-containing compounds and a control well. Nutrients and test reagents are pre-filled and dried into the 96 wells of the microplate. Iodonitrotetrazolium violet (INT) is used as a redox dye to colorimetrically measure mitochondrial activity resulting from oxidation of metabolizable carbon sources. All the wells are colorless when first inoculated. The oxidation of succinate to fumarate in the citric acid cycle, mediated by succinate dehydrogenase and FAD, causes INT to be reduced to a red-colored formazan dye with peak absorbence at 490 nm. The reduction of INT and production of colored formazan is irreversible, and the accumulation of formazan measured spectrophotometrically quantitatively reflects the oxidation of the test substrate. Absorbance readings are taken at 490 and 750 nm. The 750 nm reading measures turbidity reflecting mycelial production and assimilation of the test substrate. Since the absorbance spectrum of hyaline mycelium is essentially level over the range from 490 to 750 nm, a corrected "redox" value for the production of formazan is obtained by subtracting the 750 nm reading (490-750 nm). Trichoderma strains were grown on 2% (w/v) malt extract agar (MA) under ambient laboratory conditions of diffuse daylight and temperature (about 22ºC). Inoculum was extracted after conidial maturation (7-14 days) by rolling a sterile, wetted cotton swab over conidial areas. Conidia were suspended in 16 ml of sterile phytagel solution (0.25% phytagel, 0.03% Tween 40) in disposable borosilicate test tubes (20 x 150 mm). The suspension was agitated in a vortex mixer for about 5 sec, and additional inoculum added as required to adjust the density of the suspension to 75(±2)% transmission at 590 nm wavelength. 100 µl of conidial suspension were dispensed into each of the wells of a Biolog FF MicroPlateT (Biolog Inc., Hayward, CA). Inoculated microplates were incubated in the dark at 26°C, and percent absorbence determined after 96 h at 490 nm and 750 nm using a microplate reader. Readings at 750nm were used as a measure of mycelial production (turbidity data), and the difference in readings at the two wavelengths (490-750 nm) was used to quantify production of formazan attributable to mitochodrial activity (redox data). Analyses were performed separately on the turbidity and redox data sets, and were repeated at least three times.
posted by Alexey Kopchinskiy on 28 Sep, 2004