Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica

Abstract

Despite substantial interest investigating bacterial mechanisms of fungal growth inhibition, there are few methods available that quantify fungal cell death during direct interactions with bacteria. Here we describe an in vitro cell suspension assay using the tetrazolium salt MTT as a viability stain to assess direct effects of the bacterial antagonist Lysobacter enzymogenes on hyphal cells of the filamentous fungus Cryphonectria parasitica. The effects of bacterial cell density, fungal age and the physiological state of fungal mycelia on fungal cell viability were evaluated. As expected, increased bacterial cell density correlated with reduced fungal cell viability over time. Bacterial effects on fungal cell viability were influenced by both age and physiological state of the fungal mycelium. Cells obtained from 1-week-old mycelia lost viability faster compared with those from 2-week-old mycelia. Likewise, hyphal cells obtained from the lower layer of the mycelial pellicle lost viability more quickly compared with cells from the upper layer of the mycelial pellicle. Fungal cell viability was compared between interactions with L. enzymogenes wildtype strain C3 and a mutant strain, DCA, which was previously demonstrated to lack in vitro antifungal activity. Addition of antibiotics eliminated contributions to MTT-formazan production by bacterial cells, but not by fungal cells, demonstrating that mutant strain DCA had lost complete capacity to reduce fungal cell viability. These results indicate this cell suspension assay can be used to quantify bacterial effects on fungal cells, thus providing a reliable method to differentiate strains during bacterial/fungal interactions.

Green synthesis and antifungal mechanism of silver nanoparticles derived from chitin‐ induced exometabolites of Trichoderma interfusant

“…Known aliquot was sampled individually for each treatments and mycelial cells were collected by centrifugation (pellet), rinsing twice into 2 ml PB and re‐suspended in 900 μl of PB, to which 100 μl solution of 3‐(4,5‐dimethylthiazol‐2‐yl)2,5‐diphenyl tetrazolium bromide (MTT) was added followed by incubation at 28 °C in dark with continuous agitation up to 90 min and then centrifuged at 5000 rpm for 10 min. The MTT test performed according to Kim et al and Patel et al…”

Ferrocene‐substituted bis(ethynyl)anthracene compounds as anticancer agents

“…Cell viability assay was assessed using standard MTT assay. [43] After 24 h of incubation, 100 μl of MTT (0.5 mg ml −1 ) was added to each well and incubated for another 4 h. The medium was then removed and 100 μl of DMSO was added and was shaken for 10 min in order to dissolve formazan crystals. Absorbance at a wavelength of 570 nm was measured with a Synergy H1 Biotek microplate reader.…”

Lsp family proteins regulate antibiotic biosynthesis in Lysobacter enzymogenes OH11

“…In addition to these secondary metabolites, L. enzymogenes also produces multiple extracellular lytic enzymes that degrade fungal cell walls components and play important roles in the biological control activity of L. enzymogenes (Zhang and Yuen 2000; Palumbo et al 2003). Furthermore, L. enzymogenes produces type IV pili (T4P), which are critical to twitching motility (movement on solid surfaces) and attachment to substrates such as fungal hyphae (Kobayashi et al 2005; Patel et al 2011, 2013). …”