Uplanfl 100 oil objective

Morphology and viability of S. coelicolor M145 and ΔglnA3 mutant cells grown in a rich complex YEME–TSB (1:1) medium was analyzed in presence of polyamines. Samples were taken from every culture after 72 and168 h of growth, and obtained perpetrates were observed under phase-contrast microscope under 400× enlargement. To detect live and dead cells, SYTO9 and PI (propidium iodide) stains of the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes) were used. The SYTO 9 green fluorescent stain labels cells with intact membranes, as well as those with damaged ones. PI penetrates cells with damaged membranes, decreasing SYTO 9 stain fluorescence when both dyes are present. Thus, in the presence of both SYTO9 and PI, cells with intact cell membranes appear fluorescent green whereas cells with damaged membranes appear red. The staining solution was prepared by mixing 0.75 μl of component A and B in 500 μl of water. Stained cells were analyzed by the fluorescence microscopy using Olympus BX60 microscope with an Olympus UPlanFl 100 × oil objective and an Olympus BX-FLA reflected light fluorescence attachment. Images were taken with the F-view II camera (Olympus), using TxRed and eGFP filter sets for detection of the fluorescent markers. The ImageJ was used for image processing. Significant number of images (10) was analyzed in a minimum of three independent culture analyses.

These assays were performed with either DgDef1ΔSP, which lacks the N-terminal signal peptide, or with DgDef1ΔSPΔCTPP, which additionally lacks the acidic C-terminal domain. About 10⁻⁵ spores of S. coelicolor A3(2) M145 were plated on R5 agar [53 ] and 10 μl serial dilutions of a 9 μM peptide solution (in PBS) were spotted. 10 μl PBS was used as a negative control. Alternatively, the plates were incubated at 30°C for 24h to allow spore germination and growth of substrate mycelium before application of the peptide. After 3–7 days incubation at 30°C, plates were checked for possible effects of the peptide on morphological differentiation.
For light microscopy of substrate mycelium, aerial mycelium, and spore chains, coverslips were inserted into LB or MS agar and inoculated with a diluted spore solution (∼10³ spores) of S. coelicolor A3(2) M145 at the edge between the agar and inserted coverslips. Subsequently, 10 μl of 9 nM to 9 μM peptide solutions (in PBS) were pipetted into the gap between agar and coverslip. 10 μl PBS was used as a negative control. After 2 to 4 days of incubation at 30°C, the coverslips were placed on microscope slides coated with a thin agar pad and 1 drop of phosphate-buffered saline (PBS). Images were captured using the phase-contrast mode of an Olympus BX60 microscope equipped with an Olympus UPlanFl 100× oil objective and an F-view II camera.