Cellometer x2 image cytometer

Cyanidioschyzon merolae cells were cultured in a modified Allen’s autotrophic medium (Minoda et al., 2004 (link)) under continuous illumination at 50 μmol photon m⁻² s⁻¹ with constant bubbling of air containing 2% CO₂ at 42°C. A uracil-requiring strain M4 (Minoda et al., 2004 (link)), which was used for gene disruption, was grown in the presence of 0.5 mg ml⁻¹ uracil, while its transformants with a marker gene URA5.3 were cultured without uracil. Growth was monitored with OD₇₅₀, cell number, and chlorophyll (Chl) a. The cell number was measured by Cellometer X2 Image Cytometer (Nexcelom Bioscience, Lawrence, MA, United States), while Chl a was extracted with 100% methanol, and its concentration was determined photometrically (Arnon et al., 1974 (link)).

Endolysin activity was confirmed by examining the viability of bacterial cells population using Cellometer X2 image cytometer (Nexcelom) as described by Hodgkin et al. [53 (link)]. Ten-microliter of OD₆₀₀ = 0.5 L. fermentum 0315-25 (10⁷ CFU/mL) were mixed with 90 μL of 20 mM Tris–Cl buffer (pH 7) containing final concentration of 100 μg/mL LysMP endolysin and incubated for 30 min. Subsequently, 9 μL of sample was mixed with 1 μL of 10 mM SYTOX^® and 4 μL was pipetted into a Nexcelom counting chamber (CHT4-SD025), where the inlet and outlet ports were closed with clear tape to prevent evaporation. The bright-field and fluorescent (Excitation: 490 nm/Emission: 530 nm) images were acquired at four different locations in the chamber. The images were analysed by the Cellometer Spectrum software (version 3.2.1.2). Total bacterial concentrations were enumerated based on fluorescent intensities and cell size. Green-stained Limosilactobacilli samples were analysed to enumerate percent of dead cells based on total bacterial concentrations.