Fig. 6: Phenotype comparisons of normal colony variant (NCV) J1 and small colony variant (SCV) J9.

a Flow cytometry of strains J1 (upper panel) and J9 (lower panel) stained with rhodamine 123. Cells (1 × 10⁶ CFU/mL) were grown in 4 mL of YPD broth overnight at 30 °C. Thereafter, cells were stained with rhodamine 123, with or without 1 mM sodium azide pre-treatment. Rhodamine-123 fluorescence intensity is presented on the X-axis; intensity increases from left to right. The Y-axis presents cell numbers, as the percentage of the total number of cells. FSC and SSC density plot was performed, and the cell population of interest was identified with polygon gating. Histogram bins are normalized to peak. Unstained J1 and J9 cells (a and b, respectively) are controls. J1 and J9 cells stained with rhodamine 123 in absence of sodium azide pre-treatment are presented in c and d, respectively. In absence of sodium azide treatment, J1 (c) exhibited greater fluorescence than did J9 cells (d), consistent with higher mitochondrial activity. J1 and J9 cells stained with rhodamine 123 following sodium azide pre-treatment are presented in e and f, respectively. Set of figures below are representative images from one of two independent experiments that showed similar findings. b Transmission electron micrographs of strains J1 and J9. Representative images (from three independent experiments, with 30 images taken per experiment) shown here highlight reduced numbers and aberrant morphology of mitochondria in small colony variant strain J9, compared to normal colony variant J1. Several normal appearing mitochondria are denoted by mt. c Evaluation of respiratory status of strains J1 and J9 using electron transport inhibitors. Cells grown in synthetic completed medium for 48 h at 30° C were untreated or exposed to sodium cyanide 100 mM, salicylhydroxamic acid (SHAM) 100 mM or antimycin 1 µM. Oxygen consumption was measured as phosphorescent probe signal intensities (y-axis) versus time (x-axis). In absence of drug exposure (control), oxygen consumption by strain J1 was greater than that by strain J9. Each of the electron transport inhibitors resulted in significant reduction in J1 oxygen consumption, without impacting consumption by respiratory-deficient strain J9. Figures below are representative images from one of two independent experiments that showed similar trend of response to inhibitors.