# Age-Dependent Pleomorphism in _Mycobacterium monacense_ Cultures
## 3 Results
### Variation in Cell Morphology in Ageing Cultures  
To further understand the nature of the bi-phasic growth pattern we followed the cell shape distribution of _Mmon_T and _Mmon_RFPHyg after growth on 7H10 (solid) media at 37 \({}^{\circ}\)C for 2, 8, 14 and 30 days by microscopy. _Mmon_T cells were stained for membrane (FM4-64) and DNA [DAPI (4\({}^{\prime}\),\(\delta\)-diamidino-2-phenylindole)], while _Mmon_RFPHyg was stained with mitochondria tracking green (MTG), which stains outer as well as internal membranes [59], and DAPI (see Section 2). For each sample, we sequenced 16S rDNA as an additional control to rule out contaminations. Three different cell morphologies were observed when viewed under the microscope (Figure S3A): rods, coccoids and cells that appeared Phase Grey or phase Bright (PGB). The average cell sizes, measured for a minimum of 100 cells for each morphology type, were as follows: rods, 1.8 \(\pm\) 0.31 \(\upmu\)m (including both short rods of average length 1.6 \(\pm\) 0.26 \(\upmu\)m and longer rods of average length 1.9 \(\pm\) 0.3 \(\upmu\)m) and coccoids with 0.8 \(\pm\) 0.19 \(\upmu\)m diameters. This provided a qualitative basis for distinguishing between the different morphologies. For simplicity, rod-shaped cells detected in old cultures, although they were shorter than the exponentially growing rods, were still considered under the \"rods\" category. At early stages of growth (1-2 days), the larger fractions of _Mmon_T and _Mmon_RFPHyg cells were rod-shaped, closely followed by coccoid-shaped cells (Figure 2A-D). After three days, we detected increasing appearance of coccoid shaped cells while the cells classified as PGB started to appear at later time points (after eight days). The different time points were chosen based on their relevance to the growth phase as shown in Supplementary Figure S3B. Thus, time points from 1 to 3 days belonged to the exponential growth phase and the rest of the time points were different stages in the early (5-8 days) and late stationary phase (14 days and beyond). A heterogenous cell population of rods and coccoids was also observed when _Mmon_T cells were examined by transmission electron microscopy, TEM. For _Mmon_T, FM4-64 staining and TEM further suggested asymmetric septum formation (Figure 2E,F) in keeping with what has been reported for other mycobacteria [25; 60; 61; 62; 63; 64]. Moreover, similar changes in cell shapes as a function of time were also detected when cultivated on solid mG medium (Figure S4A,B). However, the frequency of distribution in mG medium pertaining to PGB structures differed from growth on 7H10 medium, _Mmon_T (Figure S4A) and _Mmon_RFPHyg (Figure S4B). We emphasise that a change from the rod shape to coccoid was also observed in _Mmon_RFPHyg liquid cultures (7H9 medium; Figure S4C). As for _Mmon_, the _Mboe_T cell shape changed from rod to coccoid upon prolonged incubation on 7H10 medium at 37 \({}^{\circ}\)C, and PGB cells were observed after 14 days of incubation (Figure S4D). The apparent change in the average cell size was also detected in liquid _Mboe_T cultures (7H9 medium; Figure S4E). We conclude that these two mycobacteriachange their cell shape upon ageing, as reported previously for other mycobacteria (see e.g., [33] and the refs. therein). Figure 2: Microscopy of _Mmon_T and _Mmon_RFHygR grown on 7H10 media and 37 \({}^{\circ}\)C. (**A**) Cells stained with FM4-64 (red) and DAPI (blue). Yellow arrows represent the PGB morphologies observed at different time points. Scale bar = 1 \(\upmu\)m. (**B**) _Mmon_RFHygR cells expressing RFP (red) stained with MTG (green) and DAPI (blue). Yellow arrows indicate the PGB cell morphology. Scale bar = 1 \(\upmu\)m. (**C,D**) Statistical representation of average percentage of occurrence of the various cell morphology types, _Mmon_T (**C**) and _Mmon_RFHyg (**D**). A minimum of 350 cells were analysed for each time point. “d”= number of days.