# Age-Dependent Pleomorphism in _Mycobacterium monacense_ Cultures
## 3 Results
### Bi-Phasic Pattern of Growth Curves Plotted from Absorbance Measurements  
Since _Mmon\({}^{\mathrm{T}}\)_ and _Mmon\({}^{\mathrm{RFPHyg}}\)_ grew differently in liquid medium, we compared their growth rates on solid medium, by following their growth on 7H10 plates without and with hygromycin (100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL). Identical aliquots of bacterial lawns from plates smeared with equal number of cells and maintained at 37 \({}^{\circ}\)C were collected at intervals and resuspended into 0.1 mL PBS buffer. The number of bacteria counted by plating were plotted as CFU/mL against time (see Section 2). Figure 1A shows the growth curves for _Mmon\({}^{\mathrm{T}}\)_ and _Mmon\({}^{\mathrm{RFPHyg}}\)_ grown on 7H10 agar plates over a period for >100 h. Trend lines (marked in red) were drawn to estimate generation times (GT). The growth curves are almost parallel showing very similar growth rates for the two strains; viz., with GT, 5.9 \(\pm\) 0.5 and 5.1 \(\pm\) 0.9 h for _Mmon\({}^{\mathrm{T}}\)_ and _Mmon\({}^{\mathrm{RFPHyg}}\)_, respectively. Moreover, the polymorphic cell patterns (see below) for the two strains with ageing of the plate cultures were similar. Hence, the observed _Mmon\({}^{\mathrm{RFPHyg}}\)_ growth and pleomorphism was taken to represent that of _Mmon_ in general and we used _Mmon\({}^{\mathrm{RFPHyg}}\)_ to investigate growth in liquid 7H9 medium containing 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL hygromycin (see Section 2). In order to follow the growth curve of _Mmon\({}^{\mathrm{RFPHyg}}\)_ in liquid cultures, single colonies were inoculated into 7H9 medium supplemented with hygromycin and the growth rate was measured following OD\({}_{600}\) with time for over 150 h (Figure 1B; see Section 2). Attempts to estimate GT by drawing trend lines through the time points showed two distinct rates of exponential growth with GT = 5.3 \(\pm\) 0.44 h during the early phase (Expo I) followed by a much slower exponential growth rate, GT = 22 \(\pm\) 3.1 h (Expo II) as shown in Figure 1B (see Section 2). The early growth rate is similar to GT (5.1 \(\pm\) 0.9 h and 5.9 \(\pm\) 0.5 h for _Mmon\({}^{\mathrm{RFPHyg}}\)_ and _Mmon\({}^{\mathrm{T}}\)_, respectively) for growth measured by cell counts on solid media (7H10; see Figure 1A). Upon longer incubation, the cultures appeared to reach stationary phase at OD\({}_{600}\)\(\sim\)3 (Figure 1B and Figure S2A). The bi-phasic growth could be attributed to the following: (i) depletion of a preferred nutrient such as carbon source resulting in a diauvic growth curve; (ii) accumulation of inhibitor(s) in the medium (after a critical concentration is reached) that reduced the growth rate; (iii) cell density-dependent quorum sensing; or (iv) inherent growth phase dependent heterogeneity in cell size/shape [52; 53; 54; 55; 56; 57; 58]. To distinguish between these possibilities, we tested several growth conditions for _Mmon\({}^{\mathrm{RFPHyg}}\)_ including fresh inoculation into \"spent\" media from Expo II phase cultures, cultivation of \"Expo II-cells\" in fresh media, addition of fresh carbon sources (glycerol or Tween 80) and different dilutions of \"Expo II-cells\" (for details, see Section 2). Addition of glycerol or Tween 80 to Expo II cultures did not change the growth rate. Therefore, the transition from Expo I to Expo II cannot be attributed to depletion of these carbon sources [scenario (i); Figure 1C; Table S3]; however, we cannot rule out that other carbon sources in the 7H9 media such as dextrose, which is present in the growth supplement Oleic Albumin Dextrose Catalase (OADC), might have an impact, but see below. Cells in the Expo I-phase continued to grow with \"Expo I-growth rates\" irrespective of the media added (filtered media from the Expo II-cultures, \"spent media\", or addition of fresh media; Figure 1D and Table S3; see Section 2).