# Age-Dependent Pleomorphism in _Mycobacterium monacense_ Cultures
## 2 Materials and Methods
### Media, Determination of Generation Times and bi-Phasic Growth Experiments  
MiddleBrook 7H9 (liquid) and 7H10 (solid) media were used as standard and prepared according to the manufacturer's instructions. For cultivation of \(Mmon^{\mathrm{RFPH9S}}\) and \(Mmon^{\mathrm{RFPKan}}\), hygromycin and kanamycin were added to final concentrations of 100 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\) and 25 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\), respectively. For induction of \(dnak\_3^{\mathrm{Mmon}}\) (and anti-\(dnak\)\(\mathrm{\SIUnitSymbolMicro mon}\)) carried by pBS401, 7H10 plates were supplemented with tetracycline (Tc; final concentration 20 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\)) and hygromycin 100 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\) (see also Table S2). The Arret-Kirshbaum agar (AK; BD, Bioscience, Gothenburg, Sweden), modified G media (mG; [26]), Potato Dextrose Agar (PDA; BD, Bioscience, Sweden) [27] and Luria-Bertani (LA) media were prepared according to established protocols. To determine the generation time, GT, for \(Mmon^{\mathrm{RFPH9S}}\) cultivated in liquid medium a single colony (about 5-7 days old) was used to inoculate in 10 mL 7H9 medium supplemented with hygromycin (100 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\)) and grown for 5-10 days. This primary culture was diluted 1000-fold in fresh media, incubated at \(37\ ^{\circ}\mathrm{C}\) and \(\mathrm{\SIUnitSymbolMicro OD_{600}}\) was measured at different time points (see Section 3). Growth curves were generated by plotting OD\({}_{600}\) values (\(Y\)-axis) against time (\(X\)-axis). The generation times were determined by calculating the slope for each curve using the tangent to the curve (exponential trend-line). The slopes (GT) were used to compare the growth phases (fast, slow or stationary) allowing us not to base our comparisons to the initial OD\({}_{600}\) of the culture (see Section 3). Determination of the growth curve for each condition was repeated twice with biological triplicates for each repeat. The generation times were calculated based on two repeats with biological triplicates for each repeat and given as average \(\pm\) error range. The bi-phasic growth experiments were conducted in 7H9 (see above). A 5-10 days old primary culture of \(Mmon^{\mathrm{RFPH9S}}\) grown at \(37\ ^{\circ}\mathrm{C}\) was diluted 1000-fold (final volume 120 mL per replicate). OD\({}_{600}\) was measured at regular intervals and plotted against time. From the so obtained bi-phasic growth curve, we identified a fast exponential phase Expo I, followed by a slow exponential phase Expo II and stationary phase SP (see Section 3). A fixed volume of Expo II cells (40 mL) was pelleted (in 50 mL falcon tubes) at OD\({}_{600}\approx\) 0.25-0.4 (for \(Mboe^{\mathrm{T}}\) OD\({}_{600}\approx\) 1), and subjected to different media conditions, while the remaining culture was allowed to continue to grow (see Section 3). To test for the depletion of the carbon source, Tween 80 (final concentration 0.05%) and Glycerol (final concentration 0.2%) were added individually to 40 mL cultures in the Expo II phase. Media from the Expo II growth phase cultures were filtered using sterilised filters (0.2 \(\mathrm{\SIUnitSymbolMicro m}\); referred to as \"spent media\") and used to re-suspend cells from Expo II (referred to as \"Re-sus\") and Expo I (referred to as \"EP-I in spent media\"). As a control, cells from Expo I and Expo II were re-suspended in fresh 7H9 media (supplemented with 100 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\) hygromycin), referred to as \"Expo I in fresh media\" and \"Expo II in fresh media\", respectively. Also, Expo II cells were pelleted and re-suspended in the same media (without filtration) as a control for the re-suspension step (and referred to as \"re-suspended\"). The OD\({}_{600}\) was measured at regular intervals and growth curves were generated (for details see Section 3).