Fig. 1.

Presence of spores in old stock and their reappearance in stationary phase. (A) Flow cytometric profiles covering the complete life cycle of M. marinum. An old Mm stock was used to inoculate fresh medium, and the progress of the culture to stationary phase through exponential growth was followed by flow cytometry. A 2-month-old stock was spread on 7H10 agar plates with necessary supplements; cells were harvested at different times over a period of 2 months. The harvested cells were fixed in 70% ethanol, washed and resuspended in 0.1 volume TM buffer (10 mM Tris-HCl pH 7.8; 10 mM MgCl₂), stained with mithramycin A and ethidium bromide and run in the flow cytometer (see SI Text). The profiles are histograms of cell numbers plotted against their DNA content (Left; fluorescence calibrated in chromosome number equivalents) or size (Right; light scattering measured in arbitrary scale units kept constant for all histograms). The ages of the cultures—in hours and months after inoculation into fresh medium—are shown on the left side of each row. The profiles on the top row, showing a stationary phase culture of the laboratory strain of the Gram-negative bacterium E. coli K12 (1–3 μm × 1 μm cylinders containing 4.6 Mbp DNA per chromosome), were used as a calibration standard for estimating DNA content and cell size in the Mm profiles. (B) Fluorescence microscopy of Mm at different stages of growth from fresh inoculum to stationary phase. Two-month-old stock was spread onto plates and aliquots fixed in ethanol as described in A. The fixed cells were washed in PBS, layered onto thin films of agarose (1% in 0.9% NaCl) containing 0.5 μg/mL DAPI on a microscope slide and examined under a Zeiss Axioplan 2 microscope with a CCD camera (see Materials and Methods). The frames, from top to bottom, show cells harvested at 0 h, 6 h, 12 h, 72 h, 120 h, 168 h and 336 h after inoculation into fresh medium. (Scale bars: 1 μm.) The spore size was estimated to be ≈30–60% of the size of the vegetative cells.