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.

Surface biochemistry, morphologies and internal structures of Mm cells at different stages of sporulation. (A) SEM images show isolated spores at 0 h (Upper Left); cells at germination (bulged cells) at 6 h (Upper Right); putative endospores (bulged cells) at day 5 (Lower Left); and spore with a vegetative cell at day 7 (Lower Right). (Scale bar: 1 μm.) (B) Thin-section TEM images show isolated spores (6,000 × magnification); a mature spore of the transverse section (60,000 × magnification); a mature spore of the longitudinal section (60,000 × magnification); germination 1–3 (different stages of germinating spores at 6 h) (40,000 × magnification); and a forespore and a mature endospore at day 5 (30,000 × magnification). Cells at different times after inoculation into fresh medium were prepared for SEM and TEM as described in Materials and Methods. (C) Spore-specific differential staining of Mm cells from exponential and stationary phase cultures. Cells grown and harvested at different stages of growth were heat-fixed, stained and counterstained with malachite green and safranin, respectively (see Materials and Methods), and examined under the 100 × objective of an Olympus CH30RF200 microscope.

Heat tolerance and detection of dipicolinic acid. (A) Colony-forming ability after heat treatment. Plates containing exponential (12 h after inoculation) and stationary (after 14 d of growth) phase cells with and without exposure to wet-heat treatment (15 min, 65° C). (B) Rate of killing by wet heat for Mm cells from exponential and stationary phases. Cells in exponential phase (12 h, diamonds) and stationary phase (14 d, squares) were exposed to wet heat at 65° C for different periods of time, surviving colonies were expressed as percentages against identical, unexposed cell populations, and plotted as a function of exposure times. Each point on the survival plot represents an average of at least 3 measurements [Table S5 (PDF)] with experimental variations indicated by error bars. (C) DPA released from Mm cultures at different stages of growth. Vegetative cells do not show any color relative to the cells (coloring agent: Fe(NH₄)₂(SO₄)₂.6H₂O without DPA). (Inset) Line 1, 7-week-old culture at OD₆₀₀ = 10.0; lines 2–4, purified spore suspensions at OD₆₀₀ = 2.0, 5.0, and 10.0, respectively. For details see SI Text.

Relative expression levels of putative sporulation genes from Mm genome at different stages of the life cycle. The mRNA levels of 9 genes from Mm genome, homologues of known sporulation genes from B. subtilis and S. coelicolor, were compared by using dot-blot hybridization as the culture progressed from exponential to stationary phase. Specific oligonucleotide probes [Table S6 (PDF)] were used for each mRNA candidate. The relative intensities of the dots were normalized by using corresponding 5S rRNA signals as internal standards and were plotted in arbitrary units (y axis) as Mm mRNA signals (identified as their homologues, x axis) from cultures of different ages (bars of different patterns). All samples were analyzed at least 3 times, and estimated experimental variations are indicated by error bars. The numerical values [Table S7 (PDF)] were obtained from Phosphor-Imager (ImageQuant 400, Molecular Dynamics) analysis of the dot signals.

Presence of spore particles in a culture of M. bovis bacillus Calmette–Guérin. (A) Phase fluorescence image of M. bovis cells from a 6-month-old culture. (Scale bar: 5 μm.) (B) Differentially stained, purified spore particles from bacillus Calmette–Guérin culture. (C) SEM image of purified bacillus Calmette–Guérin spores. (Scale bar: 1 μm.) (D) TEM image of thin-sectioned purified bacillus Calmette–Guérin spores. (Magnification: 60,000 ×.)