Figure 4

<p>Three independent cultures were used to determine the copy numbers of the chromosome and three additional replicons, i.e. pHS1 to pHS3, using the Real Time PCR method. One of the growth curves, the average replicon copy number per cell and the standard deviations are shown.</p

Figure 1

<p>A. Overview of the method. A culture of known cell density is embedded in low melting point agarose (step 1), agarose blocks with a defined number of cells are prepared, the cells are lysed and protein is digested (step 2). The blocks are melted and a restriction enzyme (step 3) as well as an internal standard (step 4) are added. After overnight digestion, DNA fragments are size fractionated by electrophoresis and a Southern blot is performed (step 5). A 1 kbp genomic fragment near the replication origin and the 0.9 kbp internal standard are both visualized with a single probe. Multiple aliquots containing different standard concentration are used for quantitation. B. Quantitation of the genome copy number of exponential cells. After gel electrophoresis and southern blotting, a genomic fragment (upper band) and different concentrations of an internal standard (lower band) were visualized with the same probe (step 5 in A). C. Quantitation of the genome copy number of stationary phase cells. After gel electrophoresis and Southern blotting, a genomic fragment (upper band) and different concentrations of an internal standard (lower band) were visualized with the same probe (step 5 in A). D. An example of a standard curve generated after quantitation of the bands shown in B. and C. The horizontal and vertical lines show the usage of the standard curve to determine the genome copy number in the biological replicate No. 9 (see E.). E. Summary of the results of the independent cultures that were used to determine the genome copy number with the “agarose block method”. In the first five experiments, the standard curve ranged from 0.5 molecules/cell to 10 molecules/cell (as in B.). The signal of exponential phase cells was much higher than the highest signal of the standard curve and could not be quantitated. Therefore the standard curve was chosen to range from 1 molecules/cell to 40 molecules/cell (as in C.) in subsequent experiments.</p

Figure 6

<p>A. The forward light scatter as a measure of cell size is plotted against the fluorescence of the DNA-specific dye acridine orange as a measure of the DNA content. Aliquots representing early and middle exponential phase and early stationary phase (top to bottom) were analyzed, and the optical densities are indicated. B. The fluorescence as a measure of DNA content is plotted against the fraction of the population exhibiting a specific fluorescence. The three curves from left to right represent the three aliquots shown in A from top to bottom.</p

Figure 3

<p>H. salinarum was grown by aerobic respiration at 42oC and 30 oC and by arginine fermentation at 42oC. The doubling times were 4 hours, 8 hours and 8 hours, respectively. For each condition three independent cultures were used. Aliquots representing early exponential phase (2–3×108 cells/ml), mid-exponential phase (4–5×108 cells/ml), late exponential phase (8–9×108 cells/ml) and early stationary phase (1–2×109 cells/ml) were used to determine the genome copy number using the Real Time PCR method. Average values of the three biological replicates and their standard deviation are shown.</p

Figure 5

<p>Determination of genome copy number using the agarose block method. Different aliquots from one culture were used to inoculate several new cultures that were grown overnight. Throughouht the next day aliquots were withdrawn, the cell density was determined with a counting chamber and the ploidy with the agarose block method. For each aliquot, the cell density was plotted against the genome copy number, and a trend line was calculated. B. Determination of the genome copy number using the Real Time PCR method. Three independent cultures were used to determine the genome copy number. A selected growth curve, the average ploidy values and their standard deviation are shown.</p