Three strains of Bacillus anthracis and seven strains of Bacilus cereus were grown on complex medium and on synthetic medium. Gas chromatographic analysis of whole-cell fatty acids of strains grown on complex medium gave nearly identical fatty acid patterns. Fatty acid patterns of strains grown on synthetic medium showed a high content of branched-chain fatty acids. Significant differences between the fatty acid patterns of the two species were found. Odd iso/anteiso fatty acid ratios were about equal in B. anthracis strains, whereas in B. cereus strains the fractions of iso acids were at least twice as high as the fractions of anteiso acids. The method described herein is used in our diagnostic laboratory to help differentiate between these two species.
Bacterial strains and bacteriophages. Strains of Rhodobacter sphaeroides were obtained from the German Collection of Micro-organisms (DSM), from W. R. Sistrom, University of Oregon, Ore., USA, or were isolates of this laboratory. The R. sphaeroides strains DSM 159-2 (pink) and DSM 159-1 1 (greenish) were colour variants of strain DSM 159 (red-brown). They were detected among colonies of strain DSM 159 plated on YP-medium (see below). The strains are listed in Table 1. The temperate R. sphaeroides phage 4RsG 1 is an isolate of this laboratory (Duchrow et al., 1985).Media and growth conditions. R. sphaeroides is a facultatively photosynthetic bacterium capable of growing anaerobically in the light (phototrophic) or aerobically (chemotrophic). Strains were grown at 30 "C in 20 mM-L( +)-tartrate-mineral medium (TM-medium; Rode & Gifhorn, 1983) or in rich medium (YP-medium) consisting of 0.3% yeast extract (Merck) and 0.3% trypticase peptone (Beckton Dickinson) adjusted with NaOH to pH 7.0. Stocks were maintained in liquid cultures which were incubated in completely filled screw-cap bottles (100 ml) anaerobically in the light at approximately 3000 lx and then stored at 4 "C. Bacteria on agar plates were incubated aerobically or phototrophically in Gas Pak anaerobic jars in front of a 100 W lamp.For phage growth TM-medium was supplemented with 2.5 rn~-CaCl, and YP-medium with 0.05 g CaCl, I -l , and then termed TMC-and YPC-medium respectively.Isolation and purification of phages. Phages 4RsA and 4RsD were isolated from a stock of the temperate R. sphaeroides phage 4RsG 1 (Duchrow et al., 1985) by plating on R. sphaeroides strain DSM 159-2 using the standard overlay technique and repeated single plaque isolation (Adams, 1959).Buflers. These were TE-buffer, 10 mM-Tris/HCl (pH 8*0), 1 mM-EDTA; TS-buffer, 20 mM-Tris/HCl (pH 7.0), 50 mM-NaC1; SSC-buffer, 15 mM-sodium citrate (pH 7.0), 150 mM-NaC1.Phage stocks. These were prepared by infecting a chemotrophic mid-exponential phase culture of R. sphaeroides DSM 159-2 with a filter-sterilized cleared phage lysate (m.0.i. = 0.1) and then incubating for another 48 h. Subsequently, cells and cell debris were sedimented for 10 min at lOOOOg and filter-sterilized. This procedure routinely provided stocks containing lo9 plaque-forming units (p.f.u.) per ml. Chromosomal DNA and RNA from lysed cells were removed from the stocks by incubation in the presence of DNAase (10 pg ml-I) and RNAase 50 pg ml-I) at 37 "C for 3 h. Phage particles were precipitated by addition of 6% (w/v) PEG 6000 and 0.5 M-NaCl (final concentration) and keeping the suspension at 4 "C for another 14 h. The phages were collected by centrifugation as above, resuspended in 1 ml TE-buffer and extensively dialysed against the same buffer at 4 "C.Electron microscopy. Phage particles were spread onto grids with carbon-coated Formvar support films, negatively stained with 4% (w/v) uranyl acetate (Valentine et al., 1968) and examined with a Philips EM 301 electron microscope.Preparation ofphage and bacterial DNA. Phage DN...
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