[Electron microscopy of Leptospira].

RITCHIE, A. E. (National Animal Disease Laboratory, Ames, Iowa), AND HERMAN C. ELLINGHAUSEN. Electron microscopy of leptospires. I. Anatomical features of Leptospira pomona. J. Bacteriol. 89:223-233. 1965.-Anatomical features of Leptospira pomona are described. Most cells in the logarithmic phase of growth, when examined in whole cell "negative-stained" preparations, display a homogeneous finely granular protoplasmic cylinder. Some cells contain discrete or complex lamellar structures. The cell termini are attached to the protoplasmic cylinder by a membrane system. Each organism appears to have two independent axial filaments originating from opposite ends of the cell. The axial filaments are composed of a closely knit bundle of some 12 to 15 individual strands. A simple model suggesting a locomotory function for the axial filaments is presented. A superficial callouslike mass is occasionally observed in the cell's terminal region. The enveloping sheath is similar to the cytoplasmic membrane; both are triple-layered and ca. 50 A thick. The sheath preferentially forms blebs at the cell termini and midway along the protoplasmic cylinder. Septal-wall formation occurs at the mid region of the cell, and it is concluded that simple transverse binary fission is the predominant mode of reproduction. Leptospires are currently considered to consist of at least three morphologically distinct structures: (i) a spiraling plrotolplasmic cylinder, (ii) a homogeneous axial filament (axistyle, achsenfaden) generally ly3ing, external to the cylinder and terminating near the ends of the organism with a knob-like process, and (iii) a membranous sheath enveloping the organism. G-ranular protrusions of the protoplasmic cylinder, particularly at the cell extiemities, have frequently been reported (Jacob, 1947; Czechalowski andl Eaves, 1954; Swain, 1955; Parnas et al., 1958; Varpholomeeva and Stanislavsky, 1958). In thin sections, the protoplasmic cylinder appears tubular (B3abudieri, 1959; Simpson and White, 1961) and contains osmophilic granules (AMiller and Wilson, 1962). Metal shadow-cast prleparations of osmium"fixed" organisms show that the enveloping sheath follows closelvthe contours of the protoplasmic cylinder (B3abudieri, 1948, 1949; AIlbert, 1955). Nongranular bulbous swellinogs of the sheath hav-e been observed (Kirschner, AMaguire, and l3ertaud, 1957; Simpson and White, 1961). Washing the organisms with distilled water oI physiological saline readily removes the sheath (Takeya, MIori, and Toda, 1957). Presence of an apparently sinole homogeneous axial filament in several strains of leptospires was shoNwn by B3abudieri (1948, 1949) and l3reese, G-ochenour and Yaoer (1952). The lpossibility of a multistranded structure of the axial filament Nas suggested by Mdlbert (1955). Recent experiments with trypsin digestion of "isolated axistyles" have been interpreted as evidence for a two-stranded axial filament in Leptospira canicola and L. icterohaemorrhagiae (Czechalowski, 1963). The axial filament terminal knob has been described by Czechalowski and Eaxves (1955) in shadowed )reparations of deoxycholatelysed organisms. Miller and Wgilson (1962) confirnmed its presence in thin-sectioned preparations. Extensiv-e rev-iews of the morphology of the leptospires have been published (Noguchi, 1928; Thiel, 1948; B3abudieri, 1958, 1960). Their sp)iral conformation and great lengthto-wsidth ratio (15:1 to 50:1) make leptospires difficult to examine in their entirety in thin section. In shadow-cast lpreparations, internal detail is obscured by the superficial metal layer. The method of phosphotungstate "negative staining" a)pearedl to offer advantagres over previous techniques, and a survey of the morphology of L. pomtiona was undertaken. This report describes anatomical features of L. ponmona 223 on O cber 7, 2017 by gest http/jb.asm .rg/ D ow nladed fom RITCHIE AND ELLINGHAUSEN as revealed in whole-cell mounts of "negatively stained" organisms. MATERIALS AND METHODS L. pomona was grown in an Oleic-Albumin Complex Medium, which is described in detail elsewhere (Ellinghausen and McCullough, Amer. J. Vet. Res., in press). Liquid cultures were subcultured at 3-day intervals. In most instances, the cultures examined were 48 to 72 hr old, and had a cell count of between 120 and 180 X 106 cells per milliliter. Growth curves were followed before and after sampling for electron microscopy. Where phosphate-lysed organisms were employed, the culture was sedimented, washed three times by centrifugation from sterile phosphate buffer (pH 7.0), and then incubated at 29 C in fresh sterile phosphate buffer. Potassium phosphotungstate negative-stained whole-cell mounts were prepared according to three procedures. Most preparations were made by minor modification of the frozen-suspension sectioning method (Almeida and Howatson, 1962, 1963). Modification consisted of collecting a few sections (4 to 6 i thick) of frozen-cell suspensions directly onto carbon-coated collodion-filmed grids and thawing in situ. After thawing, a droplet of 2 to 4% aqueous potassium phosphotungstate (pH 6.7 to 7.2) was applied for 0.5 to 1 min and withdrawn slowly with a torn edge of paper toweling. Other preparations were made by simple gravity sedimentation from mixtures of cell suspension and phosphotungstate, followed by withdrawal of the droplet with paper toweling as above. Currently, a more uniform dispersion of the organisms in a thin film of phosphotungstate is achieved by the "loop-film" technique recently described (Murray, 1963). In each case, the cell mounts were examined immediately in a Philips EM 200 electron microscope at 80 kv by use of double condenser illumination. For comparative purposes, organisms were prepared for thin sectioning by methods similar to those described previously (Kellenberger, Ryter, and S6chaud, 1958; Kushida, 1961). Briefly, the procedure consisted of overnight fixation of cellculture suspensions in 0.5 or 1.0% buffered (pH 7.2) osmium tetroxide containing Ca++, setting in 2% agar, treatment with aqueous uranyl acetate, and embedding in divinylbenzene cross-linked methacrylates. Sections were obtained with glass or diamond knives on an LKB Ultratome and stained with alkalinized lead solutions (Millonig, 1961).