Methyl violet: a selective agent for differentiation of Klebsiella pneumoniae from Enterobacter aerogenes and other gram-negative organisms.

Three selective media for differentiation of Klebsiella pneumoniae from Enterobacter aerogenes on the basis of colonial morphology were evaluated. Using methyl violet 2B as a selective agent, strains of K. pneumoniae isolated from urine, fresh water, and fresh produce were tested against other members of Enterobacteriaceae in addition to strains of Aeromonas hydrophila and Pseudomonas aeruginosa. Comparison of colonial morphology showed K. pneumoniae produced larger, smoother colonies than other bacteria tested. These media were developed to aid in presumptive separation of K. pneumoniae from E. aerogenes in the monitoring of bacterial quality of water.

Parasporal bodies of Bacillus laterosporus sporangia.

Intact colonies of Bacillus laterosporus examined by thin-section transmission electron microscopy revealed sporangia in various stages of development and degeneration as the endospores matured. The sporangia formed a surface layer of hexagonally arranged subunits. The variety of parasporal bodies raised questions of developmental and ecologic utility.

Electron Microscopy of the Phagocytosis of Capsulated Bacillus anthracis.

The phagocytosis of capsulated vegetative cells of Bacillus anthracis in the mouse spleen was studied by thin sectioning techniques of electron microscopy. Mice were injected with autoclaved suspensions of capsulated and noncapsulated vegetative cells via the tail vein. The animals were killed 5, 10, and 30 min and 4 hr postinjection, with the central portion of the spleen being removed and procssed for electron microscopy. Fixation was with 2% KMnO(4) for 2 hr. Results of this study indicated that phagocytosis of autoclaved, capsulated anthrax bacilli was through the normal phagocytic process. The vesicular membrane or phagosome membrane was still present around these killed, capsulated organisms 4 hr postinjection. It is concluded that such cells possess no toxic principle to account for the supposed destruction of the phagosome membrane previously reported.

Scanning electron microscopy and freeze-etching of gonorrhoeal urethral exudate.

Urethral exudates from male patients with gonorrhoea were studied by use of the critical point drying technique for scanning electron microscopy. The surfaces of phagocytes were extensively ruffled. This was interpreted to be the three-dimensional appearance of pseudopodia. Epithelial cells present in the urethral exudate showed a highly convoluted surface structure. Specimens of urethral exudate prepared by the freeze-etch technique showed that most N. gonorrhoeae were engulfed individually in phagocytes. Some phagosomes contained two or more gonococcal cells. Phagocytes containing gonococci were degranulated, but small granules were present in the phagosomes. Nuclear pores on the surface of the nuclear membrane were always associated with an indentation of the nuclear membrane. The outer layer of the nuclear membrane was smooth compared to the inner layer,

Electron microscopic study of a slime layer.

Slime layers are being studied in our laboratories in an attempt to understand their functions in the control of pollution in natural streams. A method for fixing, staining, and embedding microorganisms in the intact slime has been developed. In this method, epoxy resin discs are placed in a holder and are introduced into a simulated stream. After various periods of time the discs are punched out of the holder into the fixative. The disc with the attached slime is fixed, stained (4% osmium tetroxide plus ruthenium red), dehydrated, and embedded in epoxy resin so that thin sections can be cut through the vertical plane of the slime mass. Such thin sections permit detailed examination of the attached layer, the surface-slime interface, the spatial relationships between cells in the vertical slime structure, and the strands of extracellular material between and around cells. No special attachment structures were noted as the cells appeared to be attached to the surface by extracellular material alone. This material was observed in strands and netlike forms between cells which are positioned 1 to 4 mum apart in the slime.

Scanning electron microscopical examination of gonococcal urethral exudate.

The relationship of gonococci and phagocytes in gonococcal urethral exudate was examined using a scanning electron microscope. Several stages in the phagocytic process were evident. These included pseudopodias, microvilli, and simple attachment of gonococci to phagocytic surfaces. Epithelial cells, also a part of the exudate, were examined in this study. Parallels are drawn between this and other phagocytic systems. The technique provides excellent preservation of phagocyte-bacterial interrelationships and offers a means of investigating the nature of the resistance of Neisseria gonorrhoeae to phagocytosis.

Evaluation of double violet agar in the isolation of Klebsiella pneumoniae from river water.

A field evaluation of double violet agar for the isolation and presumptive identification of Klebsiella pneumoniae from water has been performed. Water from the North Oconee River, Clarke County, Ga., was cultured for presence of klebsiellae using the membrane filter technique. Colonies were presumptively identified as K. pneumoniae in the basis of their appearance on double violet agar. Such identifications were evaluated using appropriate biochemical tests. Once investigators have become familiar with cultural reactions on the medium, double violet agar can be used to indicate presence of K. pneumoniae in water with greater than 80% accuracy.

Scanning electron microscopy of surface and internal features of developing perithecia of Neurospora crassa.

Stages in the development of perithecia of Neurospora crassa, designated by the time elapsed after crossing, were investigated with the scanning electron microscope, from protoperithecia through perithecia. The usual examination of external features of whole specimens with this instrument was augmented by a freeze-fracture technique which allowed the viewing of development internally as well. Rapid increases in perithecial size soon after crossing were followed by the appearance, in section, of a centrum, at first undifferentiated but subsequently developing ascogenous hyphae. The perithecial beak appeared as a compact mass easily distinguishable in whole specimens from the surrounding hyphae by means of texture as well as shape. Two ascospores were photographed during emergence from an ostiole, but ostioles were found more frequently closed than open.

Freeze-etch study of Pseudomonas aeruginosa: localization within the cell wall of an ethylenediaminetetraacetate-extractable.

A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg(2+), the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system.

Airborne aflatoxin in corn processing facilities in Georgia.

Problems affecting the health of agricultural workers in processing facilities where grains are contaminated with aflatoxin have been noted previously. Airborne particulates produced during processing are reported to produce various carcinomas when inhaled by factory workers. Two corn processing plants within Georgia were surveyed during the fall of 1984 and 1985 with utilization of an Andersen 6-stage air sampler, a high-volume air sampler and a slit sampler. No airborne aflatoxin was found; however, 10% of settled dust samples were contaminated with aflatoxin. The average particles were found to be globular in shape with an effective diameter between 2 to 3 microns. Only 30% of the bulk corn samples contained aflatoxin, which was present at low levels (0.15 to 8 ppb).

Isolation of Klebsiella pneumoniae from lake water.

The question of the importance of aquatic borne Klebsiella pneumoniae to public health has been argued as the organism has appeared often in both polluted effluents and oligotrophic systems. Using a selective medium, double violet agar, which also differentiates K. pneumoniae from other organisms found in aquatic systems, we sampled water from three ponds and a large man-made lake, over a 3-month period. Results indicate that K. pneumoniae can be isolated consistently and in high numbers from eutrophic waters even when environmental stress reduces total numbers, but that isolations from cleaner waters are erratic. We conclude that mere isolation of the organism as an occasional contaminant probably does not present a hazard to public health.

Scanning electron microscopy of Thermoplasma acidophilum.

The scanning electron microscope was utilized to observe the morphology of the thermophilic, acidophilic mycoplasma, Thermoplasma acidophilum. Upon examination of the surface morphology, the size and shape of this unusual mycoplasma revealed its similarity to the other mycoplasmas that have been investigated.

Ultrastructure of lipopolysaccharide isolated from Thermoplasma acidophilum.

The fine structure of lipopolysaccharide isolated from Thermoplasma acidophilum was examined by electron microscopy. Negative staining of the lipopolysaccharide revealed long, ribbon-like structures with some branching. The average width of the lipopolysaccharide ribbons was 5 nm. Treatment of the lipopolysaccharide with 0.5% sodium dodecyl sulfate resulted in the dissociation of the ribbon-like structures to spherical- and vesicular-shaped particles and some short, rodlike structures. Results suggest that the lipopolysaccharide from T. acidophilum is morphologically similar to lipopolysaccharide isolated from gram-negative bacteria.

Flagella on Legionnaires' disease bacteria: ultastructural observations.

Scanning and transmission electron microscopic studies were done on five strains of flagellated Legionella pneumophila cultured for 1 to 3 days on charcoal yeast extract agar or yeast extract broth. Ultrastructurally, each strain consisted of pleomorphic, flagellated bacilli, many of which showed the typical pinching type of division as seen with other gram-negative bacteria. Most of the flagellated organisms, regardless of the strain, had a single, relatively straight or undulant polar flagellum, about 25 nm in diameter. In some instances, retraction of the bacterial cell membranes made the flagellar basal bodies (bulbs) visible when observed by transmission electron microscopy. The bulbous nature of these bodies appears to be different from the basal structures described for Escherichia coli and other gram-negative bacteria. Long "streamers," previously observed with the light microscope, appear to be fimbriae (or pili) that have a diameter approximatley half that of the flagella and are somewhat coiled and bent at irregular intervals.