A Technique for Precise Inoculation of the Internal Phyllosphere of Cotton with Xanthomonas campestris pv. malvacearum.

ABSTRACT A technique was developed to inoculate uniformly and gently the internal phyllosphere from the upper surface of cotton leaves with the phytopathogenic bacterium Xanthomonas campestris pv. malvacearum. The inoculum consisted of 2 to 3 x 10(7) CFU/ml in CaCO(3)-saturated sterile distilled water containing 0.02%, vol/vol, of the wetting agent Silwet L-77. A custom-made inoculation apparatus was employed to immerse a circular area of the adaxial surface of a leaf in inoculum for 90 s. This resulted in uniform, passive entry of bacteria into the substomatal chambers, producing an endophytic bacterial population of 2 x 10(4) CFU/cm(2). Microscopic signs of infection were visible 48 to 72 h after inoculation. In susceptible leaves, uniformly distributed water-soaked spots were observed 7 to 8 days after inoculation. When the technique was used on resistant leaves, the autofluorescence that is characteristic of hypersensitively necrotic cells developed in the guard cells and palisade cells lining substomatal chambers, but not in the underlying spongy mesophyll.

Modified pyrogallol-initiated immunogold-silver enhancement technique applicable to prokaryotes.

A modified immunogold-silver enhancement technique that was designed to reduce the nonspecific granular background staining, particularly for application on prokaryotic organisms, is reported. Aerial oxidation of pyrogallol contained in the commercial silver enhancer solution was effectively controlled during storage and in the reaction mixture. A combination of strategies such as storing the reagent under argon, modifying it using 0.5% (w/v) anhydrous sodium sulfite, reducing the concentration of silver ions in the reaction mixture and limiting the length of the silver enhancement reaction considerably reduced the granular background staining. The modified technique was demonstrated on the bacterium Xanthomonas campestris pv. malvacearum (Smith) Dye. A 7-min silver enhancement step produced little background staining, while optimal silver intensification of the bacterium pre-treated with the immunogold label was achieved.

Helicobacter pylori comb. nov. Exhibits Facultative Acidophilism and Obligate Microaerophilism.

Modified brucella broth medium was used to study the growth of Helicobacter pylori at varied pHs and partial pressures of oxygen and to determine the effect of urea on culture pH. Our findings suggested that the pHs of the media remained stable with or without urea and that H. pylori showed facultative acidophilism and obligate microaerophilism.

Evidence for bacterial chemotaxis to cyanobacteria from a radioassay technique.

Lyngbya birgei and Aphanizomenon flos-aquae elicited a significant chemotactic attraction of Aeromonas hydrophila compared with controls lacking cyanobacteria. There was a positive exponential relationship between biomass (chlorophyll a) of L. birgei and A. flos-aquae and chemotactic attraction of A. hydrophila. The assay equipment was simple and reliable and could be used to study bacterial chemotaxis in other species in situ.

An in situ technique to measure bacterial chemotaxis in natural aquatic environments.

A simple and reliable technique to study bacterial chemotaxis in natural aquatic environments is reported. This technique uses the test chemicals in known volumes of semi-solid agar media placed in double layered, highly porous, polyester tubes. Following in situ incubation, bacteria attracted by the test chemicals are enumerated with fluorescence microscopy following acridine orange staining. Studies in an eutrophic reservoir showed that significant numbers of bacteria were attracted to D-glucose and glycine; no significant effects were observed with L-serine, sodium succinate, or sodium chloride.