Non-specific binding of protein-stabilized gold sols as a source of error in immunocytochemistry

The observation that protein-A conjugated gold sols bound to fibronectin-collagen (FNC) fibres in human fibroblast cultures prompted a series of studies on the binding of gold particles stabilized in various ways (Staphylococcal protein A, bovine serum albumin, avidin, streptavidin, gelatin, hemoglobin, polyethylene glycol (MW 20 000), methylcellulose and the nonionic detergent Tween 20) to cell and tissue components, to protein dot blots and SDS-PAGE blots on nitrocellulose paper. We found that binding of gold particles to certain cell and tissue components and to various immobilized proteins did occur irrespective of the stabilizing agent. We argue that, albeit gold sols are stabilized against salt coagulation by adsorption of proteins and other stabilizing agents, "naked areas" are (constantly or intermittently) present on particle surfaces, available for interaction with cell and tissue components that have a high electrostatic affinity for the charged gold surface under prevailing experimental conditions. Non-specific binding may be reduced or abolished by competing proteins (i.e. proteins with a higher affinity for gold than any component in the object studied) provided the proteins and the gold conjugate are present concomitantly during incubation. We found gelatin (Bloom number 60-100) to be an effective competitive protein probably due to its high affinity for gold over a wide pH range. Further, gelatin did not appreciably inhibit the specific interaction in dot blots between SpA and IgG except at very low IgG concentrations. A protocol for the use of gold-protein conjugates to circumvent the hazards of unspecific gold binding is suggested.     

Statistic evaluation of the influence of species variation,culture conditions,surface wettability and fluid shear on attachment and biofilm development of marine bacteria

A budding coccoid bacterium, (CH1), a Vibrio sp. and a Pseudomonas sp. were investigated for factors governing their attachment to glass surfaces in static batch culture and laminar flow continuous culture systems. An analysis of variance showed that the three species exhibited very different responses. For CH1 attachment was dependent on cell density, incubation time and nutrient concentration. The Vibrio sp. was affected by nutrient concentration while the attachment of the Pseudomonas sp. was independent of cell density, incubation time and nutrient concentration. A comparison of attachment to hydrophilic and hydrophobic surfaces showed that attachment of the Vibrio sp. and CH1 to hydrophilic surfaces was 3 and 10 times greater respectively than to hydrophobic surfaces while Pseudomonas attached in equal numbers to both surfaces. The continuous culture system with defined flow hydrodynamics and growth conditions at steady state revealed a random sampling effect 3 times smaller than the batch culture system did. When the biofilm development of Pseudomonas sp. was followed during 46 h at different fluid shear under laminar and turbulent flow conditions, the former biofilm reached 3.3·10⁸ cells·cm^-2 and the latter 8.2·10⁷ cells·cm^-2.Non-common abbreviation NSS Nine salt solution     

Symbiotically defective histidine auxotrophs of Bradyrhizobium japonicum

Four histidine auxotrophs of Bradyrhizobium japonicum strain USDA 122 were isolated by random transposon Tn5 mutagenesis. These mutants arose from different, single transposition events as shown by the comparison of EcoRI and XhoI-generated Tn5 flanking sequences of genomic DNA. The mutants grew on minimal medium supplemented with l-histidine or l-histidinol but failed to grow with l-histidinol phosphate. While two of the muants were symbiotically defective and did not form nodules on Glycine max cvs. Lee and Peking and on Glycine soja, the other two mutants were symbiotically competent. Reversion to prototrophy occurred at a frequency of about 10^-7 on growth medium without added antibiotics, but prototrophs could not be isolated from growth medium containing 200 g/ml kanamycin and streptomycin. The prototrophic revertants formed nodules on all the soybean cultivars examined. When histidine was supplied to the plant growth medium, both nodulation deficient mutants formed effective symbioses. On histidine unamended plants, nodules were observed infrequently. Three classes of bacterial colonies were isolated from such infrequent nodules: class 1 were kanamycin resistant-auxotrophs; class 2 were kanamycin sensitive-prototrophs; and class 3 were kanamycin-sensitive auxotrophs. Our results suggest that two Tn5 insertion mutations in B. japonicum leading to histidine auxotrophy, affect nodulation in some way. These mutations are in regions that show no homology to the Rhizobium meliloti common nodulation genes.     

Immunochemical studies on shigella dysenteriae type 9 bacterial polysaccharide

On graded hydrolysis and Smith degradation, the O-somatic polysaccharide isolated from Shigella dysenteriae type 9 bacteria, strain NCTC 7919, yielded five oligosaccharides which were characterized. The positions of the O-acetyl and pyruvic acetal groups in the repeating unit were identified. Immunochemical studies indicated that d-galactose is the immunodominant sugar in the polysaccharide, and one of the oligomers, having the structure Gal-(1→3)-GlcNAc-(1→3)-Gal-(1→4)-Man, showed maximum inhibition of the homologous precipitation.     

Intergeneric transformation betweenRhizobium andAzotobacter

Attempts were made to transform two strains ofAzotobacter, viz.A. chroococcum andA. vinelandii with the DNA ofRhizobium leguminosarum, R. japonicum, R. meliloti andR. trifoli. Resistance to streptomycin or crystal violet was used as marker for screening purposes. The frequency of transformation varied between 0.05 to 0.95 percent. The transformants also acquired a few other characters of the donor strains.