Assessment of cell proliferation on porous microcarriers by means of image analysis

Spherical porous microcarriers (PMCs) made from collagen-glycosaminoglycan crosslinked copolymers have exhibited considerable promise as growth surfaces for the proliferation of anchorage-dependent mammalian cell lines and have demonstrated the ability to entrap anchorage-independent cells. However, quantification of cell growth on PMCs has proved difficult. A method of measuring the proliferation of PMCs, based on image analysis, is presented. Using CV1 and CHO cell lines, samples of PMCs were removed from culture at various times, fixed, embedded and sectioned. The 2 microns sections were stained, photographed and digitized in three colors. A computer program was developed to evaluate digitized PMC cross-sections and to classify pixels as conforming to either background, cytoplasmic, matrix or nuclear parameters, based on a set of classification rules determined by statistical analysis. Growth curves were generated by relating the number of pixels occupied by cellular material to the total number of pixels in the PMC cross-section. The PMCs were found to foster cell proliferation, with cell densities approaching 100% occupancy.     

A multiple chemostat system for consortia studies on microbially influenced corrosion

A multiple chemostat system has been developed in which metal specimens can be exposed to a consortium of bacteria. The system comprises a single test chemostat containing the test specimen operated at a high dilution rate to facilitate the wash out of planktonic bacteria, selecting for attached or biofilm growth. This chemostat is fed at a steady low rate by a number of separate chemostats each of which contains a pure axenic culture of one member of the consortium being tested. This system has the advantage of providing a continual inoculum of the test species to the test specimen allowing both aerobic and anaerobic bacteria to be grown in the same system. Constant levels of three bacterial types were maintained in the system: Pseudomonas aeruginosa, Thiobacillus ferrooxidans and Desulfovibrio vulgaris. Exposure of 316L stainless steel electrodes to this system resulted in increased corrosion of coupons exposed biotically, as compared to those exposed abiotically. A current monitoring technique and electrochemical impedance spectroscopy were used to evaluate effects of bacteria on metallic corrosion.     

Selective staining of nucleic acids by osmium-ammine complex in thin sections from lowicryl-embedded samples

Osmium-ammine (OA)/SO2 selectively contrasted RNA- and DNA-containing structures in thin sections from Lowicryl-embedded samples. No cell structures were stained after Epon embedding. RNAse and DNAse digestion experiments demonstrated that only RNA and DNA were stained in Lowicryl thin sections. Protease digestion did not modify the staining reaction. The very fine end-reaction produced a very high resolution of the stained structures. The staining reaction was not due to the presence of SO2 but to the low pH of the solution (ranging from 1.5-2.2). OA in glycine buffer, pH 1.5, selectively contrasted nucleic acids. Electrostatic bonds between nucleic acids and OA complex were probably involved in the staining reaction. Increasing the pH value of the staining medium resulted in loss of OA specificity for nucleic acids. The high electrolyte concentration of the staining medium hindered the staining reaction.     

Towards interbreed IBD fine mapping of the mh locus: Double-muscling in the Asturiana de los Valles breed involves the same locus as in the Belgian Blue cattle breed

The Spanish ``Asturiana' cattle breed is characterized by the segregation of a genetically determined muscular hypertrophy referred to as double-muscling or ``culones'. We demonstrate by linkage analysis that this muscular hypertrophy involves the mh locus previously shown to cause double-muscling in the Belgian Blue cattle breed, pointing towards locus homogeneity of this trait across both breeds. Moreover, using a twopoint and multipoint maximum likelihood approach, we show that flanking microsatellite markers are in linkage disequilibrium with the mh locus in both breeds albeit with different alleles. Finally, we discuss how allelic homogeneity across breeds might be exploited to achieve efficient genetic fine-mapping of the mh locus. Received: 13 September 1996 / Accepted: 20 January 1997