UBC Theses and Dissertations
A strategy for the quantitative analysis of fungal cell walls Cameron, Donald Stewart
Cultures of Tremella mesenterica grown as haploid yeast-like unicells and Saprolegnia diclina were grown on incompletely defined media and the procedures for isolating cell wall preparations from each, free of cytoplasmic and capsular material, are described. A strategy for quantitative analysis of these cell wall preparations was devised and tested, selecting from the numerous procedures available: extraction and gravimetry for lipids; hydrolysis followed by gas-liquid chromatography for neutral sugars (as trimethyIsiIyI derivatives) and automatic amino acid analysis for amino acids and amino sugars. Problems of degradation as a result of hydrolysis have been considered. The extent to which degradation occurs is difficult to estimate because each constituent polymer is 'contaminated' with the others. This may accelerate degradation, particularly of amino acids. Analytical procedures were reproducible but only 90% of the weight of the cell wall was recovered. The remaining 10% is probably the result of degradation losses that have not been accounted for, and further studies are required to improve the estimation of this error. Even under carefully standardized conditions, cell wall preparations show variable composition. A complete analysis was therefore performed on a single cell wall preparation of each of the two species. Analyses were performed on other cell wall preparations of the' two species and they showed general similarities; the ratios of components were similar, although, for example one preparation, of S. diclina contained more than twice as much total protein as. another. Similar recoveries of constituents suggest that the strategy is appropriate for quantitative analysis. However, alternative methods for lipid analysis that provide more specific information are available and should be adapted for cell wall analysis. Quantitative recovery of uronic acids provides substantial difficulties, and improved methods are required.
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