The Estimation of Glucose-Containing Substances in Micro-Organisms from the Rumen of the Sheep

Before studying glucose-containing substances in the micro-organisms from the rumen of sheep fed on hay, it was necessary to develop a method of estimation specific for glucose.Methods for the estimation of reducing sugars depend largely either on direct determinations of the reducing values of solutions or on such determinations before and after treatment of the solutions with bacteria or yeasts specifically fermenting single sugars or a group of sugars.Since such values are usually expressed in terms of an arbitrarily chosen sugar, the errors resulting when mixtures of reducing sugars are present, or when a large amount of non-sugar reducing material is present, can be considerable.In addition, the interpretation of the value of the non-sugar reducing materials is rendered difficult because different values are obtained depending on the method of estimation employed.The procedure which has been devised involves quantitative paper-partition chromatography (Flood, Hint & Jones, 1947) of hydrolysates of the rumen microorganisms.The results of this method have been compared with the loss in reducing substances in the hydrolysate after fermentation with baker's yeast (The United Yeast Co. Ltd. (D.C.L.), King Street, Aberdeen). EXPERIMENTAL AND RESULTS Paper chromatographic methodApparatus.All-glass tanks and troughs were used, since quantitative recovery of glucose was not obtained with a stainless steel trough.The paper used was Whatman no.54, since on this paper the sugars are maintained as compact 'spots' and a more regular solvent front is obtained than with the coarser-fibred Whatman no.I paper.Since complete extraction was not always obtained by using the extractor originally described by Flood et al. (1947) the all-glass extractor shown in Fig.I was devised.T h i s type of extractor was found highly satisfactory.It permits total extraction in 2-30 min.Chromatographic technique.The method used was essentially that described by Flood st al. (1947) as modified by Jermyn & Isherwood (1949).A horizontal micro-capillary burette was used to apply the solution to the paper.The sheets were run in a mixture containing n-butanol(40 parts), acetic acid (10 parts) and water (50 parts) for 24-48 hr.(Partridge, 1948), after which they were dried at 105' and the 'side-strips' removed and sprayed as described by Flood et d.(1947).In all these operations forceps were used to handle the cut paper strips.