KETOGLUCONATE BROTH

Description

Gluconate oxidation was originally used by Haynes to differentiate the Pseudomonads, but other organisms, mainly those among the Enterobacteriaceae, are now known to possess this ability. Gluconate is one of the oxidation products formed from glucose by aerobic microorganisms that metabolize carbohydrates by the Entner-Doudoroff pathway. Bacteria metabolize carbohydrates by either fermentation or oxidation. In fermentation, glucose catabolism involves initial phosphorylation, then a splitting into two triose molecules. However, when glucose is metabolized oxidatively to gluconic acid, no initial phosphorylation occurs and only organisms capable of oxidative metabolism can use potassium gluconate as their sole carbon sources. These oxidative organisms are obligate aerobes. Ketogluconate Broth is used for testing the ability of an organism to oxidize gluconate to 2-ketogluconate, which subsequently accumulates in the medium. The basis of the test is the change from gluconate (a nonreducing compound) to 2-ketogluconate (a reducing compound) when tested with a suitable reagent. Inoculate heavy inoculum into 1ml of the sterile, dispensed medium. Incubate at 37?C for 48 hrs. Then add 1ml of Benedicts reagent for reducing sugars, place the tube in a boiling water bath for 10 minutes. Observe for the production of a coloured precipitate of cuprous oxide. Organisms capable of oxidative metabolism use potassium gluconate as their sole carbon source, leading to the accumulation of 2-ketogluconate in the medium. 2-ketogluconate reduces copper sulphate, when heated, to an insoluble cuprous oxide, which is precipitated out as yellow to orange-to-orange red precipitate. The colour produced depends on the amount of 2- ketogluconate accumulated, the greater the amount, the more orange-to-orange red the colour becomes. However, any reducing activity with colours ranging from slight green to deep orange indicates oxidation.