Diauxie and determination of sugars: a laboratory classroom experiment

Abstract

We have found the following experiment to be an interesting way of giving students practise with quantitative colorimetric assays. They repeat an observation that was historically important in the discovery of catabolite repression. The experiment can be performed in two 3.5 hour sessions. During the first session, Escherichia coli is cultured in liquid medium containing both glucose and lactose. The students measure culture turbidity as a function of time and observe the diauxic growth pattern (Fig 1A) that was first reported by Monod and Audureau. 1' 2 The sequential utilization of the two sugars I led to the discovery that during growth on preferred carbon sources such as glucose, many enzymes that function in the catabolism of other carbon sources are repressed. This control mechanism was named 'catabolite repression' and has been found to be mediated by 3', 5' cyclic AMP. 3, 4, s Our students confirm this sequential utilization of the two sugars. They periodically withdraw samples of the culture, remove bacteria by membrane filtration, and freeze the filtrates. During the second laboratory session they determine the concentrations of glucose and lactose in the filtrates by means of a coupled glucose oxidase-peroxidase assay for glucose and the anthrone assay for hexose. The glucose assay iUustrates the high specificity characteristic of enzyme assays. Since the anthrone reaction is positive for both glucose and lactose, the contribution by glucose to the anthroue result must be calculated and subtracted to obtain the concentration of lactose.