Colorimetric Method for the Estimation of Growth in Cup Assays

Abstract

While investigating the enzymatic formation of acetohydroxybutyrate by extracts of Escherichia coli, it became necessary to devise an assay for the quantitation of this early intermediate in isoleucine synthesis. A microbiological assay, based on the reversal by acetohydroxybutyrate (Umbarger, 1958; Umbarger and Brown, 1958) of the inhibitory effect of L-valine on E. coli strain K-12, provided the necessary tool to accomplish the desired quantitations. Although such an assay would ordinarily require that the reaction mixtures be sterilized by filtration because of the lability of acetohydroxybutyrate to heat, it was possible to eliminate sterilization by employing an Oxford cup assay. However, in the initial attempts to apply the cup assay to the determination of acetohydroxybutyrate, it was clear that the measurement of the zone diameters was not sufficiently precise for quantitating growth. For example, the zones resulting from higher concentrations of acetohydroxybutyrate were not only wider but also denser than those from lower concentrations. Often, two zones would differ only slightly in their diameters although simple visual comparison showed clearly that the two zones differed considerably in the amount of growth. In addition, their margins were often not clearly delineated. These difficulties were overcome by measuring the protein of the bacterial cells in the growth zone. Because the method may be useful in other systems it is described in this paper. MATERIALS AND METHODS The assay organism was strain K-12 of E. coli. The mineral medium employed was that of Davis and Mingioli (1950) modified by the omission of citrate. The solid medium (valine agar) contained 1.5 per cent agar and was supple-' This work was supported in part by U. S.

Preliminary Enzymatic Events in Asparagine-Dependent Denitrification by Pseudomonas perfectomarinus

“…Acetolactate was determined as acetoin by the method of Westerfeld (1945) after decarboxylation by heating in the presence of 1.8 N H2SO4. Acetohydroxy-butyrate was determined by the method described previously (Leavitt and Umbarger, 1960).…”

ISOLEUCINE AND VALINE METABOLISM IN ESCHERICHIA COLI XI. K-12

“…Acetolactate was determined as acetoin by the method of Westerfeld (1945) after decarboxylation by heating in the presence of 1.8 N H2SO4. Acetohydroxybutyrate was determined by the method previously described (Leavitt and Umbarger, 1960).…”

“…a-Aminobutyrate was identified bv comparison with a known standard. The amino acid was determined microbiologically (Leavitt and Umbarger, 1960). The assay organism was strain 1\97-21, an isoleucine auxotroph of E. coli W lacking L-threonine deaminase.…”

“…Unfortunately, this examination could not be undertaken until a specific method for the determination of acetohydroxybutyrate was developed. The recent development of such an assay (Leavitt and Umbarger, 1960) has made it possible to obtain evidence that acetohydroxybutyrate and acetolactate are formed by the same enzyme system (Leavitt and Umbarger, 1961).…”

ISOLEUCINE AND VALINE METABOLISM IN ESCHERICHIA COLI XII

“…Acetolactate was determined as acetoin by the method of Westerfeld (1945) after decarboxylation by heating in the presence of 1.8 N H2SO4. Acetohydroxy-butyrate was determined by the method described previously (Leavitt and Umbarger, 1960).…”