Understanding the cause of false negative β- d-glucuronidase reactions in culture media containing fermentable carbohydrate

• Published in: Letters in applied microbiology Vol. 50; no. 6; pp. 547 - 551
• Main Authors: Fricker, C.R, Warden, P.S, Eldred, B.J
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01-06-2010; Blackwell Publishing Ltd; Blackwell
• Subjects: Biological and medical sciences; Culture Media - metabolism; E. coli; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - enzymology; Escherichia coli - isolation & purification; Escherichia coli - metabolism; Escherichia coli Proteins - analysis; Escherichia coli Proteins - metabolism; false negative reactions; Fermentation; Fresh Water - microbiology; Fundamental and applied biological sciences. Psychology; glucuronidase; Glucuronidase - analysis; Glucuronidase - metabolism; Lactose - metabolism; Microbiology; Culture medium; β-Glucuronidase; Enzyme; Applied microbiology; glucuronidase; false negative reactions; Glycosylases; False negative; Glycosidases; pH; Hydrolases; Carbohydrate; E. coli
• ISSN: 0266-8254; 1472-765X
• DOI: 10.1111/j.1472-765X.2010.02834.x

To explain the basis for false negative β-glucuronidase reactions seen with culture media containing lactose as a carbon and energy source. Escherichia coli strains were assessed for their reactions in culture media containing a β- d-glucuronidase substrate either with or without lactose. An assay was developed to test for the expression of β- d-glucuronidase at pH 5·0 and pH 7·2. Strains of E. coli that gave false negative glucuronidase reactions on media containing lactose generally expressed lower concentrations of the enzyme β- d-glucuronidase than strains that gave positive results, although the difference was by no means consistent. Most strains that were negative on lactose-containing media expressed virtually no β- d-glucuronidase activity at pH 5·0. Examination of colonies on Membrane lactose glucuronide agar (MLGA) from lightly polluted water showed that c. 10% of the E. coli present failed to yield green colonies on MLGA. E. coli that failed to produce green colonies on MLGA produced lower levels of β- d-glucuronidase than did strains that formed green colonies, the difference being greater at pH 5·0 than pH 7·2. The false negative rate for E. coli 10% which is similar to that experienced in the study that originally described MLGA. Strains of E. coli that fail to produce typical colonies on MLGA might produce lower concentrations of the enzyme β- d-glucuronidase. Whilst the enzyme activity is sufficient to be detected at pH 7·2, fermentation of lactose significantly lowers the pH of the medium and can result in reduced enzyme activity and therefore lack of detection. The false negative rate of c. 10% would be difficult to detect in routine laboratories as it would represent 1% or less of yellow colonies being identified as E. coli (assuming E. coli accounts for 10% of the total coliform population in drinking water).