The rapid potentiometric detection of catalase positive microorganisms.
The rate of fluoride ion release from the enzymatic cleavage of fluoride ion from 4-fluorophenol by horseradish peroxidase, in the presence of hydrogen peroxide, was measured using a fluoride ion selective electrode. Monitoring the utilisation of hydrogen peroxide by catalase (intracellularly present in almost all aerobic microorganisms) in the presence of 4-fluorophenol demonstrated the inhibition of the enzyme. Horseradish peroxidase appeared to impart a partial protective mechanism of this inhibition. The development of a sequential assay demonstrated the applicability of the proposed method in the assessment of aerobic microorganism numbers. The judicious variation of three parameters, the length of incubation, the concentration of the primary substrate (hydrogen peroxide) and the indicator enzyme activity (horseradish peroxidase), affected both the detection limit and the sensitivity of the assay. Typically with a 15 minute incubation, a detection limit for catalase activity of 1.5 x 10(-6) Uml-1 was obtained together with a sensitivity of 2.42 mumol l-1 s-1 per decade change in activity. Application of the developed catalase assay to the detection of Escherichia coli achieved a detection limit of 1 x 10(2) colony forming units (cfu) ml-1 with a sensitivity of 3.26 mumol l-1 s-1 per decade change in intact microorganisms. By lysis of the microorganisms the detection limit was further reduced to less than 10 cfu ml-1, indicating the future possibilities of the assay.
University of the West of England, Bristol, Faculty of Applied Sciences, UK.
D C
DC
Cowell
A A
AA
Dowman
R J
RJ
Lewis
R
R
Pirzad
S D
SD
Watkins
D001420:Bacteria, Aerobic / Q000302:isolation & purification*; D002374:Catalase / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology / Q000302:isolation & purification; D005459:Fluorides; D011199:Potentiometry
D002374:Catalase; D005459:Fluorides