Determination of viable Escherichia coli using antibody-coated paramagnetic beads with fluorescence detection.
A rapid and convenient assay system was developed to detect viable Escherichia coli in water. The target bacteria were recovered from solution by immunomagnetic separation and incubated in tryptic soy broth with isopropyl-beta-D-thiogalactopyranoside, which induces formation of beta-galactosidase in viable bacteria. Lysozyme was used to lyse E. coli cells and release the beta-galactosidase. Beta-galactosidase converted 4-methylumbelliferyl-beta-D-galactoside to 4-methylumbelliferone (4-MU), which was measured by fluorescence spectrophotometry using excitation and emission wavelengths of 355 and 460 nm, respectively. Calibration graphs of 4-MU fluorescence intensity versus E. coli concentration showed a detection range between 8 x 10(4) and 1.6 x 10(7) cfu mL(-1), with a total analysis time of less than 3 h. The advantage of this method is that it detects viable cells because it is based on the activity of the enzyme intrinsic to live E. coli.
Dudak
F Ceyda
FC
Department of Food Engineering, Hacettepe University, Ankara, 06532, Turkey.
Boyaci
Ismail H
IH
Jurkevica
Agnese
A
Hossain
Mahmud
M
Aquilar
Zoraida
Z
Halsall
H Brian
HB
Seliskar
Carl J
CJ
Heineman
William R
WR
D000907:Antibodies, Bacterial / Q000276:immunology*; D002138:Calibration; D004926:Escherichia coli / Q000201:enzymology / Q000276:immunology* / Q000302:isolation & purification*; D005453:Fluorescence; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry; D018189:Immunomagnetic Separation / Q000379:methods*; D050296:Microbial Viability; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D000907:Antibodies, Bacterial; D006923:Hymecromone; D001616:beta-Galactosidase