Rapid detection of fluorescent and chemiluminescent total coliforms and Escherichia coli on membrane filters.
The detection of fluorescent colonies of Escherichia coli/total coliforms (TC) on a membrane filter is currently carried out using 4-methylumbelliferyl-beta-D-glycosides as enzyme substrates and a UV-lamp for visualization. The most rapid procedures based on this approach for the demonstration of these indicator bacteria in water take 6-7.5 h to complete. As part of efforts to further reduce the detection time, an improved two-step procedure for the fluorescence or chemiluminescence labelling of microcolonies of E. coli/TC on a membrane filter has been developed. Essential features of this approach include a separation of the bacterial propagation and target enzyme induction from the actual enzymatic labelling, the use of improved fluorogenic, i.e., 4-trifluoromethylumbelliferyl-beta-D-glycosides and fluorescein-di-beta-D-glycosides, or chemiluminogenic (i.e., phenylglucuronic- or galactose-substituted adamantyl 1,2-dioxetanes) substrates for beta-glucuronidase/beta-galactosidase, of enzyme inducers, of special membrane filters and of polymyxin B to promote the cellular uptake of the substrate. This labelling procedure has been applied in conjunction with different detection devices including a UV-lamp, CCD-cameras, X-ray film and the ChemScan((R)) RDI. Using the former three, microcolonies of pure cultures could be detected within 5.5-6.5 h, but waterborne E. coli/TC may fail to form microcolonies in this short time period, thus yielding poor sensitivity and a high false-negative rate. In contrast, a quantitative enumeration was feasible in less than 4 h with the ChemScan((R)) RDI, owing to its ability to detect both microcolonies and non-dividing single cells.
Van Poucke
S O
SO
Laboratory for Pharmaceutical Microbiology, University of Ghent, Harelbekestraat 72, B-9000, Ghent, Belgium.
Nelis
H J
HJ
D015169:Colony Count, Microbial / Q000379:methods*; D003470:Culture Media; D004755:Enterobacteriaceae / Q000254:growth & development* / Q000302:isolation & purification; D004926:Escherichia coli / Q000254:growth & development* / Q000302:isolation & purification; D005188:False Negative Reactions; D005374:Filtration / Q000295:instrumentation; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D005697:Galactosides / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism; D020719:Glucuronides / Q000378:metabolism; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D008163:Luminescent Measurements; D012680:Sensitivity and Specificity; D013997:Time Factors; D014871:Water Microbiology; D001616:beta-Galactosidase / Q000378:metabolism
C419874:4-trifluoromethylumbelliferylgalactopyranoside; C419873:4-trifluoromethylumbelliferylglucuronide; D003470:Culture Media; D005456:Fluorescent Dyes; D005697:Galactosides; D020719:Glucuronides; D006923:Hymecromone; C026845:4-methylumbelliferyl-galactopyranoside; C034888:4-methylumbelliferyl glucuronide; D001616:beta-Galactosidase; D005966:Glucuronidase