Fluorogenic Membrane Overlays to Enumerate Total and Fecal Escherichia coli and Total Vibrionaceae in Shellfish and Seawater
Figure 1
Examples of representative fluorescent foci from membrane overlays of overnight colonies on plates of tryptic soy agar containing 1% NaCl. (a–d) represent plates spotted with E. coli and vibrios, incubated overnight, and then overlaid with membranes saturated with fluorescent substrate(s). Specific bacterial strains in panels (a–d) are as follows: (left to right, top row), E. coli serotypes O5:NM, O9:H21, and O22:H8; (left to right, bottom row), V. cholerae O1, V parahaemolyticus O3:K6, and V. vulnificus VV1003. Individual panels are as follows: (a) MUG assay for total E. coli (blue fluorescent foci) with incubation at overnight, (b) MUG overlay for fecal E. coli after incubation at for 2 hours followed by overnight, and (c) COPP overlay for total Vibrionaceae (green fluorescent foci) after overnight incubation at . The arrow signifies weak fluorescence that is occasionally observed in E. coli and other bacteria due to the presence of small quantities of lysyl aminopeptidases and is especially noticeable if overlays exceed 10 minutes. (d) Multiplex assay for total E. coli (blue) and total Vibrionaceae (green) after overnight incubation at . Arrow depicts a V. parahaemolyticus colony which displays weak fluorescence due to the unintentional sticking of most of the colony to the membrane and subsequent blockage of UV light penetration. (e and f) Multiplex assay for total E. coli and total Vibrionaceae performed on cultures of oyster homogenate naturally contaminated with a low level (e) and a high level (f) of E. coli after overnight incubation at . Arrows in (e) indicate three of the four blue fluorescent foci representing E. coli. All overlays were performed with cellulose membranes.
