Colilert 18

Detection and quantification of E. coli and Enterococcus were performed using the Colilert®-18 / Quanti-Tray® 2000 system and Enterolert® / Quanti-Tray® 2000 systems, respectively according to the manufacturer’s instructions (IDEXX Laboratories (Pty) Ltd., Johannesburg, South Africa). Positive results for both organisms were denoted by fluorescent blue wells under UV light at 300 nm. Positive wells were then used to determine the most probable number (MPN) of each organism in 100 mL of water sample (MPN/100 mL). Both Quanti-Tray methods had a limit of detection equivalent to 4.84E+ 06/100 ml.

Filtered samples were used for Colilert-18 analysis without pre-treatment (IDEXX Laboratories Italia S.r.l.). Serial dilutions were performed in filtered MilliQ water to find the optimal test concentration for quantification. Total coliforms and E. coli cells were quantified using Colilert-18 and Quanti-Tray/2000 (IDEXX), according to manufacturer’s instructions.
Briefly, 100 mL of sample was mixed with Colilert-18 reagent until complete dissolution of the powder and transferred to the Quanti-Tray. Bubbles were removed before sealing the tray. A negative control was performed using filtered MilliQ water. Samples were incubated up to 20 h at 35 °C and quantified using the statistical matrix to find the most probable number of cells per 100 mL of sample (MPN/100 mL), as supplied by the company. Colilert-18 can be used to target fecal contamination, at 44 °C, however, in order to compare it with the used antibodies and qPCR primers, we chose not to restring the analysis and therefore used 35 °C to screen for total coliforms.

Samples (2.5 g) were homogenized in 22.5 mL of buffered peptone water (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The homogenates were plated on CHROMagar™ ECC agar (CHROMagar, Paris, France) without and with 100 μg mL^-1 ampicillin (Sigma-Aldrich, St. Louis, MO, USA) to determine the abundance (CFU g^-1) of Escherichia coli and coliforms (excluding E. coli), as well as β-lactam-resistant E. coli and coliforms (excluding E. coli), respectively [17 (link)]. After incubation at 37 °C for 20 h, E. coli and coliform colonies were counted according to their colony colors (E. coli, blue colony; other coliforms, red colony) and morphologies. Up to three E. coli or coliform colonies were isolated from each agar plate. Means and standard errors were calculated for each type of sample. Additionally, to isolate other Escherichia species from samples wherein E. coli colonies did not grow, precultures with 2.5 and 1 g of samples were inoculated in 22.5 mL of Luria-Bertani broth (Invitrogen, Carlsbad, CA, USA) and Colilert-18 (IDEXX, Westbrook, ME, USA), respectively, and incubated at 37 °C for 20 h. Then, the preculture was streaked onto CHROMagar ECC agar without and with 100 μg mL^-1 ampicillin and incubated at 37 °C for 20 h. Up to three E. coli or coliform colonies were isolated from each agar plate.