Rainbow agar o157

Approximately 10 μl of each enrichment culture was streaked directly onto CHROMagar™ ECC agar (CH-ECC, CHROMagar, Paris, France), MacConkey agar (MAC, Land Bridge, Beijing, China), and Rainbow® Agar O157 (RBA, Biolog Inc., Hayward, CA, USA), supplemented with 10 μg/ml novobiocin and 0.8 μg/ml potassium tellurite (modified, RBA-NT), and CHROMagar™ STEC agar (CH-STEC, CHROMagar, Paris, France), respectively. After overnight incubation at 37 °C, ten presumptive colonies on each plate were picked and subjected to colony PCR to detect the stx₁ and stx₂ genes according to the method revealed in a previous study [19 (link)], including green-blue or colorless colonies on CH-ECC agar; pink or colorless colonies on MAC agar; purple, grey, or mauve colonies on RBA-NT agar; and mauve colonies on CH-STEC agar. The stx-positive colonies were then plated onto LB agar and incubated at 37 °C overnight to obtain a single colony for further identification. Only one STEC isolate from each sample was chosen for further characterization if only identical stx types/subtypes were present on the four different agars. Isolates on inclusive agars (MAC and CH-ECC agars) were selected as a priority, while other isolates with same stx types/subtypes were eliminated.

Following the incubation of TSB bags as previously described, 1 mL of enrichment was added to a 1.5 mL tube containing 20 μL of DynabeadsMAX anti E. coli O157 (Invitrogen, Frederick, MD) and vortexed for 10 s. Tubes were placed in an MPC-S rack, inverted several times, and incubated at room temperature for 10 min with gentle agitation. Following incubation, a magnetic plate was inserted into the MPC-S rack, inverted several times, and incubated for 3 min. Sample supernatant was aspirated, and the magnetic plate was removed from the MPC-S rack. One mL of 1x wash buffer (Invitrogen, Frederick, MD) was added to each tube, inverted several times, and incubated for 3 min with the magnetic plate. The wash step was repeated twice, and after the third wash the beads were re-suspended in 100 μL of wash buffer. Re-suspended cells were streaked on BBLMacConkey II Agar with Sorbitol (Becton, Dickinson and Company, Sparks, MD) supplemented with cefixime (0.05 μg/mL) (US Pharmacopeia, Rockville, MD) and potassium tellurite (2.5 μg/mL) (Chem-Impex International, Inc., Wood Dale, IL) (CT-SMAC) and Rainbow Agar O157 (BIOLOG, Inc., Hayward, CA). Plates were incubated for 24 h at 37°C, and presumptive positive E. coli O157 colonies on CT-SMAC and Rainbow Agar O157 [27 ] were re-streaked on respective plates and incubated for 24 h at 37°C.

Four types of differential chromogenic media were used to grow bacterial cultures for light-scattering experiments. The media included: BHI agar, Sorbitol MacConkey (SMAC; Becton Dickinson) agar, Rainbow Agar O157 (Biolog, Hayward, CA), BBL CHROMagar O157 (Becton Dickinson), and R&F E. coli O157:H7 medium (R&F Products, Downers Grove, IL). Agar plates were prepared as instructed by the manufacturer, tempered to 45°C, dispensed 20 mL/plate, cooled at room temperature for 10–20 min, and stored in a sealed plastic bag until use (used within 7–30 days) [54] . After plating appropriate dilutions of bacterial cultures, the plates were incubated at 37°C for 10–12 h or until the colony size reached 1.1±0.1 mm in diameter. The colony size and morphology of bacterial cultures were examined under a 10× PH1 objective of a Leica DMLB microscope (Leica Microsystems USA, Bannockburn, IL) equipped with a SPOT RT color camera (Diagnostic Instruments, Inc). Images were captured using SPOT Advanced software 4.6.4.2 (Diagnostic Instruments, Inc).