Purpose: For rapid detection there is a need to have access to internal preset concentrations of the target pathogens that can be used for assessing recovery of cells quantified by the detection method being evaluated. The aims were to compare two bacterial cell fixation methods and to assess them as internal standard cell preparations for quantifying fixed STEC cells by FCB and qPCR. The fixation methods were A) a commercial fixative (Sigma-Aldrich 4% formaldehyde/10% neutral buffer), and B) a fixative used previously (8% formaldehyde, 0.8% NaCl).
Methods: STEC E. coli strain O103:H11 was grown for 16 hours in Luria-Bertani medium then fixed for 30 minutes with periodic vortexing. Cells were centrifuged then washed in phosphate-buffered saline twice. Cell concentration was estimated using a Petroff-Hauser counter, and replicate samples were prepared by serial dilution of cells for FCB (Vivione Biosciences) or DNA extraction for qPCR (Eppendorf).
Results: The two methods did not differ significantly in cells quantified by FCB (P > 0.4). However, the qPCR method was able to detect as few as 152 cells in 5 microliters of an 8 log dilution using fixative A, but had a detection limit 10 times less sensitive in fixative B.
Significance: The results so far indicate that either fixative method will work for FCB but the fixation method appears to impact qPCR sensitivity. A whole cell-based internal standard solution will allow for comparison of very different rapid methods such as qPCR and FCB for the detection and quantification of E. coli STEC cells.