An improved direct plate method for the enumeration of stressed Escherichia coli O157:H7 from food.

The use of sorbitol MacConkey agar (SMAC) performed poorly in supporting growth of stressed Escherichia coli O157:H7 cells. Up to a 3-log difference was observed between counts on SMAC and tryptone soy agar (TSA). It is critical in the risk assessment of certain foods to be able to enumerate stressed and healthy E. coli O157:H7 in a background of potentially healthy competing bacteria. Investigations carried out to overcome the inhibitory effect of SMAC included the reduction of the selective agent concentration, inclusion of a recovery stage in broth prior to plating out, addition of recovery agents, and delayed exposure to the selective agent. The only successful approach was delayed exposure to the selective agent. This was achieved by resuscitating the stressed cells on a membrane placed on the surface of a TSA plate and, after a defined time period sufficient for full resuscitation, transferring the membrane to the surface of a SMAC plate. The choice of membrane material was critical for maintaining the positive sorbitol color change used to identify wild-type E. coli. Track-etched polycarbonate membranes allowed the typical color reactions to be visualized, whereas cellulose acetate did not. The method was validated with E. coli O157:H7 cells stressed by low pH and high salt conditions, whereby all cells that would previously be undetectable on direct inoculation of SMAC were countable.