Developing an effective thin agar layer method for the detection of injured cells of escherichia coli in milk

Injured cells of foodborne bacteria are generated by exposure to sub-lethal doses of food preservation factors. These cells maintain viability but loss resistance to selective agents in differential media used for their detection in foodstuffs, and could thus lead to false negative results. Injured cells can be reliably recovered using a 3-stage method that requires at least 4 days of lengthy culture. This study was therefore designed to develop a time and effort-effective protocol for detecting injured cells of Escherichia coli in milk. Heat-injured cells of E. coli could be generated in reconstituted skim milk (RSM) by exposure to 55 ºC for 50 min. Heat-injured cells of E. coli could be recovered using direct plating on the nonselective media of tryptone soy agar (TSA), tryptone glucose extract (TGE), and plate count agar (PCA) with TSA supplemented with 1% sodium pyrurvate (NaPyr) showing the highest recovery efficiency. None of 3 selective media of MacConkey agar (Mac), eosin methylene blue agar (EMB) and violet red bile agar (VRB) was able to recover heat-injured cells of E. coli from RSM. However, supplementing these selective media with 1% NaPyr allowed the detection of heat-injured cells of E. coli with limited recovery rates. Overlaying a thin layer of a nonselective agar over another layer of a selective medium (the thin agar layer method) improved the recovery of heat-injured cells from RSM, compared to recovery by selective media only. Supplemented TSA (TSA+) combined with EMB showed the highest recovery of injured cells from RSM, compared to other combinations of nonselective and selective media. The TSA+/EMB combination could also successfully recover heat-injured E. coli from pasteurized buffalo s milk with different fat contents. These results presented the thin agar layer method involving a combination of TSA+/EMB, as a time and effort-effective protocol for the detection of injured E. coli in milk used for preparing different dairy products.