Enumeration of Sanitizer-injured Coliform Bacteria in the Production and Harvest Environment of Vegetables

Chemical sanitizers to inactivate coliform bacteria may induce no injury (bacteria survive), sublethal injury (bacteria are injured), or lethal injury (bacteria are dead). The degree of coliforms injured sublethally by sanitizers used in the production and harvest environment of vegetables was evaluated using the thin agar layer (TAL) method. The TAL method involves laying a nonselective medium for recovering injured bacteria over a solidified selective medium containing selective agents to facilitate detection of target bacteria but inhibit the repair of injured bacteria that are sensitive to selective agents. Thus, the difference between the counts on TAL medium that includes both injured and noninjured cells and the counts on selective medium that includes noninjured cells represents the number of injured target bacteria from the background bacterial flora of samples. This number was comparable to an accurate enumeration of injured coliforms as determined by a flow cytometry that stains bacteria with permeant and impermeant DNA dyes depending on their viability. With pure cultures of Enterobacter cloacae, Escherichia coli, and E. coli O157:H7, the degree of sanitizer-injured cells was 68% to 80% for a dilute electrolyzed acidic water containing an available chlorine level of 2 ppm and 68% to 95% for a dilute alcohol agent containing an ethanol level of 1.6%. When agricultural water in a production environment was treated with electrolyzed acidic water, the percentage of chlorine-injured coliforms in agricultural water was 75%. The isolation and identification of bacteria on TAL and selective media suggested that the chlorine-stress caused injury to Ent. kobei. Sublethally injured coliform bacteria on the equipment in a harvest environment of cabbage following exposure to alcohol agent was detected with 60% of the total coliforms detected on the knife but none on the container and gloves. A comparison of isolated coliforms between TAL and selective media indicated the possibility of ethanol-injured Ent. amnigenus and Ent. asburiae. Injured bacteria were not detected on cabbage because no species of bacteria found in cabbage (14 species belonging to 11 genera) were transferred from the knife to the cabbage. These results suggest the necessity of adjusting the sanitizer concentration at level to kill coliforms in the production and harvest environment of vegetables so as not to cause sanitizer-induced cell injury.