Purpose: The purpose of this study was the creation of a protocol for the generation of sublethal injured microorganisms, which reproduces the conditions found in the food itself. These injured organisms should be stable during storage.
Methods: A 2 ml aliquot of 24h or 4h culture of Salmonella spp., Staphylococcus spp., Escherichia coli, Listeria monocytogenes or Cronobacter spp. were mixed with 40 grams of lactose or milk powder and submitted to drying in an incubator at 41°C for 24 h. The dried bacteria were stored in a desiccator at room temperature on silica gel or at 4°C and at -20°C. For heat stress, 10 ml of a bacterial culture was stressed in a water bath at 55-60°C. The cells were mixed with nutrient medium and glycerol and frozen at -20°C for long-term storage.
Results: Bacteria dried in lactose could be stored at room temperature for over a year with a slow decrease in bacterial count over time. Bacteria dried in milk powder showed a higher stability but cell count slowly decreased. Frozen heat-shock cultures could be stored for over three months without a decrease in cell count. Injury rate and stability differed depending on the bacterium.
Significance: A protocol for the in-house preparation of injured organisms and the possibility to store them afterwards for a longer time period was successfully developed. These organism can then be used for performance testing of nutrient media or test devices for microorganisms in food.