Purpose: To identify whether spot inoculation of individual analytical units or spray bulk inoculation of a large quantity of leafy greens and fresh herbs provides the most homogenous inoculation.
Methods: Ten (10) varieties of produce were used. For spot inoculation, 20 test portions, each containing 25 g of produce, were weighed out into sterile Whirl-Pak filter bags and inoculated with a single Salmonella serovar. For spray bulk inoculation, an amount of produce equivalent to at least twenty 25-g test portions was inoculated and mixed in a sterilized tote with a single Salmonella serovar. Both methods used an inoculation level of ~0.7 CFU/25 g. All inoculated samples were stored at 4°C for 2 to 3 days prior to analysis. On the day of analysis, we prepared 20 test portions (25 g) from the bulk inoculated produce. Then all 40 test portions were preenriched separately in 225 ml modified buffer peptone water for 24 h at 35°C. The BAM culture method was followed thereafter, and qPCR was performed from 24 h and 48 h enriched cultures.
Results: Both inoculation methods generated fractional results; however, spot inoculation for individual units provided better fractional results: 44% positive portions were close to the ideal fraction (50% positive and 50% negative); spray bulk inoculation provided 33% positive portions. Spot inoculation was also much less laborious to perform than spray bulk inoculation.
Significance: Using the most effective inoculation method will significantly enhance the rate of successful experiment runs to support microbial method development and validation.