The Gradient plate technique as a means of studying the recovery of heat injured Brochothrix thermosphacta

Bacterial recovery from heat injury is influenced strongly by the nature of the recover); medium used to resuscitate cells. This study used agar gradient plates, in combination with image analysis, to study the synergistic effect of NaCl concentration (% w/v) and pH on the recovery of Brochothrix thermosphacta after heat treatment. Initially, exponential, early stationary and late stationary phase cultures of B. thermosphacta grown in all-purpose tryptone (APT) broth at 25 degreesC were heat-treated at 50 degreesC to measure thermal resistance. Late stationary phase cultures were found to be 2-3 times more heat-resistant than the other two, with a D-value of 14.8 min. Exponential and early stationary phase cultures were thermally treated and inoculated onto agar gradient plates (modified APT medium) and incubated at 25 degreesC. In the instance of the late stationary phase culture, there proved to be too low a cell concentration to obtain confluent growth. These plates had gradients of pH (4.0-7.4) and NaCl (1.5-8.1% (w/v)) running at right angles across them. After 48 h incubation, bacterial growth on these plates was measured by image analysis. In all bacterial cultures (heat-treated or control), optimal growth was found at pH 6.8 and 1.5% NaCl (w/v) concentration. The range of salt concentrations and pH values over which growth could be observed was shown to be reduced as a consequence of heat treatment. Overall. it is suggested that the gradient plate technique, in combination with image analysis, could be: useful in determining combinations of different environmental factors which are effective in preventing the recovery of heat injured bacterial cells.