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 gradi ent plates, in combination with image analysis, to study the synergistic ef fect of NaCl concentration (% w/v) and pH on the recovery of Brochothrix th ermosphacta 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 me asure 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 an d 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. Thes e 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-tre ated or control), optimal growth was found at pH 6.8 and 1.5% NaCl (w/v) co ncentration. The range of salt concentrations and pH values over which grow th could be observed was shown to be reduced as a consequence of heat treat ment. Overall. it is suggested that the gradient plate technique, in combin ation with image analysis, could be: useful in determining combinations of different environmental factors which are effective in preventing the recov ery of heat injured bacterial cells.