Time-to-detection, percent-growth-positive and maximum growth rate models for Clostridium botulinum 56A at multiple...

We previously developed models for the influence of inoculum size on the growth kinetics (time-to-detection and maximum growth rate) and percent-growth-positive samples of Clostridium botulinum 56A with factors of inoculum size (1, 100, and 10,000 spores/sample). pH (5.5. 6.0 and 6.5) and sodium chloride concentration (0.5%, 2% and 4%) at 30 degrees C. In this present study, data were collected at two more temperatures (15 and 22 degrees C), making the final design a complete 3 X 3 X 3 X 3 factorial with a total of 81 conditions. Growth was followed hourly as change in A620. The Gompertz equation was fit to the growth data, and the parameters derived were used to calculate the maximum growth rate and time-to-detection. Linear regression with polynomial terms was used to analyze the effect of environmental factors on time-to-detection and maximum growth rate. Logistic regression with polynomial terms was used to analyze the data for percent-growth-positive. Despite the fact that the variance is larger in this extended data set (which includes two temperatures that are further away from the optimum), the inoculum size effect is clearly demonstrated. When inoculum size increased, the percent-growth-positive samples increased and the time-to-detection decreased. When the inoculum was 1000 spores/sample or higher, little additional effect on time-to-detection was observed. Inoculum size might influence results through simple probability or quorum sensing. Our results show that the observed effect of inoculum size from the previous report at a single temperature is not restricted to a specific growth condition, but rather a general phenomenon. The maximum growth rate was independent of inoculum levels, confirming our previous results.