Many individual samples are needed to measure cell survival following heating at multiple temperatures and multiple heating durations. For example, if eight time points are considered for each of seven treatment temperatures with three replicates at each condition, then 168 separate samples are needed. In addition, physical considerations may limit the number of points that can be measured, especially as treatment temperature increases and the heating duration decreases. For a reasonable sample size, there may be a limit to the treatment temperature as the time required to heat the culture to the target temperature becomes comparable to the treatment time. Then, using an isothermal analysis of the data introduces error and the temperature must be considered time varying, requiring estimates of the very parameters being sought. Conversely, for long treatment times, it may be difficult to insure that the temperature remains constant and that the temperature is the only modified experimental condition in the culture medium. These challenges typically lead to relatively small data sets. Furthermore, treating each temperature as a separate experiment leads to challenging statistical analysis of the data, as the few data points lead to difficulty in finding the confidence intervals of the parameters in a given model.

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