In the period of implementation of the PFCT scheme, however, the growth in total peak demand remained exponential, while the growth in domestic peak demand became somewhat linear.
This may be because S. aureus remained in exponential growth throughout the experiment, while P. acnes, an anaerobic species, began to shift to a static phase.
Total amplification of each multiplex reaction was kept below saturation levels to allow the amplifier to remain within the exponential range of the amplification curve and to provide semi-quantitative data.
The optimal number of cycles to yield sufficient cDNA for normalisation, but remain within the exponential phase of amplification, was determined by analysing aliquots of the PCR reaction after every second cycle on agarose gels.
The observed variations in BSH levels in B. subtilis do not necessarily represent what will be observed in all other BSH-producing bacteria; recent studies in S. aureus, for example, have shown that BSH levels appear to remain constant during exponential growth.
Simulated cell cultures with initial cell densities of up to the maximum simulated value of 700 cells/mm2 are sub-confluent and remain within the exponential phase of population growth (a 1 mm × 1 mm model reaches visual confluence at approximately 1000 cells).
The PCR conditions consisted of a 95°C for 1 min, followed by appropriate number of cycles of amplification at 95°C for 15 s, 65°C for 30 s, and 68°C for 6 min. The appropriate number of cycling on which the PCR product will remain in the exponential phase of amplification was determined by the cDNA amplification results of the range of PCR cycles obtained in pre-experiments.
Of the remaining two models (exponential and Michaelis-Menton), the exponential model was selected mainly because it yielded the best fit more often than did the Michaelis-Menton model and because the exponential model had been used in previous analyses of the data.
Furthermore, the time the wild-type strain remained in the exponential growth phase was longer, and the plateau of the stationary phase was significantly higher (p<0.001) for isolate E4 than the irp2-gene knockout in NB treated with 1% Chelex containing 0.54 mg/mL 80% iron-saturated lactoferrin as the main iron source (Figure 2B).
The optimal LD-PCR cycle number was determined empirically to ensure the cDNA remained in the exponential phase of amplification.
