To solve the problems of measuring the growth rates of microorganisms from optical density (OD)-growth time plots, we used relative-density (RD) plots. The relationship of OD and RD was built from the diluted grown cultures. This method was satisfactorily applied to study the growth of Escherichia coli and the cyanobacterium Anabaena spiroides.In spite of the popularity of the optical density (OD) method, the direct use of OD records of liquid cultures of microorganisms to study their growth kinetics may yield problematic results. For instance, for an Escherichia coli culture, the cell doubling time as derived from incremental OD rates varies from 46 to 38 min, depending on the wavelength of light used for measurement (Fig. 1A). Here, we report an approach to obtain more reliable results (Fig. 1B). Briefly, the OD of the liquid cell culture is recorded frequently throughout the growth period. At or near the end of cultivation, the cell density of the culture is arbitrarily defined as a relative density (RD) of 1.0, and aliquots of the culture are diluted to prepare reference samples of various RD values. For example, a reference sample of 0.3 RD is prepared by diluting 0.3 ml of grown cell culture with 0.7 ml of fresh growth medium. The ODs of the reference samples are also determined and plotted against the RD values to construct an OD-RD calibration curve using the equation OD ϭ m ϫ RD/(n ϩ RD), where m and n are empirical constants (Fig. 1B). The recorded ODs of the cell culture are converted into RD values, and the cell doubling time is determined from the RD-growth time plot (Fig. 1C). We determined the growth rates of E. coli bacteria and Anabaena spiroides cyanobacteria (Fig. 2) using this method. We found that a common laboratory E. coli strain, BL21(DE3), doubled every 31 Ϯ 3 min (mean Ϯ 1 standard deviation [SD]) (Fig. 1C), irrespective of the light wavelength used for measurement, consistent with the incremental rate of the absolute density (AD; CFU/ml) of the E. coli cells (32 Ϯ 4 min) (Fig. 1C). Furthermore, the growth rates obtained from RD-time plots and OD-time plots were statistically different (t test P values were Ͻ Ͻ0.05 for RDs versus ODs with wavelengths used). We also found that E. coli DH5␣, another common E.
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