Use of an absorbance spectrophotometer to monitor growth of bacterial cultures by measuring optical density at 600 nm (OD600) is a central technique in microbiology. A common problem, however, is that cultures must be diluted prior to measurement in order to remain within the linear dynamic range of the instrument. The increasing adoption of microvolume spectrophotometers such as a Thermo Scientific NanoDrop 2000c has raised the question of whether the shorter pathlength used in these instruments facilitates measurement of undiluted cultures.
When measuring OD600 of microbiological samples using a spectrophotometer, it is necessary to first determine the linear dynamic range for that culture. This is because in many cases the OD600 of the culture exceeds the linear range of the instrument prior to reaching the stationary phase, resulting in an inaccurate assessment of culture growth. This experiment evaluated microvolume OD600 performance, and also tested to see whether a conversion factor could be applied to data gathered using the NanoDrop 2000cTM pedestal in order to compare with a cuvette based measurement, in this case, the cuvette capability of the same instrument.
An overnight culture of E. coli JM109 was grown for 16 hr at 250 rpm, 37 °C in Luria Bertani Broth. A series of dilutions were prepared from this; each was measured using both cuvette (3 ml) and pedestal (2 μL) on a NanoDrop 2000c.
The upper limit of detection was determined by identifying the OD600 at which there ceased to be a linear correlation between the dilution factor and the measured OD600. This was found to be at an OD600 of approximately 1.4 when using a cuvette. In the case of the NanoDrop 2000c pedestal, the linear dynamic range exceeded the highest concentration of the culture; meaning that no dilution of the culture was necessary before measurement.
To compare OD600 readings between the pedestal and cuvette systems found on a NanoDrop 2000c, a conversion factor was calculated as follows:
This conversion factor was then be used to compare measurements using cuvette and pedestal (fig. 1).
|Figure 1: A dilution of a 16 hr overnight culture of E. coli measured using the NanoDrop 2000c pedestal option (red) and cuvette option (blue). Applying the conversion factor to the pedestal data (green) facilitates comparison between the two measurement methods.|
It is common for cultures to exceed the linear dynamic range of a spectrophotometer, therefore the use of a microvolume format such as the NanoDrop 2000c pedestal may present time savings and also reduce the risk of dilution error or inadvertently exceeding the instrument’s range.
The conversion factor between cuvette and microvolume formats was found to be constant throughout the linear dynamic range of the instrument, and facilitated the calculation of target OD600 values for the pedestal based on those previously established for the cuvette format.