A high-throughput-compatible method to determine the optical density of bacterial cell cultures in microplates without sample dilution

Meyers A, Furtmann C, Jose J

Abstract

A convenient and frequently applied method to determine the growth state of a bacterial cell culture is to determine the optical density (OD) spectrophotometrically. Incident light of similar wavelength to the size of a particle is scattered in suspensions like bacterial cell cultures [1], but if a beam of light is scattered through contact with more than one cell, the attenuation of light measured spectrophotometrically will not be proportional to the number of particles [2]. This results in an apparently lower OD [3]. Therefore, monitoring the OD of a bacterial culture directly in a microtiter plate without dilution, for example during cell growth or as a control in advance of enzymatic assays, is inaccurate and a dilution of the samples is necessary to measure within the linear range of a spectrophotometer. This process is time-consuming, prone to errors and not compatible with high-throughput applications. Here we present a direct method to estimate the OD at 578nm (OD578) of bacterial cultures in microplates without the need for removing aliquots and additional dilution of the samples. To establish this method, cell cultures of Pseudomonas putida KT2440 were used. A data set of 343 OD578 values which have been determined in parallel directly in the microplate without dilution and measured conventionally in a spectrophotometer after dilution, was generated. The software Origin9 was used to identify an exponential function, which allowed the reliable transformation of one dataset into the other. The formula derived thereof enabled the direct conversion of OD measurements of undiluted microplate reader-measured samples into OD values anticipated for diluted samples measured with a conventional spectrophotometer. Moreover, the applicability of an exponential fit for OD conversion was verified by a serial dilution of a formazine suspension of defined turbidity, which is usually used in water quality measurements [4]. Although our method was established with cultures of P. putida KT2440, it can be easily transferred to any other bacterial strain.