Simple and Versatile Turbidimetric Monitoring of Bacterial Growth in Liquid Cultures Using a Customized 3D Printed Culture Tube Holder and a Miniaturized Spectrophotometer: Application to Facultative and Strictly Anaerobic Bacteria

Abstract

Here we introduce a novel strategy for turbidimetric monitoring of bacterial growth in liquid culture. The instrumentation comprises a light source, a customized 3D printed culture tube holder and a miniaturized spectrophotometer, connected through optical cables. Due to its small footprint and the possibility to operate with external light, bacterial growth was directly monitored from culture tubes in a simple and versatile fashion. This new portable measurement technique was used to monitor the growth of facultative (Escherichia coli ATCC/25922, and Staphylococcus aureus ATCC/29213) and strictly (Butyrivibrio fibrisolvens JW11, Butyrivibrio proteoclasticus P18, and Propionibacterium acnes DSMZ 1897) anaerobic bacteria. For E. coli and S. aureus, the growth rates calculated from normalized optical density values were compared with those ones obtained using a benchtop spectrophotometer without significant differences (P = 0.256). For the strictly anaerobic species, a high precision (relative standard deviation < 3.5%) was observed between replicates up to 48 h. Regarding its potential for customization, this manifold could accommodate further developments for customized turbidimetric monitoring, such as the use of light-emitting diodes as a light source or flow cells.

A low-cost DIY device for high resolution, continuous measurement of microbial growth dynamics

“…As the use of complex optics contributes significantly to the disparities between commercial spectrophotometers (4, 5, 10, 11), an ideal universal measurement device should be devoid of complex optics to increase its reproducibility and cost-effectiveness. It is important to note that although spectrophotometers are capable of taking measurements at different wavelengths of light, turbidity measurements of cell cultures are widely conducted using 600nm wavelength (hence, most reports refer to the resulting turbidity measurements as OD 600 ).…”

“…The ratio between scattering and absorption of cell cultures can be affected by various factors such as cell size, type of cell membrane, growth medium, cell density, path-length, and the presence of pigments (5, 7–9). The scattering effects can also interact with the specific optical design of a given spectrophotometer, producing differential effects on the resulting measurements between spectrophotometers with different optical designs (4, 5, 7, 10, 11). As expected from these considerations, turbidity measurements of the same cell culture samples using different spectrophotometers are found to have significant variability (sometimes more than 30%) (5, 10, 11).…”

“…The scattering effects can also interact with the specific optical design of a given spectrophotometer, producing differential effects on the resulting measurements between spectrophotometers with different optical designs (4, 5, 7, 10, 11). As expected from these considerations, turbidity measurements of the same cell culture samples using different spectrophotometers are found to have significant variability (sometimes more than 30%) (5, 10, 11). The relation between the turbidity and cell concentration, while linear, is also found to vary from device to device (5–7).…”

“…In practice, however, such conversion factors are found to be highly specific to individual samples that are used for obtaining them. For example, the conversion factor obtained from a standardised solution, such as the McFarland standard (12), is not applicable in the case of corrections required to be applied when measuring cell cultures (5, 7, 10, 11). This creates a practical difficulty in establishing conversion factors between different devices, where different conversion factors would need to be calculated among all devices and on different samples.…”

A rapid and low-cost estimation of bacteria counts in solution using fluorescence spectroscopy

“…Other prototype small-scale spectrophotometers are described in the literature; however, these examples have been applied to either measuring bacterial growth by turbidity [27] or identifying the load of specific organisms by quantifying enzyme activities with fluorogenic substrates [28]. In both cases, the limit of detection and time to detection do not compare favourably with the combination of stain and spectrophotometer we describe here.…”