A Small-Volume, Low-Cost, and Versatile Continuous Culture Device

Abstract

BackgroundContinuous culture devices can be used for various purposes such as establishing reproducible growth conditions or maintaining cell populations under a constant environment for long periods. However, commercially available instruments are expensive, were not designed to handle small volumes in the milliliter range, and can lack the flexibility required for the diverse experimental needs found in several laboratories.Methodology/Principal FindingsWe developed a versatile continuous culture system and provide detailed instructions as well as a graphical user interface software for potential users to assemble and operate their own instrument. Three culture chambers can be controlled simultaneously with the proposed configuration, and all components are readily available from various sources. We demonstrate that our continuous culture device can be used under different modes, and can easily be programmed to behave either as a turbidostat or chemostat. Addition of fresh medium to the culture vessel can be controlled by a real-time feedback loop or simply calibrated to deliver a defined volume. Furthermore, the selected light-emitting diode and photodetector enable the use of phenol red as a pH indicator, which can be used to indirectly monitor the bulk metabolic activity of a cell population rather than the turbidity.Conclusions/SignificanceThis affordable and customizable system will constitute a useful tool in many areas of biology such as microbial ecology as well as systems and synthetic biology.

Development of oriC -Based Plasmids for Mesoplasma florum

“…Microplates were next incubated at 34°C for 14 h. Bacterial growth was assessed by measuring the optical density at 560 nm every hour with a microplate reader (Synergy HT; BioTek). The metabolic activity of M. florum was previously shown to result in the acidification of the ATCC 1161 growth medium, causing changes in the absorbance of phenol red at 560 nm that correlate with the number of CFU (15). The MIC of each antibiotic was defined as the lowest tested concentration that inhibited the growth of M. florum (56).…”

“…4B and C). Considering that M. florum has a doubling time of ϳ34 min in ATCC 1161 medium (14,15), this indicates that pMflT-o3 and pMflT-o4 plasmids can be stably maintained for at least 85 generations without detectable loss. Alternative transformation methods.…”

“…M. florum is closely related to the mycoides cluster of mycoplasmas, which includes Mycoplasma mycoides and Mycoplasma capricolum; these have become model organisms for whole-genome cloning (8)(9)(10), genome transplantation (8,11,12), and genome minimization (13). However, M. florum shows higher growth rates (ϳ34 min), has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest freeliving organisms (1,6,14,15). For example, M. florum L1 (RefSeq accession no.…”

Developing a low-cost milliliter-scale chemostat array for precise control of cellular growth

“…Commercially-available chemostats are expensive and are usually geared toward large culture volumes (liter-scale instead of a few milliliters). Miniature multi-chamber turbidostat/chemostat devices have been constructed previously [4][5][6] . However, the consistency and accuracy of flow rates and the operational ranges in these designs are not explicitly uncharacterized.…”

“…However, the consistency and accuracy of flow rates and the operational ranges in these designs are not explicitly uncharacterized. For example, in Matteau et al device 5 , flow rate is mediated via valve opening time. At the shortest valve opening time they tested (1 sec), actual dispensed volume (~0.49 ml) seemed to deviate from the regression by tens of percent.…”