Abstract
Batch cultures are a low maintenance and routine culturing method in microbiology. Automated tools that measure growth curves from microorganisms grown in traditional laboratory glassware, such as Balch-type tubes, are not commercially available. Here, we present a new MicrobiAl Growth Intervalometer (MAGI) that measures optical density as it correlates to microbial growth by utilizing photo-conduction as opposed to photo-attenuation used by traditional OD measurement equipment. Photo-attenuation occurs when biomass in suspension within a medium blocks and/or diffuses light directed at the detector, such that an increase in biomass results in a decrease in the measured signal. Photo-conduction differs in which the biomass contained in a medium conducts light from the emitter to the detector, where an increase in the biomass results in a corresponding increase in the measured signal. MAGI features software-driven automation that provides investigators with a highly sensitive, low-background noise growth measurement instrument with added capabilities for remote visualization and data acquisition. It is a low maintenance, cost effective, versatile, and robust platform for aerobic/anaerobic cultivation. We demonstrate the versatility of this device by obtaining growth curves from two common laboratory organisms Escherichia coli K-12 and Bacillus subtilis. We show that growth rates and generation times in E. coli K-12 are reproducible to previously published results and that morphological changes of B. subtilis during growth can be detected as a change in the slope of the growth curve, which is a function of the effects of cell size on photo-conduction through the medium. We also test MAGI to capture growth curves from an environmental organism, Intrasporangium calvum C5, under various media compositions. Our results demonstrate that the MAGI platform accurately measures growth curves in media under various redox conditions (aerobic, microaerobic, and anaerobic), complex and minimal medias, and resolving diauxic growth curves when I. calvum is grown on a disaccharide. Lastly, we demonstrate that the device can resolve growth curves for μM concentrations of resources that yield low biomass. This research advances the tools available to microbiologists aiming to monitor growth curves in a variety of disciplines, such as environmental microbiology, clinical microbiology, and food sciences.
Highlights
Microbial growth and physiology investigations rely on two cultivation techniques: batch cultivation and continuous cultivation, such as the chemostat
Operation: MicrobiAl Growth Intervalometer (MAGI) is a collection of hardware devices consisting of an Arduino Mega 2,560 microcontroller with an ethernet shield, a Raspberry Pi computer running Linux Ubuntu, an articulating arm, a power source with analog-based signal amplification for each measurement channel, and a multiplexed light emitter/detector that houses the test tubes (Figure 1)
RS-232 serial port is common to all orbital shaker manufacturers, and while command syntax varies between manufacturers, the command syntax is readily available in user manuals provided by manufacturers
Summary
Microbial growth and physiology investigations rely on two cultivation techniques: batch cultivation and continuous cultivation, such as the chemostat. For continuous culturing (Monod, 1950), μ is set a priori by the investigator through a predefined dilution rate of fresh incoming media into the culturing vessel with the cells held in a steady-state of nutrient limitation. This state of nutrient limitation is equivalent to the cusp of the growth curve at late-exponential/early-stationary phase for microorganisms growing in the same media in batch cultures that have nearly exhausted the same growth limiting nutrient (Saldanha et al, 2004). The chemostat allows investigators to focus and study microbial physiology continuously at a single-growth phase (late-exponential/earlystationary) and growth rate
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