Construction and Validation of A Low-cost, Small-scale, Multiplex Continuous Culturing System for Microorganisms

Abstract

This protocol details the construction of a simple, low-cost, small-scale, multiplex chemostat system designed for the continuous cultivation of microorganisms in suspension (i.e., bacteria, yeast, microalgae). The continuous culture device can operate at a working volume of 25 ml and allows the run of 8 chemostats in parallel by a single person. It provides a platform for parallel, long-term studies of evolution and adaptation of microorganisms under the stress of antimicrobial agents and/or toxic pollutants. The system complies with the varied needs of researchers for an accessible, highly-throughput and reliable tool that is nevertheless easy to construct, use and operate, and not demanding of space, materials, medium supply and workload. Here, we also validate the use of this system to generate de novo resistance towards a novel antimicrobial and a commonly used antibiotic in an antimicrobial-sensitive model organism. We believe that this "Do It Yourself" (DIY) system may constitute a useful tool to address the global problem of antibiotic resistance and to develop non-antibiotic based therapies.

Mesostats — A multiplexed, low-cost, do-it-yourself continuous culturing system for experimental evolution of mesocosms

“…In addition to the Miller et al . (2013) ministat system that was designed for yeast, others have applied similar designs to study nutrient limitation in yeast (Skelding et al ., 2018) and antibiotic resistance (Tonoyan et al ., 2020; Toprak et al ., 2013). Others have incorporated multiple sensors or automated controls (Ekkers et al ., 2020; Wong et al ., 2018), but these constitute a considerably larger initial investment in materials.…”

“…We used this system to test the intrinsic (see Chapter This system adds to the small, but growing, number of efforts to produce simple but scalable, multiplexed DIY chemostats from cheaper materials that are possible to build and maintain by a single person. In addition to the Miller et al (2013) ministat system that was designed for yeast, others have applied similar designs to study nutrient limitation in yeast (Skelding et al, 2018) and antibiotic resistance (Tonoyan et al, 2020;Toprak et al, 2013). Others have incorporated multiple sensors or automated controls (Ekkers et al, 2020;Wong et al, 2018), but these constitute a considerably larger initial investment in materials.…”

“…Many parts could also be easily substituted depending on availability or cost constraints. Any water-tight, sterilisable container can be used for culture chambers if suitable lids can be manufactured, such as falcon tubes (Tonoyan et al ., 2020) or commonly available lab glassware (Ekkers et al ., 2020). The controlled temperature room can be replaced with waterbaths and portable aquarium pumps can be used instead of building infrastructure.…”

“…Becks et al ., 2012; Declerck et al ., 2015; Fussmann et al ., 2000; Hiltunen et al ., 2014; Yoshida et al ., 2003). Multiplexed arrays, where the dilution rate is set by a single pump, and medium sources can be shared, further minimise variation between population chambers (Dénervaud et al ., 2013; Ekkers et al ., 2020; Miller et al ., 2013; Skelding et al ., 2018; Tonoyan et al ., 2020; Toprak et al ., 2013; Wong et al ., 2018).…”

Mesostats—A multiplexed, low-cost, do-it-yourself continuous culturing system for experimental evolution of mesocosms

“…Here we describe a multiplexed small-scale DIY chemostat array system (dubbed “mesostats”) adapted from the ministat array developed by Miller et al . [ 22 ] to suit experimental evolution of algae, in contrast to the so far described designs specifically intended for yeast [ 21 , 22 , 26 ] and bacterial cultures [ 23 , 27 ]. Our system uses common algal model species Chlamydomonas reinhardtii , with the specific goal to use it as a herbicide resistance evolution model.…”