PROTOCOL Building a morbidostat: an automated continuous- culture device for studying bacterial drug resistance under dynamically sustained drug inhibition Erdal Toprak1,2, Adrian Veres3, Sadik Yildiz2, Juan M Pedraza4, Remy Chait1, Johan Paulsson1,5 & Roy Kishony1,5 1Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. 2Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey. 3Health Sciences and Technology Program, Harvard Medical School, Boston, Massachusetts, USA. 4Department of Physics, Universidad de los Andes, Bogotá, Colombia. 5School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA. Correspondence should be addressed to E.T. (
[email protected]) or R.K. (
[email protected]). Published online 21 February 2013; doi:10.1038/nprot.2013.021 We present a protocol for building and operating an automated fluidic system for continuous culture that we call the ‘morbidostat’. The morbidostat is used to follow the evolution of microbial drug resistance in real time. Instead of exposing bacteria to predetermined drug environments, the morbidostat constantly measures the growth rates of evolving microbial populations and dynamically adjusts drug concentrations inside culture vials in order to maintain a constant drug-induced inhibition. The growth rate measurements are done using an optical detection system that is based on measuring the intensity of back-scattered light from bacterial cells suspended in the liquid culture. The morbidostat can additionally be used as a chemostat or a turbidostat. The whole system can be built from readily available components within 2–3 weeks by biologists with some electronics experience or engineers familiar with basic microbiology. INTRODUCTION Antibiotic resistance is an important public health problem, ren- at a constant rate lower than the maximal growth rate of the popu- dering currently available drugs useless and threatening millions lation, the dilution rate of the morbidostat rdilution ≅ ∆V/(V·∆t) is of lives1–4.