Maximizing cell densities in miniprep-scale cultures with H15 medium and improved oxygen transfer

H15 is a very rich growth medium for culturing bacteria that supports cell densities that are several fold higher than those obtained with more popular media like Luria-Bertani (LB) broth. The performance of H15 is dependent on adequate aeration of the growing culture. This is easily achieved when relatively large baffled flasks are used but not when standard culture tubes are used. As a result, it has been difficult to extend the superior performance of H15 to small-scale bioreactors commonly used by molecular biologists including culture tubes and microtiter plates. Here we describe a simple procedure for improving the oxygen transfer capacity (OTR) of small-scale bioreactors and modifications to the protocol for the preparation of H15 growth medium that make it possible to boost cell density by nearly 400% relative to standard LB broth. Modifications include preparation a glucose-free media stock, sterilization through a 0.45 mum filter rather than autoclaving to avoid the formation of compounds which inhibit cell growth and the introduction of "baffles" for glass culture tubes. Furthermore, these modifications support cell densities in 2ml that are statistically identical to cell densities obtained in larger baffled flasks. These improvements in the performance and reliability of H15 in small-scale cultures afford researchers the opportunity for significant time and cost savings in the production of plasmid DNA for high-throughput genetic analyses. (C) 2003 Elsevier B.V. All rights reserved.