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

摘要

H15 is a very rich growth medium for culuring bacteda that supports cell densities that are several fold higher than those obtained with more popular media like Luria-Bertani(LB)broth.The performance of H15is dependent on adequate aeration of the growing culture.This is easily achieved when relatively large bafHed Hasks are used but not when standard c111ture tubes are used.As a result, it has been dimcult to extend the superior performance of H15to small-scale bioreactors commonly used by molecular biologists in- cluding culture tubes and microtiter plates.Here we describe a simple procedure for improving the oxygen transfer capacity (OTR) of small-scale bioreactors and Inodiacations to the protocol for the preparation of H15growth medium that make it possible to boost cell density by nearly 400% relative to standard LB broth.Modiacations include preparation a glucose-free media stock,sterilization through a 0.45 mum alter rather than autoclaving to avoid the formation of compounds which inhibit cell growth and the introduction of "baffles"for glass culture tubes.Furthermore,these Inodiacations support cell densities in 2 ml that are statistically identical to cell densities obtained in larger baffled nasks.These improvements in the pedonnance and reliability of H15 in small-scale cultures afford researchers the opportunity for sigIliacant time and cost savings in the production of plasmid DNA for high-throughput genetic analyses.