72
ing nutrient across the system. The relationship between the
yield of cells using substrate, Yx/s (grams of biomass per gram
of substrate), and the limiting substrate can be calculated using
X = Yx/s (So−S), where X is the biomass at steady state (g/L),
and So and S are the concentrations of the limiting substrate in
the feed and residual substrate in the outflow respectively.- Run sheets and the Eycoferm controller data logging system
are used to record parameters on a daily basis. - The sampling regimen that has been used in previous experi-
ments is to sample at each mean generation time in steady- state
pre and post antibiotic addition; for MGT of 69.3 h (slow
growth) this is every 3 days and for a MGT of 23.1 h this is
every day. The antibiotics that have been used for previous
studies were isoniazid, rifampicin, and pyrazinamide. These
antibiotics were used at minimum inhibitory concentrations
[ 6 , 7 ]. - Plates must be preincubated at room temperature for at least
several hours (although not overnight as this causes overdrying
of the plates). - Under certain growth conditions mycobacteria will adhere to
the walls of the vessels and the probes. Once this starts to
occur, the optical density is likely to fall and the DOT and pH
levels will fluctuate. The culture will no longer be in steady
state and will need to be transferred to another vessel.
Acknowledgments
Funding was received from Department of Health Grant in Aid
and the National Institute of Health Research. The views expressed
in this publication are those of the authors and not necessarily
those of Public Health England, the National Institute for Health
Research, or the Department of Health. The research leading to
these results also received funding from the Innovative Medicines
Initiative Joint Undertaking under grant agreement n°115337,
resources of which are composed of financial contribution from
the European Union’s Seventh Framework Programme
(FP7/2007-2013) and EFPIA companies’ in-kind contribution.
The authors acknowledge Kim Hatch and Jon Allnutt for the huge
contribution they have made to the development of chemostat
models for the growth of M. tuberculosis and application to the
assessment of antibiotic activity and to Professor Philip Marsh for
his guidance and constructive input.Charlotte L. Hendon-Dunn et al.