When grown in sealed tubes with slow stirring, tubercle bacilli cease replicating when
oxygen concentrations decrease to the microaerobic level (1% oxygen saturation) and
they enter a nonreplicating persistence state (NRP-1). With continued incubation the
oxygen levels decrease to anaerobic levels (0.06% oxygen saturation) and the second
state, NRP-2 follows. This model has been used extensively as a simple model of
dormancy for M. tuberculosis. This model was developed by Lawrence Wayne here at
the VA in Long Beach and is therefore often called The Wayne Model.
The Wayne Model utilizes tightly sealed slowly stirred tubes. The stirring is
accomplished with the use of a "mag flea". This is a very small Teflon-coated magnetic
8 mm stirring bars.
The stirring is performed by a slow speed stirrer. Many stir plates designed for
mixing rotate too quickly to be used. The stirring should be just fast enough to keep
the cells from settling without disturbing the surface of the medium. We use stir
plates made for cell culture (not mixing) set at 120 rpm.
References for the Wayne Model
Wayne, L.G. and Hayes L.G. 1996. An in vitro model for sequential study of shiftdown
of Mycobacterium tuberculosis through two stages of nonreplicating persistence.
Infect Immun 64:2062-2069
Wayne, L.G. and Sohaskey, C.D. 2001. Nonreplicating persistence of Mycobacterium
tuberculosis. Annu Rev Microbiol 55:139-163.
Wayne, L.G. In Vitro model of hypoxically induced nonreplicating persistence of
Mycobacterium tuberculosis. In T. Parish and N.G. Stoker (ed.), Methods in Molecular