157Stephen H. Gillespie (ed.), Antibiotic Resistance Protocols, Methods in Molecular Biology, vol. 1736,
https://doi.org/10.1007/978-1-4939-7638-6_15, © Springer Science+Business Media, LLC 2018
Chapter 15
A Method to Evaluate Persistent Mycobacterium
tuberculosis In Vitro and in the Cornell Mouse Model
of Tuberculosis
Yanmin Hu and Anthony Coates
Abstract
Persistent Mycobacterium tuberculosis will not grow on solid or liquid media. They will, however, grow in
the presence of resuscitation promoting factors (RPF). Here we describe the production of RPF rich cul-
ture supernatants, and their use for the stimulation of growth of persisters in vitro as well as in the Cornell
model of tuberculosis.
Key words Persisters, Mycobacterium tuberculosis, Resuscitation promoting factors, Culture superna-
tants, Cornell model1 Introduction
Culture of bacteria in vitro has been the pillar of diagnosis and
research in microbiology since the days of Pasteur and Koch. These
techniques are more sensitive than microscopy alone and provide
large numbers of bacteria for subsequent antibiotic susceptibility
testing and characterization.
Traditionally, viable bacterial enumeration is based on colony
forming unit (CFU) counts on agar plates or broth counts in liquid
medium. Testing and development of novel anti-TB drug regi-
mens mainly focus on therapeutic endpoints based on sputum neg-
ative CFU counts or no mycobacterial growth in broth. However,
observation of the treatment of tuberculosis in humans [ 1 ] and in
experimental animals [ 2 , 3 ] provides strong evidence that nonmul-
tiplying persisters of M. tuberculosis are present at the beginning
and enriched during treatment. Even after 6 months of chemo-
therapy, persisters are still present and cause subsequent disease
relapse. These persistent bacteria [ 4 ] remain undetected by con-
ventional culture methods. They do not retain acid-fast stains or
multiply on agar or in broth culture medium, which makes their