85Stephen H. Gillespie (ed.), Antibiotic Resistance Protocols, Methods in Molecular Biology, vol. 1736,
https://doi.org/10.1007/978-1-4939-7638-6_8, © Springer Science+Business Media, LLC 2018
Chapter 8
Enhanced Methodologies for Detecting Phenotypic
Resistance in Mycobacteria
Robert J.H. Hammond, Vincent O. Baron, Sam Lipworth,
and Stephen H. Gillespie
Abstract
Lipid droplets found in algae and other microscopic organisms have become of interest to many research-
ers partially because they carry the capacity to produce bio-oil for the mass market. They are of importance
in biology and clinical practice because their presence can be a phenotypic marker of an altered metabo-
lism, including reversible resistance to antibiotics, prompting intense research.
A useful stain for detecting lipid bodies in the lab is Nile red. It is a dye that exhibits solvatochromism;
its absorption band varies in spectral position, shape and intensity with the nature of its solvent environ-
ment, it will fluoresce intensely red in polar environment and blue shift with the changing polarity of its
solvent. This makes it ideal for the detection of lipid bodies within Mycobacterium spp. This is because
mycobacterial lipid bodies’ primary constituents are nonpolar lipids such as triacylglycerols but bacterial
cell membranes are primarily polar lipid species. In this chapter we describe an optimal method for using
Nile red to distinguish lipid containing (Lipid rich or LR cells) from those without lipid bodies (Lipid Poor
or LP). As part of the process we have optimized a method for separating LP and LR cells that does not
require the use of an ultracentrifuge or complex separation media. We believe that these methods will
facilitate further research in these enigmatic, transient and important cell states.
Key words Tuberculosis, Dormancy, Phenotypic resistance, Lipid body1 Introduction
In recent years there has been an increasing interest in mycobacte-
ria within which lipid bodies are seen [ 1 – 3 ]. This is due to the
important association with low metabolic state and phenotypic
resistance to key anti-tuberculosis antibiotics [ 4 – 7 ]. As the goal of
improving tuberculosis treatment remains frustratingly out of
reach, it is important that we understand what the true susceptibil-
ity of M. tuberculosis is as it is clear there is a significant difference
in the susceptibility of cells with lipid bodies present in comparison
with those that are not [ 8 ]. It is of considerable importance, there-
fore, to be able to reliably separate and quantify mycobacterial cells
in different cells state. Previously published methods are effective