Preface
The mycobacteria continue to be a genus of great interest and importance, largely due to the
presence of pathogenic species. Mycobacterium tuberculosis, causing tuberculosis, continues
to be a major global health problem and is now the biggest killer from infectious disease.
Leprosy, caused by Mycobacterium leprae, has not yet been eradicated, and there has been an
increase in the number of opportunistic infections caused by nontuberculous mycobacteria.
Solving these global health problems requires an increased understanding of the basic
biology and pathogenesis of these organisms, as well as research directed toward improved
drugs and new vaccines.
Mycobacteria are difficult organisms to work with, in part due to intrinsic characteristics,
but also due to the requirement for specialized laboratories for the pathogenic organisms.
Practical difficulties include their slow growth rate, their tendency to clump in culture, the
difficulty of lysing the bacteria, and their high GC content. Despite these barriers, much
progress has been made in adapting state-of-the-art methods to be used with these organisms. In addition, much effort has been put toward developing high-throughput screening
methods that can be applied in drug discovery programs.
Perhaps it is not surprising then that we are now in our fourth edition of this book,
containing all of the tips and tricks developed by numerous researchers over the past
decades. Over the years since the first volume was published, many researchers have
expressed their gratitude for its existence. As an editor, it is rewarding to hear this, but of
course none of this would exist without the hard work of the contributors who wrote the
chapters. As new researchers come into this field, it has been encouraging to see how the
previous volumes have helped accelerate their research.
In preparing this fourth edition, we have tried to strike a balance between including
those basic methods that are still required for mycobacteriology and the newer or improved
methods that have been developed since the last volume was published. We hope these will
spark new areas of research.
We have included chapters on the basics of DNA isolation, protein isolation, and lipid
isolation, as well as more sophisticated techniques for isolation of ribosomes. Methods for
analyzing subcellular fractions—the proteome, the lipidome, and the metabolome—are
included. We have also gone back to basics and included a thorough review of culture
methods which provide the underpinning for all of the other work. Since whole genome
sequencing and its application is much more widely available, we have included a review
chapter to discuss best practices. Sequencing technology has developed rapidly, and so we
also include a chapter on whole genome sequencing from sputum samples.
A wide variety of in vitro models are available for mycobacteria. We have included
methods for macrophage and Dictyostelium infections using different mycobacterial species, as well as chapters on analysis by flow cytometry and microfluidics for single cell and
time-lapse microscopy.
Molecular methods have accelerated over the past decade, so we included basic methods
for electroporation as well as gene switching, together with newer protocols for recombineering and methods to generate knockdown strains by proteolysis or CRISPRi. Use of
fluorescent proteins has expanded greatly, and a chapter on fluorescent reporters is included
v
The mycobacteria continue to be a genus of great interest and importance, largely due to the
presence of pathogenic species. Mycobacterium tuberculosis, causing tuberculosis, continues
to be a major global health problem and is now the biggest killer from infectious disease.
Leprosy, caused by Mycobacterium leprae, has not yet been eradicated, and there has been an
increase in the number of opportunistic infections caused by nontuberculous mycobacteria.
Solving these global health problems requires an increased understanding of the basic
biology and pathogenesis of these organisms, as well as research directed toward improved
drugs and new vaccines.
Mycobacteria are difficult organisms to work with, in part due to intrinsic characteristics,
but also due to the requirement for specialized laboratories for the pathogenic organisms.
Practical difficulties include their slow growth rate, their tendency to clump in culture, the
difficulty of lysing the bacteria, and their high GC content. Despite these barriers, much
progress has been made in adapting state-of-the-art methods to be used with these organisms. In addition, much effort has been put toward developing high-throughput screening
methods that can be applied in drug discovery programs.
Perhaps it is not surprising then that we are now in our fourth edition of this book,
containing all of the tips and tricks developed by numerous researchers over the past
decades. Over the years since the first volume was published, many researchers have
expressed their gratitude for its existence. As an editor, it is rewarding to hear this, but of
course none of this would exist without the hard work of the contributors who wrote the
chapters. As new researchers come into this field, it has been encouraging to see how the
previous volumes have helped accelerate their research.
In preparing this fourth edition, we have tried to strike a balance between including
those basic methods that are still required for mycobacteriology and the newer or improved
methods that have been developed since the last volume was published. We hope these will
spark new areas of research.
We have included chapters on the basics of DNA isolation, protein isolation, and lipid
isolation, as well as more sophisticated techniques for isolation of ribosomes. Methods for
analyzing subcellular fractions—the proteome, the lipidome, and the metabolome—are
included. We have also gone back to basics and included a thorough review of culture
methods which provide the underpinning for all of the other work. Since whole genome
sequencing and its application is much more widely available, we have included a review
chapter to discuss best practices. Sequencing technology has developed rapidly, and so we
also include a chapter on whole genome sequencing from sputum samples.
A wide variety of in vitro models are available for mycobacteria. We have included
methods for macrophage and Dictyostelium infections using different mycobacterial species, as well as chapters on analysis by flow cytometry and microfluidics for single cell and
time-lapse microscopy.
Molecular methods have accelerated over the past decade, so we included basic methods
for electroporation as well as gene switching, together with newer protocols for recombineering and methods to generate knockdown strains by proteolysis or CRISPRi. Use of
fluorescent proteins has expanded greatly, and a chapter on fluorescent reporters is included
v