Novel freeze-substitution electron microscopy provides new aspects of virulent Mycobacterium tuberculosis with visualization of the outer membrane and satisfying biosafety requirements.
Transmission electron microscopy (TEM) of virulent bacteria is usually performed following chemical fixation (CF) with aldehyde fixatives such as glutaraldehyde because of the biosafety problem. However, CF may alter sample ultrastructure. In this study, we used a rapid-freeze substitution (RFS) sandwich method without pre-embedding in agar. TEM images obtained using this method were completely different from those of conventional chemically fixed samples; the bacilli cytoplasm of the RFS preparations was filled evenly with numerous ribosomes, and there was no positional variation of electron density that was obvious in those obtained with CF samples. The sandwich method is suitable for microbiological materials without expensive devices and can be easily performed in a biosafety cabinet. In future, this method coupled with novel labeling techniques may help localize structural and functional molecules throughout a bacterial cell.
Yamada
Hiroyuki
H
Bacteriology Division, Department of Mycobacterium Reference and Research, the Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24, Matsuyama, Kiyose, Tokyo, 204-8533, Japan. hyamada@jata.or.jp
Mitarai
Satoshi
S
Chikamatsu
Kinuyo
K
Mizuno
Kazue
K
Yamaguchi
Masashi
M
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000648:ultrastructure; D005404:Fixatives / Q000737:chemistry; D017110:Freeze Substitution / Q000379:methods*; D005976:Glutaral / Q000737:chemistry; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000472:pathogenicity / Q000648:ultrastructure; D014774:Virulence
D001425:Bacterial Outer Membrane Proteins; D005404:Fixatives; D005976:Glutaral