Abstract
We previously reported the exquisite preservation of the ultrastructures of virulent Mycobacterium tuberculosis cells processed through cryofixation and rapid freeze substitution. Here, we report the “structome” analysis (i.e., the quantitative three-dimensional structural analysis of a whole cell at the electron microscopic level) of virulent M. tuberculosis using serial ultrathin sections prepared after cryofixation and rapid freeze substitution and analyzed by transmission electron microscopy. Five M. tuberculosis cells, which were contained in the serial ultrathin cross sections encompassing from one end to the other, were cut into 24, 36, 69, 55, and 63 serial ultrathin sections, respectively. On average, the cells were 2.71 ± 1.05 μm in length, and the average diameter of the cell was 0.345 ± 0.029 μm. The outer membrane and plasma membrane surface areas were 3.04 ± 1.33 μm2 and 2.67 ± 1.19 μm2, respectively. The cell, outer membrane, periplasm, plasma membrane, and cytoplasm volumes were 0.293 ± 0.113 fl (= μm3), 0.006 ± 0.003 fl, 0.060 ± 0.021 fl, 0.019 ± 0.008 fl, and 0.210 ± 0.091 fl, respectively. The average total ribosome number was 1,672 ± 568, and the ribosome density was 716.5 ± 171.4/0.1 fl. This is the first report of a structome analysis of M. tuberculosis cells prepared as serial ultrathin sections following cryofixation and rapid freeze substitution and examined by transmission electron microscopy. These data are based on the direct measurement and enumeration of exquisitely preserved single-cell structures in transmission electron microscopy images rather than calculations or assumptions from indirect biochemical or molecular biological data. In addition, these data may explain the slow growth of M. tuberculosis and enhance understanding of the structural properties related to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance, particularly in regard to the ratio of target to drug concentrations.
Highlights
Bacteria can be observed with the naked eye as turbidity in a liquid medium or as colonies on the surface of a solid medium
The ribosome density for M. tuberculosis was calculated and compared to that determined for yeast cells [2, 5] as well as previously published estimations [10,11,12,13,14]. This is the first report of a structome analysis of individual M. tuberculosis cells using serial ultrathin sections and direct enumeration
We previously reported the excellent preservation of the ultrastructure of M. tuberculosis cells provided by CRF-rapid freeze substitution (RFS) [5, 7, 8]
Summary
Bacteria can be observed with the naked eye as turbidity in a liquid medium or as colonies on the surface of a solid medium. Under light or fluorescent microscopic analysis, bacteria appear as stained or fluorescence-emitting small round cocci or rod-shaped bacilli. These macroscopic and microscopic observations are equivalent to observing the biosphere from an aircraft flying at high altitude or observing large animals or tall plants on the earth’s surface from an aircraft at low altitude. Such observations provide surficial and numerical information useful for scientific and clinical investigations, they reveal only limited superficial information regarding individual bacterial cells. TEM examinations of bacteria provide a variety of information, in general these examinations are highly qualitative because the intact cytoplasmic ultrastructure is poorly preserved by conventional chemical fixation, which makes it difficult to perform quantitative analyses
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