Electron microscopy of frozen-hydrated bacteria.

Amorphous, unstained, frozen-hydrated sections of bacteria provide a faithful high-resolution image of procaryotic cells. Conventional preparation artifacts due to fixation, staining, and dehydration are nonexistent. Freezing damage is avoided by using glucose as a cryoprotectant. Cutting damage on frozen material is severe, but sectioning artifacts, being always related to the cutting direction, can be systematically recognized and thus taken into consideration. Geometry and density distribution of the bacterial envelope can be resolved to about 3 nm. The following main features have been observed. In Escherichia coli the inner and outer membranes have an approximately uniform density profile. The distance between the two membranes is constant, ca. 33 nm. In Staphylococcus aureus the cell wall is ca. 40 nm wide. It is bordered on the cytoplasmic side by an asymmetric 5.5-nm-wide bilayer. The bacterial nucleoid, clearly visible with conventional preparation methods, appears in exponentially growing bacteria as an ill-defined central region with approximately the same density as the rest of the cytoplasm. It becomes more clearly visible when bacteria are in the stationary phase, plasmolysed, fixed, or stained. We confirm that "mesosomes," hitherto quite often considered to be essential organelles in all procaryotes, are artifacts. They appear in large numbers during osmium fixation.     

Electron microscopy of methanol-utilizing bacteria

Two different groups of methanol-utilizing bacteria were studied by electron microscopy. Bacteria using the serine pathway for the assimilation of methanol were found to have a thin cell envelope (outer membrane, periplasmic area and cytoplasmic membrane). Those using the assimilatory ribulose monophosphate pathway of formaldehyde fixation had a much thicker cell envelope and in the case ofPseudomonas C protrusions of the outer membrane were found.     

Electron microscopy of some rock phosphate dissolving bacteria and fungi

BacteriaPseudomonas striata, Bacillus polymyxa, B. megaterium andB. pulvifaciens, and fungiAspergillus awamori, A. niger andPenicillium digitatum dissolve tricalcium phosphate and, much less, Mussorie and Udaipur rock phosphate. The solubilizing power of fungi was higher than that of bacteria, the highest being withA. awamori andA. niger, and withP. striata. Electron microscopy of the various cultures showed an electron-dense layer on the bacterial surface after negative staining. The size of phosphate particles decreased by the microbial action, with tricalcium phosphate from 140 — 250 to 30 — 90 nm after three weeks of incubation.     

Reduction of malachite green to leucomalachite green by intestinal bacteria.

Intestinal microfloras from human, rat, mouse, and monkey fecal samples and 14 pure cultures of anaerobic bacteria representative of those found in the human gastrointestinal tract metabolized the triphenylmethane dye malachite green to leucomalachite green. The reduction of malachite green to the leuco derivative suggests that intestinal microflora could play an important role in the metabolic activation of the triphenylmethane dye to a potential carcinogen.     

Electron microscopy of Giardia lamblia cysts.

The flagellated protozoan Giardia lamblia is a recognized public health problem. Intestinal infection can result in acute or chronic diarrhea with associated symptoms in humans. As part of a study to evaluate removal of G. lamblia cysts from drinking water by the processes of coagulation and dual-media filtration, we developed a methodology by using 5.0-microns-porosity membrane filters to evaluate the filtration efficiency. We found that recovery rates of G. lamblia cysts by membrane filtration varied depending upon the type and diameter of the membrane filter. Examination of membrane-filtered samples by scanning electron microscopy revealed flexible and flattened G. lamblia cysts on the filter surface. This feature may be responsible for the low recovery rates with certain filters and, moreover, may have implications in water treatment technology. Formation of the cyst wall is discussed. Electron micrographs of cysts apparently undergoing binary fission and cysts exhibiting a possible bacterial association are shown.     

Electron microscopy of vitrified-hydrated La Crosse virus.

La Crosse (LAC) virions were cryopreserved by rapid freezing in a thin layer of vitreous ice. The vitrified-hydrated LAC virions were subsequently imaged at -170 degrees C in a transmission electron microscope equipped with a low-temperature specimen holder. This cryoelectron microscopic technique eliminates the artifacts frequently associated with negative staining. Images of vitrified-hydrated LAC virions clearly revealed surface spikes as well as bilayer structure. Size measurements of the vitrified-hydrated LAC virions showed heterogeneity, with diameters ranging from 75 to 115 nm. Regardless of the particle size, the spike was about 10 nm long, and the bilayer was about 4 nm thick. The spikes are interpreted to be one or both of the glycoproteins, and the bilayer is interpreted to be the membrane envelope of the virus. In contrast to the pleomorphic appearance of the negatively stained LAC virions, the vitrified-hydrated LAC virions showed uniform spherical shapes regardless of their sizes.     

Electron microscopy of thin protein crystal sections.

In continuation of an earlier publication (Hoppe et al., 1968), further experiments are described here on the preparation of thin film sections of embedded protein crystals for investigation by electron microscopy and electron diffraction. Several embedding media were compared, the best being Aquon. Periodicities were observed in electron micrographs as well as in electron diffraction patterns. In diffraction experiments the best resolution observed was approximately 10 to 11 Å.