Bacterial ultrastructure

Bacterial ultrastructure is concerned with the cellular and molecular construction of bacteria . The bulk of research in bacterial ultrastructure investigates the ultrastructure of the cell wall that surrounds bacteria.

The study of bacterial ultrastructure began with the development of the staining regimen by Danish pathologist Christian Gram (1853–1938) that classifies the majority of bacteria as either Gram-negative or Gram-positive. The latter bacteria retain the crystal violet stain, while Gram-negative bacteria do not retain this stain and are stained by the second stain that is applied, safranin. While the basis for this difference was not known at first, scientists suspected that the structure of the wall surrounding the contents of the bacteria might be involved.

Subsequent to the time of Gram, scientists have discovered that the cell wall plays only a secondary role in the Gram stain reactions. However, the cell wall of Gram-positive bacteria is indeed much different than that of Gram-negative bacteria. The study of bacterial ultrastructure relates these constituent differences to the intact cell wall. In other words, ultrastructure explores the structure of each constituent and the chemical and other associations that exist between these constituents.

The exploration of bacterial ultrastructure requires samples that are as undisturbed as possible from their natural, or so-called native, state. This has been challenging, since much of the information that has been obtained has come from the use of electron microscopy. The techniques of conventional transmission electron microscopy and scanning electron microscopy require the removal of water from the sample. Because the bulk of living things, including bacteria, are comprised of water, the removal of this fluid can have drastic consequences on the structure of the bacteria. Much effort has gone into the development of regimens that chemically "fix" bacteria, so that structure is maintained following the subsequent removal of water.

Techniques have also been developed that prepare bacteria for transmission electron microscopy without the necessity of removing water from the specimen. One technique uses an embedding resin (a substance in which the bacteria are immersed and, when the resin is hardened, allows thin slices of the bacteria to be cut) that mixes with water. This resin is harder to work with than the conventional resins that are not water-soluble. Thus, while valuable information can be obtained using water-soluble resins, a great deal of experience is necessary to produce high quality results.

A second technique of sample preparation relies on the instantaneous freezing of the bacteria. Freezing is so fast that the interior water does not extensively crystallize (which would be extremely damaging to structure). Again, an experienced analyst can produce samples that information concerning the native ultrastructure of the bacteria.

In the past several decades, other tools are increasing the ultrastructure information that can be obtained. For example, the technique of atomic force microscopy can produce information on the atomic associations between adjacent molecules on the surface of bacteria. Atomic force microscopy has been very useful in ultrastructure studies of the regularly structured surface layers on bacteria.

Modern techniques of molecular genetics can also yield ultrastructure information. Mutants can be selected or designed in which a particular gene or genes has been rendered incapable of producing a protein product. If the gene is involved with cell wall constituents, the analysis of the wall can reveal the alterations that have occurred in the absence of the gene product. An example are the many mutants that are defective in the construction or assembly of lipopolysaccharide, a carbohydrate and lipid constituent of the outer membrane of Gram-negative bacteria. The loss of the carbohydrate portion of lipopolysaccharide makes the outer membrane more hydrophobic .

One approach that has been known for decades still yields useful information concerning bacterial ultrastructure. This is the substitution of the metals present in the cell wall with other metals. Metals act like glue to hold various wall components in association with one another. Examples of such metallic species include calcium and magnesium. Out-competing these species by supplying large concentrations of another metal, the influence of the normal metallic species can be assessed. For example, replacement of metals in the Gramnegative outer membrane can cause the release of lipopolysaccharide and the formation of bubbles along the surface of the membrane, where the underlying attachment to the rigid peptidoglycan layer is disrupted.

The use of specific antibodies to determine the molecular arrangement of ultrastructural constituent targets greatly enhances the effectiveness of agents to be used in drug therapy.

See also Atomic force microscope; Bacterial appendages; Bacterial surface layers; Caulobacter; Electron microscope, transmission and scanning; Electron microscopic examination of microorganisms; Sheathed bacteria