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Identifying And Classifying Bacteria

The most fundamental technique for classifying bacteria is the gram stain, developed in 1884 by Danish scientist Christian Gram. It is called a differential stain because it differentiates among bacteria and can be used to distinguish among them, based on differences in their cell wall.

In this procedure, bacteria are first stained with crystal violet, then treated with a mordant—a solution that fixes the stain inside the cell (e.g., iodine-KI mixture). The bacteria are then washed with a decolorizing agent, such as alcohol, and counterstained with safranin, a light red dye.

The walls of gram positive bacteria (for example, Staphylococcus aureus) have more peptidoglycans (the large molecular network of repeating disaccharides attached to chains of four or five amino acids) than do gram-negative bacteria. Thus, gram-positive bacteria retain the original violet dye and cannot be counterstained.

Gram negative bacteria (e.g., Escherichia coli) have thinner walls, containing an outer layer of lipopolysaccharide, which is disrupted by the alcohol wash. This permits the original dye to escape, allowing the cell to take up the second dye, or counterstain. Thus, gram-positive bacteria stain violet, and gram-negative bacteria stain pink.

The gram stain works best on young, growing populations of bacteria, and can be inconsistent in older populations maintained in the laboratory.

Microbiologists have accumulated and organized the known characteristics of different bacteria in a reference book called Bergey's Manual of Systematic Bacteriology (the first edition of which was written primarily by David Hendricks Bergey of the University of Pennsylvania in 1923).

The identification schemes of Bergey's Manual are based on morphology (e.g., coccus, bacillus), staining (gram-positive or negative), cell wall composition (e.g., presence or absence of peptidoglycan), oxygen requirements (e.g., aerobic, facultatively anaerobic) and biochemical tests (e.g., which sugars are aerobically metabolized or fermented).

In addition to the gram stain, other stains include the acid-fast stain, used to distinguish Mycobacterium species (for example, Mycobacterium tuberculosis, the cause of tuberculosis); endospore stain, used to detect the presence of endospores; negative stain, used to demonstrate the presence of capsules; and flagella stain, used to demonstrate the presence of flagella.

Another important identification technique is based on the principles of antigenicity—the ability to stimulate the formation of antibodies by the immune system. Commercially available solutions of antibodies against specific bacteria (antisera) are used to identify unknown organisms in a procedure called a slide agglutination test. A sample of unknown bacteria in a drop of saline is mixed with antisera that has been raised against a known species of bacteria. If the antisera causes the unknown bacteria to clump (agglutinate), then the test positively identifies the bacteria as being identical to that against which the antisera was raised. The test can also be used to distinguish between strains, slightly different bacteria belonging to the same species.

Phage typing, like serological testing, identifies bacteria according to their response to the test agent, in this case viruses. Phages are viruses that infect specific bacteria. Bacterial susceptibility to phages is determined by growing bacteria on an agar plate, to which solutions of phages that infect only a specific species of bacteria are added. Areas that are devoid of visible bacterial growth following incubation of the plate represent organisms susceptible to the specific phages.

Because a specific bacterium might be susceptible to infection by two or more different phages, it may be necessary to perform several tests to definitively identify a specific bacterium.

The evolutionary relatedness of different species can also be determined by laboratory analysis. For example, analysis of the amino acid sequences of proteins from different bacteria disclose how similar the proteins are. In turn, this reflects the similarity of the genes coding for these proteins.

Protein analysis compares the similarity or extent of differences between the entire set of protein products of each bacterium. Using a technique called electrophoresis, the entire set of proteins of each bacterium is separated according to size by an electrical charge applied across gel. The patterns produced when the gel is stained to show the separate bands of proteins reflects the genetic makeup, and relatedness, of the bacteria.

The powerful techniques of molecular biology have given bacteriologists other tools to determine the identity and relatedness of bacteria.

Taxonomists interested in studying the relatedness of bacteria compare the ratio of nucleic acid base pairs in the DNA of microorganisms, that is, the number of guanosine-cytosine pairs in the DNA. Because each guanosine on a double-stranded molecule of DNA has a complementary cytosine on the opposite strand, comparing the number of G-C pairs in one bacterium, with that in another bacterium, provides evidence for the extent of their relatedness.

Determining the percentage of G-C pairs making up the DNA also discloses the percentage of adenosine-thymine (A-T)—the other pair of complementary nucleic acids making up DNA (100% - [ % G-C] = % A-T).

The closer the two percentages are, the more closely related the bacteria may be, although other lines of evidence are needed to make a definitive determination regarding relationships.

The principle of complementarity is also used to identify bacteria by means of nucleic acid hybridization. The technique assumes that if two bacteria are closely related, they will have long stretches of identical DNA. First, one bacterium's DNA is isolated and gently heated to break the bonds between the two complementary strands. Specially prepared DNA probes representing short segments of the other organism's DNA are added to this solution of single-stranded DNA. The greater degree to which the probes combine with (hybridize) complementary stretches of the single stranded DNA, the greater the relatedness of the two organisms.

In addition to helping bacteriologists better classify bacteria, the various laboratory tests are valuable tools for identifying disease-causing organisms. This is especially important when physicians must determine which antibiotic or other medication to use to treat an infection.

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