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Figure 10.13 Gram stain technique. This modification (one of many) is recommended for staining of activated sludge mixed liquor.

Joachim Gram. Although the method was developed empirically and seemed to show important differences between cells, it was not until much later that it was learned that a fundamental difference in cell wall composition was being highlighted (Figure 10.14). Gram-positive cells stain blue-violet because they retain the crystal violet, gram-negative cells are red because they are decolorized by ethanol and then take up the safranin coun-terstain.

Other stains also may be of great utility in differentiating among organisms or in visualizing cell structures. Figure 10.15 shows several types of staining approaches. Various staining procedures were particularly necessary prior to development of phase contrast and electron microscopy, and still may be highly useful.

Motility Many prokaryotes have motility, the ability to move, by means of one or more flagella (singular, flagellum), long, thin, hairlike organelles that protrude from the cell. Flagella at the end of a rod-shaped cell are referred to as polar; those on the sides are peritrichous. The flagella of prokaryotes are too small to be seen with a light microscope

Figure 10.14 Bacterial cell wall and membrane: (a) gram positive; (b) gram negative.

but can be made visible through staining or observed with an electron microscope. Also, in some cases there may be visible tufts, consisting of numerous flagella. Both bacteria and archaea may be flagellated (as are some algae and protozoa). Rates of movement can exceed 50 mm/s.

Some cells are motile even without flagella. Several species, particularly of filamentous bacteria, are able to ''push'' against a surface or other object, resulting in gliding motility, or can flex the filament, leading to twitching motility. However, speeds typically are much slower (<10 mm/s) than for flagellated organisms.

Organism movement may be in response to a gradient. Chemotaxis, for example, is movement toward (or occasionally away from) a higher concentration of a chemical; phototaxis is in response to light. Magnetotaxis, response to a magnetic field, has also been observed in a few specialized species, such as Magnetospirillum (Section 10.5.6).

Figure 10.15 Staining approaches.

Pigmentation Most prokaryotes appear colorless under the microscope, and colorless or whitish in liquid culture and on agar plates. However, a number of groups are photosyn-thetic and have appropriate pigments for this purpose. There are also a number of other pigments that might be formed by particular strains, so that cells and cultures may have a variety of colors (including red, pink, orange, yellow, and purple). Usually, any pigments formed will be within the cell, but in some cases they are released and may color the growth medium.

Sporulation A number of prokaryotes produce resting stages, known as spores, that are resistant to hostile environmental conditions such as desiccation. Some gram-positive bacteria produce an especially heat-resistant structure within the cell, called an endospore. Some spores may be seen directly under a light or phase-contrast microscope, whereas others may be made visible by staining.

Intracellular Inclusions A wide variety of materials may be deposited within cells, often as a means of energy storage. Some are readily visible under a microscope, whereas others require staining before they become apparent. Examples include poly-p-hydroxy-butyrate (PHB), glycogen, polyphosphate (volutin), and elemental sulfur.

Nitrogen and Sulfur Sources Many microorganisms are able to use inorganic nitrogen in the form of ammonium and/or nitrate as their nitrogen source. Others require organic forms of nitrogen, especially certain amino acids. A relatively specialized ability is utilization, or fixation, of elemental nitrogen. Similarly, many cells can utilize sulfur in the form of sulfate, or perhaps sulfide, but others may require organic sulfur. Some organisms are able to use inorganic nitrogen or sulfur compounds as energy sources or as electron acceptors.

Carbon and Energy Sources; Terminal Electron Acceptors and Relationships to Oxygen As discussed above (Sections 10.3.1 and 10.3.2), organisms can be photo-trophic or chemotrophic, organotrophic or lithotrophic (if chemotrophs), and autotrophic or heterotrophic. The various relationships to oxygen (e.g., aerobic, anaerobic; Section 10.3.3) and the terminal electron acceptors they are able to use are also important for characterization.

Habitat Preferences/Tolerances In addition to oxygen, other environmental conditions preferred or tolerated can be useful in characterization. These include such factors as temperature, pH, and salinity, as mentioned above, but also include association with more specific habitats, such as the intestines of warm-blooded animals or plant root nodules.

Range of Substrates; Reaction Products; Presence of Specific Enzymes The range of substrates metabolizable by a microorganism, and the reaction products formed, constitute a major part of the traditional identification process. Wide ranges of substrates might be tested in what is potentially a very laborious process. Ability to grow, the presence of specific enzymes, the general nature of the products formed (e.g., acid and/or gas production), and specific chemical products might be looked for. Production of catalase, which decomposes potentially toxic hydrogen peroxide to oxygen and water, and oxidase, which mediates the oxidation of some cytochromes, are two common tests for enzymes; both are related to the ability to grow aerobically. Modern techniques include some commercial tests, such as Biolog (Figure 10.16; Biolog, Inc., Hayward, California, www.biolog.com), capable of screening many compounds at once. Other systems, such as Enterotube (Becton, Dickinson and Company, Franklin Lakes, New Jersey, www.bd.com) and API strips (bioMerieux, Inc., Durham, North Carolina, www.biomerieux-inc.com), have been developed for identifying species within a particular family, such as Enterobacteriaceae (the enteric Proteobacteria; see Section 10.5.6).

Figure 10.16 Biolog test; 95 test compounds and a control well are included in each plate. The plate shown was used to identify a gram-negative bacterium as Leminorella grimontii, based on comparing the pattern of positive (dark) and negative tests to results in a database. (Photo courtesy of Biolog, Inc.)

Figure 10.16 Biolog test; 95 test compounds and a control well are included in each plate. The plate shown was used to identify a gram-negative bacterium as Leminorella grimontii, based on comparing the pattern of positive (dark) and negative tests to results in a database. (Photo courtesy of Biolog, Inc.)

Pathogenicity Although most prokaryotes are free-living saprobes or saprophytes (organisms that feed on dead organic material), some bacteria are associated with diseases of humans, other animals, or plants (Chapter 12). Often, these pathogens (disease-causing organisms) infect a single species, although some have a relatively wide range of hosts. Bacteria are also part of the normal biota of higher organisms (e.g., on the skin or in the intestinal tract of humans) without causing disease. Occasionally, opportunistic pathogens, which normally are free-living, may attack a compromised (weakened) host.

Immunological Properties Antibodies may be used to detect specific antigens, which are typically cell surface proteins. Such testing is used widely in clinical microbiology for identification of pathogens. It can be highly specific, capable of differentiating among the sometimes numerous strains, or serotypes, of a single species.

Inhibition/Resistance Patterns Inhibition of growth or activity by certain chemicals may be a helpful diagnostic tool. The pattern of resistance to specific antibiotics, with their different modes of action, can be particularly useful.

Analysis of Fatty Acids Different species have different fatty acid compositions of their cell membrane lipids. Composition will also change based on growth conditions, but if these are standardized and the fatty acids extracted and analyzed, the resulting pattern can be highly useful in identification. The most widely used procedure involves forming the methyl ester of the fatty acid to make it readily analyzable by gas chromatography. FAME (fatty acid methyl ester) profiles (Figure 10.17) can be compared to a database for rapid identification of many isolates.

% G + C Composition Recall from Chapter 3 that guanine is always paired with cyto-sine in DNA, and adenine with thymine. Thus, the base-pair composition of an organism's

Figure 10.17 Fatty acid methyl ester (FAME) profiles showing different patterns for (a) Serratia marcescens and (b) Tsukamurella paurometabolum. (Courtesy of Michael Fleming.)

DNA can be specified either by any single nucleotide or by the sum of either A + Tor G + C. By convention, % G + C is used as an indication of genetic relatedness. Organisms with very different % G + C compositions cannot be closely related. On the other hand, % G + C does not indicate the specific sequences of the bases, so that organisms with similar % G + C values are not necessarily related.

DNA Hybridization One means of genotyping cells is by comparing the similarity of sequences of bases in their DNA. To compare two organisms, the DNA of one is first labeled (often with radioactive 32P). The DNA extracted from both organisms is then broken mechanically into small fragments and heated to separate the two complementary strands (denatured). The labeled DNA and an excess of the unlabeled DNA are next mixed together and cooled to allow them to reanneal (re-form a double strand). The extent to which the two different DNAs are able to combine with each other, or hybridize, is a measure of how similar the base sequences are. This can be determined by the amount of radioactivity present in the reannealed DNA.

Molecular Probes A large number of molecular probing techniques are now available or under development. One group of methods of particular interest is referred to as fluorescent in situ hybridization (FISH). In a generalized FISH approach, an oligonucleotide (short chain of nucleotides, typically 15 to 25 bases for FISH) containing a sequence characteristic of a particular taxon of interest is labeled with a fluorescent dye. The labeled oligonucleotide is then allowed to hybridize with the complementary sequence in the organism in situ. With fluorescence microscopy, it is then possible to see the numbers and location of the brightly colored target organism within a natural community. Depending on the degree of specificity of the sequence chosen, this approach potentially can be used to identify organisms belonging to any taxonomic level ranging from domain (e.g., Bacteria) to a particular species or even strain.

Another type of probe is used in ribotyping. Restriction enzymes break DNA at points where specific sequences of nucleotides occur. If DNA is treated with one or more such enzymes, fragments of DNA are produced. These fragments are then separated by size on a gel and probed with 16S rRNA genes. For treatment with a particular set of restriction enzymes, the resulting pattern of DNA fragment sizes will be characteristic of the organism.

Denaturing gradient gel electrophoresis (DGGE) analysis offers a characterization of the GC composition of DNA segments coding for rRNA or other genes. Following tagging and amplification using PCR (Section 6.3.3), these fragments are drawn electrophoreti-cally (i.e., attracted by their electrical charge) through gel tracks laden with a gradient matrix of a denaturing agent (e.g., urea ranging from 30 to 55%). Since the G-C triple bond is stronger than the A-T double bond, it does not denature until farther along in the gradient. Fully denatured fragments stop migrating, resulting in a linear separation of the fragments into a banding pattern that is characteristic of the organism (Figure 10.18). A similar approach is used in thermal gradient gel electrophoresis (TGGE), except that a temperature gradient is used for denaturing the DNA.

16S rRNA Analysis The primary tool used today in determining phylogeny is 16S rRNA analysis, developed largely by Carl Woese beginning in the early 1970s. Prokaryo-tic ribosomal RNA (rRNA) is composed of three molecules, referred to by their sedimentation coefficients measured in Svedberg units (S) as 5S (containing ^120 bases), 16S (~1500), and 23S (^2900). Woese initially worked with 5S fragments but found they were too small to yield sufficient information. The emphasis then shifted to 16S rRNA (and the comparable 18S rRNA found in eukaryotes) based on its larger, yet still manageable number of nucleotides.

One important advantage of using the nucleotide sequences of 16S (or 18S) rRNA for determining relationships is that these molecules occur in all organisms. Also, because of

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Figure 10.18 Denaturing gradient gel extraction track profiles.

their central role in the process of gene expression, there are strong selective pressures, making it unlikely that they will undergo rapid changes. This leads to the presence of extended regions of the molecule that are highly conserved (little changed) and which therefore are useful for establishing distant phylogenetic relationships. On the other hand, they also have an adequate number of variable regions that can be used to examine closer relationships. Thus, 16S rRNA can be used as an evolutionary clock, or chronometer, to measure the phylogenetic distance between taxa at a variety of levels based on the number of nucleotide differences—representing stable mutations—that have occurred over time.

One method to sequence 16S rRNA begins with extraction of a cell's RNA. A small DNA oligonucleotide primer (15 to 20 nucleotides in length) that is complementary in its base sequence to a conserved region of the 16S rRNA molecule is added. Reverse transcriptase can then be used to generate complementary DNA (cDNA), which in turn is amplified using PCR. The nucleotide sequences of these cDNA are then determined, and the original sequence of the rRNA is deduced from them. Another common approach is to extract and sequence the DNA of the gene that codes for the 16S rRNA rather than the RNA itself.

The 16S rRNA sequences of thousands of species have now been determined. Using advanced computer algorithms, short (6 to 10 nucleotides) signature sequences have been found that are highly consistent within groups of organisms. Also, phylogenetic trees can be generated in which the evolutionary distance between two groups is indicated by the length of the connecting lines (Figure 10.19).

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