Characterization of Prokaryotes

Before looking at the various groups of Bacteria and Archaea, it is useful to describe briefly some of the characteristics commonly used to differentiate among them. In many cases it is necessary first to isolate the organism and then to grow it in pure culture (only one species present) before testing.

Shape Prokaryotic cells show a remarkable diversity of shapes (Figure 10.2 showed a few). Most common are cylindrical rods (Figure 10.8), also called bacilli (singular,

Figure 10.8 Rod-shaped bacteria: Pseudomonas. (SEM image courtesy of the University of Iowa Central Microscopy Research Facility.)

Figure 10.8 Rod-shaped bacteria: Pseudomonas. (SEM image courtesy of the University of Iowa Central Microscopy Research Facility.)

Figure 10.9 Cocci: Staphylococcus. (SEM copyright Dennis Kunkel Microscopy, Inc.)

bacillus), and spherical cells (Figure 10.9), called cocci (singular, coccus). Variations include short rods (coccobacilli), bent or comma-shaped rods (vibrios), and greatly elongated rods. There are also a variety of spiral cells (Figure 10.10), cells with specialized appendages, and those with irregular, indefinite, or special shapes.

The shape of a cell can also change during its life cycle. For example, Arthrobacter (Section 10.5.7) cells routinely alternate between cocci and rods.

Size Typical rods may be 0.5 to 1.0 mm in diameter and 2 to 4 mm long, but size can vary tremendously, from diameters of 0.1 mm or less up to lengths of 200 mm or more. Most cocci have diameters of 0.5 to 2.0 mm, but again there is a considerable range.

Growth Form Many microorganisms grow as individual, single cells. However, some grow in chains, or filaments, composed of a single species (Figure 10.11). Others may grow in clusters, or be found in clumps (floc; also in Figure 10.11) or other associations (such as biofilms or floating mats), sometimes with characteristic shapes, and often with

Figure 10.10 Spiral-shaped bacteria: Campylobacter. (SEM copyright Dennis Kunkel Microscopy, Inc.)

Figure 10.10 Spiral-shaped bacteria: Campylobacter. (SEM copyright Dennis Kunkel Microscopy, Inc.)

Figure 10.11 A filamentous bacteria growing with floc in an activated sludge wastewater treatment plant.

multiple species. Some go through a life cycle in which their form or type of association changes in a predictable way.

Prokaryotic cells usually reproduce by binary fission, or splitting into two roughly equal "daughter" cells. However, some bacteria reproduce by budding, in which a smaller new cell separates from the original one, and some bacteria produce special structures for releasing new cells for dispersal.

Stalked bacteria (Figure 10.12) produce an appendage that usually serves for attachment to a surface. If the stalk consists of an extrusion of cytoplasm surrounded by the cell membrane and wall, it is called a prostheca (plural, prosthecae). Some cells may attach directly to a surface with a small structure known as a holdfast.

Staining The way in which their cells respond to certain colored chemicals (stains) is used to differentiate some species. The most widely used staining technique for bacteria is the Gram stain (Figure 10.13), developed in 1884 by Dutch bacteriologist Hans Christian

Figure 10.12 Stalked bacteria growing on a filament in activated sludge.

Solutions

Solution 1 : Hucker' s crystal violet

Component A: 2 g crystal violet in 20 mL 95% ethanol; Component B: 0.8 g ammonium oxalate in 80 mL distilled water; Mix A and B (lasts 3 months).

Solution 2: modified Lugol's solution

Mix 1 g iodine and 2 g potassium iodide in 300 mL distilled water; Store in dark (lasts 3 months).

Solution 3: decolorizing agent 95% ethanol

Solution 4: counterstain

Safranin O solution: 2.5 g in 100 mL 95% ethanol; Mix 10 mL in 100 mL distilled water.

Procedure

Make thin smear of culture on glass slide. Allow to thoroughly air dry. Cover with Solution 1 for 1 minute. Gently rinse with tap or distilled water. Cover with Solution 2 for 1 minute. Hold slide at angle.

Decolorize drop by drop with Solution 3 until no more color runs off. Rinse with water. Q

Counterstain with Solution 4 for 1 minute. Rinse with water, then blot dry. Put drop of immersion oil directly on slide. Observe under microscope at 1000 X.

Results

Blue/violet = Gram positive; Red = Gram negative.

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