Metabolic Differences

The identification of most prokaryotes relies on analyzing their metabolic capabilities such as the types of sugars utilized or the end products produced. In some cases these characteristics are revealed by the growth and colony morphology on cultivation media, but most often they are demonstrated using biochemical tests. ■ growth of colonies, p. 103

Culture Characteristics

Microorganisms that can be grown in pure culture are the easiest to identify, because it is possible to obtain high numbers of a single type of microorganism. Even the colony morphology can give initial clues to the identity of the organism. For example, colonies of streptococci are generally fairly small relative to many

Streptococcus pneumoniae

Neisseria gonorrhoeae (Gram-negative diplococci)

White blood cells

Figure 10.3 Gram Stains of Clinical Specimens (a) Sputum showing Gram-positive Streptococcus pneumoniae and (b) male urethra secretions showing Gram-negative Neisseria gonorrhoeae inside white blood cells.

Streptococcus pneumoniae

Neisseria gonorrhoeae (Gram-negative diplococci)

White blood cells

Figure 10.3 Gram Stains of Clinical Specimens (a) Sputum showing Gram-positive Streptococcus pneumoniae and (b) male urethra secretions showing Gram-negative Neisseria gonorrhoeae inside white blood cells.

other bacteria such as staphylococci. Colonies of Serratia marcescens are often red when incubated at 22°C due to the production of a pigment. Pseudomonas aeruginosa often produces a soluble greenish pigment, which discolors the growth medium (see figure 11.13). In addition, cultures of P. aeruginosa have a distinct fruity odor. ■ Pseudomonas aeruginosa pigment production, p. 281

The use of selective and differential media in the isolation process can provide additional information that helps identify an organism. For example, if a soil sample is plated onto medium that lacks a nitrogen source and is then incubated aerobically, any resulting colonies are likely members of the genus Azotobacter. The ability to fix nitrogen under aerobic conditions is an identifying characteristic of these bacteria. ■ Azotobacter, p. 283 ■ nitrogen fixation, p. 775 ■ selective media, p. 93 ■ differential media, p. 94

In a clinical lab, where rapid but accurate diagnosis is essential, specimens are plated onto media specially designed to provide important clues as to the identity of the disease-causing organism. For example, a specimen taken by swabbing the throat of a patient complaining of a sore throat is inoculated onto blood agar, a nutritionally rich medium containing red blood cells. This differential medium enables the detection of the characteristic ^-hemolytic colonies that are typical of Streptococcus pyogenes (see figure 4.6). Urine collected from a patient suspected of having a urinary tract infection is plated onto MacConkey agar, which is both selective and differential. MacConkey agar has bile salts, which inhibit the growth of most non-intestinal organisms, and lactose along with a pH indicator,

10.2 Using Phenotypic Characteristics to Identify Prokaryotes which differentiates lactose-fermenting organisms. E. coli, the most common cause of urinary tract infections, forms characteristic pink colonies on MacConkey agar due to its ability to ferment lactose (see figure 4.7). Other bacteria can also grow and ferment lactose on this medium, however, so colony appearance alone is not enough to conclusively identify E. coli.

■ blood agar, pp. 92,94 ■ MacConkey agar, p. 94

Biochemical Tests

Growth characteristics on culture media can narrow down the number of possible identities of an organism, but biochemical tests are generally necessary for a more conclusive identification. One of the simplest of these is to assay for the enzyme catalase (figure 10.4a). Nearly all bacteria that grow in the presence of oxygen are catalase positive. Important exceptions are the lactic acid bacteria, which include members of the genus Streptococcus. Thus, if a throat culture yields b-hemolytic colonies but further testing reveals they are all catalase positive, then Streptococcus pyogenes has been ruled out. ■ catalase, p. 89 ■ Streptococcus pyogenes, p. 565

Most biochemical tests rely on a pH indicator or chemical reaction that results in a color change when a compound is degraded. For example, to test for the ability of an organism to ferment a given sugar, a broth medium containing that sugar and a pH indicator is employed. Fermentation of the sugar results in acid production, which lowers the pH, resulting in a color change from pink to yellow; an inverted tube traps any gas that is produced (figure 10.4b). A medium designed to detect urease, an enzyme that degrades urea to produce carbon dioxide and ammonia, utilizes a different pH indicator that turns bright pink in alkaline conditions (figure 10.4c). The characteristics of these and other important biochemical tests are summarized in table 10.4.

The basic strategy for identifying bacteria based on biochemical tests relies on the use of a dichotomous key, which is essentially a flow chart of tests that give either a positive or negative result (figure 10.5). Because each test often requires an incubation period, however, it would be too time-consuming to proceed one step at a time. In addition, relying on a single biochemical test at each step could lead to misidentification. For example, if a strain that normally gives a positive result for a certain test lost the ability to produce a key enzyme, it would instead produce a negative result. Therefore, simultaneously inoculating a battery of different tests identifies the organism faster and more conclusively.

In certain cases, biochemical testing can be done without culturing the organism. Helicobacter pylori, the cause of most stomach ulcers, can be detected using the breath test, which assays for the presence of urease. The patient drinks a solution containing urea that has been labeled with an isotope of carbon. If H. pylori is present, its urease breaks down the urea, releasing labeled carbon dioxide, which escapes through the airway. Several hours after drinking the solution, the patient exhales into a balloon. The expired air is then tested for labeled carbon dioxide. This test is less invasive and, consequently, much cheaper and faster than the stomach biopsy that would otherwise need to be performed to culture the organism. ■ Helicobacter pylori, p. 605

10.2 Using Phenotypic Characteristics to Identify Prokaryotes

(a)
(b)

Figure 10.4 Biochemical Tests (a) Catalase production. Bacteria that produce catalase break down hydrogen peroxide (H2O2) to release oxygen gas (O2) which causes the bubbling. (2 H2O2 —> 2 H2O + O2). A negative catalase test is shown in the right tube. (b) Sugar fermentation.The tubes on the left and right show acid (yellow color) and gas.The center tube shows no color change, indicating that the sugar was not utilized. (c) Urease production. Breakdown of urea releases ammonia, which turns the pH indicator pink.

Figure 10.4 Biochemical Tests (a) Catalase production. Bacteria that produce catalase break down hydrogen peroxide (H2O2) to release oxygen gas (O2) which causes the bubbling. (2 H2O2 —> 2 H2O + O2). A negative catalase test is shown in the right tube. (b) Sugar fermentation.The tubes on the left and right show acid (yellow color) and gas.The center tube shows no color change, indicating that the sugar was not utilized. (c) Urease production. Breakdown of urea releases ammonia, which turns the pH indicator pink.

1 Nester-Anderson-Roberts: Microbiology, A Human Perspective, Fourth Edition

1 II. The Microbial World 1 10. Identification and 1 Classification of Prokaryotes

© The McGraw-Hill Companies, 2003

Chapter 10 Identification and Classification of Prokaryotes

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