Oxidation-Fermentation Tests

Oxidation-Fermentation Tests

Determining a bacterium’s oxidation-fermentation (0/F) pattern is an important test in the identification of bacteria. In particular it is helpful in differentiating members of the family Enterobacteriaceae, glucose fermenters, from the aerobic pseudo monads and similar gram-negative bacteria, which are nonfermenters.

Carbohydrate fermentation tests determine the ability of a microorganism to ferment a specific carbohydrate incorporated into a basal medium. During fermentation, glucose enters the glycolysis pathway resulting in the formation of pyruvic acid, which can be further oxidized to other acids. The end product of carbohydrate fermentation is acid or acid with gas. Acid formation is detected with pH indicators added to the medium. Some bacteria produce primarily a single acid, such as the streptococci, which are homolactic acid fermenters. Other bacteria produce several different acids, including lactic acid, propionic acid, and succinic acid. These organisms are referred to as mixed acid fermenters.

Oxidation also begins by glucose entering the glycolysis pathway; however, the pyruvic acid formed from glycolysis is further oxidized to CO2,Oxidation requires oxygen (aerobic respiration) or another inorganic molecule (anaerobic respiration), such as nitrate (NO~ as a terminal electron acceptor. Higher acidity is produced during fermentation. The same medium is used for both oxidative and fermentative tests. Characteristically, oxidizers and fastidious fermenters often produce either weak or small amounts of acids from carbohydrates. In media that contain large amounts of peptones (2.0%), such as triple sugar iron (fSI) agar, whatever acids produced are neutralized or masked by the alkaline reaction from peptone utilization. To detect small amounts of acids produced, whether fermentatively or oxidatively, Hugh and Leifson developed an OfF basal medium (OFBM) that contains the same concentration of carbohydrates (1 %) found in the TSI medium but a lower concentration of peptones (0.2%). The pH indicator is bromthymol blue. Uninoculated medium is green; in an acid environment the indicator is yellow, and it is blue in an alkaline environment (Figure 9-2). Table 9-1 shows the differences in reactions among

different groups of organisms. When OfF tests are preformed, two tubes of Hugh-Leifson OFBM are inoculated; one is overlayed with sterile mineral oil to create an anaerobic environment (closed), and the other tube is left aerobic (open), without mineral oil overlay.

When acid is produced in both tubes, the isolate is an oxidizer and fermenter. The presence of acid in the closed tube indicates only that the organism is a fermenter, whereas the presence of acid in the open tube indicates an oxidizer. The open tube mayor may not show acidity. No acid production in the open tube may indicate that the organism is a nonoxidizer; in fact many nonoxidizers will produce an alkaline reaction from peptone utilization.

figure 9-2

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