Background
This laboratory exercise illustrates the capability of some bacteria to produce the enzyme cytochrome oxidase which is essential for the electron transport chain (ETC) of cellular respiration. The enzyme is needed to catalyze the oxidation reaction of the cytochrome molecule at the end of the ETC. The electron acceptor in the reaction is oxygen and the product is water (Lammert 152). Bacteria that are aerobic, facultative anaerobic, or microaerophilic show a positive test result when the reagent p-Aminodimethylaniline oxalate is applied to a growing colony of the organism. In the presence of cytochrome oxidase, the reagent changes color to purple. If no cytochrome oxidase is present, the reagent color does not change (Cappuccino 205).
Materials and Methods
The materials for this laboratory experiment include: cultures of Escherichia coli, Lactococcus lactis, Pseudomonas aeruginosa; Trypticase soy agar plates; p-Aminodimethylaniline oxalate reagent; Bunsen burner; inoculating loop; glassware marking pencil.
The procedure consists of the following steps. Using the glass marking pencil, mark the bottom of one agar plate with a dividing line and write E. coli on one side of the line and L. lactis on the other side. Using the glass marking pencil, mark the bottom of a second agar plate with a dividing line and write P. aeruginosa on one side of the line and “Blank” on the other side. Using aseptic technique, inoculate the E. coli side of the plate with the E. coli culture by making a vertical line. Similarly, inoculate the L. lactis side of the plate with the L. lactis culture. Repeat the technique for the second plate using the P. aeruginosa culture. The side marked “Blank” is not inoculated
All plates are to be incubated in an inverted position at 37 degrees Celsius for a period of 48 hours.
After the incubation period is completed, remove the plates from the incubator. Apply three drops of the reagent to the surface growth of each organism. Observe each plate for a period of 30 seconds and note any changes to the color of the reagent.
Results
The reagent applied to the plate with the E. coli organism did not show a color change. The reagent applied to the plate with the P. aeruginosa did show a slight color change. The reagent applied to the plate with L. lactis did show a change of color to purple.
Figure 1.1 shows an image of the agar plate results for the E. coli and L. lactis. Figure 1.2 shows an image of the agar plate result for P. aeruginosa.
Figure 1.1 Agar plates inoculated with E. coli and L. lactis.
Figure 1.2 Agar plates inoculated with P. aeruginosa.
Conclusions
The test result of the oxidase test for E. coli was negative and matched the expected result (Holt 179). The bacteria of the Genus Escherichia are facultative anaerobes. They can use aerobic respiration or fermentation metabolic pathways.
The test result of the oxidase test for P. aeruginosa was not as expected. There was a slight color change which indicates the presence of the cytochrome oxidase enzyme. The Genus Pseudomonas has the characteristic of being strict aerobes with oxygen as the terminal acceptor (Holt 93).
The test result of the oxidase test for L. lactis was not as expected. There was a noticeable color change which indicates the presence of the cytochrome oxidase enzyme. The Genus Lactococcus has the characteristics of using fermentation as their metabolic pathway (Holt 528).
The fact that the oxidase results for P. aeruginosa and L. lactis are wrong seems to indicate that a procedural error might have been made during inoculation or labeling of the plates.
Review questions:
1. What is the function of cytochrome oxidase?
The function of cytochrome oxidase is to catalyze the oxidation reaction of cytochrome c molecules in the membrane of bacteria and the mitochondria of eukaryote organisms. It is the last enzyme used in the chain of reactions of the electron transport chain. The cytochrome oxidase protein transfers electrons from cytochrome c molecules to molecules of oxygen to produce molecules of water.
2. Why are strict aerobes oxidase-positive?
Strict aerobes are oxidase-positive because their means of cellular metabolism is aerobic respiration using the electron transport chain mechanism. The cytochrome oxidase is an essential component of the electron transport chain mechanism.
3. The oxidase test is used to differentiate among which groups of bacteria?
The oxidase test is used to differentiate bacteria that use aerobic respiration in their metabolism from those that rely on other means of energy generation such as fermentation.
4. What is the function of the test reagent in this procedure?
The test reagent is an electron donor and mimics cytochrome c. The cytochrome oxidase enzyme can also catalyze the reaction of the reagent and oxygen. The main purpose of the reagent is to detect the presence of the cytochrome oxidase enzyme that is produced by the bacterial organisms in this test.
5. Your instructor asks you to isolate and identify the organisms in an unknown culture. You find that the culture contains two gram-negative bacilli that produce swarming colonies. What biochemical test would you use to identify the bacilli? Justify your answer.
Gram-negative bacilli can be differentiated on the basis of the metabolic pathway that they use to produce energy. They can be differentiated as fermenters or non-fermenters. The oxidase test should be run to determine if the organism uses aerobic respiration or not.
Works cited
Holt, John G., et. al. Bergey’s manual of determinative bacteriology – 9th ed. Baltimore, MD: Williams & Wilkins, 1994. Print
Cappuccino, James G. Microbiology: a laboratory manual – 10th ed. Glenview, IL: Pearson, 2014. Print.
Lammert, John M. Techniques in Microbiology. San Francisco, CA: Pearson Education Inc., 2007. Print.
Last updated on 7-April-2014 at 2:43 PM
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