Background
The purpose of this experiment is to test whether or not a microorganism produces the catalase enzyme. If an organism can produce catalase, it can break down hydrogen peroxide to water and free oxygen. The test is important because it can differentiate between aerobic and anaerobic organisms.
Hydrogen peroxide, H2O2, is formed as a byproduct of aerobic respiration. This substance has undesirable effects since it can damage cell membranes and affect the active sites of some enzymes (Lammert 150).
Materials and Methods
The materials for this laboratory experiment include: cultures of S. aureus, M. luteus, L. lactis; Trypticase soy agar plates; 3% H2O2 solution; Bunsen burner; inoculating loop; glassware marking pencil.
The procedure consists of the following steps (Cappuccino 201). Using the glass marking pencil, mark the bottom of one agar plate with a dividing line and write S. aureus on one side of the line and M. luteus on the other side. Using the glassware marking pencil, mark the bottom of a second agar plate with a dividing line and write L. lactis on one side of the line and “Blank” on the other side. Using aseptic technique, inoculate the S. aureus side of the plate with the S. aureus culture by making a vertical line. Similarly, inoculate the M. luteus side of the plate with the M. luteus culture. Repeat the technique for the second plate using the L. lactis 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 complete, remove the plates from the incubator. Apply three drops of the 3% H2O2 to the surface growth of each organism. Observe each plate for a period of 30 seconds and note the appearance of bubbling as an indicator that catalase is present.
Results
Figure 1.1 L. lactis and control
Figure 1.2 S. aureus and M. luteus
The plate containing the S. aureus organism showed a moderate amount of bubbling The plate containing the M. luteus organism showed a moderate amount of bubbling. The plate containing the L. lactis organism showed a minor amount of bubbling. Figure 1.1 shows an image of the agar plate result for L. lactis. Figure 1.2 shows an image of the agar plate results for S. aureus and M. luteus.
Conclusion
The test result of the catalase test for S. aureus was positive and matched the expected result (Holt 544). The bacteria of the Genus Staphylococcus are facultative anaerobes. They can use aerobic respiration or fermentation metabolic pathways (Holt 532).
The test result of the catalase test for M. luteus was positive and matched the expected result (Holt 530. The bacteria of the Genus Micrococcus are strict aerobes. They use aerobic respiration as their metabolic pathway (Holt 530).
The test result of the catalase test for L. lactis was not conclusive as there was some noticeable bubbling present. The expected result for L lactis is negative for catalase. The bacteria of the Genus Lactococcus are facultative anaerobes (Holt 528). They use fermentation as their metabolic pathway (Holt 529). There is no clear explanation for the unexpected false positive catalase reaction. It is possible that the sample culture had been contaminated with a substance that reacts with H2O2.
Review Questions:
1. Explain the toxic effect of O2 on strict anaerobes.
With the absence of catalase production in strict anaerobes, these organisms can suffer damage from reactive oxygen species such as H2O2. Their cell membranes and DNA can get damaged resulting in death of the organism.
2. Illustrate the chemical reaction involved in the degradation of hydrogen peroxide in the presence of catalase.
2 H2O2 —— catalase —– > 2 H2O + O2
3. Would catalase be classified as an endoenzyme or an exoenzyme?
Catalase would be classified as an endoenzyme since it is needed to act on the products of aerobic respiration which take place within the cell.
4. Account for the ability of streptococci to tolerate O2 in the absence of catalase.
Streptococci are facultative anaerobes. They have peroxidase which is used to convert hydrogen peroxide to water and oxygen.
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 7-April-2014 2:25 PM
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