Chapter 5.6 Catalase Test
Chapter 5.7 Oxidase Test
BMED-4440-DB
Daisy Vela
Introduction
We tested a variety of bacteria in this lab using the Oxidase and Catalase tests. (Leboffe
and Pierce 2015), the catalase test is used to determine whether the bacteria have the enzyme that
"converts hydrogen peroxide into water and gaseous oxygen." In the electron transport chain,
oxygen is the last electron acceptor used by most facultatively anaerobic and aerobic
microorganisms. Hydrogen peroxide is produced when the ETC molecule flavoprotein transports
electrons to oxygen directly, avoiding the subsequent electron carrier. Since hydrogen peroxide
is harmful to cells, catalase must transform it into gaseous oxygen and water. (Leboffe and
Pierce 2015), the oxidase test is now "designed to identify the presence of cytochrome c
oxidase." Glucose is oxidized by an electron transport chain found in the majority of aerobes and
some anaerobes. Numerous co-enzymes are decreased when glucose is oxidized. If the bacterium
possesses a mitochondrial ETC that is similar to that of eukaryotes, cytochrome c oxidase will
oxidize these coenzymes to return them to their oxidized forms. Although cytochrome c oxidase
can be oxidized by the oxidase test, tetramethyl-p-phenylenediamine can also lower it.
Objectives
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Determine whether a bacterium has catalase.
Determine whether a bacterium has cytochrome C oxidase.
Perform the catalase test.
Perform the oxidase test.
Data Sheet
Post Lab Questions 5-6
1. Think about the advice given in the procedure to test a known catalase positive organisms
along with an unknown.
a. Is this a positive or negative control?
Positive
b. What information is provided by the results?
This demonstrates what a true positive indicates when a bacterial sample has catalase.
The unknown should behave similarly to the known catalase-positive creature if it
possesses catalase.
2. Consider the step in the tube test where hydrogen peroxide is added to the uninoculated
tube.
a. Is this a positive or a negative control?
negative control
b. What information is provided by the results?
This demonstrates the expected appearance of a negative reaction with hydrogen
peroxide. This will show that catalase is not present. This further demonstrates that
our reagent will only bubble in the presence of catalase in the sample.
3. When flavoprotein transfers electrons directly to oxygen, hydrogen peroxide is produced.
What other consequences might result from electron carriers in the ETC being bypassed?
Because the electrons won't reach the last electron acceptor, oxygen, which is the step
before ATP creation, there may be a decrease in ATP production. The proton gradient
may be upset as a result, which may interfere with the synthesis of ATP. Hydrogen
peroxide can also interfere with normal cell activity because it is harmful to cells.
4. Would a false positive from the reaction between the inoculating loop and hydrogen
peroxide be caused by poor specificity or poor sensitivity? Explain.
The low specificity would be the cause. The loop produces identical results as the
catalase enzyme.
Post Lab Question 5-7
1. Think about the advice given in the procedure to test a known oxidase positive organism
along with an unknown.
a. Is this a positive or negative control?
Positive
b. What information is provided by the results?
This demonstrates what a true positive indicates when a bacterial sample has cytochrome
c oxidase. The unknown should behave similarly to known oxidase-positive creatures if it
possesses cytochrome c oxidase.
2. Think about the 20-second time limit on the oxidase test.
a. What happens to the oxidase reagent after 20 seconds?
The bacterial enzyme cytochrome oxidase oxidizes tetramethyl-p-phenylenediamine
(TMPD), which is present in the oxidase reagent used in the test. This reaction causes the
reagent to turn purple, indicating the presence of the enzyme. Although the reaction is
time-sensitive, the reagent begins to lose its color after 20 seconds as a result of exposure
to light and air. Because of this, the test has a 20-second validity period; results obtained
beyond this time may not be trustworthy. It is essential to follow the test instructions and
review the results within the allocated time in order to ensure accurate interpretation.
b. Does this only happen after 20 seconds? If not, why is a 20-second time limit set?
No, the reaction occurs before 20 seconds. The 20-second time limit is in place to ensure
that the test is given correctly and suitably. If the reaction is allowed to run for more than
20 seconds, it may produce false positives or non-specific reactions. The exam is more
exact and precise because the reaction time is limited to 20 seconds. Furthermore, the
sample or the reagent may deteriorate if the reaction is let to go on for an extended period
of time, leading to unreliable results.
3. Provide a possible explanation as to why this test identifies the presence of cytochrome c
oxidase and not other oxidases (such as those of E. coli shown in Fig. 5.24)
This oxidase test uses a reducing substance that is unique to cytochrome c oxidase. ETC
in certain bacterial species and eukaryotes contains cytochrome c oxidase. Because they
have either cytochrome o oxidase or cytochrome d oxidase, E. coli will not produce a
positive response in this oxidase test.
Conclusion
To sum up, we completed the catalase and oxidase test satisfactorily. According to the results,
the existence of bubbles when hydrogen peroxide is introduced indicates a positive catalase test.
Although both SE and EC are catalase positive, the EC sample appeared to convert hydrogen
peroxide to water and gaseous oxygen more quickly. This is because bubbles formed in the EC
sample much more quickly than in the SE sample. We examined the presence of cytochrome c
oxidase in SE and NS using the oxidase test. The reducing plate instantly became light purple
when a sample of NS was introduced. NS has cytochrome c oxidase, which indicates that it is
oxidase positive.
Bibliography
Leboffe, M. J., & Pierce, B. E. (2015). Microbiology Laboratory Theory & Application (4th ed.).
Morton Publishing.