Ex. 5-4 Catalase test

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Ex. 5-4 Catalase test

• MCB 1000 Lab. Prof. Meléndez

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Catalase test

• The catalase test is used to detect the presence
  of the enzyme catalase in bacteria. Catalase
  serves to neutralize the bactericidal effects of
  hydrogen peroxide. Its concentration in bacteria
  has been correlated with pathogenicity. This
  enzymatic test is essential in the scheme of
  identification for gram-positive organisms and
  certain gram-negative organisms. It is a primary
  test used in the differentiation of staphylococci
  and streptococci.

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Catalase test

• TheoryThe catalase enzyme serves to neutralize
  the bactericidal effects of hydrogen peroxide .
  Catalase expedites the breakdown of hydrogen
  peroxide (H2O2- (toxic) into water (H2O) and
  oxygen(O2)
• 2H2O2 Catalase ? 2H2O O2(gas bubbles)
• This reaction is evident by the rapid formation
  of bubbles

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Catalase test

• The catalase test facilitates the detection of
  the enzyme catalase in bacteria. It is essential
  for differentiating catalase-positive
  Micrococcaceae from catalase-negative
  Streptococcaceae.
• The catalase test is also valuable in
  differentiating aerobic and obligate anaerobic
  bacteria, as anaerobes are generally known to
  lack the enzyme . In this context, the catalase
  test is valuable in differentiating aerotolerant
  strains of Clostridium, which are catalase
  negative, from Bacillus, which are catalase
  positive

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Slide catalase test results. Hydrogen peroxide
was added directly to the culture on a microscope
slide. A positive reaction produced by
Staphylococcus aureus is indicated by bubbling a
negative reaction produced by Streptococcus
pyogenes is indicated by lack of bubbling. (Karen
Reiner, Andrews University, Berrien Springs, MI)
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Practice questions

• Read lab. manual pages 243-246 and fill out
  following chart Determine if situation on first
  column will cause a false positive or a false
  negative. Explain why?

Description of situation Effect on test Why?
1. Adding hydrogen peroxide to slide first, then adding cells and mixing cells with hydrogen peroxide using loop. False positive Metal wire of loop can react with hydrogen peroxide causing bubbles due to oxidation of metal, not the enzyme activity.
2. Hydrogen peroxide is expired (old)
3. Cells are heat fixed into slide
4. Cells are very old
5. Contamination of culture with aerobic microorganism
6. Dirty slide was used
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References

• http//www.microbelibrary.org/index.php/library/la
  boratory-test/3226-catalase-test-protocol
• Leboffe, Michael J. , and Burton E. Pierce.
  Microbiology Laboratory, Theory and application
  (brief edition). Colorado Morton Publishing
  Company, 2008.