Laboratory Investigations in Microbiology

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Chapter 19: Special enzymes

Aside from purely metabolic reactions designed to obtain food, many bacteria produce extracellular enzymes for other functions related to survival in their environment:

Catalase test (Atlas p. 44)Catalase test. Notice bubbles in slants on left and right. For bacteria living in oxygen-rich environs, the enzyme catalase is essential for disposing of toxic hydrogen peroxide. Together with the enzyme superoxide dismutase, catalase can dispose of oxygen free radicals. Bacteria that are obligate aerobes (e.g. Micrococcus) or facultative anaerobes (e.g. Staphylococcus) are almost always catalase-positive. Bacteria that are aerotolerant, such as Enterococcus and Streptococcus, are usually found in anaerobic environments and lack the enzyme catalase. Likewise. obligate anaerobes, such as Clostridium, do not produce this enzyme. Catalase perform the following reaction:

2H2O2 + catalase 2H2O + O2 

When hydrogen peroxide is added to a bacterial lawn or colony, bubbles of oxygen gas are given off.  This bubbling indicates a positive catalase test.

Urease test (Atlas p. 81): Several bacteria strains have the ability to break down the nitrogen-containing compound urea. The products of urea hydrolysis are carbon dioxide and ammonium:

Urea + urease + H2O CO2 + 2 NH3

The ammonia generated can benefit bacteria in two ways. First, ammonia is a base, since it buffers free hydrogen ions (acid): Urease test: negative result (left) and positive result (right)

NH3 + H+ NH4+

Bacteria that live in acidic environments, such as the pathogen Helicobacter pylori (the cause of Peptic Ulcer Disease), use urease to buffer the acid around them, enabling them to survive and grow. Other bacteria use urease to obtain ammonia as a source of nitrogen. Ammonia is useful for the synthesis of amino acids and nucleotides from precursor molecules:

Pyruvate +NH3  alanine 

Urease activity is detected by culturing bacteria in a broth containing urea and a pH indicator. Hydrolysis of urea results in an increase in pH of the medium and a change in the medium's color to purple. This indicates a positive urease test. A negative urease test is observed when the color of the broth remains red/orange.

Gelatinase test (Atlas p. 54): When collagen is boiled, gelatin is produced. Gelatin is a protein thGelatinase test. Notice the liquid medium in the tube on the solidifies at room temperature, but is liquid at 35C. Gelatin (collagen), of course, is a major component of connective tissue in animals, forming a defensive barrier between the outer layers of skin and the body's interior. Pathogenic bacteria sometimes produce an enzyme to break down (hydrolyze) this protein, allowing them to spread further in the body they infected. Like DNAse, gelatinase is a virulence factor that makes the bacteria more harmful. We can test for the presence of gelatinase by incubating bacteria in a gelatin deep. During incubation, the gelatin melts, forming a liquid for bacterial growth. If bacteria hydrolyze the gelatin, producing amino acids, the protein can no longer gel when cooled down. A gelatin deep that remains liquid after cooling down indicates a positive test for gelatinase (EA). If the deep hardens again after cooling, the test is negative (EC, PV).  

Coagulase test (Atlas p. 46): Yet another enzyme that aids pathogenic bacteria is coagulase.  Staphylococcus aureus is perhaps the best-known bacterium with this enzyme. Coagulase causes plasma to clot (coagulate). bacteria that are threatened by white blood cells can secrete this enzyme, producing a clot around themselves that the white blood cells cannot penetrate. This gives the bacteria time to multiply. We can test for this enzyme by inoculating a small volume of rabbit serum with bacteria. If a clot forms in the serum, the test is positive for coagulase. If the medium remains liquid, the test is negative.

Materials and Methods

Materials per lab group
Catalase test (EF, EC, PA)

Reagent: Hydrogen peroxide 3%

  1. Label 3 sterile TSA slants
  2. Inoculate the slants with EF, EC, and PA
  3. Bring tubes up front to be incubated

Next lab period:

  1. Add a dropper full of hydrogen peroxide to each slant. 
  2. Record your data. 
Urease test:
  1. Label 3 urease tubes 
  2. Inoculate the urease broths with PV, EF, and EC
  3. Bring tubes up front to be incubated

Next lab period:

  1. Observe broths for a color change. 
  2. Record data:
Gelatinase test (inoculations from last week)

Next lab period

  1. Allow tubes to cool down (they may already be cool) until the gelatin deeps labeled CONTROL (provided by instructor) are solid
  2. Tilt tubes to determine whether the gelatin has solidified or remains liquid. Record data.
Coagulase test
  1. Label 2 coagulase (rabbit serum) test tubes "SA" and "SE"
  2. Inoculate each with a loop full of the appropriate culture
  3. Incubate tubes

Next lab period

  1. Tilt tubes to determine if the serum is completely liquid or if a clot is present. Record data



2003 - 2019 Jos de Ondarza, Ph.D.