BISC209: Enzyme tests

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Wellesley College-BISC 209 Microbiology -Spring 2010

Enzyme Tests for Differentiation of Microorganisms by Metabolic Processes

Microorganisms vary in their ability to process nutrients and to make energy. Many of these differences stem from variations in metabolic pathways. These pathways use different enzymatically catalyzed reactions. One way to differentiate microorganisms is to test for particular enzymes that are charactistically present or absent in certain groups.
For more information on the formulations and types of media available in microbiology see: BD diagnostice Systems Difco catalog of media

Catalase test

Catalase protects aerobic bacteria from the accumulation of hydrogen peroxide, an extremely toxic form of oxygen, by catalyzing the reaction:

H2O2 --------> H2O + O2

Place a drop of fresh 3 % hydrogen peroxide (H2O2) on a clean glass slide.
Using aseptic technique, take a loopful of bacteria that you want to test and use the loop to quickly and briefly mix the cells in the peroxide.
Remove the loop and immediately look for the formation of bubbles in the emulsion.
Since the reaction of peroxide with catalase liberates bubbles of oxygen, it is easy to see the product of the reaction as escaping gas.
A positive test is indicated by the evolution of oxygen bubbles.
Bacillus or Staphylococcus species are good positive controls.

Since hydrogen peroxide is formed by the interaction of molecular oxygen with a variety of cellular reaction systems, most aerobic bacteria are catalase positive while most anaerobes are catalase negative. Lactic acid bacteria are unusual. This group lacks catalase, but sometimes forms tiny or small colonies in aerobic conditions.

When performing a catalase test, if a colony is taken from a blood-agar plate, you must keep in mind that the enzyme catalase is present in red blood cells; therefore, any carry-over of blood cells with the colony can give a false-positive reaction.

Oxy-Swab Oxidase Test

The Oxidase test is used to detect the presence of the enzyme cytochrome c oxidase. Organisms such as Micrococcus luteus, Neisseria, and Pseudomonas species are positive for this enzyme. If cytochrome c oxidase is produced by the organism, cytochrome c will be present in the oxidized form and can react with either substrate, dimethyl or tetramethyl-p-phenylenediamine dihydrochloride, to produce a purple colored product, indophenol.

Using aseptic technique, obtain an Oxy-Swab and use it to pick up a small amount of a pure colony of bacteria to be tested.
Press the swab against the sterile inside wall of the slant or sterile part of the inside surface of the plate to diffuse the innoculum into the fibers of the swab.
Look for the appearance of a purple or black color within 10-30 seconds on the swab in the area containing the bacteria.
Purple or black color is a positive result indicating the organism has the enzyme cytochrome c oxidase.
Good positive controls for this test are Bacillus spp, Neisseria, Micrococcus or Pseudomonas
No color change is a negative result (-).
Discard used swabs in an autoclave bag.

SIM TEST: Hydrogen Sulfide Production / Indole Formation / Motility using SIM medium (this test is also included in Motility Tests)

The ingredients in SIM (sulfate/ indole/ motility) medium enable the determination of three activities by which Gram-negative rods of the Enterobactericaea group of enteric bacteria can be differentiated using Sulfur Reduction/Indole Production/Motility media (SIM tubes). SIM medium contains nutrients, iron, and sodium thiosulfate.

The indole test is used for detecting tryptophanase. Casein peptone is rich in tryptophan, which is attacked by certain microorganisms resulting in the production of indole. Bacteria possessing the enzyme tryptophanase cleave trytophan, producing three end products. One of these endproducts is indole, produced in aerobic conditions; another is skatole, produced in anaerobic conditions. Amyl alcohol in Kovacs reagent acts as a solvent for indole, which then reacts with p-dimethylaminobenzaldehyde to produce a red rosindole dye. (Skatole will also give a positive indole reaction.) Organisms which do not produce tryptophanase produce no color change in SIM medium when Kovacs is added. Bacteria positive for tryptophanase do produce a red color when Kovacs reagent is added.

The hydrogen sulfide test relies on the use of sodium thiosulfate and ferrous ammonium sulfate as indicators of hydrogen sulfide production. Ferrous ammonium sulfate reacts with H2S gas to produce ferrous sulfide, a black precipitate.


Using aseptic technique, take a small amount of bacteria to be tested from a pure colony with a sterile inoculating needle or loop and make a stab inoculation into SIM agar tubes. Make sure that your needle or loop goes straight into the center of the medium almost all the way to the bottom and that you pull the needle or loop straight out in the same path as the entry (no mixing). This procedure is called "making a soft agar deep inoculation". Incubate for 24 to 48 hours.


  • Indole test: To detect indole production due to the enzyme tryptophanase, add three or four drops of Kovacs’ reagent and observe the fluid for development of a ring of red color(positive reaction)at the top of the tube.
  • Hydrogen Sulfide Test. When hydrogen sulfide gas is produced, a precipitation reaction will occur with the ferrous ammonium sulfate. An insoluble black precipitate is seen as a positive result.
  • Motility detection is possible due to the semisolid nature (low concentration of agar) of the SIM medium. Growth radiating out from the central stab inoculation line indicates that the test organism is motile. The motility test should be assessed first. Motile organisms will exhibit growth radiating from the stab inoculation line. Non motile organisms will exhibit growth only along the stab inoculation line.

Link to Motility Tests.

SIM agar:
Approximate Formula* Per Liter
Pancreatic Digest of Casein - 20.0 g
Peptic Digest of Animal Tissue - 6.1 g
Ferrous Ammonium Sulfate - 0.2 g
Sodium Thiosulfate - 0.2 g
Agar - 3.5 g

Kovac's reagent: (per liter)
p-Dimethylaminobenzaldehyde 50,0g
Amyl Alcohol 750.0 ml
Hydrochloric acid 250.0 ml

An alternative Indole test - not used in 2010 Indole Spot Test Reagent
p-Dimethylaminocinnamaldehyde 10.0 g
Hydrochloric acid 100.0ml
Water, deionized 900.0 ml

Methyl Red/Voges-Proskauer Tests (MR-VP)

The Methyl Red (MR) test is used to identify mixed acid fermenting bacteria that yield a stable acid end product. The Voges-Proskauer (VP) test is used to identify bacteria capable of 2,3 butanediol fermentation following mixed-acid fermentation. MRVP media contains glucose, peptone and phosphate buffer. Many organisms can overcome the buffering capacity of the media by producing large quantities of a stable acid end product, thus lowering the pH. Acid produciton is detected using the pH indicator methyl red (red pH<4.4, yellow pH > 6). In order to insure that the acid is stable, the indicators should be added a minimum of 48 hrs. post inoculation of the broth test media. Some organisms do not produce stable acid end products and, instead, further metabolize acids to more neutral end products like 2,3 butanediol. The reagents used, however, don't test for 2,3 butanediol but, rather, its precursor acetoin.

You can do both of these tests from one tube of dextrose peptone broth ( 0.7% peptone, 0.5% dextrose, 0.5% dipotassium phosphate) that you inoculate with a small amount of a pure colony of bacteria you want to test.

Incubate the broth culture(s) for at least 48 hours and not longer than 1 week.

Label 2 13mm glass testtube (do not need to be sterile), one with MR and one with VP.
a.Methyl red tests for acid production from dextrose:
Remove 1ml of the broth culture and place it into the small glass tube labeled MR Add 5 drops of methyl red indicator.
Read immediately for red color (positive test) or yellow color (negative test).
If negative, the rest of the sterile broth culture can be incubated for 24-48 hours and the test repeated.

b. Voges-Proskauer tests for the production of acetylmethylcarbinol from dextrose:
Remove 1ml of the broth culture and place it into the small glass tube labeled VP.
Add 15 drops of 5% alphanaphthol in absolute ethyl alcohol and 10 drops of 40% KOH. Do not mix contents after addition of test reagents!
A positive test is indicated by the development of a red color at the surface layer in the tube in 15-30 minutes.

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab 12