BISC209: Enzyme tests

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.

Catalase test

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

H₂O₂ --------> H₂O + O₂

Procedure
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 mix the cells in the peroxide until you have created a circular emulsion about the size of a quarter.
Since the reaction of peroxide with catalase liberates bubbles of oxygen, it is easy to see the product of the reaction.
Remove the loop and look for the formation of bubbles in the emulsion.
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

Background
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.

Procedure
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.

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

Background
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 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. Black color is a positive test for hydrogen sulfide production.

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.
To detect indole production due to the enzyme tryptophanase, add three or four drops of Kovacs’ reagent and observe for a red color(positive reaction).

SIM agar and Reagent recipes:

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.

Procedure
Using aseptic technique, take a small amount of bacteria to be tested from a pure colony with a sterile inoculating needle and make a stab inoculation into SIM agar tubes. Inoculate a positive control tube using the same technique.(See the chart to follow for a list of appropriate control bacteria.) Incubate at 37C for 24 to 48 hours.

Motile organisms will exhibit growth radiating from the stab inoculation line. Non motile organisms will exhibit growth only along the stab inoculation line.

A blackening of the medium, either along the stab line or throughout the medium, indicates the production of hydrogen sulfide by the microorganisms.

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

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab11
Lab 12