oxidase and catalase test

OXIDASE TEST

AIM:

To demonstrate the production of cytochrome oxidase C by the given bacterial culture

PRINCIPLE:

The oxidase test is used to differentiate between the families of Pseudomonadaceae (ox +) and Enterobacteriaceae (ox -), and is useful for characterization of many other bacteria, those that have to use oxygen as the final electron acceptor in aerobic respiration. The enzyme cytochrome oxidase is involved with the reduction of oxygen at the end of the electron transport chain. There may be different types of oxidase enzymes produced by bacteria. The colorless redox reagent, tetramethyl-p-phenylenediamine dihydrochloride (or dimethyl can be used) used in the test will detect the presence of the enzyme oxidase and, reacting with oxygen, turn a color. The oxidase reagent contains a chromogenic reducing agent, a compound indophenol blue that changes color when it becomes oxidized, so it acts as an artificial electron acceptor for the enzyme oxidase. This test is used for the screening of Pseudomonas, Vibrio, Neisseria, Brucella and Pasteurella, which give positive test. Enterobacteriaceae are oxidase negative.

MATERIALS EQUIRED:

Oxidase reagent, Sterile wooden sticks, Bacterial culture

Reagent preparation:

Oxidase reagent is specially prepared as 10g/l or 1% solution of tetramethyl-p-phenylene diamine dihydrochloride.

PROCEDURE:

1. A good amount of fresh inoculums was taken by non metal stick from a plate culture or liquid culture

2. It was inoculated on Oxidase disc (supplied by HiMedia).

3. The occurrence of color change was observed.

RESULT:

 A positive reaction was occurred within 5 seconds and there was no color occurred in negative culture.

Interpretation

Oxidase positive organisms give blue color within 5-10 seconds, and in oxidase negative organisms, color does not change. The reagent acts as an artificial electron acceptor for the enzyme oxidase and is oxidized to form the colored compound Wurster’s blue. Wurster’s blue is a purple compound that is readily visible and signifies a positive reaction. A positive reaction will usually occur within 10-15 seconds, and will be a bluish-purple color that progressively becomes purpler.

CATALASE TEST

AIM:

 To demonstrate the production of catalase enzyme by the given bacterial culture.

PRINCIPLE:

The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. The culture should not be more than 24 hours old.

Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is accumulated as an end product of aerobic carbohydrate metabolism.

PROCEDURE:

TUBE METHOD:

1. 1-2 ml of hydrogen peroxide solution was poured into a test tube.
2. A sterile wooden stick or a glass rod was used to take several colonies of the 18 to 24 hours test organism and immersed in the hydrogen peroxide solution.
3. The immediate bubbling was observed.

SLIDE METHOD:

1. A loop or sterile wooden stick was used to transfer a small amount of colony in the surface of a clean, dry glass slide.
2. A drop of 3% H₂O₂ was placed in the glass slide.
3. The evolutions of oxygen bubbles were observed.

PLATE CATALASE TEST:

1. One drop of freshly prepared 3% H₂O₂ was carefully placed over a single colony with the help of a dropper.

2. The lid of the plate must be closed immediately.

3. Production of effervescence (bubbles) in 5-10 seconds should be a positive test. This method must not be performed on blood agar plates.

RESULT:

Catalase Positive reactions: Evident by immediate effervescence (bubble formation)

Catalase Negative reaction: No bubble formation (no catalase enzyme to hydrolyze the hydrogen peroxide)