Biochemical test-2

Citrate utilization test:

The basic principle of citrate utilization test is to determine the ability of an organism to utilize citrate as the sole source of carbon for metabolism with resulting alkalinity.

Normally, citrate metabolism involves a condensation of acetyl, with coenzyme A and oxaloacetate to enter the Krebs's cycle. Citrate metabolism by most bacteria is rapid via the tricarboxylic acid cycle or the citrate fermentation pathway. In bacteria, the cleavage of citrate involves an enzyme system without the intervention of the coenzyme A. this enzyme is called citrate (citrate oxaloacetate-lase) or citrate demolase. The enzyme requires a divalent cation for its activity which is supplied by magnesium or manganese. Thus, the utilization of citrate depends on the presence of enzyme produced by organisms that break down the citrate to oxaloacetic acid and acetic acid as intermediate products. These products are later converted to pyruvic acid and carbon dioxide.

Citrate → oxaloacetate + acetate

↓

Pyruvate + CO₂

The medium used in this experiment is Simmon’s citrate agar which contains sodium citrate as the only source of carbon and energy. The indicator used in it is bromothymol blue. When the citrate is metabolized by the organisms, the CO₂ generated combines with sodium and water to form sodium carbonate, which is an alkaline product. The carbonate so produced changes the colour of the indicator from green to blue which is a positive test.

CO₂ + 2Na⁺ + H₂O → Na₂CO₃

(Alkaline)

The medium used for citrate fermentation also contains inorganic ammonium salts. An organism that is capable of utilizing citrate as its sole source of carbon also utilizes the ammonium salts as its sole nitrogen source. Ammonium salts are broken down to ammonia (NH₃) with resulting alkalinity.

Sulphide indole motility (Sim) test:

It is a test where a semisolid agar called Sulphide Indole motility (Sim) medium is inoculated with a bacteria to test for hydrogen sulphide, indole production and motility of the organism. The medium is inoculated by a swab and stab method (rub some bacteria on the surface of the medium and stab the straight hole through the medium using a straight wire) or simply a stab method. After incubation at 37⁰C for 24 hours, if hydrogen sulphide is present, it will react with sodium thiosulphate in the medium and the indicator, ferric ammonium citrate, to produce ferrous sulphide which falls out of solution as a black precipitate. The presence of hydrogen sulphide typically means that the bacteria produces the enzyme cysteine desulfurase, which breaks up the cysteine in the medium into, among other components, hydrogen sulphide. The indole test is performed by adding Kovac's reagent to the inoculated medium. The Kovac's reagent reacts with indole (if present ) to produce a pinkish red or reddish purple ring at the top of the test tube. If indole is not present, there will be no colour change. The presence of indole means that the bacteria produces tryptophanase, an enzyme which breaks down tryptophan into smaller components, one of which being indole.

Catalase test:

The basic principle of catalase test is to determine the presence of “catalase” an enzyme in micro-organism that decomposes hydrogen peroxide to release oxygen.

The enzyme catalase is present in most cytochrome-containing aerobic and facultative anaerobic bacteria with the main exception of Streptococcus sps. Usually, the organism which lacks the cytochrome system also lack the catalase enzyme and therefore are unable to break down hydrogen peroxide.

Catalase is a hemeprotein, the prosthetic group of it is made up of four atoms of trivalent iron (ferric-Fe⁺⁺⁺) per molecule, which retains its oxidized state during enzyme activity.

In microorganism, hydrogen peroxide is formed as an oxidative end product of the aerobic breakdown of sugars. Reduced flavoprotein reacts directly with gaseous oxygen by way of electron reduction to form hydrogen peroxide and not by the direct action between hydrogen and molecular oxygen.

Hydrogen peroxide, if allowed to accumulate, is toxic to bacteria, resulting in their death. It has been postulated that the death of strict anaerobes in the presence of oxygen is due to the suicidal act of H₂O₂ production in the absence of catalase production.

Oxidase test:

The basic principle of this test is to determine the presence in bacteria of oxidase enzyme that will catalyse the transport of electrons between electron donors in the bacteria and a redox dye-tetramethyl p-phenylenediamine.

The oxidase reaction is due to the presence of a cytochrome oxidase system which activates the oxidation of reduced cytochrome by molecular oxygen, which in turn acts as an electron acceptor in the terminal stage of the electron transfer system.

During respiration, molecular oxygen oxidizes a substrate through the intervention of the electron transport system. The cytochrome system is usually present only in aerobic organisms which make them capable of utilizing oxygen as a final hydrogen acceptor to reduce molecular oxygen to hydrogen peroxide. All oxidase positive organisms are either aerobic or facultatively anaerobic, with the exception of vibrio fetus which requires a microaerophilic oxygen requirement. A positive oxidase reaction is also limited to those organisms capable of growing in the presence of oxygen and at the same time producing the enzyme catalase. Obligate anaerobes lack oxidase activity since they are unable to live in the presence of atmospheric oxygen and do not possess a cytochrome oxidase system.

All pseudomonas and Neisseria sps. Produce an oxidase enzyme which when in the presence of atmospheric oxygen, cytochrome C, and an oxidase reagent, oxidize the reagent to form a coloured compound, indophenol.

References

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.