Question

Describe the purpose, principle, testing method and interpretation for the following tests:

ONPG

Oxidase

indole

MR-methyl red

VP- voges proskauer

Nitrate reduction

Urea agar

Citrate agar

phenylalanine Deaminase

Motility

Decarboxylase media

TSI - Triple Sugar iron agar

Kia - Kligler iron agar

OF- oxidase fermentation agar

Answer 1

ortho-Nitrophenyl-β-galactoside (ONPG) is a colourimetric and spectrophotometric substrate for detection of β-galactosidase activity. This compound is normally colourless. However, if β-galactosidase is present, it hydrolyzes the ONPG molecule into galactose and ortho-nitrophenol.

The oxidase test is a test used in microbiology to determine if a bacterium produces certain cytochrome c oxidases. It uses disks impregnated with a reagent such as N′, N′-tetramethyl-p-phenylenediamine which is also a redox indicator. The reagent is a dark-blue to maroon colour when oxidized, and colourless when reduced. Oxidase-positive bacteria possess cytochrome oxidase or indophenol oxidase (an iron-containing hemoprotein). These both catalyze the transport of electrons from donor compounds (NADH) to electron acceptors (usually oxygen). The test reagent, TMPD dihydrochloride acts as an artificial electron donor for the enzyme oxidase. The oxidized reagent forms the coloured compound indophenol blue.

The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to convert tryptophan into indole. This division is performed by a chain of a number of different intracellular enzymes, a system generally referred to as "tryptophanase."

Methyl Red (MR) test is a biochemical test performed on bacterial species to detect the ability of an organism to produce stable acids end products (Mixed-acid fermentation) from supplied glucose. When methyl red is added to MR-VP broth that has been inoculated with Escherichia coli, it stays red. This is a positive result for the MR test. When methyl red is added to MR-VPbroth that has been inoculated with Enterobacter cloacae, it turns yellow. This is a negative MR result.

VP is a test used to detect acetoin in bacterial broth culture. The test is performed by adding alpha-naphthol and potassium hydroxide to the Voges-Proskauer broth which has been inoculated with bacteria. The Voges-Proskauer (VP) test is used to determine if an organism produces acetyl methyl carbinol from glucose fermentation. If present, acetyl methyl carbinol is converted to diacetyl in the presence of ∝- naphthol, strong alkali (40% KOH), and atmospheric oxygen. A cherry red colour indicates a positive result, while a yellow-brown colour indicates a negative result. The test depends on the digestion of glucose to acetylmethylcarbinol.

The nitrate reductase test is a test to differentiate between bacteria based on their ability or inability to reduce nitrate to nitrite using anaerobic respiration. A heavy inoculum of test organism is incubated in a broth containing nitrate. The organisms capable of producing the nitrate reductase enzyme reduce the nitrate, present in the broth, to nitrite which may then be further reduced to nitric oxide, nitrous oxide, or nitrogen. Therefore, if the medium turns red after the addition of the nitrate reagents, it is considered a positive result for nitrate reduction. No colour change means that no nitrate was present.

Urea Agar contains urea and phenol red as the pH indicator. Organisms capable of hydrolyzing urea form ammonia as a by-product, thus turning the medium alkaline. The pH indicator turns from pale yellow to pink-red in colour in these conditions.

Simmons Citrate Agar is used to test an organism's ability to utilize citrate as a source of energy. Ammonium Dihydrogen Phosphate is the sole source of nitrogen. Dipotassium Phosphate acts as a buffer.  Inoculate Simmons citrate agar lightly on the slant by touching the tip of a needle to a colony that is 18 to 24 hours old. Incubate at 35^oC to 37^oC for 18 to 24 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium. When Simmons Citrate agar is inoculated with Salmonella typhimurium, the medium turns royal blue. This is a positive result for the citrate test. When Simmons Citrate agar is inoculated with Escherichia coli, the medium remains green. This is a negative result for the citrate test.

Phenylalanine deaminase medium tests the ability of an organism to produce the enzyme deaminase. This enzyme removes the amine group from the amino acid phenylalanine and releases the amine group as free ammonia. As a result of this reaction, phenyl pyruvic acid is also produced. If the medium remains a straw colour, the organism is negative for phenylalanine deaminase production. When 10% of ferric chloride is added to phenylalanine deaminase medium inoculated with Proteus mirabilis, the presence of phenyl pyruvic acid causes the media to turn dark green. This is a positive result.

Motility is the ability of an organism to move by itself by means of propeller-like flagella unique to bacteria or by special fibrils that produce a gliding form of motility. Bacterial motility is evident by a diffuse zone of growth extending out from the line of inoculation. A positive motility test is indicated by a diffuse zone of growth flaring from the line of inoculation. A negative motility test is indicated by growth confined to the stab line. A positive motility test is indicated by a pink colour diffusing from the line of inoculation.

The decarboxylase test is useful for differentiating the Enterobacteriaceae. Each decarboxylase enzyme produced by an organism is specific to the amino acid on which it acts. Ornithine decarboxylation yields putrescine. Lysine decarboxylation results in cadaverine. These byproducts are sufficient to raise the pH of the media so that the broth turns purple. If the inoculated me

Triple sugar iron agar (TSI) is a differential medium that contains lactose, sucrose, a small amount of glucose (dextrose), ferrous sulfate, and the pH indicator phenol red. It is used to differentiate enterics based on the ability to reduce sulfur and ferment carbohydrates. As with the phenol red fermentation broths, if an organism can ferment any of the three sugars present in the medium, the medium will turn yellow.

Kligler Iron Agar (KIA) combines features of Kligler's Lead Acetate medium and Russell's Double Sugar Agar. Phenol red is added as the colour indicator. The basal medium of KIAis composed of casein and meat peptones with the addition of lactose and dextrose.

The oxidative-fermentative test determines if certain gram-negative rods metabolize glucose by fermentation or aerobic respiration (oxidatively). The high concentration of acid produced during fermentation will turn the bromthymol blue indicator in OF media from green to yellow in the presence or absence of oxygen.