Saran's Learn Microbiology

PHENYLALANINE DEAMINASE TEST AIM       To differentiate the Proteus sp., Morganella sp. and Providencia sp. based on their ability to produce Phenylpyruvic/ Pyruvic acid by Oxidative deamination form other members of Enterobacteriaceae family. PRINCIPLE              In 1950, Hendriksen demonstrated that  Proteus  spp. were able to convert the amino acid phenylalanine to phenylpyruvic acid. Later, Buttiaux in 1954 developed a culture medium for detecting the formation of Phenylpyruvic acid from Phenylalanine by members of the  Proteus ,  Providencia and  Morganella  groups. This medium was further modified by Ewing in 1957 by omitting Proteose peptone.          Phenylalanine Deaminase Test is used to differentiate members of the Gram negative bacterial genera  Proteus, Morganella  (which were originally classified under the genus  Proteus ), and  Providencia  from other Enterobacteriaceae. Phenylalanine agar, also known as Phenylalanine deaminase medium, contains nutrients

GELATIN HYDROLYSIS TEST AIM       To determine the ability of an organism to produce proteolytic enzyme Gelatinase that liquefy Gelatin. PRINCIPLE                 Gelatin is a protein derived from the animal protein collagen– component of vertebrate connective tissue. It has been used as a solidifying agent in food for a long time. Robert Koch used nutrient gelatin as an early type of solid growth medium. Gelatin dissolves in water at 50 °C and exists as liquid above 25 °C and solidifies or gels when cooled below 25 °C.          In 1926, Frazier described the very first method to detect the ability of microorganisms to liquefy gelatin. Clarke (1953) described a simplified plate method using 10 % leaf gelatin and HgCl  2 –HCl solution for the detection of gelatin-liquefying bacteria and compared it with the gelatin stab method and the Frazier’s plate method. This comparison showed that hydrolysis of gelatin was generally more accurate and rapid in the simplified plate method (3

CASEIN HYDROLYSIS TEST AIM             To demonstrate the ability of the bacteria to produce an exoenzyme Caseinase and degrade milk protein Casein. PRINCIPLE                    Casein is a macromolecule composed of amino acids linked together by peptide bonds, CO-NH. Some microorganism have the ability to degrade the Casein protein by producing proteolytic exoenzyme, called Proteinase (Caseinase) which breaks the peptide bond CO-NH by introducing water into the molecule, liberating smaller chains of amino acids called peptides, which are later broken down into free amino acids by extracellular or intracellular peptidases. These amino acids are transported through the cell membrane into the intracellular amino acid pool for use in the synthesis of structural and functional cellular proteins.              The protein casein is a large polymer of amino acids that make around 85 % of the protein found in milk. Casein is a large protein that is responsible for the white color of m

STARCH HYDROLYSIS TEST AIM             To differentiate bacteria based on their ability to hydrolyze starch with the enzyme α –amylase. PRINCIPLE                   Starch is a complex carbohydrate (polysaccharide), composed of two constituents –amylose, a straight-chain polymer of 200-300 glucose units, and amylopectin, a larger branched polymer groups. The α -D-glucose molecules in both amylose and amylopectin are bonded by 1,4- α -glycosidic (acetal) linkages. The two forms differ in that the amylopectin contains polysaccharide side chains connected to approximately every 30th glucose in the main chain. These side chains are identical to the main chain except that the number 1 carbon of the first glucose in the side chain is bonded to carbon number 6 of the main chain glucose. The bond is, therefore, a 1,6- α -glycosidic linkage. Starch is too large to pass through the bacterial cell membrane. Therefore, to be of metabolic value to the bacteria it must first be split into s

TRIPLE SUGAR IRON (TSI) TEST   AIM          To check whether Gram negative bacilli particularly Enterobacteriaceae family to utilize Dextrose (or Glucose), Lactose and Sucrose fermentatively, and produce Hydrogen sulfide. PRINCIPLE              In 1917, Sulkin and Willett described a medium containing the carbohydrates, Glucose, Lactose and Sucrose, and Iron salts. The medium showed fermentation of these carbohydrates, as well as Hydrogen sulfide production. Hajna modified the medium in 1945 to contain phenol red as the pH indicator for identifying Enterobacteriaceae, and is the formulation still in use today.       Triple sugar iron agar (TSI) is a Differential medium that contains three Carbohydrate sugars, Lactose (10 %), Sucrose (10 %), a small amount of Glucose or Dextrose (1 %), Ferrous sulfate, and the pH indicator Phenol red.  It is used to differentiate Enterobacteria based on the ability to reduce Sulfur and ferment Carbohydrates. As with the Phenol red, if an organi