Biochemical Test Lab Report

Biochemical tests are the tests used for the identification of bacterial species based on the differences in the biochemical activities of different bacteria. Bacterial physiology differs from one species to the other. These differences in carbohydrate metabolism, protein metabolism, fat metabolism, production of certain enzymes and ability to utilize a particular compound help them to be identified by the biochemical tests. Gram’s stain was originally devised by histologist Hans Christian Gram in 1884. Gram-positive bacteria stain purple, while Gram-negative bacteria stain pink when subjected to Gram staining. Approximately 60-90% of the Gram-positive bacterial cell wall is made up of peptidoglycan and interwoven teichoic acid, while only

It is often used in the selective identification of enteric bacteria including Salmonella and Shigella. The TSI agar has glucose, lactose and sucrose as the sources of carbohydrates. Phenol red is the acid base indicator incorporated in the medium. The TSI medium indicates whether the bacteria ferments glucose only, or lactose and sucrose with or without production of gas. Nitrate serves as a source of nitrogen for many bacteria. They can also act as a final electron acceptor. Many bacteria can be differentiated and are identified by their capacity to reduce nitrates to nitrites. Most of the bacteria belonging to the family Enterobacteriaceae reduce nitrates [165]. OF test is used to differentiate those organisms that utilize carbohydrates aerobically (Oxidation) such as P. aeruginosa, from those that utilize carbohydrates anaerobically (Fermentation) such as members of the Enterobacteriaaceae. The OF medium contains peptone, test carbohydrate and bromothymol blue as indicator. When carbohydrate is utilized, acids are formed which changes the colour of the medium from green to yellow

In order to utilize casein, bacteria cells secrete proteolytic exoenzymes (amylases, proteases, pectinases, lipases, xylanases and cellulases) outside of the cell that hydrolyze the protein to amino acids. The amino acids can then be used by cells after crossing the cell membrane via transport proteins [169]. Starch hydrolysis test is used to differentiate bacteria based on their ability to hydrolyze starch with the enzyme α-amylase or oligo-l, 6-glucosidase. These enzymes hydrolyze starch by breaking the glycosidic linkages between the sugar subunits. It aids in the differentiation of species from the genera Corynebacterium, Clostridium, Bacillus, Bacteroides, Fusobacterium and members of Enterococcus [170]. Gelatin hydrolysis test is used to detect the ability of an organism to produce gelatinase (proteolytic enzyme) that liquefy gelatin. This process takes place in two sequential reactions. In the first reaction, gelatinase degrade gelatin to polypeptides. Then, the polypeptides are further converted into amino acids. The bacterial cells can then take up these amino acids and use them in their metabolic processes. Gelatin hydrolysis test is helpful in identifying and differentiating species of Bacillus, Clostridium, Proteus, Pseudomonas and Serratia [171]. Hydrogen sulphide (H2S) production test is used for the detection of H2S gas produced by an organism. It is used