Saran's Learn Microbiology

UREASE TEST AIM             To determine the ability of an organism to produce an exoenzyme Urease and degrade Urea. PRINCIPLE          The alkaline fermentation of urine (urea) with the resultant production of ammonia was hypothesized to occur due to the action of microorganisms by Reoch in 1875. Christensen later formulated a medium that would allow growth of other members of the Enterobacteriaceae that cannot utilize the by-product of urea hydrolysis (ammonia) as a sole nitrogen source for growth.        Urea is a nitrogen containing compound that is produced during Decarboxylation of the amino acid Arginine in the Urea cycle. Urea is a diamide of Carbonic acid. Many organisms ( Proteus mirabilis , Proteus vulgaris, Klebsiella pneumoniae and Helicobacter pylori ) especially those that infect the Urinary tract have Urease enzyme which is able to split Urea in the presence of Water to release Ammonia and Carbon-di-oxide. The Ammonia combines with Carbon-di-oxide and Water t

OXIDASE TEST AIM             To test the Oxidase enzyme producing efficiency of Bacteria. PRINCIPLE                 The Oxidase test is used to identify bacteria that produce Cytochrome - c oxidase, an enzyme of the bacterial Electron transport chain. In 1928, Gordon and McLeod introduced the use of a dimethyl-pphenylenediamine dihydrochloride solution to test for the presence of oxidase systems.  When the enzyme is present, the Cytochrome - c oxidase oxidizes the reagent Tetramethyl-p-phenylenediamine dihydrochloride to Indophenols (purple color end product). When the enzyme is not present, the reagent remains reduced and is colorless.            Bacteria which contain Cytochrome - c as part of their respiratory chain are Oxidase positive and turn the reagent Blue/purple. Organisms lacking Cytochrome - c as part of their respiratory chain do not oxidize the reagent, leaving it colorless within the limits of the test, and are Oxidase negative. Examples for Oxidase positive bac

CATALASE TEST AIM             To test the Catalase enzyme producing efficiency of Bacteria. PRINCIPLE         Catalase test is used to differentiate the bacteria that produce an enzyme Catalase, such as  Staphylococcus sp. from Non-catalase producing bacteria such as  Streptococcus sp. It is also used for differentiating Bacillus sp. from Clostridium sp. In 1893, a publication by Gottstein brought attention to bacterial catalase, making it one of the first bacterial enzymes.            Aerobic and facultative anaerobic organisms produce two toxins during normal metabolism. One is Hydrogen peroxide (H 2 O 2 ) and another one is Superoxide radical (O 2 − ). These bacteria have two enzymes that detoxify the products of normal metabolism. One of these enzymes, Catalase, is capable of converting Hydrogen peroxide to Water and Oxygen. Normally, 5 % H 2 O 2  is used for the detection of Catalase in bacteria. The presence of the Catalase enzyme in a bacterial isolate is evident when

CARBOHYDRATE FERMENTATION TEST AIM             To test the ability of the microorganisms to utilize and digest several Carbohydrate sugars. PRINCIPLE            Carbohydrates are biological molecules which are made up of carbon, hydrogen and oxygen molecules. There are three types of Carbohydrates based on their structure and number of carbon atoms present, they are Monosaccharides (simple sugar containing 3-7 carbon atoms), Disaccharides (comprising of two monosaccharides linked together by the glycosidic bond) and Polysaccharides (containing 8 or more monosaccharide molecules). Carbohydrate fermentation is the process by which the microorganism utilizes the Carbohydrate sugars to produce energy in the form of ATP, the ultimate energy source of the organism.  The purpose of the Carbohydrate fermentation test is to check whether the bacteria can ferment a specific carbohydrate or not. Carbohydrate fermentation patterns are useful in differentiating among bacterial groups or spec

CITRATE UTILIZATION TEST AIM          To detect the ability of the bacteria to utilize Sodium citrate as sole carbon source and Ammonium salt as sole source of nitrogen. PRINCIPLE            Citrate utilization test is used to determine the ability of bacteria to utilize Sodium citrate as its only Carbon source and Inorganic ammonium salts as its only Nitrogen source. Citrate utilization test can be used to differentiate the coliforms such as  Klebsiella  (formerly  Enterobacter )  aerogenes  (Positive) which occur naturally in the Soil and Aquatic environments from the Fecal coliforms such as  Escherichia coli  (Negative) whose presence would be indicative of fecal contamination. Simmon’s Citrate agar is used for the Citrate utilization test. The medium contains Citrate as the sole Carbon source and inorganic Ammonium salts (NH 4 H 2 PO 4 ) as the sole source of Nitrogen. Bacteria that can grow on this medium produce an enzyme, Citrate permease which is capable of converting C