Bacteriology

Bacteriology

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  RANI LAKSHMI BAICENTRAL AGRICULTURAL UNIVERSITY JHANSI,UP Course:Plant Bacteriology APP503(2+1) Topic:Biochemical tests and Physiological tests for various groups of Bacteria. Submitted to, Dr.P.P.Jambhulkar HOD,Plant pathology, RLBCAU,Jhansi Submitted by, Harish J Ag/Pg/0020/19 M.sc (Agri) Plant pathology, RLBCAU,Jhansi
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  Biochemical Tests • Eachdifferent species of bacterium has a different molecule of DNA (i.e., DNA with a unique series of nucleotide bases). • Since DNA codes for protein synthesis, then different species of bacteria must, by way of their unique DNA, be able to synthesize different protein enzymes. • Enzymes catalyze all the various chemical reactions of which the organism is capable. This in turn means that different species of bacteria must carry out different and unique sets of biochemical reactions.
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  Types of Biochemicaltests • IMVIC a.Indole test b.Citrate utilization test • Sulphate reduction test • Catalase production test • Urease test • Fermentation of carbohydrates • Gelatin liquefaction test • Oxidase test • Starch hydrolysis • Protein hydrolysis • KOH solubility test
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  IMVIC • This testimportant for coliform group of bacteria. • The coliform group of bacteria includes all the aerobic and facultatively aerobic bacteria. Indole test Principle:Tryptophan is an essential amino acid, which is oxidized by some bacteria resulting in the formation of indole, pynivic acid and ammonia. The indole test is done by inoculating the test organism into tryptophan broth, which contain tryptophan. The indole which is produced is detected by adding KOVAC’s reagent which produced cherry red colored ring.
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  Citrate utilization test Principle:Citratetest is used to differentiate among enteric bacteria on the basis of their ability to utilize as their soul carbon source. The utilization of citrate depends upon the presence of enzyme “Citrate Permease” produced by organism that helps its transports into the cell.
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  Sulphate reduction test Principle:H2Sis formed by some bacteria by reduction of sulphure containing amino acids (Cystein), Cysteine and metheonine or through reduction of inorganic sulphure compounds like thiosulphates (S2O3-)or sulphates (SO4 --) or sulphite (SO3-- ).The H2S production can be detected by incorporating a heavy metal salt containing ion (Fe++) or lead (Pb++) ion as a H2S indicator to neutrient culture medium containing cystein and sodiumthiosulphate as the sulphure substance.H2S a colourless gas when produced react with metal salts (FeSO4) forming visible insoluble black Ferrous Sulphide precipitates. Production of H2S from systein and Na2S2O3.
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  Catalase Production test Principle:Theoxidation of flavoproteins invariably result in the formation of Hydrogen peroxide as one major product. In addition this oxidation (and other oxygenation) produce small quantities of and even more toxic radical-The superoxide.In aerobes and aerotolerant aerobes the potentially leathal accumulation of oxygen is prevented by the enzyme superoxide dismutase which catalysis it to hydrogen peroxide and oxygen.
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  Urease test Principle:Urea isa major organic waste production of protein digestion by most vertebrates and is excreted in urine.Some microorganisms have the ability to produce the enzyme urease. Urease is a hydrolytic enzyme, which attack the amide linkage liberating Ammonia. Fermentation of Carbohydrates Principle:Fermentation can be defined as an energy yielding process that does not involve an electron transport chain and exogenous terminal electrons acceptors instead it relies on substrate level. Phasphorylation and an endogenously generated electron acceptor.
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  Starch Hydrolysis: Principle:Amylase isan exoenzyme that hydrolysis starch, a polysaccharides into monosaccharide and disaccharides such as glucose.these mono and disaccharide enters into cell cytoplasm of bacteria through the semi-permeable membrane and their by attacked by endoenzymes.Starch is a complex carbohydrate composed of glucose molecules linked together by α-1,4 and α-1,6 glycosidic bonds.
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  Casein hydrolysis Principle:Casein isa major protein found in milk. It is a macromolecule composed of amino acid linked together by peptide bond. Some microorganism have the ability to decrease the protein casein by producing proteolytic exo-enzymes called protease. The process breake down the peptide bond by introducing water into the molecule liberating the soluble amino acid pool for use in the synthesis of structural and functional cellular protein.
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  Oxidase test: Principle:Cytochrome areheam containing catalytic enzyme which are tightly bound to (prokaryotic cells) cells of plasma membrane.they are concerned with later stages of biochemical oxidation.They act as electron or H2 carries of biological oxidation by virtue of their availability valence by heam ions i.e., they can undergo reduction and oxidation . During period of great activity they are reduced. Cytochrome C is more abundant and freely soluble in water. Cytochrome C does not react directly with O2 but reduced form will be oxidized by cytooxidase with which it is closely associated. Cyto a1,a3 is called cytooxidase or indophenolase or endophenol oxidase or ferro cytochrome C or oxygen oxido reductase.
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  Protein hydrolysis Principle:Proteins aremade up of various amino acids linked together in long chains by means of peptide bonds. Many bacteria can hydrolyze a variety of proteins into peptides (short chains of amino acids) and eventually into individual amino acids. They can then use these amino acids to synthesize their own proteins and other cellular molecules or to obtain energy. The hydrolysis of protein is termed proteolysis and the enzyme involved is called a protease. Nitrate reduction Many organisms can respire anaerobically by using NO3- as a terminal electron acceptor for an electron transport system (nitrate respiration or dissimulator nitrate reduction).Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase. It also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen.
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  Gelatin Liquification test Principal:Gelatinis a protein derived form collagen,which is insoluble in cold water but insoluble in cold water but soluble in hot water and form gel on cooling it. gelatin is liquid at room temperature that solidify on cooling up to the temperature of -4oc for bacteriological use and edible grade of gelatin is used since it is free of preservatives and inhibitory amount of heavy metals.
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  Lipase test Lipolytic organismssplit off the fatty acid, and the calcium salts of the fatty acids produce opaque zones around the colonies.
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  Physiological methods • Temperature •pH range • Utilization of carbon source • NaCl range • Utilization of nitrogen source • Relation to Oxygen Temperature range Incubate cultures at a range of temperatures; using constant temperature incubators or water baths, measure the growth response of the actinobacteria. The tested temperature range is usually from 0°C to 75°C. In general, temperature experiments employ solid medium instead of broth in order to better observe. The basal medium is Bennett's medium or YIM38 medium or nutrient medium.
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  pH range An essentialpart of the description of any actinobacteria is the range of pH values at which it can grow, as well as the optimal pH for growth. Measure growth responses from a standarisedinoculum using basic medium (Bennett's medium or YIM38 medium or nutrientmedium) at various pH values. Liquid medium is to be used for pH tests, to measure thegrowth responses turbidimetrically. The selection of buffer is critical. A buffer should be usedin most media to maintain a stable pH for growth of the test strain. Buffers are most effectiveat their pKa values and should be chosen with this in mind.Some buffers such as citrate, succinate, or glycine may be metabolized by the test organism.
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  Ultilization of Carbonsource Utilization of carbon source tests usually uses turbidimetric method. Use a chemically defined basal medium that lacks a carbon source, but otherwise is suitable for growth of the actinobacteria being tested. The basal medium is Pridham and Gottlieb carbon utilization medium [8]. Add carbon sources to a concentration (sugar alcohols 0.5–1%, others 0.10.2%). After growth has occurred, measure the growth response turbidimetrically with a spectrophotometer.
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  Nacl range Salt toleranceexperiments mainly test the tolerance ability of the organism to NaCl and other salts, and determine the optimum concentration for growth. Inoculate liquid media containing a range of NaCl (usually 0–30%, W/V, or relative molar concentrations) concentrations and measure the growth response turbidimetrically. Bennett's medium or YIM38 medium or nutrient medium can be used as basal medium. Utilization of nitrogen source Use the turbidimetric method to test the utilization of nitrogen source, especially sole nitrogen sources. Basal medium that omit nitrogen source but include a suitable carbon source are used. Add nitrogen source to a concentration (usually 0.5%).