Oxidative Fermentative (OF) Test: Carbohydrate catabolism and fermentation review, Microbiology PDF

Carbohydrate Catabolism & Fermentation Review  Metabolism ◈ Chemical reactions that occur within living organism ◈ Metabolic Process involve enzymes ◈ Enzymes – proteins that catalyze biological reactions ◈ Endoenzymes- enzymes function inside a cell ◈ Ex...

Carbohydrate Catabolism & Fermentation Review  Metabolism ◈ Chemical reactions that occur within living organism ◈ Metabolic Process involve enzymes ◈ Enzymes – proteins that catalyze biological reactions ◈ Endoenzymes- enzymes function inside a cell ◈ Exoenzymes – release from the cell to catalyze reactions outside the cell  Catabolism ◈ All decomposition reactions in living organism; the breakdown of complex organic compounds into simpler ones ◈ A chemical reactions that release energy from decomposition of complex organic molecules  Carbohydrates ◈ Organic molecules that contain carbon, hydrogen and oxygen in the ratio (CH2O)n ◈ Classified based on size Monosaccharides – simple sugar Oligosaccharides – 2 to 20 monosaccharides molecules Polysaccharides – 20 or more monosaccharides molecules  Starch Hydrolysis ◈ What is the purpose of the test? ◈ The ability to degrade starch is used as a criterion for the determination of amylase production by a microbe. ◈ Starch agar is a differential medium that tests the ability of an organism to produce certain exoenzymes, including a-amylase and oligo-1,6-glucosidase, that hydrolyze starch. ◈ Exoenzymes are mainly Hydrolytic Enzymes that leave the cell ◈ Breakdown large substrate by the addition of water into smaller components.  Starch Hydrolysis ◈ Starch is a complex polysaccharide found abundantly in plants and usually deposited in the form of large granules in the cytoplasm of the cell ◈ Starch molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized by the organism. ◈ The major component of starch can be hydrolyzed by a-amylase, which is present in some bacteria while well known in case of fungi Procedure ◈ Divide the starch agar into 3 sectors by labelling the bottom plate. ◈ Streak a single line of the bacteria sample ◈ Incubate the plate, inverted, at 35 C for 24 hours ◈ Record the bacterial growth ◈ Flood the plate with Gram’s iodine (indicator) ◈ Examine for the clear zone around the line of bacterial growth  Starch Hydrolysis ◈ Starch agar is a simple nutritive medium with starch added. Since no color change occurs in the medium when organisms hydrolyze starch, we add iodine to the plate after incubation. ◈ Iodine turns blue, purple, or black (depending on the concentration of iodine) in the presence of starch. ◈ A clearing around the bacterial growth indicates that the organism has hydrolyzed starch.  Results ◈ Positive test: a clear zone around the line of growth after addition of iodine solution. The clear zone indicates hydrolysis of starch into monosaccharides which cannot bind the iodine molecule and appear as clear zone around the bacterial growth. ◈ Negative test: dark blue coloration of the medium Starch in the presence of iodine produces dark blue coloration of the medium as iodine is trapped in the helical structure of starch Iodine has been added to this starch agar plate. The zone of clearing surrounding Bacillus megaterium and Bacillus subtilis indicate that both were able to hydrolyze starch. Iodine has been added to this starch agar plate. The absence of any clearing indicates that neither Streptococcus agalactiae nor Staphylococcus epidermidis were able to hydrolyze starc  Oxidative Fermentative (OF) Test ◈ The oxidative-fermentative (OF) test was developed by Hugh and Leifson in 1953. ◈ They developed OF media to differentiate between oxidative bacteria (that produces acid from carbohydrates under aerobic condition only) and fermentative bacteria (that produces acid both under aerobic and anaerobic conditions).  Oxidative Fermentative (OF) Test ◈ Saccharolytic microorganisms degrade glucose either fermentatively or oxidatively. ◈ The end products of fermentation are relatively strong mixed acids that can be detected in a conventional fermentation test medium. ◈ The acids formed in oxidative degradation of glucose are extremely weak and less, and the more sensitive oxidation fermentation medium of Hugh and Leifson’s OF medium is required for the detection. ◈ The medium was made by increasing the amount of glucose above that found in medium used to detect fermentation and by decreasing the amount of peptone.  Procedures ◈ Two tubes are required for interpretation of the OF test. ◈ OF-glucose deeps contain glucose as a carbohydrate, peptones, bromothymol blue (indicator) ◈ Both are inoculated, and one tube is overlaid with mineral oil, producing an anaerobic environment. ◈ Oil is not added to the other tube to allow for aerobic conditions. ◈ The tubes are then incubated for 24–48 hours at 35 C ◈ Record the following: presence of growth, whether glucose was use, and the type of metabolism.  Oxidative Results ◈ This result is indicated by a small amount of acid production in the open tube. ◈ The acid produced (pH 6.0) changes the pH indicator, bromothymol blue, from green to yellow. ◈ After a 24-hour incubation a change in pH is observed at the surface of the open tube where growth in the presence of oxygen is observed. ◈ With prolonged incubation (more than 48 hours), the reduced concentration of agar in the medium allows for the eventual diffusion of the weak acid throughout the whole tube. ◈ No color change or reaction occurs in the oil-covered tube. ◈ e.g. Pseudomonas aeruginosa Fermentative Results Bacteria that can ferment glucose give a fermentative result as indicated by acid production in both the open (aerobic) and oil covered (anaerobic) tube. The acid produced (pH 6.0) changes the pH indicator, bromothymol blue, from green to yellow. e.g. Escherichia coli  Negative results ◈ Non-sacchrolytic bacteria give a negative OF result. ◈ The negative result is indicated by no color change in the oil-covered tube and in some cases an increase in pH (pH 7.6) changing the bromothymol blue from green to blue in the top of the open tube. ◈ The increase in pH is due to amine production by bacteria that break down the peptone (protein) in the medium. ◈ Other bacteria give a negative result indicated by no growth or color change in the medium. ◈ e.g. Alcaligenes faecalis. Open (Aerobic) Tube Covered (Anaerobic) Tube Metabolism Acid (yellow) Alkaline (green) Oxidative Acid (yellow) Acid (yellow) Fermentative Non saccharolytic (glucose not Alkaline (green) Alkaline (green) metabolized) CARBOHYDRATE FERMENTATION  The carbohydrate fermentation test is used to determine whether bacteria can ferment a specific carbohydrate. Carbohydrate fermentation patterns are useful in differentiating among bacterial groups or species. FERMENTATI Tests for the presence of acid and/or gas produced ON TUBES from carbohydrate fermentation. Basal medium containing a single carbohydrate source such as Glucose, Lactose, Sucrose or any other carbohydrate is used for this purpose.  A pH indicator (Phenol red) is present in the medium; which will detect the lowering of the pH of the medium due to acid production. Phenol red ( Red color- neutral, acidic - yellow) FERMENTATI Small inverted tubes called Durham tube is also immersed in the medium to test the production of gas ON TUBES If the test organism produce gas (hydrogen or carbon dioxide), the gas displaces the media present inside the tube and get trapped producing a visible air bubble.  When microorganisms ferment carbohydrate an acid or acid with gas are produced. Depending up on the organisms involved and the substrate being fermented, the end products may varie. Common end-products of bacterial fermentation include lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, carbon dioxide and hydrogen. PRINCIPLES The production of the acid lower the pH of the test medium, which is detected by the color change of the pH indicator. Color change only occurs when enough acid is produced, as bacteria may utilize the peptone producing alkaline by products. BASED ON THE CHARACTERISTICS REACTIONS OBSERVED, BACTERIA CAN BE CLASSIFIED AS: Fermenter with acid production only Fermenter with acid and gas production Non-fermenter  This test is used to determine which fermentation pathway is used to utilize glucose. It is a simple broth that contains peptone, buffers, and dextrose or glucose. METHYL RED Different bacteria convert dextrose and glucose to pyruvate using different metabolic pathways. (MR) AND Some of these pathways produce unstable acidic products which quickly convert to neutral VOGES-PROS compounds. KAUER (VP) Some organisms use the butylene glycol pathway, which produces neutral end products, including BROTH acetoin and 2,3-butanediol. Other organisms use the mixed acid pathway, which produces acidic end products such as lactic, acetic, and formic acid. These acidic end products are stable and will remain acidic.  METHYL RED (MR) TEST The Methyl Red test involves adding the pH indicator methyl red to an inoculated tube of MR-VP broth. If the organism uses the mixed acid fermentation pathway and produces stable acidic end-products, the acids will overcome the buffers in the medium and produce an acidic environment in the medium. When methyl red is added, if acidic end products are present, the methyl red will stay red.  The VP test detects organisms that utilize the butylene glycol pathway and produce acetoin. When the VP reagents are added to MR-VP broth VOGES-PROS that has been inoculated with an organism that uses the butylene glycol pathway, the acetoin KAUER (VP) end-product is oxidized in the presence of TEST potassium hydroxide (KOH) to diacetyl. Creatine is also present in the reagent as a catalyst. Diacetyl then reacts to produce a red color.  VP TEST RESULT The Voges-Proskauer test detects the presence of acetoin, a precursor of 2,3 butanediol. If the culture is positive for acetoin, it will turn “brownish-red to pink” (tube on the left in the second picture). If the culture is negative for acetoin, it will turn “brownish-green to yellow” (tube on the left in the second picture). Note: A culture will usually only be positive for one pathway: either MR+ or VP+. Escherichia coli is MR+ and VP-. In contrast, Enterobacter aerogenes and Klebsiella pneumoniae are MR- and VP+. Pseudomonas aeruginosa is a glucose nonfermenter and is thus MR- and VP-.  This is a defined medium used to determine if an organism can use citrate as its sole carbon source. It is often used to differentiate between members of SIMMON’S Enterobacteriaceae. CITRATE In organisms capable of utilizing citrate as a carbon source, the enzyme citrate lyase hydrolyzes citrate AGAR into oxaloacetic acid and acetic acid. The oxaloacetic acid is then hydrolyzed into pyruvic acid and CO2.  SIMMON’S CITRATE AGAR If CO2 is produced, it reacts with components of the medium to produce an alkaline compound (e.g. Na2CO3). The alkaline pH turns the pH indicator (bromthymol blue) from green to blue. This is a positive result (the tube on the right is citrate positive). Klebsiella pneumoniae and Proteus mirabilis are examples of citrate positive organisms. Escherichia coli and Shigella dysenteriae are citrate negative.  Citrate positive: growth will be visible on the slant surface and the EXPECTED ◾ medium will be an intense Prussian blue. The alkaline carbonates and RESULTS IN bicarbonates produced as by- products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green CITRATE ◈ color to blue. Citrate negative: trace or no growth will be visible. No color change UTILIZATIO will occur; the medium will remain the deep forest green color of the uninoculated agar. Only bacteria that can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium, thus a citrate-negative test culture will be virtually N TEST: indistinguishable from an uninoculated slant