Microbial Metabolism and Carbohydrate Catabolism

An exoenzyme is an enzyme that is secreted by a cell and functions outside the cell. It can also be called an extracellular enzyme. It is most commonly used to breakdown large macromolecules into smaller units so they can be taken up by the cell. An endoenzyme is an enzyme that functions within the cell. It can also be called an intracellular enzyme. It commonly functions in intracellular metabolic pathways. Purpose: To see if the bacteria can use starch as a source of carbon and energy. Test the activity of alpha amylase. Starch molecules are too large to enter bacterial cells so they release exoenzymes that digest the starch into smaller subunits that can then enter the cell. Medium containing starchstarch agar. (Differential) Tryptone Soytone Sodium Chloride Dipotassium phosphate Agar + Soluble starch Iodine test: Iodine reagent is used to flood the plate. Clear regions around the area of growth indicate that the organism is able to digest starch. negative positive http://www.austincc.edu/microbugz/starch_hydrolys Fermentation of Carbohydrates: Fermentation is the process of converting carbohydrates into organic acids or alcohols with production of gases like carbon dioxide under anaerobic conditions.

Endoenzymes are involved in the biochemical processes that ferment sugars. Multiple fermentation pathways exist that result in the generation of different end products. Fermentation tests that detect the different substrates and end products are used to determine which fermentation pathway is being utilized by the organism. Purpose: Test for fermentation of different sugars by bacteria. Tubes contain peptone, one carbohydrate source (glucose, lactose, sucrose, etc.), phenol red (pH indicator). Fermentation generates different acids, ethanol, hydrogen gas, carbon dioxide gas, and other organic compounds, depending on the specific reactions. In this test the production of acids can be observed by a change in color of the medium to yellow. Each tube should also have an inverted and filled Durham tube which is used to look for the production of gas(es). Acid/ no Gas Acid/ Gas No Acid/ no Gas http://www.blinn.edu/natscience/phillips/micropictures Protein catabolism: Unit: amino acid. Multiple amino acids are held by peptide bonds. biologia.laguia2000.com Large polypeptides are hydrolyzed by enzymes that cleave the peptide bonds and release smaller products that can cross the cell membrane and be used by the cell.

Gelatin is one such polypeptide that is cleaved by exoenzyme gelatinase To test for gelatinase activity, bacteria are grown in tubes containing gelatin instead of agar. When the gelatin is hydrolyzed, it cannot solidify again. The areas of solid gelatin media where the organism grows will turn into liquid. Even if you refrigerate this medium, the media will remain liquid. positive negative quizlet.com Urea broth is a differential medium used to identify bacteria that can produce the exoenzyme urease that can breakdown urea and convert it into ammonia and carbon dioxide. The medium contains peptone, glucose, urea and phenol red indicator. Phenol red turns yellow in an acidic environment and bright pink when alkaline. If the urea is hydrolyzed then ammonia is released; an alkaline environment is created, turning the media pink. Members of the genus Proteus, can degrade urea rapidly. control positive negative These are known as “rapid urease-positive” organisms. http://www.blinn.edu/natscience/phillips/micro%20pictures.htm Microbial Metabolism 1 Carbohydrate catabolism Starch Hydrolysis Sugar fermentations reactions. 1. Starch hydrolysis Monosaccharides Amylase Disaccharides Starch (polysaccharide) Starch hydrolysis test  Starch molecules are too large to enter bacterial cells so these bacteria release an enzyme- amylase- that digest the starch into smaller subunits that can then enter the cell.  Purpose: To see if the bacteria can use starch as a source of carbon and energy.

Test the activity of alpha amylase. Test medium Starch agar plate Performing the test… • Inoculate given organisms on a starch agar plate • Incubate at 37℃. Bacillus subtilis E. coli Starch Agar plate 2. Sugar fermentation reactions Sucrose/Lactose/Maltose Glucose Pyruvate Fermentation Acid (acidic pH) Alcohol (neutral pH) Gas Phenol Red test  Fermentation generates different acids, ethanol, hydrogen gas, carbon dioxide gas, and other organic compounds, depending on the specific reactions.  Purpose: Test for fermentation of different sugars by bacteria. Test medium Tubes contain peptone, carbohydrate source (glucose, lactose, sucrose), phenol red (pH indicator). Medium containing phenol red Inverted Durham tube In this test the production of acids can be observed by a change in color of the medium to yellow. Each tube should also have an inverted and filled Durham tube which is used to look for the production of gas. Durham tube filled with medium Performing the test… • Incubate at 37℃. Control tube (uninoculated) Glucose PR medium Alcaligenes faecalis Escherichia coli Providencia stuartii Protein catabolism Gelatin hydrolysis Urea hydrolysis 3. Gelatin hydrolysis Gelatin Polypeptides Amino acids Complex, folded protein Chains of amino acids Individual amino acid units Gelatin hydrolysis test  Large polypeptides are hydrolyzed by enzymes that cleave the peptide bonds and release smaller products that can cross the cell membrane and be used by the cell.  Gelatin is one such polypeptide that is cleaved by gelatinase.  Purpose: To see if the bacteria can breakdown gelatin into individual amino acids using the enzyme gelatinase.

Test medium A semi solid medium containing gelatin as the solidifying agent instead of agar. Performing the test… • Incubate at 37℃. Escherichia coli Bacillus subtilis 4. Urea hydrolysis H2O Urea Ammonia + CO2 Urea hydrolysis test  This test helps in identifying bacteria in the Enterobacteriaceae family especially of the Proteus genus that can rapidly hydrolyze urea.  Purpose : to identify bacteria that can produce the enzyme urease that can breakdown the urea and convert into ammonia and carbon dioxide. Test medium The medium contains peptone, glucose, urea and phenol red indicator. Phenol red turns yellow in an acidic environment and bright pink when alkaline. If the urea is hydrolyzed then ammonia is released, an alkaline environment is created, turning the media pink. Performing the test… • Incubate at 37℃. Uninoculated control Escherichia coli Proteus vulgaris Week 9: Microbial Metabolism 1: Day 1 1. Starch agar plates help us distinguish between those organisms that can breakdown starch and those that cannot. The components of the starch agar plates are: Beef extract, soluble starch, agar and water. What type of media do you think this isselective/differential? 2. Pseudomonas aeroginosa is a strict aerobe. Microbial Metabolism and Carbohydrate Catabolism

A glucose fermentation test with this organism would show which if the following results? a. Red broth with a gas bubble b. Yellow broth with a gas bubble c. Red broth with no gas bubble d. Yellow broth with no gas bubble 3. What is the purpose of placing the gelatin deeps in the ice bucket after it is removed from the incubator? (Hint: think about how incubating at 37 degrees would affect gelatin) 4. Phenol red is used as an indicator both in the fermentation tests as well as the urea hydrolysis test. Fill in the blanks based on your knowledge of how phenol red acts as a pH indicator in each of these tests. a. In the sugar fermentation tests the phenol red broth is used to test for the production of ______________. The broth is originally __________ in color and turns ___________ in color if the result is positive. b. In the urea hydrolysis test the urea broth is used to test for the production of _______________. The broth is originally ___________ in color and turns __________ in color if the result is positive. …