Fermentation and MR-VP Tests

• Carbohydrate catabolism and fermentation are being reviewed

Metabolism

• Metabolism consists of chemical reactions occurring within living organisms
• Metabolic process requires enzymes
• Enzymes are proteins that catalyze biological reactions
• Endoenzymes function inside a cell
• Exoenzymes are released from the cell to catalyze reactions outside the cell

Catabolism

• Catabolism is a decomposition reaction in a living organism; the breakdown of complex organic compounds into simpler ones
• A chemical reaction releases energy from the decomposition of complex organic molecules

Carbohydrates

• Carbohydrates are organic molecules that contain carbon, hydrogen, and oxygen in the ratio of (CH2O)n
• Carbohydrates are classified based on size
• Monosaccharides are simple sugars
• Oligosaccharides consist of 2 to 20 monosaccharide molecules
• Polysaccharides consist of 20 or more monosaccharide molecules

Starch Hydrolysis

• The ability to degrade starch is a criterion for determining amylase production by a microbe
• Starch agar is a differential medium that tests an organism's ability to produce certain exoenzymes, including a-amylase and oligo-1,6-glucosidase which hydrolyze starch
• Exoenzymes are mainly hydrolytic enzymes that leave the cell
• Breakdown large substrates occur by adding water into smaller components
• Starch is a complex polysaccharide found abundantly in plants, usually deposited as large granules in the cytoplasm
• Some bacteria secrete exoenzymes to degrade starch into subunits, as starch molecules are too large to enter the bacterial cell
• The major starch component can be hydrolyzed by a-amylase, common in some bacterias and fungi

Starch Hydrolysis Procedure

• Divide the starch agar into 3 sectors by labeling 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 Results

• Starch agar is a simple nutritive starch medium
• Iodine is added to the plate after incubation, as since there is no color change in the medium when organisms hydrolyze starch
• Iodine will turn blue, purple, or black depending on its concentration where there is starch
• A clearing around the bacterial growth indicates the organism hydrolyzed the starch

Starch Hydrolysis Results Interpretation

• A positive test is identified with a clear zone around the line of growth after adding the iodine solution
• The clear zone indicates starch hydrolysis into monosaccharides, which cannot bind the iodine molecule, appearing as a clear zone around bacterial growth
• A negative test is identified with a dark blue coloration of the medium
  • Starch in the presence of iodine produces dark blue coloration, because iodine is trapped in the helical starch structure

Oxidative Fermentative (OF) Test

• Hugh and Leifson developed the oxidative-fermentative (OF) test in 1953
• OF media differentiates between oxidative bacteria (acid from carbohydrates under aerobic conditions only) and fermentative bacteria (acid under both aerobic and anaerobic conditions)
• Saccharolytic microorganisms degrade glucose either fermentatively or oxidatively
• Fermentation end products are relatively strong mixed acids detectable in a conventional fermentation test medium
• Acids formed in oxidative glucose degradation are extremely weak, requiring Hugh and Leifson's sensitive oxidation fermentation medium for detection
• The medium increases glucose amount above that in fermentation detection mediums, and decreases the amount of peptone

OF Test Procedure

• Two tubes are required for interpreting the OF test
• OF-glucose deeps contain glucose as a carbohydrate, peptones, and bromothymol blue (indicator)
• Both tubes are inoculated, and mineral oil overlays one tube, producing an anaerobic environment
• Oil is not added to the other tube to allow for aerobic conditions
• The tubes are incubated for 24–48 hours at 35°C
• Records show any growth, glucose usage, and metabolism type

Oxidative Results of OF Test

• A small amount of acid production in the open tube indicates the result
• The produced acid (pH 6.0) changes the pH indicator, bromothymol blue, from green to yellow
• After 24 hour incubation, there is a pH change at the surface of the open tube where growth in the presence of oxygen is observed
• With prolonged incubation (over 48 hours), the medium's reduced agar concentration allows the diffusion of weak acid throughout the tube
• There is no color change or reaction occurs in the oil-covered tube
• Pseudomonas aeruginosa is an example

Fermentative Results of OF Test

• Bacteria that ferments glucose gives a fermentative result, indicated by acid production in both the open (aerobic) and oil-covered (anaerobic) tube
• The produced acid (pH 6.0) changes the pH indicator, bromothymol blue, from green to yellow
• Escheria coli is an example

Negative Results of OF Test

• Non-sacchrolytic bacteria give a negative OF result
• The negative result is indicated by no color change in the oil-covered tube, and sometimes an increase in pH (pH 7.6) changing the bromothymol blue from green to blue in the top of the open tube
• The pH increase is due to amine production from bacteria breaking down peptone (protein) in the medium
• Other bacteria give a negative result, indicated by no growth or color change in the medium
• Alcaligenes faecalis is an example

Outcomes of OF Tests

• O-/F- is Non-sacchrolytic
• O+/F+ Fermentative is Enterobacteriaceae
• O+/F- Oxidative is Pseudomonas

Outcomes of OF Tests by Tube and Metabolism

• If the Open (aerobic) Tube is Acid (yellow), and the Covered (Anaerobic) Tube is Alkaline (green), the Metabolism state is Oxidative
• If both tubes (Open (aerobic) Tube and the Covered (Anaerobic) Tube) and are Acid (yellow), the Metabolism state is Fermentative
• If both tubes (Open (aerobic) Tube and the Covered (Anaerobic) Tube) and are Alkaline (green), the result is Non saccharolytic (glucose not metabolized)

Carbohydrate Fermentation

• Determines whether bacteria can ferment a specific carbohydrate
• Patterns are useful in differentiating among bacterial groups or species
• Tests are run for the presence of acid and/or gas produced from carbohydrate fermentation
• A basal medium containing a single carbohydrate, such as Glucose, Lactose, Sucrose, etc. is used

Carbohydrate Fermentation Tubes

• 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 appears Red in a neutral state, and yellow in an acidic state
• Small inverted Durham tubes are also immersed in the medium to test for gas production
• If the test organism produces gas (hydrogen or carbon dioxide), the gas displaces the media inside the tube as a visible air bubble is trapped

Principles of Carbohydrate Fermentation

• Microorganisms ferment carbohydrate where acid or acid with gas are produced
• Depending on the organisms involved and the substrate being fermented, products may vary
• Common end-products include lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, carbon dioxide, and hydrogen
• Acid production lowers the test medium's pH, detected by the pH indicator's color change
• The color only changes when enough acid is produced, as bacteria utilize peptone, producing alkaline byproducts

Carbohydrate Fermentation Results

• Bacteria are classified based on the characteristics and reactions observed in the tests as follows
• Fermenter with acid production only
• Fermenter with acid and gas production
• Non-fermenter

Methyl Red (MR) and Voges-Proskauer (VP) Broth

• The test determines which fermentation pathway utilizes glucose
• It is a simple broth containing peptone, buffers, and dextrose or glucose
• Different bacteria convert dextrose and glucose to pyruvate using different metabolic pathways
• Some of these pathways produce unstable acidic products that convert to neutral compounds quickly
• Some organisms use the butylene glycol pathway, producing neutral end products, including acetoin and 2,3-butanediol
• Other organisms use the mixed acid pathway, producing acidic end products, such as lactic, acetic, and formic acid, and remain stable

Methyl Red (MR) Test

• Methyl Red involves adding the pH indicator methyl red to an MR-VP broth tube
• If the organism uses the mixed acid fermentation pathway and produces stable acidic end-products, the acids overcome the buffers in the medium and produce an acidic environment
• When methyl red is added, it will stay red where acidic end-products are present

Voges-Proskauer (VP) Test

• It detects organisms that utilize the butylene glycol pathway and produce acetoin
• When VP reagents add to MR-VP broth that has an organism that uses the butylene glycol pathway, the acetoin end-product oxidizes in potassium hydroxide (KOH) to diacetyl
• Creatine is also present in the reagent as a catalyst
• Diacetyl reacts to produce a red color

Catalytic Breakdown of Voges-Proskauer

• First the Embden-Myerhoff pathway converts a-D-glucose into pyruvic acid
• The pyruvic acid is converted via glycol pathway into butylene glycol pathway, acetoin (AMC) then carbon dioxide
• Next KOH converts acetoin into diacetyl
• The diacetyl catalyses ammonium (NH) to condensation into a pink-to-red product

Voges-Proskauer (VP) Test Result

• It detects the presence of acetoin, a precursor to 2,3 butanediol
• The culture will turn “brownish-red to pink" if positive for acetoin
• For example, the tube on the left when positive in the second picture
• If the culture is negative for acetoin, it will turn "brownish-green to yellow"
• The tube is on the left and is not positive for the test
• It is positive for one pathway either MR+ or VP+
• Escherichia coli is MR+ and VP-
• Enterobacter aerogenes and Klebsiella pneumoniae are MR- and VP+
• Pseudomonas aeruginosa is a glucose nonfermenter and is thus MR- and VP-

Simmon's Citrate Agar

• It determines if an organism can use citrate as its sole carbon source
• It differentiates between members of Enterobacteriaceae
• In organisms capable of utilizing citrate as a carbon source, the enzyme citrate lyase hydrolyzes citrate into oxaloacetic acid and acetic acid
• The oxaloacetic acid is then hydrolyzed into pyruvic acid and CO2

Simmon's Citrate Agar Results

• If CO2 is produced it reacts with components f the medium to an alkaline compound
  • For example Na2CO3 where alkaline pH turns the pH indicator (bromthymol blue) from green to blue
• The result is a positive test (tube on the right is citrate positive).
  • Klebsiella pneumoniae and Proteus mirabilis are examples of positive citrate organisms
• Escherichia coli and Shigella dysenteriae are citrate negative
• The tube shown on the right is the tube that indicates the citrate is positive

Expected Results in Citrate Utilization Test

• Citrate results are classified as
• Growth visible and identifiable as an intense Prussian blue
• Alkaline Carbonates raise the pH of the medium above 7.6
• The bromothymol blue will change from the original green to blue in the medium with the sample
• Citrate can also be trace where there is trace growth, the culture is virtually indistinguishable from an uninoculated slant
• 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 indistinguishable from an uninoculated slant

Questions about fermentation tests, including gas production and pH indicators. Covers the Methyl Red and Voges-Proskauer tests, metabolic pathways, and the role of buffers. Explores mixed acid and butylene glycol pathways.