Biochemical characteristics
Most important criteria for identification of unknown organism
Enzymes
Responsible for catalyzing biochemical reactions in a microbe.
Hydrolysis reaction
Chemical reaction that require hydrolases and water to breakdown and split large macromolecules into smaller molecules.
Gelatin Hydrolysis
Medium: nutrient gelatin deep tubes |
Substrate: Gelatin |
Enzyme: Gelatinase |
Indicator/Reagent: None |
End-Product: Amino Acids |
Positive Reaction: Liquifaction |
Negative Reaction: Non-liquifaction |
Incubation Time: 25C for 48 hours
Starch Hydrolysis
Medium: starch agar plate |
Substrate: Starch |
Enzyme: Amylase |
Indicator/Reagent: Iodine |
End-Product: Glucose |
Positive Reaction: Clear area after Iodine added (Glucose + Iodine) |
Negative Reaction: Purple-Black area after Iodine added (Starch + Io
Casein Hydrolysis
Medium: Milk Agar Plate |
Substrate: Casein |
Enzyme: Caseinase |
Indicator/Reagent: None |
End-Product: Amino Acids |
Positive Reaction: Clear area around colony |
Negative Reaction: No clear area around colony |
Incubation Time: 37C for 24-48 hours
Biooxidation Reactions
In microorganisms, refers to methods of obtaining energy utilizing Aerobic Respiration and Fermentation
Oxidative Reactions
Common to aerobic microbes that have an energy yielding metabolic process (Aerobic Respiration) in which organic compounds are broken down, have an electron transport chain (cytochrome system), the final electron acceptor is oxygen and their end products
Fermentative Reactions
Microbes that have an energy yielding metabolic process (Fermentation) in which organic compounds are broken down anaerobically, lack an electron transport chain (cytochrome system), the final electron acceptor is an organic molecule and the end products
Phenol Red Broth Sugar Fermentation Test
Medium: Phenol Red Fermentation Broth Tubes |
Substrates: Glucose, Lactose, Sucrose |
Enzymes: Fermentation Set of Enzymes |
Indicator/Reagent: Phenol Red (initial pH 7.4) |
End-Products: Acids, Alcohols, Gases (CO2 and H2) |
Positive Reaction: Fermentati
Starting pH of Phenol Red Broth Fermentation Tube
pH 7.4
Durham Tube
Phenol Red Broth Fermentation Tube
Phenol Red
Acid-Base Indicator |
Indicates presence of small amounts of acids
Inverted Vial (Durham Tube)
Collects and measures gas production (CO2 and H2) |
One bubble equals Positive Reaction
What is required by Non-Fermenting Bacteria in the Phenol Red Broth Fermentation Test?
Protein (Beef Extract and Peptone) as a nitrogen source and for minerals
Proteolysis
Breaking down of protein to make ATP | * End-product: NH3 (Ammonia)
Protein Sparing Mechanism
Bacteria ferments sugars first, causing an acidic pH. When the sugars are completely used up, proteins are then utilized as an energy source which results in a shift in pH to basic or alkaline |
Result: Color of the broth tube goes from Yellow to Red
Litmus Milk
Medium: Litmus Milk (initial neutral starting pH 6.8) |
Substrates: Casein (protein), Lactose (sugar) |
Enzymes: Lactase, Caseinase |
Indicator: Litmus Blue |
End-product: Fermentaing (Acids, Alcohols, Gases (CO2, H2)), Non-fermenting (Ammonia), Soft curd
Tryptophan Hydrolysis (Indole Test)
Medium: 1% Tryptone Broth Tube |
Substrate: Trytophan (Amino Acid) |
Enzyme: Tryptophanase |
Indicator/Reagent: Kovac's Reagent |
End-product: Indole |
Positive Reaction: Red Ring at top of tube |
Negative Reaction: Yellow Ring at top of tube |
Incubation
Use of Methyl Red Test
Methyl Red Test is used to detect the production of large amounts of acids from glucose fermentation, measured by Methyl Red Indicator. |
It is employed to help identify Coliform Mixed Acid fermenters from non-coliforms.
Methyl Red Test
Medium: MR-VP Broth Tube (Initial pH 6.9) |
Substrate: Glucose |
Enzyme: Complete Fermentation Set of Enzymes |
Indicator/Reagent: Methyl Red |
End-product: Large amount of acids (Acetic, Lactic, and Formic acids) |
Positive Reaction: Red Tube (pH ? 4.5)
Use of Voges-Proskauer Test
* The Voges-Proskauer Test is used to detect the production of Acetoin (Acetylmethylcarbinol), which is a neutral end-product from incomplete glucose fermentation. The incomplete fermentation of glucose results in the accumulation of equal amount of neutr
Voges-Proskauer Test
Medium: MR-VP Broth Tube |
Substrate: Glucose |
Enzyme: Incomplete Fermentation Set of Enzymes |
Indicator/Reagent: VP Reagent I (Alpha-Naphthol Solution), VP Reagent II (Potassium Hydroxide (KOH)) |
End-product: Acetoin (Neutral end-product) |
Positive R
Use of Citrate Test
The Citrate test measures growth or no growth which is measured by a color change of the indicator brom thymol blue. |
If the organism can utilize citrate as its sole carbon source, then it will grow, produce an alkaline end-product called sodium carbonat
Citrate Test
Medium: Simmon's Citrate Agar Slant (Initial pH 6.9) |
Substrate: Citrate |
Enzyme: Citrase |
Indicator/Reagent: Brom Thymol Blue |
End-product: Sodium Carbonate (Alkaline/Basic pH) |
Positive Reaction: Blue Slant (Growth) | * Negative Reaction: Green Sla
Test that uses Iodine as Indicator
Starch Hydrolysis
Test that uses Phenol Red as Indicator
Phenol Red Broth Sugar Fermentation Test
Test that uses Litmus Blue as Indicator
Litmus Milk
IMViC Series Tests
A group of tests used for analysis of water, food, food products and sewage for determination of Coliform contamination
Test that uses Kovac's Reagent as Reagent
Tryptophan Hydrolysis (Indole Test)
Test that uses Methyl Red as Indicator
Methyl Red Test
Test that uses VP Reagent I/II as Reagents
Voges-Proskauer Test
Test that uses Brom Thymol Blue as Indicator
Citrate Test
Initial pH of Phenol Red Broth
pH 7.4
Initial pH of Litmus Milk
pH 6.8
Initial pH of MR-VP Broth
pH 6.9
Initial pH of Simmon's Citrate Agar Slant
pH 6.9
Results of Gelatin Hydrolysis
Positive: Liquifaction of Nutrient Gelatin |
Negative: Non-liquifaction of Nutrient Gelatin
Results of Starch Hydrolysis
Positive: Glucose + Iodine = Clear area around colony |
Negative: Starch + Iodine = Purple-black color around colony
Results of Casein Hydrolysis
Positive: Clear area around colony |
Negative: No clear area around colony
Results of Phenol Red Broth Fermentation Test
Positive: Yellow Color = Acidic pH (< 7.0), Gas bubble in inverted vial |
Negative: Red Color = Basic pH (>7.0)
Results of Litmus Milk
Acid Reaction: Litmus Blue turns pink with acid production, soft curd formation |
Alkaline Reaction: Litmus Blue turns purple with ammonia (NH3) production, soft curd formation
What causes soft curd formation?
Precipitation of casein protein due to extreme pH shift
Results of Tryptophan Hydrolysis (Indole Test)
Positive: Red Ring at top of tube |
Negative Test: Yellow Ring at top of tube
Results of Methyl Red Test
Positive: Red Tube |
Negative: Yellow Tube
Results of Voges-Proskauer Test
Positive: Red Tube |
Negative: Yellow Tube
Results of Citrate Test
Positive: Blue Slant, Growth |
Negative: Green Slant, No Growth