뒤로Identifying Bacteria Based on Enzymatic Activity and Differential Media
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Identifying Bacteria Based on Enzymes and Multiple Test Media
Aim and Overview
Microbiologists often identify bacteria by detecting the presence of specific enzymes that catalyze unique biochemical reactions. These enzymatic activities can be revealed using specialized media and reagents, allowing for the differentiation and identification of bacterial species based on their metabolic capabilities.
Key enzymes detected: Amylase, Protease, Lipase, Tryptophanase, Urease, Phenylalanine deaminase
Purpose: To determine which enzymes are present in an unknown bacterial sample by observing changes in selective and differential media.
Enzyme Detection Tests
Summary Table: Enzyme Tests and Expected Results
Enzyme (Example Organism) | Medium Used | Color Change/Observation | Byproduct Detected |
|---|---|---|---|
Amylase (B. subtilis) | Starch agar plate | Add iodine; look for clear zone due to starch degradation | Clear zone (starch hydrolysis) |
Protease (B. subtilis) | Skim milk plate | Look for clear zone due to degradation of casein protein | Clear zone of degradation |
Lipase (S. aureus) | Spirit blue agar | Dark blue precipitation or depletion of fat droplets (pH drop from fatty acids) | Blue precipitation or fat droplet depletion |
Tryptophanase (E. coli) | Tryptone broth | Add Kovac's reagent; red ring indicates indole production | Indole (from tryptophan breakdown) |
Urease (P. vulgaris) | Urea agar slant | Color change from yellow to bright pink (phenol red indicator) | Ammonia (pH increase) |
Phenylalanine deaminase (P. vulgaris) | Phenylalanine agar | Greenish precipitate after adding 10% ferric chloride | Phenylpyruvic acid (green color) |
Detailed Explanations of Enzyme Tests
Amylase Test
Amylase is an enzyme that hydrolyzes starch into simpler sugars. Bacteria that produce amylase can be detected on starch agar plates. After incubation, iodine is added; a clear zone around the bacterial growth indicates starch degradation.
Key Point: Clear zone after iodine addition = positive for amylase.
Example: Bacillus subtilis is amylase-positive.
Protease Test
Proteases degrade proteins such as casein in milk. On skim milk agar, a clear zone around colonies indicates proteolysis.
Key Point: Clear zone = casein hydrolysis (protease activity).
Example: Bacillus subtilis is protease-positive.
Lipase Test
Lipases hydrolyze lipids. On spirit blue agar, lipase activity is indicated by a dark blue precipitate or depletion of fat droplets, due to fatty acid release lowering the pH.
Key Point: Blue precipitation or fat droplet depletion = positive for lipase.
Example: Staphylococcus aureus is lipase-positive.
Tryptophanase (Indole) Test
Tryptophanase breaks down tryptophan into indole, pyruvate, and ammonia. Indole is detected by adding Kovac's reagent, which forms a red ring if indole is present.
Key Point: Red ring after Kovac's reagent = positive for indole (tryptophanase activity).
Example: Escherichia coli is indole-positive.
Equation:
Urease Test
Urease hydrolyzes urea to ammonia and carbon dioxide. The release of ammonia increases the pH, causing phenol red in the medium to turn bright pink.
Key Point: Pink color = positive for urease.
Example: Proteus vulgaris is urease-positive.
Equation:
Phenylalanine Deaminase Test
Phenylalanine deaminase removes the amino group from phenylalanine, producing phenylpyruvic acid and ammonia. Addition of ferric chloride yields a green color if phenylpyruvic acid is present.
Key Point: Green color after ferric chloride = positive for phenylalanine deaminase.
Example: Proteus vulgaris is phenylalanine deaminase-positive.
Multiple Testing Media
Some media are designed to test for several metabolic properties simultaneously, saving time and resources in bacterial identification.
Examples: Kligler's Iron Agar, SIM medium, Litmus Milk
Kligler's Iron Agar
Kligler's Iron Agar is a differential medium containing 1.0% lactose, 0.1% glucose, cysteine, peptones, ferrous salts, and phenol red. It is used to detect glucose and lactose fermentation, as well as hydrogen sulfide (H2S) production.
Key Point: Color changes and black precipitate indicate fermentation and H2S production, respectively.
Inoculation: Streak and stab the medium with the test organism.
Example: P. vulgaris and E. coli are used as controls.
SIM Medium (Sulfide, Indole, Motility)
SIM medium is semi-solid (0.7% agar) and tests for three properties: motility, hydrogen sulfide production, and tryptophan breakdown (indole production).
Key Point: Motility is indicated by diffuse growth; blackening indicates H2S; red ring after Kovac's reagent indicates indole.
Inoculation: Stab inoculation is required.
Controls: S. aureus, P. vulgaris, E. coli
Litmus Milk (Optional)
Litmus milk is a complex medium that can detect multiple metabolic activities:
Fermentation of sugar: Pink color (acid formation)
Protein breakdown (alkaline reaction): Blue color (ammonia formation)
Litmus reduction: White color (reduction of dye)
Coagulation: Curd formation
Peptonization: Medium becomes translucent or brown
Ropiness: Thick slime due to waste accumulation
Inoculate a tube of litmus milk with the unknown organism only.
Summary Table: Multiple Testing Media
Medium | Properties Detected | Key Observations |
|---|---|---|
Kligler's Iron Agar | Glucose fermentation, lactose fermentation, H2S production | Color change (yellow/red), black precipitate |
SIM | Motility, H2S production, indole production | Diffusion, blackening, red ring after Kovac's |
Litmus Milk | Sugar fermentation, protein breakdown, reduction, coagulation, peptonization, ropiness | Pink, blue, white, curd, translucent/brown, slime |
Conclusion
Enzyme-based tests and multiple testing media are essential tools in microbiology for the identification and differentiation of bacteria. By observing specific color changes, precipitates, or zones of clearing, microbiologists can infer the metabolic capabilities of unknown organisms and classify them accordingly.
