L7 - Protein Binding; Introduction to Enzymes

# EMB (Eosin Methylene Blue) Agar **Purpose:** Selective and differential medium. * **Selective:** Inhibits Gram-positive bacteria. * **Differential:** Differentiates lactose fermenters. ### Green metallic sheen * Strong lactose fermentation * Classic organism: **E. coli** ### Colony colors * **Green metallic sheen:** Strong lactose fermenter (E. coli) * **Purple/black colonies:** Lactose fermenter (Enterobacter, Klebsiella) * **Colorless colonies:** Non-lactose fermenter (Salmonella, Shigella) --- # MacConkey Agar ### Contains * Lactose * Neutral red indicator * Crystal violet * Bile salts ### Purpose * Selects for Gram-negative bacteria * Differentiates lactose fermenters ### Results **Pink/red colonies** * Lactose fermenter **Colorless colonies** * Non-lactose fermenter --- # TSIA (Triple Sugar Iron Agar) ### Tests for * Glucose fermentation * Lactose fermentation * Sucrose fermentation * Gas production * Hydrogen sulfide (H₂S) ### Sugars present * Glucose (0.1%) * Lactose (1%) * Sucrose (1%) ### Color meanings Yellow = Acid (A) Red = Alkaline (K) Black = H₂S production Cracks/lifting = Gas production ### Interpretations **A/A** * Yellow slant * Yellow butt * Ferments glucose + lactose and/or sucrose **K/A** * Red slant * Yellow butt * Ferments glucose only **K/K** * Red slant * Red butt * No sugar fermentation ### Proper inoculation * Stab the butt once. * Streak the slant as you remove the needle. --- # Mannitol Salt Agar (MSA) ### Purpose Selective and differential. ### Selective * 7.5% salt selects for **Staphylococcus**. ### Differential * Mannitol fermentation ### Results Yellow media * Mannitol fermented * Acid produced Red/Pink media * No mannitol fermentation ### Species **Staphylococcus aureus** * Yellow * Pathogenic **Staphylococcus epidermidis** * Pink/red * Usually non-pathogenic --- # Blood Agar ### Classification **Both enriched and differential** ### Hemolysis **Beta** * Complete hemolysis * Clear zone **Alpha** * Partial hemolysis * Green zone **Gamma** * No hemolysis * No color change --- # Kirby-Bauer Test Purpose: Determine antibiotic susceptibility. ### Measure Diameter of the **zone of inhibition** (mm). Large zone * Sensitive Small/no zone * Resistant --- # UV Light ### Mechanism UV damages DNA by forming **thymine dimers**. ### Most harmful wavelength **254 nm (UV-C)** ### Interpretation Less bacterial growth = UV more effective. --- # Urease Test ### Purpose Detect urease enzyme. ### Reaction Urea → Ammonia + CO₂ Ammonia raises pH. ### Color Pink = Positive Yellow/orange = Negative ### Strong urease-positive genus **Proteus** --- # Catalase Test ### Reagent Hydrogen peroxide (3%) ### Positive Bubbles immediately ### Negative No bubbles Reaction: 2H₂O₂ → 2H₂O + O₂ The bubbles are oxygen gas. --- # Passive (Indirect) Agglutination ### Definition Antibodies bind to antigen-coated particles, causing visible clumping. Positive = Clumping Negative = Smooth suspension --- ## High-Yield Organisms to Memorize | Organism | Key Feature | | ------------------------------ | ------------------------------------------------- | | **E. coli** | Green metallic sheen on EMB | | **Proteus** | Strong urease positive | | **Staphylococcus aureus** | MSA turns yellow (mannitol fermenter) | | **Staphylococcus epidermidis** | MSA stays pink | | **Salmonella** | Non-lactose fermenter; often H₂S positive on TSIA | | **Shigella** | Non-lactose fermenter; H₂S negative | ## Last-minute memorization * **EMB** → Green sheen = **E. coli** * **MacConkey** → Pink = lactose fermenter * **MSA** → Yellow = mannitol fermenter (**S. aureus**) * **Blood agar** → Beta = clear, Alpha = green, Gamma = none * **TSIA** → Yellow = acid, Red = alkaline, Black = H₂S * **Kirby-Bauer** → Bigger zone = more susceptible * **Urease** → Pink = positive (**Proteus**) * **Catalase** → Bubbles = positive * **UV** → 254 nm causes thymine dimers

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