Microbiology Staining Techniques and Identification

Gram Stain Identification

  • Identify Gram stain & morphology using slides.

  • Why?

    • Gram-positive: Appears purple due to thick peptidoglycan retaining crystal violet.

    • Gram-negative: Appears pink/red because the thin cell wall loses crystal violet and picks up safranin.

  • Morphology:

    • Cocci: round shape.

    • Bacilli: rod-shaped.

    • Spirilla: spiral shape.

Identify Staining Technique

  • Endospore stain:

    • Endospores appear green, while vegetative cells appear pink.

  • Capsule stain:

    • Clear halo around the cell against a dark background.

  • Gram stain:

    • Purple for Gram-positive and pink for Gram-negative.

EMB Agar & MacConkey Agar

  • a. Type & purpose

    • EMB (Eosin Methylene Blue) Agar:

    • Selective for Gram-negative bacteria.

    • Differential for lactose fermentation.

    • MacConkey Agar:

    • Selective for Gram-negative bacteria.

    • Differential for lactose fermentation.

  • b. Interpret results:

    • Lactose fermenter:

    • EMB: Produces dark purple or black colonies, or a metallic green sheen.

    • MacConkey: Produces pink colonies.

    • Non-lactose fermenter:

    • EMB: Produces colorless colonies.

    • MacConkey: Produces colorless colonies.

    • Coliform: Gram-negative lactose-fermenting rod.

  • c. Identify E. coli:

    • EMB: Metallic green sheen indicates E. coli.

    • MacConkey: Bright pink colonies with pink precipitate in the agar indicate E. coli.

  • d. Gram reaction:

    • E. coli is identified as a Gram-negative rod.

MSA (Mannitol Salt Agar)

  • a. Type & purpose:

    • Selective for Staphylococcus due to high salt concentration (7.5% salt).

    • Differential for mannitol fermentation.

  • b. Interpret results:

    • Growth: Indicates the organism tolerates salt (Staphylococcus).

    • No growth: Indicates the organism is not Staphylococcus.

    • Yellow agar: Indicates mannitol fermentation through acid production.

    • Red agar: Indicates no fermentation.

  • c. Identify S. aureus:

    • Growth with yellow agar indicates the presence of Staphylococcus aureus.

  • d. Identify Staph that isn’t S. aureus:

    • Growth with red agar indicates presence of other Staphylococcus species (e.g., S. epidermidis).

Blood Agar

  • a. Type & purpose:

    • Enriched and differential medium.

    • Utilized for detecting hemolysis.

  • b. Identify hemolysis types:

    • Alpha hemolysis: Produces a green zone due to partial breakdown of RBCs.

    • Beta hemolysis: Produces a clear zone indicating complete breakdown of RBCs (more pathogenic).

  • c. Meaning of hemolysis types:

    • Alpha hemolysis: Indicative of partial red blood cell (RBC) breakdown.

    • Beta hemolysis: Indicative of complete RBC breakdown, suggests greater pathogenicity.

Catalase & Oxidase Tests

  • Catalase test:

    • Positive result: Formation of bubbles indicates the presence of catalase.

    • Negative result: No bubbles are produced, indicating no catalase present.

  • Oxidase test:

    • Positive result: Develops a purple/blue color within 10 seconds, indicating presence of cytochrome C oxidase.

    • Negative result: No color change, indicating absence of cytochrome C oxidase.

Biochemical Tests

  • A. Phenol Red Sugar Broths (PRG, PRL, PRS):

    • Yellow: Indicates acid production (positive result).

    • Yellow + gas bubble: Indicates acid and gas production.

    • Red: Indicates negative result for acid production.

    • Pink: Indicates an alkaline result due to protein use.

  • B. Citrate test:

    • Blue: Positive result indicating utilization of citrate.

    • Green: Negative result indicating no citrate utilization.

  • C. SIM (Sulfur, Indole, Motility):

    • Sulfur:

    • Black: Indicates positive result for hydrogen sulfide production.

    • Not black: Indicates negative result.

    • Indole:

    • Red ring formation (with Kovac’s reagent): Indicates positive result.

    • No red ring: Indicates negative result.

    • Motility:

    • Growth spreading out: Indicates positive motility.

    • Growth only along the stab line: Indicates negative motility.

  • D. Urease test:

    • Hot pink color: Positive result indicating urease production.

    • Yellow/no change: Negative result indicating no urease production.

  • E. Gelatinase test:

    • Liquid when chilled: Indicates positive result indicating gelatinase production.

    • Solid state: Indicates negative result for gelatinase production.

  • F. Phenylalanine Deaminase test:

    • Green color: Positive result indicating phenylalanine deaminase presence.

    • Yellow color: Negative result indicating absence of phenylalanine deaminase.