Gram Positive Lab-1

Gram Positive Tests Overview

1. Catalase Test (Ex 22)

  • Purpose: Determines the presence of catalase enzyme which converts hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2).

  • Differentiation:

    • Staphylococcus: Catalase positive, facultatively anaerobic.

    • Streptococcus: Catalase negative, fermentative.

  • Procedure: Use an inoculating loop to add bacteria to hydrogen peroxide and observe for bubbles (oxygen gas).

2. Coagulase Test

  • Purpose: Detects coagulase enzyme that causes plasma coagulation.

  • Differentiation:

    • Pathogenic Staphylococcus aureus: Coagulase positive.

    • Non-pathogenic staphylococci: Coagulase negative.

3. Mannitol Salt Agar

  • Characteristics: Selective and differential media.

  • Components:

    • High salt content selects for Gram positive organisms.

    • Mannitol fermentation indicates acid production, turning the agar yellow.

  • Organisms:

    • Staphylococcus aureus: Mannitol positive, agar turns yellow.

    • Staphylococcus epidermidis: Mannitol negative, agar remains red.

4. Phenyl Ethyl Alcohol (PEA) Plate

  • Characteristics: Selective media for Gram positive bacteria.

  • Function: Inhibits growth of Gram negative bacteria, allowing cultivation of Gram positive.

5. Oxidase Test

  • Purpose: Tests for cytochrome oxidase in bacteria.

  • Procedure: Apply oxidase reagent; color change indicates positive result.

  • Positive Reaction: Blue/purple color indicates presence of enzyme (e.g., Micrococcus luteus).

  • Negative Reaction: No color change indicates absence of enzyme.

6. Bile Esculin Test

  • Media Components: Esculin, bile, ferric citrate.

  • Function: Bile inhibits growth of all Gram positive except Group D Streptococci and enterococci.

  • Positive Reaction: Black precipitate indicates conversion of Esculin to esculetin.

  • Example: Enterococcus faecalis shows positive result.

7. Blood Agar Plate (BAP)

  • Components: Contains 5-10% sheep blood.

  • Purpose: Differentiates bacteria based on hemolytic properties.

  • Hemolysis Types:

    • Beta Hemolysis: Complete lysis of red blood cells.

    • Alpha Hemolysis: Partial lysis with green discoloration around colonies.

    • Gamma Hemolysis: No lysis, remain unchanged (e.g., Enterococcus faecalis).

8. Hemolysis Types

  • Beta Hemolysis: e.g., Streptococcus agalactiae; clear zones indicate complete lysis.

  • Alpha Hemolysis: Indicative of partial lysis, producing a green discoloration.

  • Gamma Hemolysis: No change in surrounding medium.

9. Streptococcus Species

Streptococcus pyogenes

  • Hemolysis: Beta type.

  • Antigens: C carbohydrate and teichoic acid.

  • Virulence Factors: M protein, tissue-digesting enzymes.

  • Pathogenicity: Skin and throat infections; long-term infection can lead to rheumatic fever.

Streptococcus pneumoniae

  • Staining: Gram positive.

  • Hemolysis: Alpha type, greenish zones on BAP.

  • Virulence Factor: Capsule; causes bacterial pneumonia.

10. Gram-Positive Cocci and Bacilli

Catalase Positive

  • Micrococcus luteus: Positive oxidase test; yellow pigmentation may affect oxidase test appearance.

  • Staphylococcus aureus: Coagulase positive, growth and yellow color change on Mannitol Salt Agar.

Catalase Negative

  • Enterococcus faecalis: Gamma hemolytic on BAP.

Bacillus cereus

  • Catalase Positive: Produces dull, irregular colonies.