Testing for Gram Positive and Gram Negative Organisms

Gram Positive Cocci

1. Catalase Test

  • Purpose: Test for the presence of the enzyme catalase.

  • Substrate: Hydrogen peroxide.

  • Reaction: If the organism contains the enzyme catalase, the reaction produces bubbles through the following equation:

  • 2 H₂O₂ (hydrogen peroxide) + catalase → 2 H₂O + O₂ (oxygen gas), indicating a positive result.

  • Relevance: During metabolism, bacteria that utilize oxygen produce hydrogen peroxide, a toxic byproduct to bacterial cells unless degraded enzymatically by catalase.

  • Differentiation:

    • Staphylococcus spp.: Catalase positive (produces bubbles).

    • Streptococcus spp.: Catalase negative (no bubbles).

2. Coagulase Test

  • Purpose: Test for the presence of the enzyme coagulase.

  • Function of Coagulase: The enzyme converts fibrinogen to fibrin, allowing bacteria to evade phagocytosis by forming a protective clot.

  • Procedure:

    • Uses rabbit plasma (as the reagent).

    • A portion of the bacterial colony is incubated in rabbit plasma for a minimum of 4 hours at 37°C.

    • If the coagulase enzyme is present, clotting will occur.

    • If no clot forms after 4 hours, incubation can be extended up to 24 hours before reporting a negative result.

  • Differentiation:

    • Staphylococcus aureus: Coagulase positive.

    • Other Staphylococcus spp.: Coagulase negative.

Gram Negative Organisms

1. Oxidase Test

  • Purpose: Test for the presence of the enzyme oxidase in gram-negative organisms.

  • Role of Oxidase Enzyme: Involves chemiosmosis during aerobic respiration, catalyzing the reduction of oxygen to water in certain gram-negative bacteria.

  • Outcome: If the oxidase enzyme is present, a sample from the colony will turn the oxidase reagent purple (positive).

  • Examples of Oxidase Positive Organisms:

    • Pseudomonas spp.: Gram-negative bacilli, known to be detrimental for burn patients.

    • Neisseria spp.: Gram-negative diplococci.

2. API Test Strip

  • Purpose: Used to identify enteric gram-negative organisms.

  • Methodology:

    • The API test is based on the specific nutritional requirements for bacterial growth.

    • It consists of a series of 21 tests that evaluate different biochemical properties.

    • The results of these tests yield a unique 7-digit number.

    • This number is compared against a database to identify the organism.