Dichotomous Key for Unknown Bacterial Identification

Dichotomous Key for Unknown Bacterial Identification Exercise 11

Overview

  • Dichotomous Key Purpose: Used for the identification of unknown bacteria based on the results of various biochemical tests.
  • Source of Organisms: Derived from previous exercises (9 and 10) focusing on Gram-negative (GNR) and Gram-positive (GP) organisms.

Step-by-Step Process

  1. Initial Gram Staining:

    • Result: Gram-positive bacteria appear purple under a microscope.

  2. Morphology of Gram-Positive Organisms:

    • Identified as Cocci (spherical bacteria).

  3. Biochemical Tests and Media: The following tests are used for further classification:

    • Mannitol Salt Agar (MSA): A selective and differential medium for Staphylococcus species.
    • Catalase Test: Determines whether an organism can break down hydrogen peroxide (H₂O₂).
    • Hemolysis Test: Identifies the ability of bacteria to lyse red blood cells on blood agar.
    • Amylase/Starch Test: Checks for the presence of amylase enzyme via iodine clearing zone.

Key Tests and Possible Outcomes

Mannitol Salt Agar (MSA) Test:
  • Growth on MSA: Indicates ability to tolerate high salt concentration.
  • Mannitol Fermentation:
    • (+) Yellow Color Change: Indicates mannitol fermentation.
    • (-) No Color Change: Indicates no fermentation.
Catalase Test:
  • (+) Bubbles: Positive for catalase (organism breaks down H₂O₂).
  • (-) No Bubbles: Negative for catalase.
Hemolysis Patterns on Blood Agar:
  • Beta Hemolysis: Clear zone indicating complete lysis of red blood cells.
  • Alpha / Gamma Hemolysis: Partial or no lysis of red blood cells.
Amylase / Starch Test:
  • (+) Clear Zone: Indicates presence of amylase; the starch has been broken down.

Final Organism Identification

Specific Results and Identifications:
  • Gram-Positive Organisms and Corresponding Results:
    • Staphylococcus aureus:
      • MSA: Growth, Mannitol: (+) Yellow, Catalase: (+) Bubbles.
    • Enterococcus faecalis:
      • MSA: No Growth, Catalase: (-) No Bubbles, No Color Change.
    • Staphylococcus epidermidis:
      • MSA: Growth, Mannitol: (-) No Color Change, Catalase: (+) Bubbles.
    • Micrococcus luteus:
      • MSA: No Growth, Catalase: (-)
    • Streptococcus pyogenes:
      • Hemolysis: Beta (clear zone).
    • Streptococcus agalactiae:
      • Hemolysis: Alpha / Gamma.
    • Bacillus subtilis:
      • Amylase Test: (+) Clear zone indicating amylase production.

Diagram Explanation

  • Figure 1: Illustrates the dichotomous key process where each node (test) branches into two potential outcomes.
  • Test Outcomes: Includes crystal violet staining, safranin application, and interpretation of growth appearance on selective media, catalase activity, and hemolytic patterns.
    • Growth indications include MSA color change to yellow, presence of bubbles upon H₂O₂ addition, and clear zones for amylase activity.
Pathway Indicators:
  • Identify the pathway by marking the sequence of results leading to the final identification based on observed characteristics and testing outcomes.