Lab Exercise 22: The Staphylococcal Unknown

Background

  • Bacterial Genus: Staphylococcus includes three medically important species:
    • S. aureus: Pathogenic in the community.
    • S. epidermidis: Common in nosocomial infections.
    • S. saprophyticus: Less common but notable.
  • Colonization: All three species can be found on the skin and mucous membranes of healthy adults.
  • MRSA: Methicillin-resistant S. aureus has become prevalent in community settings.
  • Halophiles: Staphylococci can thrive in saline conditions, growing in media with up to 10% NaCl, suggesting they are adapted for skin environments.

Laboratory Features

  • Testing for Staphylococci: Before biochemical testing, validate Gram positive, catalase-positive staphylococci.
  • Catalase Test Distinction: Staphylococci are distinguished from streptococci by their catalase-positive nature.
  • Culture Medium: Mannitol Salt Agar (MSA) is used for isolation because:
    • Selective due to 7.5% NaCl (only halotolerant organisms grow).
    • Differential for mannitol fermentation via phenol red, signaling a color change from red to yellow in acidic conditions.

Identification Protocol Overview

Introduction
  • Objective: Identify an unknown Staphylococcus species.
    • Utilize MSA for mannitol fermentation.
    • Assess Novobiocin susceptibility.
    • Evaluate coagulase production using rabbit plasma.
    • Examine alpha-toxin production on Blood Agar (BAP).
    • Compare results to theoretical characteristics in a reference table.
Test Characteristics (Table 12)
OrganismMannitol FermentationNovobiocin SusceptibilityCoagulaseAlpha-toxinPigmentation
S. aureusPositiveSensitivePositivePositiveNegative
S. epidermidisNegativeSensitiveNegativeWeak PositiveNegative
S. saprophyticusVariable (+/-)ResistantNegativeWeak PositiveNegative
S. xylosusPositiveResistantNegativeWeak PositivePositive
Day One Protocol
  1. Mannitol Fermentation Test: Streak unknown on MSA plate.
  2. Novobiocin Susceptibility: Inoculate nutrient agar plate; bring for disk placement.
  3. Coagulase Production: Inoculate rabbit plasma.
  4. Alpha-toxin Production: Streak onto BAP.
  5. Incubation: All media at 37°C.
  6. Storage: Label and refrigerate unknown sample.
Day Two Protocol
  1. Assess Mannitol Fermentation: Analyze MSA results.
  2. Measure Novobiocin Zone of Inhibition: >16 mm indicates susceptibility.
  3. Pigmentation on TSA: Evaluate for color characteristics.
  4. Coagulase Test Results: Determine coagulation.
  5. Alpha-toxin Production Check: Inspect BAP results.
  6. Record Data: Enter all findings in analysis section.
  7. Waste Disposal: Discard all inoculated media.

Data Collection and Analysis

  • Maintain organized data table for tests performed.
  • Include results for:
    • Mannitol fermentation, Novobiocin susceptibility, pigmentation, coagulase activity, and alpha-toxin production.
Test Descriptions
  1. Mannitol Salt Agar (MSA): Detects mannitol fermentation and salt tolerance with phenol red indicator.
  2. Rabbit Plasma Coagulase Test: Measures coagulation indicating presence of coagulase enzyme.
  3. Sheep Blood Agar (BAP): Identifies alpha-toxin production through hemolysis patterns.
  4. Pigmentation: Observed on slants at varying temperatures; helps in identifying specific strains.