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Microbiology

Colony Morphology & ID

The rapid and accurate identification of the causative agents of acute meningitis is crucial for initiating prompt and appropriate antimicrobial therapy, which is critical for patient survival and minimizing neurological sequelae. Understanding the colony morphology and the identification methods of the major pathogens associated with this life-threatening condition is essential for clinical microbiology laboratory personnel

General Principles

  • CSF Examination: A comprehensive CSF analysis is essential for the diagnosis of meningitis and to identify the pathogen
  • Initial Tests
    • Gram Stain: Performed immediately to detect bacteria and provide rapid presumptive identification
    • Cell Count and Differential: Determines the number and type of cells (WBCs, RBCs)
    • Protein and Glucose: Measure of protein and glucose levels
  • Culture: The gold standard for diagnosis. The most important test to determine the cause of the infection
    • Media: CSF is typically inoculated onto:
      • Blood Agar (BAP): Non-selective medium. Supports growth of most bacteria
      • Chocolate Agar (CHOC): Enriched medium. Supports the growth of fastidious organisms, such as Haemophilus influenzae and Neisseria meningitidis
      • Other media: May use other selective/differential media (e.g., MacConkey agar)
    • Incubation: Cultures are incubated at 35-37°C in an incubator with a 5% CO2 atmosphere
    • Incubation Time: Cultures are typically incubated for at least 48-72 hours, or longer if slow-growing organisms are suspected (e.g., Listeria monocytogenes)
    • Daily Examination: Colonies examined for morphology, Gram stain
  • Additional Tests
    • Antigen Detection Tests: Rapid tests to detect specific bacterial antigens in the CSF. Can provide rapid results
    • Molecular Methods: PCR and other molecular tests provide a rapid and sensitive method to identify organisms directly from CSF
    • Susceptibility Testing: Performed on all significant isolates to guide antimicrobial therapy

Major Pathogens: Colony Morphology, Gram Stain, and Identification

Streptococcus pneumoniae

  • Colony Morphology
    • BAP: Small, gray, mucoid, glistening colonies. May have a characteristic “draughtsman” appearance (a central raised area and a flattened peripheral edge)
    • Hemolysis: Alpha-hemolytic (greening around the colonies)
  • Gram Stain: Gram-positive, lancet-shaped diplococci (pairs of cocci)
  • Identification
    • Catalase: Negative
    • Optochin Susceptibility: Susceptible (zone of inhibition around the optochin disk)
    • Bile Solubility: Positive (colonies dissolve in bile or a bile salt solution)
    • Pneumococcal Antigen Test: Rapid test to detect pneumococcal capsular antigen
    • Quellung Reaction: (Serotyping). Capsular swelling when exposed to specific antibody

Haemophilus influenzae

  • Colony Morphology
    • CHOC: Small, gray, translucent, slightly mucoid colonies. “Mousy” odor
    • BAP: Will only grow with V factor (NAD) and X factor (hemin)
  • Gram Stain: Gram-negative coccobacilli or pleomorphic rods
  • Identification
    • X and V Factor Requirement: Requires both X factor (hemin) and V factor (NAD) for growth (use of factor strips or a quad plate)
    • Latex Agglutination: (or other rapid antigen tests). For capsular polysaccharide (type b H. influenzae)
    • Commercial Identification Systems

Neisseria meningitidis

  • Colony Morphology
    • CHOC: Small, gray, non-hemolytic, translucent colonies. Can be mucoid
    • Appearance: Can develop a distinctive blue or purple color
  • Gram Stain: Gram-negative diplococci (kidney bean-shaped)
  • Identification
    • Oxidase: Positive
    • Carbohydrate Utilization: Utilizes glucose and maltose, but not lactose
    • Rapid Antigen Tests: Tests for capsular antigens
    • Commercial Identification Systems

Escherichia coli

  • Colony Morphology
    • BAP: Large, gray colonies, often with a metallic sheen
    • MacConkey Agar: Pink, lactose-fermenting colonies
  • Gram Stain: Gram-negative rods
  • Identification
    • Oxidase: Negative
    • Lactose Fermentation: Positive
    • IMViC Tests: ++ –
    • Commercial Identification Systems

Listeria monocytogenes

  • Colony Morphology
    • BAP: Small, translucent, bluish-gray colonies
    • Appearance: Often has a “dewdrop” appearance
    • Hemolysis: Beta-hemolytic (narrow zone of hemolysis)
  • Gram Stain: Gram-positive short rods or coccobacilli
  • Identification
    • Catalase: Positive
    • Motility: Motile at room temperature (tumbling motility)
    • Hippurate Hydrolysis: Positive
    • Commercial Identification Systems

Enterobacteriaceae (Other than E. coli Examples: Klebsiella, Proteus, etc.)

  • Colony Morphology
    • BAP: Large, gray colonies
    • MacConkey Agar: Pink, lactose-fermenting colonies
  • Gram Stain: Gram-negative rods
  • Identification
    • Oxidase: Negative
    • Lactose Fermentation: Variable
    • IMViC Tests: (Indole, Methyl Red, Voges-Proskauer, Citrate)
    • Commercial Identification Systems

Staphylococcus aureus

  • Colony Morphology
    • BAP: Medium to large, circular, opaque, smooth, golden-yellow or cream-colored colonies
    • Hemolysis: Usually beta-hemolytic
  • Gram Stain: Gram-positive cocci in clusters
  • Identification
    • Catalase: Positive
    • Coagulase: Positive

Beta-Hemolytic Streptococci (e.g. Streptococcus agalactiae, Group B)

  • Colony Morphology
    • BAP: Small, translucent or gray colonies
    • Hemolysis: Beta-hemolytic
  • Gram Stain: Gram-positive cocci in chains
  • Identification
    • Catalase: Negative
    • Lancefield Grouping: (Group B)
    • CAMP Test: Positive

Additional Considerations

  • Fastidious Organisms: Some organisms, such as Haemophilus influenzae and Neisseria meningitidis, require enriched media (e.g., chocolate agar) for optimal growth
  • Rapid Antigen Detection Tests: Can provide rapid presumptive identification. Have variable sensitivity
  • Molecular Methods (PCR)
    • High Sensitivity and Specificity
    • Can detect organisms directly from CSF, even if they are not culturable
    • Multiplex PCR assays: Allow for the simultaneous detection of multiple pathogens
  • Reporting: Report results promptly to the physician, including Gram stain results, preliminary identification, and any preliminary susceptibility results
  • Quality Control: Follow established quality control procedures for all media, reagents, and tests

Key Terms

  • Acute Meningitis: Rapid onset of inflammation of the meninges
  • Cerebrospinal Fluid (CSF): The fluid that surrounds the brain and spinal cord
  • Gram Stain: A staining technique that differentiates bacteria based on cell wall characteristics
  • Culture: The growth of microorganisms in a laboratory setting for identification and susceptibility testing
  • Blood Agar (BAP): A general-purpose, enriched, non-selective medium
  • Chocolate Agar (CHOC): An enriched medium for fastidious organisms
  • Alpha-Hemolysis: Partial lysis of red blood cells
  • Beta-Hemolysis: Complete lysis of red blood cells
  • Optochin Susceptibility: Used to differentiate Streptococcus pneumoniae from other alpha-hemolytic streptococci
  • Quellung Reaction: (capsular swelling). Serotyping
  • Oxidase Test: Used to identify Neisseria spp
  • Lactose Fermentation: Ability to ferment lactose
  • IMViC Tests: A series of biochemical tests for identifying Enterobacteriaceae
  • Hippurate Hydrolysis: Test used to identify Listeria monocytogenes
  • CAMP Test: Test used to identify Group B streptococci
  • Commercial Identification System: System that uses pre-packaged biochemical tests for identifying microorganisms
  • PCR (Polymerase Chain Reaction): A molecular method used to amplify specific DNA or RNA sequences
  • Antigen Detection Test: A test that detects specific antigens of a microorganism
  • Antimicrobial Susceptibility Testing: Laboratory tests performed to determine the susceptibility of a bacterial isolate to various antibiotics
  • Enriched Media: Culture media containing extra nutrients to support the growth of fastidious microorganisms
  • Fastidious Organism: A microorganism with complex nutritional requirements
  • Capsule: A polysaccharide layer that surrounds some bacteria, protecting them from phagocytosis
  • Diplococci: Cocci arranged in pairs