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Microbiology

Colony Morphology & ID

The accurate identification of major pathogens in body fluids from normally sterile sites is critical for the rapid diagnosis and appropriate treatment of serious infections. The laboratory plays a vital role in distinguishing between these potentially life-threatening infections, and contamination and proper methods must be followed. Knowing the colony morphology, Gram stain characteristics, and identification methods for these organisms is essential for clinical microbiology

General Principles

  • Sterility and Rapid Results: Body fluids from normally sterile sites are expected to be sterile. The presence of any microorganism requires quick analysis
  • Specimen Handling: The laboratory must have strict protocols for handling, processing, and culturing these specimens to ensure rapid and accurate diagnosis
  • Culture Media: Multiple types of media are used
    • Blood Agar (BAP): Used for general growth
    • Chocolate Agar (CHOC): For fastidious organisms
    • Anaerobic Media: To cultivate anaerobic organisms
    • Selective and Differential Media: To enhance recovery of pathogens and aid in differentiation
  • Incubation
    • Incubation temperature is typically 35-37°C
    • CO2 enhanced environment for some media, or organisms (e.g., 5% CO2)
    • Incubation times will vary
  • Microscopic Examination: Gram stains are performed on all suspicious specimens to provide preliminary results and guide further testing
  • Colony Morphology: Colonies are examined and described for size, shape, color, texture, and any hemolytic reactions
  • Identification
    • Colony morphology, Gram stain results, and patient history provide clues
    • Biochemical tests, rapid tests, and commercial identification systems are used for definitive identification
  • Reporting: Results are reported promptly, including Gram stain results, preliminary identification, and any preliminary susceptibility results
  • Quality Control: Quality control procedures are followed for all media and reagents

Major Pathogens: Colony Morphology, Gram Stain, and Identification

Streptococcus pneumoniae

  • Source: Pleural, peritoneal, pericardial, synovial fluids, etc
  • 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

Haemophilus influenzae

  • Source: Pleural, peritoneal, pericardial, synovial fluids, etc
  • 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: For capsular polysaccharide (type b H. influenzae)
    • Commercial Identification Systems

Neisseria spp. (e.g., N. meningitidis, N. gonorrhoeae)

  • Source: Pericardial, synovial fluids, etc
  • Colony Morphology
    • CHOC: Small, gray, non-hemolytic, translucent colonies. Can be mucoid
  • Gram Stain: Gram-negative diplococci (kidney bean-shaped)
  • Identification
    • Oxidase: Positive
    • Carbohydrate Utilization
      • N. meningitidis: Glucose and maltose
      • N. gonorrhoeae: Glucose only
    • Rapid Antigen Tests: Tests for capsular antigens
    • Commercial Identification Systems

Escherichia coli

  • Source: Pleural, peritoneal, pericardial, synovial fluids, etc
  • 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

  • Source: Pleural, peritoneal, pericardial, synovial fluids, etc
  • 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.)

  • Source: Pleural, peritoneal, pericardial, synovial fluids, 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

  • Source: Pleural, peritoneal, pericardial, synovial fluids, etc
  • 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; Streptococcus pyogenes, Group A)

  • Source: Pleural, peritoneal, pericardial, synovial fluids, etc
  • Colony Morphology
    • BAP: Small, translucent or gray colonies
    • Hemolysis: Beta-hemolytic
  • Gram Stain: Gram-positive cocci in chains
  • Identification
    • Catalase: Negative
    • Lancefield Grouping
      • Group A (Streptococcus pyogenes): Bacitracin susceptible, PYR positive
      • Group B (Streptococcus agalactiae): CAMP test positive
    • Commercial Identification Systems

Enterococcus spp.

  • Source: Pleural, peritoneal, pericardial, synovial fluids, etc
  • Colony Morphology
    • BAP: Grayish, medium-sized colonies
    • Hemolysis: Non-hemolytic or alpha-hemolytic
  • Gram Stain: Gram-positive cocci in pairs or short chains
  • Identification
    • Catalase: Negative
    • Growth on Bile Esculin Agar: Positive (turns the agar black)
    • 6.5% NaCl Tolerance: Positive (growth in broth containing 6.5% NaCl)
    • PYR test: Positive
    • Commercial Identification Systems

Pseudomonas aeruginosa

  • Source: Pleural, peritoneal, pericardial, synovial fluids, etc
  • Colony Morphology
    • BAP: Large, flat, spreading colonies
    • Pigment Production: Often produces a blue-green pigment (pyocyanin) and/or a yellow pigment (pyoverdine)
    • Appearance: Can have a distinct fruity odor (grape-like)
  • Gram Stain: Gram-negative rods
  • Identification
    • Oxidase: Positive
    • Glucose Fermentation: Oxidative
    • Motility: Motile
    • Commercial Identification Systems

Acinetobacter spp.

  • Source: Pleural, peritoneal, pericardial, synovial fluids, etc
  • Colony Morphology
    • BAP: Gram-negative, oxidase-negative, non-fermentative
    • Appearance: Can vary, but often appear as plump, opaque, smooth, slightly raised, and gray-white to tan
  • Gram Stain: Gram-negative coccobacilli or short rods
  • Identification
    • Oxidase: Negative
    • Glucose Fermentation: Negative
    • Motility: Non-motile
    • Commercial Identification Systems or MALDI-TOF: Used for species-level identification

Clostridium perfringens

  • Source: Pleural, peritoneal fluids, etc
  • Colony Morphology
    • Anaerobic media: Large, gray, flat colonies
    • Hemolysis: Double-zone beta-hemolysis
  • Gram Stain: Gram-positive rods
  • Identification
    • Anaerobic culture required
    • Commercial Identification Systems

Bacteroides fragilis group

  • Source: Pleural, peritoneal fluids, etc
  • Colony Morphology
    • Anaerobic media: Gray, non-hemolytic colonies
    • Appearance: Can be small, convex and circular
  • Gram Stain: Gram-negative rods or coccobacilli
  • Identification
    • Anaerobic culture required
    • Commercial Identification Systems

Additional Considerations

  • Fastidious Organisms
    • Some organisms (e.g., Haemophilus influenzae) require enriched media (e.g., chocolate agar)
  • Specimen Source
    • S. aureus and Streptococcus pyogenes can be present
  • Reporting
    • Preliminary results (Gram stain, morphology)
    • Species level identification
    • Antimicrobial susceptibility
  • Prompt Communication
    • Communicate results to clinicians

Key Terms

  • Body Fluids: Fluid from normally sterile sites
  • Pleural Fluid: Fluid from the pleural space
  • Peritoneal Fluid: Fluid from the peritoneal cavity
  • Pericardial Fluid: Fluid from the pericardial space
  • Vitreous Humor: Fluid of the eye
  • Aqueous Humor: Fluid of the eye
  • Synovial Fluid: Fluid from a joint
  • Amniotic Fluid: Fluid that surrounds the fetus
  • Gram Stain: Staining technique to differentiate bacteria
  • Culture: Growth of organisms in the laboratory
  • Alpha-Hemolysis: Partial lysis of red blood cells
  • Beta-Hemolysis: Complete lysis of red blood cells
  • Optochin Susceptibility: Used to differentiate Streptococcus pneumoniae
  • 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 or automated methods for identifying microorganisms
  • MALDI-TOF Mass Spectrometry: A proteomic-based technique that provides rapid and accurate bacterial identification based on protein profiles
  • Beta-Lactamase: An enzyme produced by some bacteria that breaks down beta-lactam antibiotics (e.g., penicillins, cephalosporins)
  • Carbapenemase: An enzyme that inactivates carbapenem antibiotics
  • MRSA (Methicillin-Resistant Staphylococcus aureus): Staphylococcus aureus that is resistant to methicillin and other beta-lactam antibiotics
  • VRE (Vancomycin-Resistant Enterococci): Enterococci that are resistant to vancomycin
  • ESBL (Extended-Spectrum Beta-Lactamase): An enzyme produced by some bacteria that inactivates a broad range of beta-lactam antibiotics, including penicillins, cephalosporins, and monobactams
  • Antimicrobial Stewardship: A coordinated program that promotes the appropriate use of antibiotics, improving patient outcomes and reducing antimicrobial resistance
  • 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