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Microbiology

Colony Morphology & ID

The ability to recognize and identify major bacterial and fungal pathogens from blood and bone marrow cultures is paramount for timely and accurate diagnosis, crucial for guiding antimicrobial therapy and improving patient outcomes

General Principles

  • Culture Media: Blood and bone marrow cultures are typically inoculated onto a variety of media
    • Blood Agar (BAP): A general-purpose, enriched, non-selective medium that supports the growth of most bacteria
    • Chocolate Agar (CHOC): An enriched medium that supports the growth of fastidious organisms and enhances the recovery of Haemophilus influenzae and Neisseria meningitidis
    • Anaerobic Media: Used for blood cultures to support the growth of anaerobic organisms
    • Selective and Differential Media: Media used when specific organisms are suspected
  • Incubation: Cultures are typically incubated at 35-37°C in an incubator with a 5% CO2 atmosphere
  • Daily Examination: Cultures are examined daily for the presence of bacterial or fungal growth. The technologist examines plates for
    • Colony Morphology (size, shape, color, texture, hemolysis)
    • Gram Stain Characteristics
  • Reporting: All isolates are identified. A Gram stain of the isolate is performed, which provides the initial information for a potential identification
  • Identification Methods
    • Conventional biochemical tests
    • Commercial identification systems
    • MALDI-TOF mass spectrometry
    • Molecular methods
  • Antimicrobial Susceptibility Testing: Performed on all clinically significant isolates to guide antibiotic therapy

Major Pathogens: Colony Morphology, Gram Stain, and Identification

Staphylococcus aureus

  • Colony Morphology
    • BAP: Medium to large, circular, opaque, smooth, golden-yellow or cream-colored colonies
    • Hemolysis: Usually beta-hemolytic (complete clearing of red blood cells)
    • Appearance: Often described as “buttery” in texture
  • Gram Stain: Gram-positive cocci in clusters (grape-like)
  • Identification
    • Catalase: Positive
    • Coagulase: Positive (confirmatory test, usually tube coagulase)
    • Other tests: Catalase, coagulase, and other tests are often performed using commercial identification kits or automated systems

Other Staphylococcus spp. (Including Coagulase-Negative Staphylococci)

  • Colony Morphology
    • BAP: Colonies vary in size, shape, and pigmentation depending on the species. Most are white or cream-colored
    • Hemolysis: Usually non-hemolytic or gamma-hemolytic (no hemolysis)
  • Gram Stain: Gram-positive cocci in clusters (grape-like)
  • Identification
    • Catalase: Positive
    • Coagulase: Negative (CoNS)
    • Further Identification: Differentiation of CoNS species often requires:
      • Susceptibility to Novobiocin: Used to differentiate S. saprophyticus (resistant) from other CoNS
      • Commercial Identification Systems: Used to identify individual CoNS species, such as S. epidermidis, S. haemolyticus, S. hominis, S. capitis, etc

Beta-Hemolytic Streptococci

  • Colony Morphology
    • BAP: Small to medium, translucent or gray colonies
    • Hemolysis: Beta-hemolytic (complete clearing of red blood cells)
  • 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
      • Other Beta-Hemolytic Streptococci: May require serological or biochemical tests for identification (e.g., group C, group G)

Enterococcus spp.

  • Colony Morphology
    • BAP: Grayish, medium-sized colonies
    • Hemolysis: Non-hemolytic or alpha-hemolytic (greening)
  • 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
    • Species Identification: E. faecalis and E. faecium are the most clinically significant species and may require additional testing

Candida spp.

  • Colony Morphology
    • BAP: Creamy, white, or off-white colonies. Can have a “yeasty” odor
    • CHOC: Similar to BAP
    • Appearance: Colonies may be smooth, mucoid, or wrinkled
    • Germ tube test: A presumptive identification test. A positive test results in the formation of germ tubes (hyphae-like structures) when the yeast is incubated in serum
  • Gram Stain: Gram-positive budding yeast cells and/or pseudohyphae
  • Identification
    • Germ Tube Test: Candida albicans is often germ tube positive
    • Other Tests: Additional biochemical tests are often used to differentiate Candida species (e.g., C. albicans, C. glabrata, C. parapsilosis, C. tropicalis)
    • Commercial Identification Systems: Often used for rapid and accurate 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

Acinetobacter baumannii

  • 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
    • Colonies can be small and non-hemolytic
  • 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

Enterobacteriaceae (e.g., Escherichia coli, Klebsiella pneumoniae, Proteus spp.)

  • Colony Morphology
    • BAP: Large, gray, mucoid, often with a distinct odor
    • Appearance
      • E. coli: Gray, often with a metallic sheen
      • Klebsiella pneumoniae: Mucoid (especially on BAP)
      • Proteus spp.: Swarming colonies
  • Gram Stain: Gram-negative rods
  • Identification
    • Oxidase: Negative
    • Glucose Fermentation: Positive
    • Lactose Fermentation: Variable (important for differentiating species)
    • IMViC Tests: (Indole, Methyl Red, Voges-Proskauer, Citrate):
      • E. coli: + + - -
      • Klebsiella pneumoniae: - - + +
      • Proteus mirabilis: + - + +
    • Commercial Identification Systems or MALDI-TOF: Used for species-level identification

Pseudomonas spp. (Pseudomonas aeruginosa)

  • 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 (uses glucose but does not ferment it)
    • Motility: Motile
    • Commercial Identification Systems or MALDI-TOF: Used for species-level identification

Additional Considerations

  • Specimen Source: The clinical significance of an isolate is highly dependent on the source of the specimen. For example, the isolation of S. aureus from a blood culture is concerning
  • Antimicrobial Resistance: Always perform antimicrobial susceptibility testing and report results according to established guidelines
  • Reporting: Promptly report all significant isolates 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

  • Colony Morphology: The visual characteristics of bacterial colonies on culture media (size, shape, color, texture, hemolysis)
  • Gram Stain: A staining technique that differentiates bacteria based on cell wall composition (Gram-positive or Gram-negative)
  • Catalase Test: A biochemical test that detects the presence of the enzyme catalase
  • Coagulase Test: A biochemical test that detects the presence of the enzyme coagulase
  • Hemolysis: The lysis (breakdown) of red blood cells by bacterial enzymes
  • Alpha-Hemolysis: Partial lysis of red blood cells, producing a green discoloration around the colonies
  • Beta-Hemolysis: Complete lysis of red blood cells, producing a clear zone around the colonies
  • Non-Hemolytic: No lysis of red blood cells
  • Oxidase Test: A biochemical test that detects the presence of the enzyme cytochrome c oxidase
  • Glucose Fermentation: The ability of a bacterium to break down glucose with the production of acid and/or gas
  • IMViC Tests: A series of biochemical tests used to identify Enterobacteriaceae (Indole, Methyl Red, Voges-Proskauer, Citrate)
  • Antimicrobial Susceptibility Testing: Laboratory tests performed to determine the susceptibility of a bacterial isolate to various antibiotics
  • Commercial Identification System: A system that uses pre-packaged biochemical tests or automated methods for identifying microorganisms (e.g., API, Vitek)
  • 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