Colony Morphology & ID

This section provides a details on the key aspects of analyzing urine cultures in the clinical microbiology laboratory, focusing on the colony morphology and identification of the most common urinary pathogens. Accurate identification is crucial for appropriate antimicrobial therapy and patient management

Overview

Purpose: To identify and characterize the bacteria and yeast present in urine samples, enabling the diagnosis of urinary tract infections (UTIs) and guiding treatment decisions
Key Concepts
- Colony Morphology: The visual characteristics of bacterial colonies on agar plates, including size, shape, color, texture, and hemolysis, provide initial clues for identification
- Differential and Selective Media: Media used to isolate and differentiate bacterial species based on their growth characteristics and biochemical properties
- Identification Methods: A variety of techniques used to confirm the identity of bacterial isolates, including biochemical tests, rapid tests, automated systems, and molecular methods
- Antimicrobial Susceptibility Testing (AST): Testing to determine the susceptibility of the identified pathogen to various antibiotics, guiding treatment choices
- Reporting: Accurate and timely reporting of results to clinicians, including the identification of the organism, the quantity of organisms, and the antimicrobial susceptibility profile

Procedure

Specimen Processing

Inoculation: Urine specimens are typically inoculated onto agar plates using a calibrated loop (e.g., 0.001 or 0.01 mL). This allows for the quantification of bacterial growth
Media: Common media used include:
- Blood Agar Plate (BAP): A general-purpose, non-selective, enriched medium that supports the growth of most bacteria. Used for observing colony morphology and hemolysis
- MacConkey Agar (MAC): A selective and differential medium that inhibits the growth of Gram-positive bacteria and differentiates lactose fermenters (pink colonies) from non-fermenters (colorless colonies)
- Chromogenic Media: Media that incorporate chromogenic substrates, producing colonies with distinct colors based on the organism’s enzymatic activity. These are useful for presumptive identification and can aid in differentiating multiple organisms
Incubation: Plates are incubated at 35-37°C (95-98.6°F) in an aerobic atmosphere for 18-24 hours

Colony Examination

Macroscopic Observation: After incubation, examine the plates for the presence of bacterial growth. Note:
- Number of Colonies: Estimate the number of colonies and calculate the colony-forming units per milliliter (CFU/mL) based on the loop size (e.g., 0.001 mL loop: multiply the colony count by 1000)
- Colony Morphology: Observe and describe the colony characteristics for each distinct colony type:
  - Size: (e.g., pinpoint, small, medium, large)
  - Shape: (e.g., circular, irregular, filamentous)
  - Color: (e.g., white, gray, yellow, pink, red)
  - Texture: (e.g., smooth, rough, mucoid, dry, wrinkled)
  - Elevation: (e.g., flat, raised, convex, umbonate)
  - Opacity: (e.g., transparent, translucent, opaque)
  - Hemolysis (on BAP)
    - Alpha (α) hemolysis: Partial hemolysis, producing a greenish discoloration around the colonies
    - Beta (β) hemolysis: Complete hemolysis, producing a clear zone around the colonies
    - Gamma (γ) hemolysis: No hemolysis, no change in the agar around the colonies
Microscopic Examination (Gram Stain): Perform a Gram stain from representative colonies to determine the Gram reaction (positive or negative), cell shape (cocci, bacilli, etc.), and arrangement (e.g., chains, clusters). This provides further clues for identification

Identification of Major Urinary Pathogens

Here’s a breakdown of the key colony morphology and identification characteristics of common urinary pathogens:

Enterobacteriaceae (Gram-negative bacilli)

General Characteristics: Gram-negative rods or bacilli, ferment glucose, oxidase-negative
Colony Morphology
- BAP: Generally large, gray, or white colonies. May be mucoid or dry
- MAC: Most species ferment lactose, producing pink colonies. Non-fermenters appear as colorless colonies
Common Genera and Species
- Escherichia coli
  - Colony Morphology: Large, gray, mucoid colonies on BAP. Pink, lactose-fermenting colonies on MAC. Often has a characteristic “fried egg” appearance
  - Identification: Oxidase-negative, indole-positive, lactose-fermenting, motile
  - Significance: The most common cause of UTIs
- Klebsiella pneumoniae
  - Colony Morphology: Large, mucoid, encapsulated colonies on BAP. Pink, lactose-fermenting colonies on MAC
  - Identification: Oxidase-negative, indole-negative, urease-positive, lactose-fermenting, non-motile
  - Significance: Can cause UTIs, pneumonia, and other infections
- Proteus mirabilis
  - Colony Morphology: Swarming, spreading colonies on BAP, often forming a thin film. Colorless colonies on MAC
  - Identification: Oxidase-negative, indole-negative, urease-positive, motile
  - Significance: Can cause UTIs, often associated with catheter-associated UTIs
- Enterobacter spp. (e.g., E. cloacae)
  - Colony Morphology: Similar to E. coli on BAP, may be more mucoid. Pink, lactose-fermenting colonies on MAC
  - Identification: Oxidase-negative, indole-variable, motile
  - Significance: Can cause UTIs, pneumonia, and other infections
- Citrobacter spp. (e.g., C. freundii)
  - Colony Morphology: Similar to E. coli on BAP. Pink, lactose-fermenting colonies on MAC
  - Identification: Oxidase-negative, indole-negative, citrate-positive, motile
  - Significance: Can cause UTIs, often associated with nosocomial infections
- Serratia marcescens
  - Colony Morphology: Often produces a red pigment on BAP, especially after prolonged incubation. Pink, lactose-fermenting colonies on MAC
  - Identification: Oxidase-negative, indole-negative, DNase-positive, motile
  - Significance: Can cause UTIs, pneumonia, and other infections

Enterococcus spp. (Gram-positive cocci in chains)

General Characteristics: Gram-positive cocci, catalase-negative, facultative anaerobes
Colony Morphology
- BAP: Small to medium, gray, or white colonies. May exhibit alpha, beta, or gamma hemolysis
- MAC: Variable growth, may produce small, pink colonies if they ferment lactose
Common Species
- Enterococcus faecalis
  - Colony Morphology: Generally non-hemolytic or alpha-hemolytic on BAP
  - Identification: Catalase-negative, PYR-positive, bile esculin-positive, growth in 6.5% NaCl
  - Significance: A common cause of UTIs, often associated with catheter-associated UTIs
- Enterococcus faecium
  - Colony Morphology: Generally non-hemolytic or alpha-hemolytic on BAP
  - Identification: Catalase-negative, PYR-positive, bile esculin-positive, growth in 6.5% NaCl. Often resistant to ampicillin and vancomycin
  - Significance: Can cause UTIs, often more resistant to antibiotics than E. faecalis

Streptococcus agalactiae (Group B Streptococcus, GBS) (Gram-positive cocci in chains)

General Characteristics: Gram-positive cocci, catalase-negative, beta-hemolytic
Colony Morphology
- BAP: Small, gray, translucent colonies with beta-hemolysis (complete hemolysis)
Identification: Catalase-negative, beta-hemolytic, CAMP-positive, hippurate hydrolysis-positive
Significance: Can cause UTIs, especially in pregnant women and newborns

Candida spp. (Yeast)

General Characteristics: Eukaryotic, oval-shaped yeast cells
Colony Morphology
- BAP: Creamy, white, or slightly colored colonies. May have a “yeasty” odor
- MAC: Colonies appear as pale pink or colorless, as they do not ferment lactose
Common Species
- Candida albicans
  - Colony Morphology: Creamy, white colonies
  - Identification: Germ tube-positive, produces chlamydospores on cornmeal agar
  - Significance: A common cause of yeast UTIs, especially in patients with diabetes, indwelling catheters, or those taking antibiotics
- Candida glabrata
  - Colony Morphology: Small, white, or cream-colored colonies
  - Identification: Germ tube-negative, urease-negative
  - Significance: Increasingly common cause of yeast UTIs, often resistant to azole antifungals
- Candida parapsilosis
  - Colony Morphology: Creamy white to slightly pink colonies
  - Identification: Germ tube-negative
  - Significance: Can cause yeast UTIs, often associated with indwelling devices

Staphylococcus saprophyticus (Gram-positive cocci in clusters)

General Characteristics: Gram-positive cocci, catalase-positive, coagulase-negative
Colony Morphology
- BAP: Medium to large, white or cream-colored colonies
- MAC: Usually non-fermenting, producing non-pigmented colonies
Identification: Catalase-positive, coagulase-negative, novobiocin-resistant
Significance: A common cause of UTIs, particularly in young, sexually active women

Further Identification Methods

Biochemical Tests: If presumptive identification cannot be achieved based on colony morphology and Gram stain, perform additional biochemical tests, such as:
- Catalase Test: Differentiates catalase-positive (e.g., Staphylococcus) from catalase-negative (e.g., Streptococcus, Enterococcus)
- Oxidase Test: Differentiates oxidase-positive (e.g., Pseudomonas, Moraxella) from oxidase-negative (e.g., Enterobacteriaceae)
- Indole Test: Detects the production of indole from tryptophan (e.g., E. coli is indole-positive)
- Urease Test: Detects the production of urease, which hydrolyzes urea to ammonia (e.g., Proteus, Klebsiella are urease-positive)
- Citrate Utilization Test: Determines the ability of an organism to utilize citrate as a sole carbon source
- Sugar Fermentation Tests: Differentiates bacteria based on their ability to ferment various sugars (e.g., glucose, lactose, sucrose)
- PYR Test: Detects the enzyme L-pyrrolidonyl arylamidase (PYR), used to identify Enterococcus and Streptococcus pyogenes
- CAMP Test: Tests for the enhanced hemolysis of S. aureus by a factor released by Streptococcus agalactiae
- Bile Esculin Test: Differentiates Enterococcus from other Gram-positive cocci
- Growth in 6.5% NaCl: Differentiates salt-tolerant organisms like Enterococcus from other Gram-positive cocci
Rapid Identification Systems: Commercial systems (e.g., API, Vitek, MicroScan) can rapidly identify bacteria based on a panel of biochemical tests
Automated Systems: Automated systems, such as MALDI-TOF mass spectrometry, use mass spectrometry to analyze the protein profile of the organism, providing rapid and accurate identification
Molecular Methods: PCR-based assays and gene sequencing can be used for definitive identification, particularly for organisms that are difficult to identify using conventional methods

Antimicrobial Susceptibility Testing (AST)

Method: Perform AST using standardized methods (e.g., disk diffusion, broth microdilution) according to CLSI guidelines
Interpretation: Interpret results based on CLSI breakpoints, reporting the organism’s susceptibility or resistance to various antibiotics
Reporting: Report the results, including the identified organism, the quantity of organisms (CFU/mL), and the antibiotic susceptibility profile, to the clinician

Safety Precautions

Standard Precautions: Follow standard precautions, including hand hygiene, use of personal protective equipment (PPE), and safe handling of sharps
Biohazard Waste Disposal: Dispose of all biohazardous waste (e.g., culture plates, used swabs) in appropriate containers
Spill Control: Have a spill kit available to clean up any spills of potentially infectious material
Training: Ensure that all personnel receive appropriate training in laboratory safety procedures

Key Terms

Colony Morphology: The visual characteristics of bacterial colonies on agar plates, including size, shape, color, texture, and hemolysis
Blood Agar Plate (BAP): A general-purpose, non-selective, enriched medium that supports the growth of most bacteria
MacConkey Agar (MAC): A selective and differential medium that inhibits the growth of Gram-positive bacteria and differentiates lactose fermenters from non-fermenters
Gram Stain: A differential staining technique used to classify bacteria based on their cell wall structure
Catalase Test: A biochemical test that identifies bacteria that produce the enzyme catalase
Oxidase Test: A biochemical test that identifies bacteria that produce the enzyme cytochrome c oxidase
Indole Test: A biochemical test that detects the production of indole from tryptophan
Urease Test: A biochemical test that detects the production of urease
Antimicrobial Susceptibility Testing (AST): Testing to determine the susceptibility of a bacterial isolate to various antibiotics
Colony-Forming Units per milliliter (CFU/mL): A unit of measurement used to quantify the number of bacteria in a urine sample
Beta-Hemolysis: Complete lysis of red blood cells, producing a clear zone around bacterial colonies on blood agar
Alpha-Hemolysis: Partial lysis of red blood cells, producing a greenish discoloration around bacterial colonies on blood agar
Gamma-Hemolysis: No hemolysis, no change in the agar around the bacterial colonies
Germ Tube Test: A test used to differentiate Candida albicans from other Candida species
CAMP Test: A test used to differentiate Streptococcus agalactiae (Group B Streptococcus) from other beta-hemolytic streptococci
PYR Test: A test used to identify Enterococcus and Streptococcus pyogenes
Bile Esculin Test: A test used to differentiate Enterococcus from other Gram-positive cocci
Novobiocin Resistance: A test used to differentiate Staphylococcus saprophyticus from other coagulase-negative staphylococci
Lactose Fermentation: The process by which bacteria break down lactose, producing acid and often changing the color of the media
Motility: The ability of bacteria to move independently
Encapsulated: Bacteria with a protective outer layer (capsule) that can affect colony morphology

Specimen Sources

Correlation of Colony Counts