Colony Morphology & ID

This section provides details on colony morphology and rapid tests used for the presumptive identification of BSL-3 pathogens and Select Agents, focusing on Bacillus anthracis, Yersinia pestis, Brucella spp., and Francisella tularensis. It is crucial that all procedures are performed in a BSL-3 laboratory with strict adherence to safety protocols

General Safety and Procedural Considerations

Biosafety Level 3 (BSL-3) Laboratory: All procedures must be conducted in a certified BSL-3 laboratory, equipped with:
- Proper ventilation (negative pressure)
- Sealed windows and doors
- Access control
- Appropriate personal protective equipment (PPE)
Personal Protective Equipment (PPE): Mandatory use of:
- Gloves (double gloving recommended)
- Gowns (impermeable)
- Respiratory protection (N95 respirator or higher; powered air-purifying respirator - PAPR - may be required)
- Eye protection (face shield or goggles)
Biosafety Cabinet (BSC): All manipulations of cultures or potentially infectious materials must be performed within a certified Class II BSC
Training: Personnel must receive comprehensive training in:
- BSL-3 laboratory practices
- Handling of Select Agents
- Use of PPE
- Emergency procedures
Decontamination
- Work surfaces must be disinfected with an appropriate disinfectant (e.g., 10% bleach solution, approved sporicidal agent) before and after use, and after any spill
- All waste materials (cultures, used PPE, etc.) must be autoclaved or appropriately decontaminated before disposal
Chain of Custody: Maintain meticulous chain of custody documentation for all specimens and isolates
Reporting: Immediately report any suspected Select Agent isolates or positive results to the appropriate authorities (e.g., local and state health departments, CDC)
Standard Operating Procedures (SOPs): Adhere to detailed, written SOPs for all procedures

Colony Morphology and Presumptive Identification

This section outlines the key colony characteristics and rapid tests used for presumptive identification. Remember that these are presumptive and must be confirmed by more definitive methods

Bacillus anthracis (Anthrax)

Specimen Sources: Blood, sputum, tissue (skin lesions), other body fluids
Colony Morphology
- Media: Blood agar is the primary medium
- Appearance
  - Non-hemolytic (no clearing of blood agar around the colonies)
  - Large (2-5 mm in diameter) colonies
  - Grayish-white to slightly opaque
  - “Ground glass” or “frosted glass” appearance
  - Irregular, “comma-shaped” or “medusa head” projections at the colony edges (often described as “whipped egg white”)
  - May have a tenacious, stringy consistency when touched with a loop (“string of pearls” test - see below)
Rapid Tests
- Gram Stain
  - Large, Gram-positive rods
  - Often arranged in chains (bamboo cane appearance)
  - Spores may be visible (but not always) as refractile, oval structures
- “String of Pearls” Test
  - A drop of a broth culture is mixed with a drop of bacteriophage gamma (specific for B. anthracis)
  - A loopful of the mixture is streaked onto a microscope slide
  - After 1-2 hours of incubation at 37°C, the presence of long chains of bacterial cells (resembling a string of pearls) is a positive result
- Catalase Test: Positive
- Motility: Non-motile
- Gamma Phage Lysis: B. anthracis is susceptible to lysis by gamma phage
- Direct Fluorescent Antibody (DFA): DFA tests using antibodies specific to B. anthracis are available for rapid identification in clinical samples

Yersinia pestis (Plague)

Specimen Sources: Blood, sputum, lymph node aspirate/biopsy, tissue
Colony Morphology
- Media: Blood agar, chocolate agar
- Appearance
  - Small (pinpoint to 1 mm in diameter) colonies
  - Grayish-white or translucent
  - Non-hemolytic
  - “Fried egg” appearance (may be seen with prolonged incubation)
  - May become more mucoid with prolonged incubation
Rapid Tests
- Gram Stain
  - Small, Gram-negative coccobacilli or short rods
  - May show bipolar staining (safety pin appearance) with special stains (e.g., Wayson stain)
- Catalase Test: Positive
- Oxidase Test: Negative
- Urease Test: Negative
- Motility: Non-motile
- Rapid Antigen Tests: Rapid lateral flow immunoassays are available for the detection of Y. pestis F1 antigen in clinical samples
- DFA: DFA tests using antibodies specific to Y. pestis are available for rapid identification in clinical samples

Brucella spp. (Brucellosis)

Specimen Sources: Blood, bone marrow, other body fluids, tissue
Colony Morphology
- Media: Blood agar (requires prolonged incubation), chocolate agar
- Appearance
  - Small (0.1-1 mm in diameter) colonies
  - Non-hemolytic
  - Translucent to slightly opaque
  - Smooth, glistening colonies
  - May take several days to weeks to appear (slow-growing)
  - Older colonies may become slightly yellow or brownish
Rapid Tests
- Gram Stain
  - Small, Gram-negative coccobacilli or short rods
  - Often difficult to visualize in Gram stains due to their small size and pleomorphism
- Catalase Test: Positive
- Oxidase Test: Positive (variable, may be weak)
- Urease Test: Positive
- H2S Production: Positive
- Motility: Non-motile
- Rapid Agglutination Tests: Rapid agglutination tests (e.g., card agglutination test) are available for the detection of Brucella antibodies in serum. However, these are not direct identification methods
- DFA: DFA tests using antibodies specific to Brucella spp. are available for rapid identification in clinical samples

Francisella tularensis (Tularemia)

Specimen Sources: Blood, sputum, tissue (ulcers, lymph nodes), lymph node aspirate/biopsy
Colony Morphology
- Media: Requires enriched media containing cysteine or cystine (e.g., chocolate agar with cysteine, buffered charcoal yeast extract (BCYE) agar)
- Appearance
  - Very small (pinpoint to 0.5 mm in diameter) colonies
  - Grayish-white or translucent
  - Non-hemolytic
  - Smooth, slightly mucoid colonies
  - May take several days to appear (slow-growing)
Rapid Tests
- Gram Stain
  - Small, Gram-negative coccobacilli or short rods
  - Often difficult to visualize in Gram stains due to their small size
- Catalase Test: Negative
- Oxidase Test: Negative
- Motility: Non-motile
- DFA: DFA tests using antibodies specific to F. tularensis are available for rapid identification in clinical samples
- Serological Tests: Antibody detection (e.g., ELISA, microagglutination) can be helpful, but are not direct identification methods
- PCR: Molecular methods (e.g., PCR) are highly sensitive and specific and are often used for rapid detection and confirmation

Confirmation and Further Identification

Definitive Identification: Presumptive identification based on colony morphology and rapid tests must be confirmed by more definitive methods
Biochemical Tests: Standard biochemical tests (e.g., carbohydrate fermentation, enzyme production) are used to further characterize the isolates
Molecular Methods: PCR, real-time PCR, and sequencing are increasingly used for rapid and definitive identification of these pathogens
Serotyping/Phage Typing: May be used to further characterize and differentiate isolates
Reporting: Report all confirmed or suspected Select Agent isolates to the appropriate authorities

Important Considerations

Specimen Quality: The quality of the specimen is critical for successful identification. Ensure proper collection, transport, and storage of specimens
Communication: Maintain open communication with clinicians, public health officials, and the laboratory team
Documentation: Thoroughly document all procedures, results, and communications
Training and Competency: Ensure that all personnel are properly trained and demonstrate competency in the procedures
Emerging Technologies: Be aware of and adopt new technologies (e.g., MALDI-TOF MS) for improved identification and characterization

Key Terms

Bioterrorism Agent: A biological agent (bacteria, virus, toxin, etc.) intentionally used to cause harm or death to humans, animals, or plants. These agents are often chosen for their ease of production, dissemination, and potential for causing widespread illness or panic
BSL-3 Laboratory: A laboratory designed to handle infectious agents that can cause serious or potentially lethal disease through aerosol transmission. This requires specific engineering controls (e.g., directional airflow, sealed windows/doors), specialized equipment (e.g., biosafety cabinets), and stringent safety practices
Select Agent: A biological agent or toxin that has been identified by the U.S. government as posing a severe threat to public health and safety, animal or plant health, or to the safety of animal or plant products. Handling, storage, and transfer of Select Agents are strictly regulated
Colony Morphology: The visual characteristics of bacterial colonies grown on solid media, including size, shape, color, texture, edge, and elevation. These characteristics provide valuable clues for presumptive identification
Gram Stain: A differential staining technique used to classify bacteria based on their cell wall structure. Gram-positive bacteria stain purple, while Gram-negative bacteria stain pink/red. It’s a crucial first step in identifying an unknown bacterial isolate
Presumptive Identification: A preliminary identification of a microorganism based on initial tests, such as colony morphology, Gram stain, and rapid biochemical tests. Presumptive results must be confirmed by more definitive methods
Biosafety Cabinet (BSC): A ventilated cabinet designed to protect the user, the environment, and the materials being handled from exposure to infectious agents. BSCs are essential for working with BSL-3 pathogens, providing a primary containment barrier
Aerosol Transmission: The spread of infectious agents through the air in the form of small droplets or particles. Many BSL-3 pathogens are transmitted via aerosol, making it a significant risk in the laboratory
Rapid Test: A quick diagnostic test used to provide preliminary identification or detection of a microorganism or its products (e.g., antigens, toxins) within a short timeframe (minutes to hours). Examples include catalase test, oxidase test, or rapid antigen tests
Chain of Custody: A documented process that tracks the movement of a specimen or isolate from collection to disposal, ensuring its integrity and providing a record of who handled it and when. This is essential in cases of suspected bioterrorism or Select Agent investigations to maintain accountability

Specimen sources

Laboratory Response Network