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Microbiology

Sample Sources

Body fluids from normally sterile sites, such as the pleural, peritoneal, pericardial, vitreous and aqueous humor, synovial, and amniotic fluids, are considered sterile under normal circumstances. The presence of microorganisms in these fluids indicates infection, which requires rapid and accurate diagnosis for effective patient management. Obtaining, handling, and processing these samples with the appropriate protocols is essential for the laboratory’s role

General Principles

  • Sterile Environments: These body fluids are typically sterile, meaning they should not contain any microorganisms in a healthy individual
  • Infection Indication: The presence of microorganisms in these fluids is indicative of infection and usually warrants aggressive therapy
  • Specimen Collection
    • Aseptic Technique: Strict aseptic technique is absolutely critical during collection to avoid contamination
    • Physician Procedure: Typically performed by a physician (e.g., pulmonologist, gastroenterologist, cardiologist, ophthalmologist, obstetrician, orthopedic surgeon)
    • Invasive Procedures: Collection often involves invasive procedures, such as:
      • Thoracentesis: Pleural fluid
      • Paracentesis: Peritoneal fluid
      • Pericardiocentesis: Pericardial fluid
      • Vitreous/Aqueous Tap: Vitreous/Aqueous fluid
      • Arthrocentesis: Synovial fluid
      • Amniocentesis: Amniotic fluid
  • Laboratory Procedures
    • Rapid and accurate testing to identify or rule out infection is paramount
    • Initial tests are performed (e.g., Gram stains) to provide preliminary information
    • Cultures are performed to identify the infecting organism
    • Other tests (e.g., cell counts, chemistry) are done to provide additional information

Specific Specimen Sources

Pleural Fluid (Thoracentesis)

  • Source: The space between the lung and the chest wall
  • Indications
    • Pleural effusion (fluid accumulation in the pleural space)
    • Suspicion of empyema (pus in the pleural space)
    • Suspicion of lung infection with pleural involvement
  • Procedure
    • Performed by a physician. Patient is positioned for easy access to the pleural space
    • The site is prepped with an antiseptic solution
    • A needle is inserted into the pleural space
    • Fluid is aspirated
  • Collection Considerations
    • Sufficient volume is needed for various tests
    • Sterile collection containers are used

Peritoneal Fluid (Paracentesis)

  • Source: The abdominal cavity
  • Indications
    • Ascites (fluid accumulation in the peritoneal cavity)
    • Suspicion of peritonitis (inflammation of the peritoneum)
    • Suspected infection in the abdominal cavity
  • Procedure
    • Performed by a physician. Patient is positioned for easy access to the abdomen
    • The site is prepped with an antiseptic solution
    • A needle is inserted into the peritoneal space
    • Fluid is aspirated
  • Collection Considerations
    • Sufficient volume is needed for various tests
    • Sterile collection containers are used

Pericardial Fluid (Pericardiocentesis)

  • Source: The space surrounding the heart
  • Indications
    • Pericardial effusion (fluid accumulation around the heart)
    • Suspicion of pericarditis (inflammation of the pericardium)
    • Suspected infection around the heart
  • Procedure
    • Performed by a physician, with ECG guidance
    • The site is prepped with an antiseptic solution
    • A needle is inserted into the pericardial space
    • Fluid is aspirated
  • Collection Considerations
    • A small volume is usually collected
    • Sterile collection containers are used

Vitreous and Aqueous Humor (Eye)

  • Source: Fluids of the eye
    • Vitreous humor: The gel-like substance that fills the eye behind the lens
    • Aqueous humor: The fluid that fills the space between the cornea and the lens
  • Indications
    • Endophthalmitis (inflammation of the inside of the eye), especially following eye surgery or trauma
    • Suspicion of intraocular infection
  • Procedure
    • Performed by an ophthalmologist
    • Under sterile conditions. The site is prepped with an antiseptic solution
    • A needle is inserted into the eye to aspirate fluid
  • Collection Considerations
    • Very small volumes are collected
    • Requires immediate processing
    • Sterile collection containers are used

Synovial Fluid (Arthrocentesis)

  • Source: Fluid from a joint space
  • Indications
    • Arthritis (inflammation of a joint)
    • Suspicion of septic arthritis (joint infection)
  • Procedure
    • Performed by a physician. The joint is prepared with antiseptic
    • A needle is inserted into the joint space
    • Fluid is aspirated
  • Collection Considerations
    • Sufficient volume is needed
    • Sterile collection containers are used

Amniotic Fluid (Amniocentesis)

  • Source: Fluid that surrounds the fetus in the amniotic sac
  • Indications
    • Suspicion of intrauterine infection
    • Premature rupture of membranes (PROM)
  • Procedure
    • Performed by an obstetrician
    • The site is prepped with antiseptic
    • A needle is inserted into the amniotic sac
    • Fluid is aspirated
  • Collection Considerations
    • Sterile collection containers are used
    • The sample should be transported to the laboratory as quickly as possible

Specimen Handling and Processing

  • Rapid Transport: Transport specimens to the laboratory as quickly as possible after collection
  • Prompt Processing: Processing should be done as soon as possible after receipt in the laboratory
  • Specimen Volume: Collect adequate volumes
  • Initial Processing
    • Visual Inspection: Note the appearance of the fluid (e.g., clear, cloudy, purulent, bloody)
    • Cell Counts: Perform total cell count and differential cell count, if requested
    • Gram Stain: Perform immediately
  • Culture
    • Inoculation: Inoculate the fluid onto appropriate media
      • Blood agar
      • Chocolate agar
      • Anaerobic media, if appropriate
    • Incubation: Incubate cultures at 35-37°C in a CO2 incubator
    • Incubation Time: Incubate cultures for at least 5-7 days
    • Observation: Examine cultures daily for growth

Factors Affecting Specimen Quality

  • Aseptic Technique
    • Essential
    • Prevent Contamination
  • Traumatic Tap
    • Can cause blood
    • May interfere with Gram staining and culture
  • Anticoagulants
    • Usually not needed
    • Can be used if delayed processing
  • Specimen Volume
    • Sufficient volume is needed
  • Transportation
    • Prompt transport
    • Optimal conditions

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
  • Thoracentesis: Procedure to collect pleural fluid
  • Paracentesis: Procedure to collect peritoneal fluid
  • Pericardiocentesis: Procedure to collect pericardial fluid
  • Arthrocentesis: Procedure to collect synovial fluid
  • Amniocentesis: Procedure to collect amniotic fluid
  • Aseptic Technique: Procedure to prevent contamination
  • Gram Stain: Staining technique to differentiate bacteria
  • Culture: Growth of organisms in the laboratory
  • Pleural Effusion: Fluid in the pleural space
  • Empyema: Pus in the pleural space
  • Peritonitis: Inflammation of the peritoneum
  • Endophthalmitis: Inflammation of the eye
  • Arthritis: Inflammation of a joint
  • Septic Arthritis: Joint infection
  • Intrauterine infection: Infection of the fetus
  • PROM: Premature rupture of membranes
  • Turbid: Cloudy
  • Purulent: Containing pus
  • Cytology: Evaluation of cells