Laboratory Diagnosis of Bacterial Infections

Lecture 9: Laboratory Diagnosis of Bacterial Infections (I)

• The lecture covers laboratory diagnosis of bacterial infections, focusing on specimen collection, bacterial identification, and result interpretation.

Specimen Collection & Transport

• Samples must be collected before antimicrobial therapy begins.
• Samples should be representative of the infection's source (e.g., CSF for meningitis, sputum for respiratory tract infections).
• Samples should be collected in an appropriate sterile container.
• Adequate sample volume is essential.
• Complete request forms containing pertinent information (date, sample type, department, etc.) must be included.
• Samples should be transported rapidly to the lab. Transport media should be used if delay is anticipated.

Bacterial Identification

• Direct Detection: Methods for directly identifying the causative bacteria, their antigens, or genetic material.
  • Microscopic examination (using various types of microscopes like ordinary light, dark field, fluorescence, and electron).
  • Culture and antibiotic sensitivity testing to identify specific bacteria and their susceptibility to different antibiotics.
  • Antigen detection to identify specific bacterial antigens.
  • Molecular tests (like PCR) to detect bacterial genetic material.
• Indirect Detection: Detects antibodies produced in response to a bacterial infection, not the bacteria itself.
  • Serology, which detects specific antibodies against the bacteria in blood.

Microscopic Examination

• Types of microscopes:
  • Ordinary light microscope, dark field microscope, fluorescence microscope, and electron microscope.
  • Dark field microscopes are used to detect Treponema pallidum (syphilis).
  • Fluorescence microscopes use UV light to detect Mycobacteria, certain intracellular organisms (e.g., Chlamydia), and viruses.
  • Electron microscopes are used to visualize viruses.
• Unstained (Wet Mount) Preparations: used to observe bacterial motility (e.g., Vibrio cholerae, Salmonella, E. coli, Proteus).
• Stained Preparations: used to determine bacteria's staining reaction, shape, and arrangement.
  • Gram stain: classifies bacteria into Gram-positive (purple) and Gram-negative (red) based on cell wall structure.
  • Ziehl-Neelsen stain (Acid-Fast Stain): detects bacteria like Mycobacterium spp. (e.g., those causing tuberculosis) which retain the stain during decolorization due to their waxy cell walls.

Gram Stain Steps

• Fix the specimen on a glass slide (heating or alcohol).
• Apply crystal violet (primary stain).
• Apply iodine (mordant).
• Decolorize with alcohol (95% ethanol).
• Apply safranin (counter stain).
• Examine stained smear using oil immersion lens (100x magnification).

Bacterial Culture

• Clinical samples are inoculated onto various culture media under specific temperature and atmospheric conditions.
• Media are classified by consistency: liquid (broth) and solid/semi-solid (agar plates).
• Media are also classified by ingredients and usage:
  • Transport media maintain viability during transport. Examples include Stuart, Amies, and Carry & Blair media.
  • Culture media support bacterial growth. Examples include simple (nutrient agar and nutrient broth), enriched (blood agar, chocolate agar, Loffler's serum agar), and selective (media that inhibit growth of unwanted organisms). -Differential media, such as blood agar, help classify bacteria based on hemolysis (breakdown of red blood cells) β (complete), α (partial), γ (no hemolysis).

Ziehl-Neelsen Stain

• Used for identifying Mycobacteria species (like Mycobacterium tuberculosis) with high lipid content in their cell walls.
• Primary stain: Carbol fuchsin.
• Decolorization: Sulfuric acid with alcohol.
• Counterstain: Methylene blue.
• Examine under oil immersion.

Case Study

• A 45-year-old man with a six-month history of night sweats, fever, and cough producing sputum, and weight loss, likely has Mycobacterium tuberculosis infection.
• Ziehl-Neelsen stain is crucial for diagnosis of Mycobacterium tuberculosis.

Important Note: This summary emphasizes concepts for understanding the methodology. Visual components, details about microbes, and specific results from a sample are necessary for a full lab report.