00 Review on diagnostics microbiology

Detection of Bacteria in Clinical Specimens

• General procedures include:
  • Appropriate specimen collection
  • Microscopy analysis
  • Cultivation in artificial media to isolate pure cultures and evaluate biochemical activities
  • Use of lab animals and cell cultures for certain bacteria and viruses
  • Isolation of pure cultures
  • Identification through biochemical assays, serology, and phage typing
  • Antimicrobial susceptibility testing
  • Interpretation of results

Structure of Gram-Positive and Gram-Negative Bacteria

• Gram-Positive Bacteria:

  • Thick multilayered cell wall consisting mainly of peptidoglycan (up to 95%) surrounding the cytoplasmic membrane.
  • Peptidoglycan is essential for survival, structure, and replication under hostile conditions.
  • Teichoic acids are present, contributing to cell viability and virulence. Includes lipoteichoic acids as surface antigens.

• Gram-Negative Bacteria:

  • More complex and includes two layers external to the cytoplasmic membrane.
  • Thin peptidoglycan layer without teichoic or lipoteichoic acids.
  • Outer membrane contains lipopolysaccharides (LPS), acting as endotoxins and damaging to the host.
  • Periplasmic space lies between the cytoplasmic and outer membranes.

Microscopic Slide Preparations

• Native (Wet Mount) Slides:
  • Used for observing alive bacteria, morphologies, motility, and cell division.
• Dry (Permanent, Stained) Slides:
  • Used for morphological studies and differential diagnoses.
• Dry Slide Preparation: Steps include smear preparation, drying, fixation, and staining.

Gram Staining

• Overview:
  • Developed by Hans Christian Gram in 1885.
  • Bacteria classified as:
  • Gram-Positive: Blue-violet
  • Gram-Negative: Pink/red
  • Gram-Variable: Mixed appearance.
• Principle:
  • Based on the retention of crystal violet dye during solvent treatment.
  • Gram-positive bacteria, with higher peptidoglycan and lower lipid content, retain the dye after treatment, while Gram-negative bacteria do not.

Gram Staining Procedure

1. Prepare a smear and heat-fix it.
2. Apply the primary stain (crystal violet).
3. Add iodide to bind and trap crystal violet in cells.
4. Decolorize using alcohol or acetone.
5. Rinse with water.
6. Counterstain using safranin or carbol fuchsin.

Important Rules for Gram Staining

• Old Gram-positive cultures may resemble Gram-negative.
• Ensure proper concentration of ethanol for decolorization (below 95% may not remove dye).
• Maintain even smear consistency.
• Follow timing for each step strictly.

Acid-Fast Bacteria (e.g., Mycobacteria)

• Distinctive thick, waxy cell walls rich in mycolic acids.
• Staining: Utilizes Ziehl-Neelsen method due to their acid resistance.

Spores (Endospores) Formation in Gram-Positive Bacteria

• Dormant, tough structures primarily found in Bacillus and Clostridium species.
• Formed during nutrient depletion and consist of DNA, ribosomes, and dipicolinic acid.
• Differ in shape, form, and internal location within bacteria.

Microscopy for Volutin Granules (Neisser's Method)

• Utilizes specific staining techniques to visualize granules in bacteria (e.g., Corynebacterium diphtheriae).
• Result: Cytoplasm appears clear; granules appear blue-black under oil immersion lens.

Types of Culture Media

• Minimal Essential: Basic media only with primary nutrients required for growth.
• Enriched Media: Contains additional nutrients (blood, serum) to promote growth of specific organisms.
• Selective Media: Contains inhibitors to suppress unwanted organisms and promote specific growth.
• Differential Media: Used to distinguish between organisms based on biochemical activities.

Antimicrobial Susceptibility Testing

• Necessary for managing infections, involves testing microorganisms against antibiotics.
• Common antibiotics tested include:
  • β-lactams (e.g., penicillins, cephalosporins)
  • Macrolides (e.g., erythromycin)
  • Aminoglycosides (e.g., gentamicin)
  • Quinolones (e.g., ciprofloxacin)

Testing Methods for Antibiotic Susceptibility

• Diffusion Tests: e.g., Kirby-Bauer disk diffusion to assess bacterial sensitivity to antimicrobial agents.
  • Measured by zones of inhibition around antibiotic disks after incubation.
• Dilution Tests: Variety includes broth dilution methods to determine Minimum Inhibitory Concentration (MIC).

Conclusion: Principles of Microbiological Diagnosis

• Identification of etiologic agents of disease through:
  • General characteristics
  • Morphology
  • Cultural features
  • Biochemical properties
  • Virulence factors
  • Epidemiology and pathogenicity
  • Immunological responses
  • Microbiological diagnostics and treatment options.