Characterization and identification of microorganisms

Microorganisms are extremely diverse and often invisible to the naked eye. To study them scientifically, we need to characterize (describe their properties) and identify (determine their exact taxonomic position).

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1. Characterization of Microorganisms

Characterization means describing the morphological, physiological, biochemical, genetic, and ecological features of microbes.

a) Morphological Characteristics

• Cell shape: cocci (spherical), bacilli (rod), spirilla (spiral), vibrios, filamentous.
• Cell size: nanobacteria (<0.3 µm) to giant bacteria (>500 µm).
• Colony characteristics: shape, size, margin, elevation, pigmentation, opacity.
• Cell structures:
  • Cell wall type (Gram-positive / Gram-negative by Gram staining).
  • Presence of spores, capsules, flagella, pili.
  • Ultrastructure by electron microscopy.

b) Physiological and Metabolic Characteristics

• Nutritional requirements: autotrophs, heterotrophs.
• Oxygen requirement: aerobic, anaerobic, facultative, microaerophilic.
• Temperature range: psychrophiles, mesophiles, thermophiles, hyperthermophiles.
• pH tolerance: acidophiles, neutrophiles, alkaliphiles.
• Salt tolerance: halophiles.
• Metabolic activities: fermentation, respiration, nitrogen fixation, photosynthesis.

c) Biochemical Characteristics

• Enzyme production: catalase, oxidase, urease, amylase, protease.
• Carbohydrate utilization (sugar fermentation tests).
• Production of acids, gases, alcohols.
• Antibiotic resistance/sensitivity patterns.

d) Genetic and Molecular Characteristics

• DNA base composition (G+C content).
• Nucleic acid hybridization (DNA-DNA relatedness).
• Sequencing of conserved genes (e.g., 16S rRNA for prokaryotes, 18S rRNA for eukaryotes).
• Whole-genome sequencing and comparative genomics.

e) Ecological Characteristics

• Natural habitat: soil, water, host-associated, extreme environments.
• Symbiotic or pathogenic relationships.
• Role in biogeochemical cycles (carbon, nitrogen, sulfur).

2. Identification of Microorganisms

Identification = Determining the name and taxonomic group of an isolate.

a) Classical Methods

1. Staining techniques
   1. Gram staining → Gram-positive or Gram-negative.
   1. Acid-fast staining (Ziehl–Neelsen) → e.g., Mycobacterium.
   1. Endospore, capsule, flagella staining.
2. Culture methods
   1. Isolation of pure cultures using streak plate, spread plate, or pour plate methods.
   1. Use of selective/differential media (e.g., MacConkey agar, Mannitol salt agar).
3. Biochemical tests
   1. IMViC tests (Indole, Methyl Red, Voges-Proskauer, Citrate).
   1. Triple sugar iron (TSI) test.
   1. API strips (commercial biochemical test panels).

b) Molecular Methods

• 16S rRNA gene sequencing: Standard method for bacterial identification.
• PCR-based methods: Amplification of specific genes for rapid identification.
• DNA hybridization: Determines genetic relatedness.
• Whole-genome sequencing: High-resolution identification and classification.
• MALDI-TOF Mass Spectrometry: Identifies microbes based on protein fingerprints.

c) Immunological Methods

• Use of specific antibodies to detect microbes (serotyping, ELISA).
• Agglutination reactions for bacterial strains.
• Immunofluorescence microscopy.

d) Automated and Computational Tools

• Computer-based numerical taxonomy (phenetic analysis).
• Bioinformatics tools for sequence analysis.
• Databases (NCBI, RDP, Bergey’s Manual, etc.) for reference identification.

3. Bergey’s Manual of Systematic Bacteriology

• Standard reference for classification and identification of bacteria.
• Provides morphological, biochemical, and molecular characteristics for each genus/species.
• Used worldwide for microbial taxonomy.

4. Significance of Characterization and Identification

• Medical microbiology: Identification of pathogens for diagnosis and treatment.
• Food industry: Detection of spoilage and foodborne pathogens.
• Agriculture: Identifying beneficial microbes (biofertilizers, biocontrol agents).
• Environmental studies: Characterizing microbial roles in ecosystems.
• Biotechnology: Selection of microbial strains for industrial production.