Microbiology – Culture, Media & Microscopy

Inoculation & Incubation

  • Inoculation: introduction of microbes onto/into media for culture.
  • Culture: propagation of microorganisms on nutrient medium.
  • Medium (media): nutrient-containing environment for microbial multiplication.
  • Sterile instruments: free of all life (incl. spores & viruses) before sampling.
  • Incubation: maintain cultures at 20!{-}!45\, ^\circ!C; may control O2 & CO2.
    • Liquid growth → cloudiness, sediment, scum, color.
    • Solid growth → discrete colonies.

Media Classification

  • Classified by:
    • Physical state
    • Chemical composition
    • Functional type (purpose)
  • Defined (synthetic): exact chemical formula known.
  • Complex: at least one component not chemically defined (extracts, digests).

Functional Media Types

  • General purpose: support wide range; e.g., brain-heart infusion.
  • Enriched: add complex organics (blood, serum, hemoglobin, growth factors) for fastidious spp.
  • Selective: agents inhibit many microbes allowing selected ones; e.g., mannitol salt agar.
  • Differential: all grow but show visible differences (color, size); blood agar → hemolysis patterns:
    • \beta: complete lysis
    • \alpha: partial
    • \gamma: none

Solid Media & Agar

  • Solid media: firm surface for colony isolation.
  • Agar: complex polysaccharide from red alga Gelidium; melts \approx100\, ^\circ!C, solidifies \approx40\, ^\circ!C.

Microscopy Essentials

  • Microbial sizes:
    • Viruses: 20{-}800\,\text{nm}
    • Smallest bacteria: \approx200\,\text{nm}
    • Largest bacteria: \approx750\,\mu m
    • Yeasts: 3{-}4\,\mu m; Protozoa: 100{-}300\,\mu m
  • Magnification: interaction of light waves with lens curvature.
  • Refraction: bending of light when passing mediums, forms image.
  • Student scope objectives: 4\times,\;10\times,\;40\times,\;100\times.
  • Oil immersion (100×):
    • Oil matches glass refractive index → prevents light scatter.
    • Resolves objects \ge0.2\,\mu m apart.
  • Resolution: ability to separate two close points.
  • Light microscope types: bright-field, dark-field, phase-contrast, interference.
  • Most powerful: electron microscope.

Slide Preparation & Staining

  • Wet/hanging drop: cells in fluid (water, broth, saline); shows true size, shape, motility.
  • Fixed smear: thin film of cells, air-dried, heat-fixed.
  • Simple stain: one dye (e.g., methylene blue).
  • Gram stain:
    • Gram-positive → purple.
    • Gram-negative → pink.
  • Acid-fast stain:
    • Acid-fast bacteria → pink.
    • Non-acid-fast → blue.
    • Detects Mycobacterium (TB, leprosy).

Inspection & Identification Methods

  • Phenotypic, genotypic, immunologic testing.
  • Analyses: macroscopic (colony) & microscopic (cell) observations.