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 $O<em>2$ & $CO</em>2$.
  • 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.